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[ { "id": "PMC-1310901-00-TIAB__text", "type": "abstract", "text": [ "Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region \nAlthough the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia. \n" ], "offsets": [ [ 0, 1788 ] ] } ]

[ { "id": "PMC-1310901-00-TIAB_T1", "type": "Protein", "text": [ "interferon regulatory factor 4" ], "offsets": [ [ 19, 49 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T2", "type": "Protein", "text": [ "bcr" ], "offsets": [ [ 159, 162 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T3", "type": "Protein", "text": [ "abl" ], "offsets": [ [ 163, 166 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T4", "type": "Protein", "text": [ "interferon regulatory factor 4" ], "offsets": [ [ 368, 398 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 400, 405 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T6", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 659, 664 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 735, 740 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T8", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 867, 872 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T9", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 912, 917 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T10", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 983, 988 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1024, 1029 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1195, 1200 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T13", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1340, 1345 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T14", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1366, 1371 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T15", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1457, 1462 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T16", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1688, 1693 ] ], "normalized": [] }, { "id": "PMC-1310901-00-TIAB_T17", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1757, 1762 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-00-TIAB_E1", "type": "Negative_regulation", "trigger": { "text": [ "Down-regulation" ], "offsets": [ [ 0, 15 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E2" } ] }, { "id": "PMC-1310901-00-TIAB_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 55, 65 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T1" } ] }, { "id": "PMC-1310901-00-TIAB_E3", "type": "Regulation", "trigger": { "text": [ "deregulation" ], "offsets": [ [ 306, 318 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T4" } ] }, { "id": "PMC-1310901-00-TIAB_E4", "type": "Regulation", "trigger": { "text": [ "regulation" ], "offsets": [ [ 721, 731 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E5" } ] }, { "id": "PMC-1310901-00-TIAB_E5", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 741, 751 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T7" } ] }, { "id": "PMC-1310901-00-TIAB_E6", "type": "Regulation", "trigger": { "text": [ "altered" ], "offsets": [ [ 859, 866 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E7" } ] }, { "id": "PMC-1310901-00-TIAB_E7", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 873, 883 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T8" } ] }, { "id": "PMC-1310901-00-TIAB_E8", "type": "Regulation", "trigger": { "text": [ "influence" ], "offsets": [ [ 973, 982 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E9" } ] }, { "id": "PMC-1310901-00-TIAB_E9", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 989, 1002 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T10" } ] }, { "id": "PMC-1310901-00-TIAB_E10", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 1030, 1038 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T11" } ] }, { "id": "PMC-1310901-00-TIAB_E11", "type": "Positive_regulation", "trigger": { "text": [ "resulted" ], "offsets": [ [ 1135, 1143 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E12" } ] }, { "id": "PMC-1310901-00-TIAB_E12", "type": "Positive_regulation", "trigger": { "text": [ "increase" ], "offsets": [ [ 1183, 1191 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T12" } ] }, { "id": "PMC-1310901-00-TIAB_E13", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 1346, 1354 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T13" } ] }, { "id": "PMC-1310901-00-TIAB_E14", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1372, 1380 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T14" } ] }, { "id": "PMC-1310901-00-TIAB_E15", "type": "Negative_regulation", "trigger": { "text": [ "down-regulation" ], "offsets": [ [ 1463, 1478 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T15" } ] }, { "id": "PMC-1310901-00-TIAB_E16", "type": "Negative_regulation", "trigger": { "text": [ "down-regulated" ], "offsets": [ [ 1742, 1756 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_E17" } ] }, { "id": "PMC-1310901-00-TIAB_E17", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1763, 1773 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-00-TIAB_T17" } ] } ]

[ { "id": "PMC-1310901-00-TIAB_1", "entity_ids": [ "PMC-1310901-00-TIAB_T4", "PMC-1310901-00-TIAB_T5" ] } ]

[]

[ { "id": "PMC-1310901-01-INTRODUCTION__text", "type": "abstract", "text": [ "INTRODUCTION\nChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with a typical three phased course (chronic, accelerated and blastic phase) reflecting the loss of differentiation and malignant progress which inevitably leads to death after the blastic phase (1,2). The hallmark genetic aberration of CML is a reciprocal chromosomal translocation t(9;22) leading to expression of a bcr-abl fusion gene, an aberrant activated tyrosine kinase (2). Treatment with interferon alpha (IFN-alpha) prolongs survival of CML patients and is associated with a complete cytogenetic response in 5-33% of CML patients (1,2). Recently, we described an impaired expression of the interferon regulatory factor 4 (IRF-4) in CML, correlating with poor response to IFN-alpha treatment (3). The cause of the silencing of IRF-4 level remained unclear. \nInterferon regulatory factors (IRFs) are a family of transcriptional regulators defined by a characteristic homology in their DNA-binding domain. They play an important role in the regulation of various genes (such as IFNs, interleukins, MHC class I/II), apoptosis and differentiation/maturation (4-6). IRF-4 (ICSAT/Pip/MUM1/LSIRF) is one member with very restricted expression pattern: Predominately B- and activated T-lymphocytes are IRF-4 positive (7-11). In contrast to other IRFs, expression of IRF-4 cannot be induced by IFNs, but by antigen stimulation, crosslinking of T- or B-cell receptors or phorbol-myristate-acetate (10,11). Consistent with the restriction of expression to immunocompetent cells, mice with deletion of IRF-4 failed to develop mature and functionally active B- and T-lymphocytes (12), and the impaired expression of IRF-4 in CML was predominately found in T-cells (3). These data suggest a crucial role for IRF-4 in the function of immune cells. \nMethylation of dinucleotide cytosine-guanosine motifs (CpG), especially in CpG islands located in promoter regions, is one of the mechanisms of gene regulation in mammals and a common event of gene silencing in human neoplasias (13,14). As opposed to normal cells, hypermethylation of CpG islands is a frequently observed phenomenon in every cancer type. De novo DNA methylation of genes such as cell cycle, DNA repair, apoptosis and tumor suppressor genes is therefore thought to be involved in tumorigenesis (15-17). Examples for such aberrated genes are MGMT, DAPK, p14ARF, p15INK4b, p16INK4a, BRCA1, CDH13 and APAF-1 (17-19). In CML, methylation is known to regulate expression of the c-abl, the bcr gene and others (20-23), and the extent of methylation in the c-abl promoter has been shown to be associated with advanced disease (24). Hypermethylation due to overexpression of DNA methyltransferases (DNMTs) remains one possible explanation for de novo methylation in tumorigenesis. Recently, DNMTs have been shown to be up-regulated in hematopoietic malignancies (25). Methyl-CpG-binding proteins (MBPs) are thought to inhibit the binding of transcriptional factors to the promoter and are therefore discussed as one mechanism of transcription inhibition by hypermethylation (26). \nIn this work, we studied mechanisms of IRF-4 gene expression silencing in leukemic cells. We analyzed the IRF-4 promoter region for genetic aberrations and methylational status in IRF-4-positive and -negative hematopoietic cells. \n" ], "offsets": [ [ 0, 3346 ] ] } ]

[ { "id": "PMC-1310901-01-INTRODUCTION_T1", "type": "Protein", "text": [ "bcr-abl fusion gene" ], "offsets": [ [ 401, 420 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T2", "type": "Protein", "text": [ "interferon alpha" ], "offsets": [ [ 480, 496 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T3", "type": "Protein", "text": [ "IFN-alpha" ], "offsets": [ [ 498, 507 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T4", "type": "Protein", "text": [ "interferon regulatory factor 4" ], "offsets": [ [ 683, 713 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 715, 720 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T6", "type": "Protein", "text": [ "IFN-alpha" ], "offsets": [ [ 764, 773 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 819, 824 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T8", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1153, 1158 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T9", "type": "Protein", "text": [ "ICSAT" ], "offsets": [ [ 1160, 1165 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T10", "type": "Protein", "text": [ "Pip" ], "offsets": [ [ 1166, 1169 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T11", "type": "Protein", "text": [ "MUM1" ], "offsets": [ [ 1170, 1174 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T12", "type": "Protein", "text": [ "LSIRF" ], "offsets": [ [ 1175, 1180 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T13", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1286, 1291 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T14", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1350, 1355 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T15", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1582, 1587 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T16", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1695, 1700 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T17", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1786, 1791 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T18", "type": "Protein", "text": [ "MGMT" ], "offsets": [ [ 2383, 2387 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T19", "type": "Protein", "text": [ "DAPK" ], "offsets": [ [ 2389, 2393 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T20", "type": "Protein", "text": [ "p14ARF" ], "offsets": [ [ 2395, 2401 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T21", "type": "Protein", "text": [ "p15INK4b" ], "offsets": [ [ 2403, 2411 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T22", "type": "Protein", "text": [ "p16INK4a" ], "offsets": [ [ 2413, 2421 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T23", "type": "Protein", "text": [ "BRCA1" ], "offsets": [ [ 2423, 2428 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T24", "type": "Protein", "text": [ "CDH13" ], "offsets": [ [ 2430, 2435 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T25", "type": "Protein", "text": [ "APAF-1" ], "offsets": [ [ 2440, 2446 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T26", "type": "Protein", "text": [ "c-abl" ], "offsets": [ [ 2515, 2520 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T27", "type": "Protein", "text": [ "bcr" ], "offsets": [ [ 2526, 2529 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T28", "type": "Protein", "text": [ "c-abl" ], "offsets": [ [ 2592, 2597 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T29", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 3154, 3159 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T30", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 3221, 3226 ] ], "normalized": [] }, { "id": "PMC-1310901-01-INTRODUCTION_T31", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 3295, 3300 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-01-INTRODUCTION_E1", "type": "Positive_regulation", "trigger": { "text": [ "leading" ], "offsets": [ [ 374, 381 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E2" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 385, 395 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T1" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E3", "type": "Negative_regulation", "trigger": { "text": [ "impaired" ], "offsets": [ [ 656, 664 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E4" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E4", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 665, 675 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T4" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E5", "type": "Negative_regulation", "trigger": { "text": [ "silencing" ], "offsets": [ [ 806, 815 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T7" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E6", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1217, 1227 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T8" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E7", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1292, 1300 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T13" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E8", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1336, 1346 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T14" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E9", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 1366, 1373 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E8" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E10", "type": "Negative_regulation", "trigger": { "text": [ "deletion" ], "offsets": [ [ 1570, 1578 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T15" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E11", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1681, 1691 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T16" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E12", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 2488, 2496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E13" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E14", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 2488, 2496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E15" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E13", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2497, 2507 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T26" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E15", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2497, 2507 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T27" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E16", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 3165, 3175 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T29" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E17", "type": "Negative_regulation", "trigger": { "text": [ "silencing" ], "offsets": [ [ 3176, 3185 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_E16" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E18", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 3301, 3309 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T31" } ] }, { "id": "PMC-1310901-01-INTRODUCTION_E19", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 3315, 3323 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-01-INTRODUCTION_T31" } ] } ]

[ { "id": "PMC-1310901-01-INTRODUCTION_1", "entity_ids": [ "PMC-1310901-01-INTRODUCTION_T2", "PMC-1310901-01-INTRODUCTION_T3" ] }, { "id": "PMC-1310901-01-INTRODUCTION_2", "entity_ids": [ "PMC-1310901-01-INTRODUCTION_T4", "PMC-1310901-01-INTRODUCTION_T5" ] }, { "id": "PMC-1310901-01-INTRODUCTION_3", "entity_ids": [ "PMC-1310901-01-INTRODUCTION_T8", "PMC-1310901-01-INTRODUCTION_T10", "PMC-1310901-01-INTRODUCTION_T11", "PMC-1310901-01-INTRODUCTION_T9", "PMC-1310901-01-INTRODUCTION_T12" ] } ]

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[ { "id": "PMC-1310901-02-MATERIALS_AND_METHODS-01__text", "type": "abstract", "text": [ "Cell lines\nK-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative. \n" ], "offsets": [ [ 0, 341 ] ] } ]

[ { "id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 324, 329 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_E1", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 330, 338 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-02-MATERIALS_AND_METHODS-01_T1" } ] } ]

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[ { "id": "PMC-1310901-03-MATERIALS_AND_METHODS-02__text", "type": "abstract", "text": [ "Cell culture and stimulation\nAll cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany). When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods. Owing to their chemical instability fresh substances were re-added every 24 h. \n" ], "offsets": [ [ 0, 480 ] ] } ]

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[ { "id": "PMC-1310901-04-MATERIALS_AND_METHODS-03__text", "type": "abstract", "text": [ "Sequencing of the IRF-4 promoter\nFor analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended. The primers were 1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', 1-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3'; 2-forward: 5'-AGCGGTGAAACTGAGAGTGCGAGGT-3', 2-reverse: 5'-GCCACATCGCTGCAGTTTAG-3'. The products were cloned with the 'TOPO TA cloning kit' (Invitrogen, Groningen, The Netherlands). After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer. \n" ], "offsets": [ [ 0, 837 ] ] } ]

[ { "id": "PMC-1310901-04-MATERIALS_AND_METHODS-03_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 18, 23 ] ], "normalized": [] }, { "id": "PMC-1310901-04-MATERIALS_AND_METHODS-03_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 53, 58 ] ], "normalized": [] } ]

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[ { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04__text", "type": "abstract", "text": [ "Expression analysis\nTo analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany). An aliquot of 1 mug total RNA was used for cDNA synthesis as described previously (27). RNA expression analysis for IRF-4 and the reference gene beta-actin was carried out by semi-quantitative PCR as described previously (3,27). PCR products were verified by automated sequencing. PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28). \nFor analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29). Briefly, protein lysates were generated by incubating 1 x 106 cells in 100 microl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 microg/ml phenylmethylsulfonyl fluoride, 10 microl/ml protease-inhibitory-mix, 1 micromol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice. After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended. Protein lysates (70-100 microg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 microm; Millipore, Eschborn, Germany). Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris-HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 microl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively. The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany). The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-beta-actin (AC-74) (Sigma). \n" ], "offsets": [ [ 0, 1944 ] ] } ]

[ { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 35, 40 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 271, 276 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T3", "type": "Protein", "text": [ "beta-actin" ], "offsets": [ [ 300, 310 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T4", "type": "Protein", "text": [ "(DNMT1" ], "offsets": [ [ 502, 508 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T5", "type": "Protein", "text": [ "DNMT3A" ], "offsets": [ [ 509, 515 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T6", "type": "Protein", "text": [ "DNMT3B" ], "offsets": [ [ 517, 523 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T7", "type": "Protein", "text": [ "MeCP" ], "offsets": [ [ 525, 529 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T8", "type": "Protein", "text": [ "MBD1" ], "offsets": [ [ 531, 535 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T9", "type": "Protein", "text": [ "MBD2" ], "offsets": [ [ 537, 541 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T10", "type": "Protein", "text": [ "MBD4" ], "offsets": [ [ 546, 550 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 600, 605 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1838, 1843 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T13", "type": "Protein", "text": [ "ICSAT" ], "offsets": [ [ 1844, 1849 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T14", "type": "Protein", "text": [ "M-17" ], "offsets": [ [ 1851, 1855 ] ], "normalized": [] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T15", "type": "Protein", "text": [ "beta-actin" ], "offsets": [ [ 1915, 1925 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E1", "type": "Transcription", "trigger": { "text": [ "transcriptional" ], "offsets": [ [ 41, 56 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T1" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E2", "type": "Transcription", "trigger": { "text": [ "RNA expression" ], "offsets": [ [ 243, 257 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T2" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E3", "type": "Transcription", "trigger": { "text": [ "RNA expression" ], "offsets": [ [ 243, 257 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T3" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E4", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T4" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E5", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T5" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E6", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T6" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E7", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T7" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E8", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T8" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E9", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T9" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E10", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 467, 477 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T10" } ] }, { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_E11", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 614, 624 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_T11" } ] } ]

[ { "id": "PMC-1310901-05-MATERIALS_AND_METHODS-04_1", "entity_ids": [ "PMC-1310901-05-MATERIALS_AND_METHODS-04_T12", "PMC-1310901-05-MATERIALS_AND_METHODS-04_T14", "PMC-1310901-05-MATERIALS_AND_METHODS-04_T13" ] } ]

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[ { "id": "PMC-1310901-06-MATERIALS_AND_METHODS-05__text", "type": "abstract", "text": [ "Methylation-specific restriction-PCR-assay\nDNA was extracted with a commercial kit (Qiagen) as recommended. Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24). As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used. DNA (0.8 microg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h. Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A). The sequences of the primers were F1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3' (341 bp); F2-forward: 5'-AAGGTGAACTGAAGTCTGGAAGTGA-3', F2-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3' (474 bp). The PCR conditions were described elsewhere (3). PCR was performed with an annealing temperature of 62degreesC and 35 cycles. When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated. The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed. PCR products were verified by automated sequencing. \n" ], "offsets": [ [ 0, 1495 ] ] } ]

[ { "id": "PMC-1310901-06-MATERIALS_AND_METHODS-05_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 726, 731 ] ], "normalized": [] } ]

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[ { "id": "PMC-1310901-07-MATERIALS_AND_METHODS-06__text", "type": "abstract", "text": [ "Bisulfite treatment\nDNA was extracted as described above. Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows. Briefly, 1 microg of DNA and 2 microg of poly(dA-dT)(poly(dA-dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42degreesC in 0.3 M NaOH in a volume of 50 microl. Fresh solutions of 30 microl of 10 mM hydrochinon (Sigma) and 530 microl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12-13 h at 50degreesC. The aqueous phase was recovered using the 'Wizard DNA clean-up system' (Promega, Mannheim, Germany). The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37degreesC to ensure complete desulfonisation. DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 microl H2O. Subsequent PCR amplification of 4 microl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the 'TOPO TA cloning kit' (Invitrogen) (see Figure 3A). The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5'-TATTTGGATTTTTAGGGAGTTTTTTTT-3', BS-I-reverse 5'-ACCCAACTCCCTTAAACTATTAAACT-3' (187 bp); BS-II-forward 5'-AGTTTAATAGTTTAAGGGAGTTGGGT-3', BS-II-reverse 5'-CTCACCCTAAACTCAAAACTAAAAAC-3' (674 bp). After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems). \n" ], "offsets": [ [ 0, 1900 ] ] } ]

[ { "id": "PMC-1310901-07-MATERIALS_AND_METHODS-06_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1171, 1176 ] ], "normalized": [] } ]

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[ { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07__text", "type": "abstract", "text": [ "In vitro methylation and reporter gene assays\nThe IRF-4 promoter-reporter gene construct was generously provided by J.Hiscott (31). Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A). Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29). Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transiently co-expressed via electroporation. The control construct served as an internal reference for transfection efficiency. Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany). IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu). Each experiment was carried out at least three times. \n" ], "offsets": [ [ 0, 992 ] ] } ]

[ { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 50, 55 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T2", "type": "Protein", "text": [ "CpG Methylase" ], "offsets": [ [ 173, 186 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T3", "type": "Protein", "text": [ "M.Sss I" ], "offsets": [ [ 188, 195 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T4", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 356, 366 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 534, 544 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T6", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 722, 732 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 820, 825 ] ], "normalized": [] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T10", "type": "Entity", "text": [ "promoter" ], "offsets": [ [ 826, 834 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E1", "type": "Gene_expression", "trigger": { "text": [ "expressing" ], "offsets": [ [ 511, 521 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5" } ] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E2", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 570, 579 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T5" } ] }, { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_E3", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 835, 845 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T7" }, { "role": "Site", "ref_id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_T10" } ] } ]

[ { "id": "PMC-1310901-08-MATERIALS_AND_METHODS-07_1", "entity_ids": [ "PMC-1310901-08-MATERIALS_AND_METHODS-07_T2", "PMC-1310901-08-MATERIALS_AND_METHODS-07_T3" ] } ]

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[ { "id": "PMC-1310901-09-RESULTS-01__text", "type": "abstract", "text": [ "Absence of IRF-4 expression in leukemia cells is not due to promoter alterations\nWe have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3). Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D). After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations. However, 2 bp changes (nucleotide -1081, T-->C and -1068, A-->C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B). At position -116 an A-->C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B). Consequently, these alterations are unlikely to affect IRF-4 expression. \n" ], "offsets": [ [ 0, 1274 ] ] } ]

[ { "id": "PMC-1310901-09-RESULTS-01_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 11, 16 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 123, 128 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T3", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 234, 239 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T4", "type": "Protein", "text": [ "bcr-abl" ], "offsets": [ [ 333, 340 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T5", "type": "Protein", "text": [ "bcr-abl" ], "offsets": [ [ 372, 379 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T6", "type": "Protein", "text": [ "bcr-abl" ], "offsets": [ [ 452, 459 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T7", "type": "Protein", "text": [ "bcr-abl" ], "offsets": [ [ 505, 512 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T8", "type": "Protein", "text": [ "bcr-abl" ], "offsets": [ [ 608, 615 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T9", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 682, 687 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T10", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 735, 740 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 910, 915 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 940, 945 ] ], "normalized": [] }, { "id": "PMC-1310901-09-RESULTS-01_T13", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1255, 1260 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-09-RESULTS-01_E1", "type": "Negative_regulation", "trigger": { "text": [ "Absence" ], "offsets": [ [ 0, 7 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_E2" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 17, 27 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T1" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E3", "type": "Positive_regulation", "trigger": { "text": [ "due" ], "offsets": [ [ 53, 56 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_E1" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E4", "type": "Negative_regulation", "trigger": { "text": [ "lack" ], "offsets": [ [ 115, 119 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_E5" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E5", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 129, 139 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T2" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E6", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 240, 250 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T3" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E7", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 341, 349 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T4" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E8", "type": "Gene_expression", "trigger": { "text": [ "positve" ], "offsets": [ [ 380, 387 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T5" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E9", "type": "Gene_expression", "trigger": { "text": [ "positve" ], "offsets": [ [ 460, 467 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T6" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E10", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 513, 521 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T7" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E11", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 616, 624 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T8" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E12", "type": "Negative_regulation", "trigger": { "text": [ "absence" ], "offsets": [ [ 724, 731 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_E13" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E13", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 741, 751 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T10" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E14", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 916, 924 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T11" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E15", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 946, 954 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T12" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E16", "type": "Regulation", "trigger": { "text": [ "affect" ], "offsets": [ [ 1248, 1254 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_E17" } ] }, { "id": "PMC-1310901-09-RESULTS-01_E17", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1261, 1271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-09-RESULTS-01_T13" } ] } ]

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[ { "id": "PMC-1310901-10-RESULTS-02__text", "type": "abstract", "text": [ "Increase of IRF-4 expression in hematopoietic cells after demethylating treatment\nWe next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression. A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A). Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33). We used AzadC to generate unmethylated DNA. A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A). IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B). Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown). In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C). Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D). There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown). This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC). \n" ], "offsets": [ [ 0, 1646 ] ] } ]

[ { "id": "PMC-1310901-10-RESULTS-02_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 12, 17 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 172, 177 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T3", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 222, 227 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T4", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 613, 618 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 722, 727 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T6", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 929, 934 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1139, 1144 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T8", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1207, 1212 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T9", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1287, 1292 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T10", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1400, 1405 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1510, 1515 ] ], "normalized": [] }, { "id": "PMC-1310901-10-RESULTS-02_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1619, 1624 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-10-RESULTS-02_E1", "type": "Positive_regulation", "trigger": { "text": [ "Increase" ], "offsets": [ [ 0, 8 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E2" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 18, 28 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T1" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 178, 188 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T2" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E4", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 599, 609 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E5" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E5", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 619, 632 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T4" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E6", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 728, 741 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T5" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E7", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 746, 753 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E6" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E8", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 846, 855 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E6" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E9", "type": "Transcription", "trigger": { "text": [ "transcripts" ], "offsets": [ [ 935, 946 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T6" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E10", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1213, 1221 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T8" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E11", "type": "Regulation", "trigger": { "text": [ "effect" ], "offsets": [ [ 1277, 1283 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E12" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1293, 1303 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T9" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E13", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 1386, 1395 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E14" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E14", "type": "Transcription", "trigger": { "text": [ "mRNA level" ], "offsets": [ [ 1406, 1416 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T10" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E15", "type": "Regulation", "trigger": { "text": [ "control" ], "offsets": [ [ 1502, 1509 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_E16" } ] }, { "id": "PMC-1310901-10-RESULTS-02_E16", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1516, 1526 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-10-RESULTS-02_T11" } ] } ]

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[ { "id": "PMC-1310901-11-RESULTS-03__text", "type": "abstract", "text": [ "Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells\nTo further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa. Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II. EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls. Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A). After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C). Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2. In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B). This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells. \n" ], "offsets": [ [ 0, 1564 ] ] } ]

[ { "id": "PMC-1310901-11-RESULTS-03_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 63, 68 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 174, 179 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T3", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 567, 572 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T4", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 856, 861 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1046, 1051 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T6", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1173, 1178 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1385, 1390 ] ], "normalized": [] }, { "id": "PMC-1310901-11-RESULTS-03_T8", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1541, 1546 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-11-RESULTS-03_E1", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 185, 195 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T2" } ] }, { "id": "PMC-1310901-11-RESULTS-03_E2", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 862, 870 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T4" } ] }, { "id": "PMC-1310901-11-RESULTS-03_E3", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1052, 1060 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T5" } ] }, { "id": "PMC-1310901-11-RESULTS-03_E4", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1179, 1187 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T6" } ] }, { "id": "PMC-1310901-11-RESULTS-03_E5", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 1391, 1404 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T7" } ] }, { "id": "PMC-1310901-11-RESULTS-03_E6", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 1547, 1555 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-11-RESULTS-03_T8" } ] } ]

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[ { "id": "PMC-1310901-12-RESULTS-04__text", "type": "abstract", "text": [ "Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells\nIn order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34). This technique is especially useful for detection of unknown methylation patterns. PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1). In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative). Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1). A 5'-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3'-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1). \nIntriguingly, a stretch of 13 CpG sites (#10-22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B). Furthermore, three CpG sites at the 5' end (#54, 56, 58) and two CpG motifs at the 3' end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression. In addition, two CpG sites located in a NFkappaB (#48) and a SP1element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones: 1/8 versus 8/8). These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells. \n" ], "offsets": [ [ 0, 2222 ] ] } ]

[ { "id": "PMC-1310901-12-RESULTS-04_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 26, 31 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 136, 141 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T3", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 766, 771 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T4", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 788, 793 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 831, 836 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T6", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 857, 862 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 930, 935 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T8", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 950, 955 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T9", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1012, 1017 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T10", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1561, 1566 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1675, 1680 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1891, 1896 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T13", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 2010, 2015 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T14", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 2033, 2038 ] ], "normalized": [] }, { "id": "PMC-1310901-12-RESULTS-04_T15", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 2185, 2190 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-12-RESULTS-04_E1", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 794, 802 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T4" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E2", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 863, 871 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T6" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E3", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 956, 964 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T8" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E4", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 1018, 1026 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T9" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E5", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 1567, 1575 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T10" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E6", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 1681, 1689 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T11" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E7", "type": "Negative_regulation", "trigger": { "text": [ "absence" ], "offsets": [ [ 1880, 1887 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_E8" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E8", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1897, 1907 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T12" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E9", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 2016, 2024 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T13" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E10", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 2039, 2047 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T14" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E11", "type": "Regulation", "trigger": { "text": [ "regulation" ], "offsets": [ [ 2171, 2181 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_E12" } ] }, { "id": "PMC-1310901-12-RESULTS-04_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2191, 2201 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-12-RESULTS-04_T15" } ] } ]

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[ { "id": "PMC-1310901-13-RESULTS-05__text", "type": "abstract", "text": [ "In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity\nTo provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation. A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A). Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B). The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C). In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown). These data proved a direct association between methylation and activity of the IRF-4 promoter. \n" ], "offsets": [ [ 0, 1030 ] ] } ]

[ { "id": "PMC-1310901-13-RESULTS-05_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 27, 32 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 151, 156 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T3", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 214, 219 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T4", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 450, 455 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T5", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 510, 515 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T6", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 620, 625 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T7", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 719, 724 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T8", "type": "Protein", "text": [ "FasL" ], "offsets": [ [ 844, 848 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T9", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 1013, 1018 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T11", "type": "Entity", "text": [ "promoter" ], "offsets": [ [ 489, 497 ] ], "normalized": [] }, { "id": "PMC-1310901-13-RESULTS-05_T15", "type": "Entity", "text": [ "promoter" ], "offsets": [ [ 698, 706 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-13-RESULTS-05_E1", "type": "Negative_regulation", "trigger": { "text": [ "decreased" ], "offsets": [ [ 479, 488 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-13-RESULTS-05_T4" }, { "role": "Site", "ref_id": "PMC-1310901-13-RESULTS-05_T11" } ] }, { "id": "PMC-1310901-13-RESULTS-05_E2", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 516, 524 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-13-RESULTS-05_T5" } ] }, { "id": "PMC-1310901-13-RESULTS-05_E3", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 626, 634 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-13-RESULTS-05_T6" } ] }, { "id": "PMC-1310901-13-RESULTS-05_E4", "type": "Negative_regulation", "trigger": { "text": [ "abrogation" ], "offsets": [ [ 684, 694 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-13-RESULTS-05_T7" }, { "role": "Site", "ref_id": "PMC-1310901-13-RESULTS-05_T15" } ] }, { "id": "PMC-1310901-13-RESULTS-05_E5", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 725, 733 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-13-RESULTS-05_T7" } ] } ]

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[ { "id": "PMC-1310901-14-RESULTS-06__text", "type": "abstract", "text": [ "mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation\nSince abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter. To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D). In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status. These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription. \n" ], "offsets": [ [ 0, 879 ] ] } ]

[ { "id": "PMC-1310901-14-RESULTS-06_T1", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 101, 106 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T2", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 339, 344 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T3", "type": "Protein", "text": [ "DNMT1" ], "offsets": [ [ 420, 425 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T4", "type": "Protein", "text": [ "DNMT3A" ], "offsets": [ [ 427, 433 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T5", "type": "Protein", "text": [ "DNMT3B" ], "offsets": [ [ 438, 444 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T6", "type": "Protein", "text": [ "MBD1" ], "offsets": [ [ 454, 458 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T7", "type": "Protein", "text": [ "MBD2" ], "offsets": [ [ 460, 464 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T8", "type": "Protein", "text": [ "MBD4" ], "offsets": [ [ 466, 470 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T9", "type": "Protein", "text": [ "MeCP" ], "offsets": [ [ 475, 479 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T10", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 505, 510 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T11", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 695, 700 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T12", "type": "Protein", "text": [ "IRF-4" ], "offsets": [ [ 783, 788 ] ], "normalized": [] }, { "id": "PMC-1310901-14-RESULTS-06_T13", "type": "Entity", "text": [ "promoter" ], "offsets": [ [ 174, 182 ] ], "normalized": [] } ]

[ { "id": "PMC-1310901-14-RESULTS-06_E1", "type": "Regulation", "trigger": { "text": [ "regulation" ], "offsets": [ [ 183, 193 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T2" }, { "role": "Site", "ref_id": "PMC-1310901-14-RESULTS-06_T13" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E2", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T3" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E3", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T4" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E4", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T5" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E5", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T6" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E6", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T7" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E7", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T8" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E8", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 481, 496 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T9" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E9", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 511, 519 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T10" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E10", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 525, 533 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T10" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E11", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T3" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E12", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T4" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E13", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T5" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E14", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T6" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E15", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T7" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E16", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T8" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E17", "type": "Transcription", "trigger": { "text": [ "mRNA levels" ], "offsets": [ [ 602, 613 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T9" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E18", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T3" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E19", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T4" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E20", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T5" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E21", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T6" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E22", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T7" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E23", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T8" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E24", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 659, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T9" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E25", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 789, 797 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T12" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E26", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 803, 811 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T12" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E27", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T3" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E28", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T4" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E29", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T5" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E30", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T6" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E31", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T7" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E32", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T8" } ] }, { "id": "PMC-1310901-14-RESULTS-06_E33", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 863, 876 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-14-RESULTS-06_T9" } ] } ]

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[]

[ { "id": "PMC-1310901-15-DISCUSSION__text", "type": "abstract", "text": [ "DISCUSSION\nMany genetic lesions are known to influence gene expression of tumor suppressor genes. Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively. We studied a putative cause for absent IRF-4 expression in leukemia cells and first focused on genetic aberrations of the promoter. We observed no genetic alterations in the IRF-4 promoter, which can account for the lack of IRF-4 expression: The detected base pair changes at position -1081 (T-->C substitution), at position -1068 (A-->C substitution) and at position -116 (A-->C substitution) are unlikely responsible for absent IRF-4-expression since the first two mutations were found both in IRF-4-positive and -negative cells whereas the latter change was not detected consistently in all IRF-4-negative or -positive cells and may thus be a polymorphism. All three substitutions did not change any known putative transcription factor binding sites (30,31) and also do not affect any restriction sites or primer binding sites of the used assays. However, permanent genetic variations in the IRF-4 coding sequence, such as deletions or mutations resulting in stop-codons have not been excluded by sequence analysis. Since IRF-4 expression in cell lines and CML can be induced by demethylation and successful IFN-alpha therapy (3), respectively, the existence of such genetic aberrations seems unlikely. \nWe then investigated whether the previously described down-regulation of IRF-4 expression in human myeloid leukemias was due to a differential hypermethylation of the promoter, since the presented re-expression due to AzadC-treatment might also be a result of activation of positive transcriptional regulators of IRF-4. Methylation of CpG sites is a common mechanism of silencing genes in leukemia and has also been shown for another IRF, IRF-7 (35) and for PU.1 (36), an interacting partner of IRF-4. To elucidate the relevance of this mechanism for the regulation of IRF-4 expression, various leukemic cells were treated with demethylating agents and promoters were sequenced after bisulfite treatment. We found that IRF-4 expression could indeed be connected to the methylation status of distinct CpG motifs in the IRF-4 promoter. In Figure 4A, those CpG sites are shown (bottom line), whose hypermethylation may account for the absence of IRF-4 expression in the respective cells. One of them (#54) is adjacent to an identified regulatory element (NFkappaB-site), indicating a possible involvement of this site. At two further CpG sites (#48, 45) the methylation status in IRF-4-positive was lower than that of IRF-4-negative cells. These CpG sites are located in an NFkappaB and an SP1 element (31) and thus may also play a role in regulation of IRF-4 expression. It has been shown that NFkappaB elements play an important role in IRF-4 induction as IRF-4 expression depends on binding of the transactivator c-Rel to these elements in the IRF-4 promoter (31,37). Furthermore, methylation of the central CpG in the NFkappaB element inhibits binding of the NFkappaB protein complexes (38), promoting the significance of the observed methylation differences in IRF-4-positive and -negative cells. \nVia in vitro methylation and reporter gene assays we could clearly appoint the silencing of the IRF-4 promoter to a methylation effect, which may thus be the mechanism of IRF-4 deregulation in vivo. One possible cause for the aberrant methylation in tumorigenesis is an increased level of DNMTs during the pathogenetic process. In colon, lung and hematologic malignancies, overexpression of DNMT1, a maintenance DNMT, has been detected (39-41). Furthermore, it has been shown that CML cells in the acute phase exhibited elevated levels of the three known DNMTs, while CML cells in chronic phase expressed normal levels of DNMTs if compared with normal bone marrow cells (25). Interestingly, a positive correlation between DNMT1 expression levels and hypermethylation of p15INK4b has been detected in AML (25). In this work, we did not detect significant mRNA expression differences of selected DNMT or MBP, making it an unlikely cause for the observed methylation and thus IRF-4 expression differences in leukemia cells. \nThe finding that IRF-4 expression is silenced by promoter hypermethylation might represent a mechanism that accounts for the previously observed loss of IRF-4 expression in CML. Indeed, several clinical trials with leukemia patients and patients with myelodysplastic syndromes demonstrated the potential clinical benefit of a treatment with demethylating agents (42-45). \nThe expression of another IRF, IFN consensus sequence binding protein (ICSBP/IRF-8), is impaired in myeloid leukemias especially CML (27,46,47). But in contrast to IRF-4, the loss of this IRF could not be reverted in ICSBP-negative cell lines (EM-2, CML-T1, K-562 and LAMA-84) by treatment with AzadC (Figure 6) and AzadC has no effect on ICSBP levels in ICSBP-positive U-937 cells (Figure 6). These data suggest a distinct regulatory mechanism for these two IRFs. \nIRF-4, similar to many other classical tumor suppressor genes p15INK4b, p16INK4a or p53, may thus be a subject of alterations in the promoter methylation status leading to expression changes, which might contribute to the initiation and/or progression of cancer. Still, the obvious functional diversity of IRF-4 remains remarkable and cannot be fully explained by the IRF-4 promoter methylation status. For example, IRF-4 is primarily known for its oncogenic features. In multiple myeloma (MM) a translocation on chromosome 14q was reported to lead to a fusion gene of immunoglobulin heavy-chain (IgH) and IRF-4 resulting in a subsequent overexpression of IRF-4 (48,49). In addition, abundant IRF-4 expression was found to be a marker for various subsets of lymphomas, such as diffuse large B-cell lymphomas, primary effusion lymphoma, and marginal zone lymphoma, and adult T-cell leukemia (11,31,50-52). This draws a more complex picture of the role of IRF-4. Down-regulation of IRF-4 may promote leukemogenesis in myeloid cell context (3), which was recently confirmed in IRF-4-/- ICSBP-/- double knock-out mice (53), while IRF-4 up-regulation may induce a growth advantage in lymphomas or MM (48). \nTaken together, our data suggest that IRF-4 promoter methylation regulates IRF-4 expression, and that aberrant expression of IRF-4 in certain types of leukemia may be a consequence of IRF-4 promoter hypermethylation. \n" ], "offsets": [ [ 0, 6568 ] ] } ]

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3096, 3099 ] ], "normalized": [] }, { "id": "PMC-1310901-15-DISCUSSION_T104", "type": "Entity", "text": [ "NFkappaB element" ], "offsets": [ [ 3107, 3123 ] ], "normalized": [] }, { "id": "PMC-1310901-15-DISCUSSION_T110", "type": "Entity", "text": [ "promoter" ], "offsets": [ [ 3390, 3398 ] ], "normalized": [] } ]

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[ 688, 699 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E6" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E6", "type": "Negative_regulation", "trigger": { "text": [ "absent" ], "offsets": [ [ 704, 710 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E7" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E7", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 717, 727 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T4" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E8", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 783, 791 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T5" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E9", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 797, 805 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T5" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E10", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 881, 889 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T6" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E11", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 894, 902 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T6" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E12", "type": "Regulation", "trigger": { "text": [ "change" ], "offsets": [ [ 973, 979 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T4" }, { "role": "Site", "ref_id": "PMC-1310901-15-DISCUSSION_T75" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E13", "type": "Regulation", "trigger": { "text": [ "affect" ], "offsets": [ [ 1058, 1064 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T4" }, { "role": "Site", "ref_id": "PMC-1310901-15-DISCUSSION_T77" } ] }, { "id": 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"PMC-1310901-15-DISCUSSION_E18" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E18", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1567, 1577 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T10" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E19", "type": "Positive_regulation", "trigger": { "text": [ "due" ], "offsets": [ [ 1609, 1612 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E17" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E20", "type": "Gene_expression", "trigger": { "text": [ "re-expression" ], "offsets": [ [ 1685, 1698 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T10" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E21", "type": "Positive_regulation", "trigger": { "text": [ "result" ], "offsets": [ [ 1738, 1744 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E20" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E22", "type": "Binding", 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"arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T35" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E51", "type": "Negative_regulation", "trigger": { "text": [ "impaired" ], "offsets": [ [ 4770, 4778 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E50" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E52", "type": "Negative_regulation", "trigger": { "text": [ "loss" ], "offsets": [ [ 4857, 4861 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T35" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E53", "type": "Negative_regulation", "trigger": { "text": [ "reverted" ], "offsets": [ [ 4887, 4895 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E52" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E54", "type": "Gene_expression", "trigger": { "text": [ "negative" ], "offsets": [ [ 4905, 4913 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T39" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E55", "type": "Regulation", "trigger": { "text": [ "effect" ], "offsets": [ [ 5011, 5017 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E56" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E56", "type": "Gene_expression", "trigger": { "text": [ "levels" ], "offsets": [ [ 5027, 5033 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T40" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E57", "type": "Gene_expression", "trigger": { "text": [ "positive" ], "offsets": [ [ 5043, 5051 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T41" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E58", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 5320, 5330 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T42" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E59", "type": "Regulation", "trigger": { "text": [ "changes" ], "offsets": [ [ 5331, 5338 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E58" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E60", "type": "Positive_regulation", "trigger": { "text": [ "resulting" ], "offsets": [ [ 5760, 5769 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E61" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E61", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 5786, 5800 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T52" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E62", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 5847, 5857 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T53" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E63", "type": "Negative_regulation", "trigger": { "text": [ "Down-regulation" ], "offsets": [ [ 6109, 6124 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T55" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E64", "type": "Gene_expression", "trigger": { "text": [ "knock-out" ], "offsets": [ [ 6247, 6256 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T56" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E65", "type": "Gene_expression", "trigger": { "text": [ "knock-out" ], "offsets": [ [ 6247, 6256 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T57" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E66", "type": "Positive_regulation", "trigger": { "text": [ "up-regulation" ], "offsets": [ [ 6280, 6293 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_T58" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E67", "type": "Regulation", "trigger": { "text": [ "regulates" ], "offsets": [ [ 6415, 6424 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1310901-15-DISCUSSION_E68" } ] }, { "id": "PMC-1310901-15-DISCUSSION_E68", "type": "Gene_expression", "trigger": { "text": [ "expression" ], 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[ { "id": "PMC-1447668-00-TIAB__text", "type": "abstract", "text": [ "Foxp3 Represses Retroviral Transcription by Targeting Both NF-kappaB and CREB Pathways \nForkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis. Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-kappaB (NF-kappaB) activation. Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-kappaB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells. We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-kappaB-dependent and NF-kappaB-independent mechanisms. Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3. Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease. These results suggest an expanded role for Foxp3 in regulating NF-kappaB- and CREB-dependent cellular and viral gene expression. \n" ], "offsets": [ [ 0, 1599 ] ] } ]

[ { "id": "PMC-1447668-00-TIAB_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 197, 202 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 335, 340 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T4", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 411, 416 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T5", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 442, 445 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T6", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 719, 724 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T7", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 810, 813 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T8", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 852, 857 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T9", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1160, 1165 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T10", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1202, 1207 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T11", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1208, 1211 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T12", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 1212, 1216 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1391, 1396 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T14", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1512, 1517 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T17", "type": "Entity", "text": [ "carboxyl-terminal forkhead (FKH) domain" ], "offsets": [ [ 593, 632 ] ], "normalized": [] }, { "id": "PMC-1447668-00-TIAB_T19", "type": "Entity", "text": [ "nuclear" ], "offsets": [ [ 647, 654 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-00-TIAB_E1", "type": "Positive_regulation", "trigger": { "text": [ "Overexpression" ], "offsets": [ [ 393, 407 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_T4" } ] }, { "id": "PMC-1447668-00-TIAB_E2", "type": "Negative_regulation", "trigger": { "text": [ "Deletion" ], "offsets": [ [ 577, 585 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_T6" }, { "role": "Site", "ref_id": "PMC-1447668-00-TIAB_T17" } ] }, { "id": "PMC-1447668-00-TIAB_E3", "type": "Positive_regulation", "trigger": { "text": [ "critical" ], "offsets": [ [ 634, 642 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_E4" }, { "role": "Cause", "ref_id": "PMC-1447668-00-TIAB_T6" }, { "role": "CSite", "ref_id": "PMC-1447668-00-TIAB_T17" } ] }, { "id": "PMC-1447668-00-TIAB_E5", "type": "Positive_regulation", "trigger": { "text": [ "critical" ], "offsets": [ [ 634, 642 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_E6" }, { "role": "Cause", "ref_id": "PMC-1447668-00-TIAB_T6" }, { "role": "CSite", "ref_id": "PMC-1447668-00-TIAB_T17" } ] }, { "id": "PMC-1447668-00-TIAB_E4", "type": "Localization", "trigger": { "text": [ "localization" ], "offsets": [ [ 655, 667 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_T6" }, { "role": "ToLoc", "ref_id": "PMC-1447668-00-TIAB_T19" } ] }, { "id": "PMC-1447668-00-TIAB_E6", "type": "Binding", "trigger": { "text": [ "binding" ], "offsets": [ [ 676, 683 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_T6" } ] }, { "id": "PMC-1447668-00-TIAB_E7", "type": "Positive_regulation", "trigger": { "text": [ "high" ], "offsets": [ [ 1386, 1390 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_E8" } ] }, { "id": "PMC-1447668-00-TIAB_E8", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1397, 1407 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-00-TIAB_T13" } ] } ]

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[ { "id": "PMC-1447668-01-Synopsis__text", "type": "abstract", "text": [ "Synopsis\nOver the past several years, mounting evidence has shown that immune tolerance in healthy individuals can be maintained by a population of T lymphocytes known as regulatory T cells (Tregs). As a component of this system, a protein known as Foxp3 has been shown to be absolutely required for the development and function of Tregs. While Foxp3 plays an important role in maintaining immune tolerance by blocking T cell proliferation and production of inflammatory proteins known as cytokines, little is known about the molecular mechanisms that are used by Foxp3 to accomplish these events. The present study expands our understanding of how Foxp3 maintains a check on inappropriate immune responses by demonstrating that Foxp3 can block activation of key inducible proteins such as nuclear factor kappaB (NF-kappaB) and cAMP-responsive element binding protein (CREB). Since NF-kappaB and CREB are integrally involved in controlling cell cycle progression, inflammatory cytokine production, and the replication of numerous viruses at the level of transcription, understanding the mechanisms by which Foxp3 functions to regulate cellular and viral gene expression may aid in the discovery of therapeutic approaches designed to rescue the expression and/or function of Foxp3, which have been found to be deficient in several autoimmune diseases and virus-induced disorders. \n" ], "offsets": [ [ 0, 1380 ] ] } ]

[ { "id": "PMC-1447668-01-Synopsis_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 249, 254 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 345, 350 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 564, 569 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T4", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 649, 654 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 729, 734 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T6", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1107, 1112 ] ], "normalized": [] }, { "id": "PMC-1447668-01-Synopsis_T7", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1274, 1279 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-01-Synopsis_E1", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1244, 1254 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-01-Synopsis_T7" } ] } ]

[]

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[ { "id": "PMC-1447668-02-Introduction__text", "type": "abstract", "text": [ "Introduction\nImmunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1]. Suppression of pathogenic T cell responses is mediated by naturally arising CD4+CD25+ T regulatory cells (Tregs) [2,3]. Deficiencies in Treg development and function have been linked to the severe autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [4]. In addition, recent studies have provided strong evidence that dysregulation of Treg development and/or function may be a significant factor in the pathogenesis of several autoimmune disorders (e.g., multiple sclerosis [5], myasthenia gravis [6], and type 1 diabetes [7]) and virus-induced immunologic disorders (e.g., human T lymphotropic virus type I [HTLV-I]-associated myelopathy/tropical spastic paraparesis [HAM/TSP], and HIV-induced AIDS [8-10]). \nThe transcription factor Foxp3 is a 431-amino acid (48-kDa) protein expressed at very high levels in CD4+CD25hi T cells and has previously been shown to be absolutely critical for Treg development and function [11-14]. Foxp3 contains a proline-rich amino-terminal domain reported to function as a nuclear factor of activated T cells (NF-AT) and nuclear factor-kappaB (NF-kappaB) binding domain, a central region containing a zinc finger and leucine zipper potentially important for protein-protein interactions, and a carboxyl-terminal forkhead (FKH) domain required for nuclear localization and DNA-binding activity [14-16]. Functional inactivation of Foxp3 by genetic mutations affecting the Foxp3 coding region, as demonstrated in IPEX, or repression of Foxp3 expression by the HTLV-I-encoded transactivator protein Tax, as recently reported in patients with HAM/TSP, results in loss of regulatory activity in CD4+CD25hi T cells [4,8,17]. Although it is clear that Foxp3 regulates T cell proliferation and cytokine production, very little is known concerning the molecular mechanisms of Foxp3 function. \nThe first evidence to indicate how Foxp3 promotes the development and function of regulatory T cells came from a report by Ziegler and colleagues [16], which suggested that Foxp3 could inhibit transcriptional activation by physically interacting with forkhead binding sites located immediately adjacent to critical cis-acting NF-AT binding sites found in various cytokine promoters (e.g., IL-2 promoter). That study also demonstrated that Foxp3 could repress activation of a synthetic reporter vector containing an SV40 promoter and three tandem copies of a forkhead binding site. These results provided additional evidence suggesting that Foxp3 transcriptional repression was mediated by binding in a sequence-specific manner to promoters containing forkhead binding sites. A recent study by Bettelli and colleagues [15] further demonstrated that Foxp3 could inhibit NF-AT as well as NF-kappaB activation, although the mechanism of suppression was shown to involve direct protein-protein interactions between NF-AT or NF-kappaB and Foxp3 rather than binding of Foxp3 to promoter elements adjacent to cis-acting NF-AT or NF-kappaB sites. Collectively, these data suggested that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions. \nIn the present study, we expanded upon these observations by defining additional requirements of Foxp3-mediated repression of NF-kappaB activation, and investigated whether Foxp3 could target additional signaling pathways by examining transcriptional activation of NF-kappaB-dependent and NF-kappaB-independent retroviral pathogens. The characterization of the molecular targets of Foxp3 and the mechanism(s) utilized by Foxp3 to support Treg development and function will aid in our understanding of the role Tregs play in the pathogenesis of human autoimmune disease. \n" ], "offsets": [ [ 0, 4033 ] ] } ]

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"text": [ "expression" ], "offsets": [ [ 1800, 1810 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-02-Introduction_T9" } ] }, { "id": "PMC-1447668-02-Introduction_E12", "type": "Negative_regulation", "trigger": { "text": [ "inhibit" ], "offsets": [ [ 2329, 2336 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-02-Introduction_E13" }, { "role": "Cause", "ref_id": "PMC-1447668-02-Introduction_E14" } ] }, { "id": "PMC-1447668-02-Introduction_E13", "type": "Positive_regulation", "trigger": { "text": [ "transcriptional activation" ], "offsets": [ [ 2337, 2363 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-02-Introduction_T17" }, { "role": "Site", "ref_id": "PMC-1447668-02-Introduction_T45" } ] }, { "id": "PMC-1447668-02-Introduction_E14", "type": "Binding", "trigger": { "text": [ "interacting" ], "offsets": [ [ 2378, 2389 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-02-Introduction_T16" } ] }, { "id": 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[ { "id": "PMC-1447668-03-Results-01__text", "type": "abstract", "text": [ "Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation\nTo ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. \n" ], "offsets": [ [ 0, 2346 ] ] } ]

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[ { "id": "PMC-1447668-04-Results-02__text", "type": "abstract", "text": [ "The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-kappaB Activation in T Cells\nTo define the requirements of Foxp3 with respect to inhibition of NF-kappaB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and a mutant Foxp3 protein from a patient with IPEX [4,11,14,17]. Unlike full-length Foxp3, which localizes almost exclusively to the nucleus and can bind in a sequence-specific manner to forkhead binding sites, the DeltaFKH mutant fails to localize to the nucleus and thus cannot interact with promoter elements or nuclear proteins [16]. Therefore, we utilized the DeltaFKH mutant to determine whether nuclear localization (or other function associated with the FKH domain) of Foxp3 was a prerequisite for inhibition of NF-kappaB activation. Although Foxp3 interaction with NF-kappaB presumably takes place in the nucleus, it may also be possible for a cytoplasmic Foxp3 protein to bind to NF-kappaB in the cytoplasm and prevent localization to the nucleus following an activation stimulus. Overexpression of full-length Foxp3, but not of DeltaFKH, was able to suppress activation of a cotransfected NF-kappaB reporter vector in HEK 293T cells (Figure 2B). Both Foxp3 and DeltaFKH were expressed at very high levels following transfection as detected by real-time RT-PCR (unpublished data). These data appear to suggest that the carboxyl-terminal FKH domain is critically important for Foxp3 to down-regulate NF-kappaB-dependent transcription. However, NF-kappaB activation was blocked to a similar extent by both full-length Foxp3 and DeltaFKH in Jurkat T cells (Figure 2C) and primary human CD4+ T cells (Figure 2D). Western blot analysis of NF-kappaB p65 expression demonstrated that Foxp3 and DeltaFKH does not block NF-kappaB activation at the level of p65 protein expression (Figure 2E). These results are very interesting with respect to Foxp3 function, because they suggest that the carboxyl-terminal FKH domain, and possibly nuclear localization, are dispensable for Foxp3 function in T cell populations. Alternative interpretations may include the possibility that the localization of DeltaFKH differ between epithelial cells and T cells. In either case, these results suggest a cell type-specific mechanism of action for this Foxp3 mutant. \n" ], "offsets": [ [ 0, 2369 ] ] } ]

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[ { "id": "PMC-1447668-05-Results-03__text", "type": "abstract", "text": [ "Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-kappaB\nIf Foxp3 functions as a repressor of NF-kappaB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-kappaB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-kappaB binding sites located between positions -102 and -81 with respect to the transcription initiation site [21]. NF-kappaB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not DeltaFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-kappaB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and DeltaFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-kappaB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-kappaB sites located between -102 and -81 (HIV-1 Delta-kappaB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-kappaB binding sites. Residual levels of inhibition of the HIV-1 Delta-kappaB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-kappaB sites within the HIV-1 LTR [22,23]. \n" ], "offsets": [ [ 0, 2076 ] ] } ]

[ { "id": "PMC-1447668-05-Results-03_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 91, 96 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 188, 193 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T4", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 653, 656 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 702, 707 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T6", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 717, 725 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T7", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 978, 983 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T8", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 988, 996 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T9", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1048, 1053 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T10", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1458, 1461 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T11", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1533, 1538 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T12", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1619, 1624 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1787, 1792 ] ], "normalized": [] }, { "id": "PMC-1447668-05-Results-03_T14", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1965, 1970 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-06-Results-04__text", "type": "abstract", "text": [ "The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3\nPrevious studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-kappaB activation as well as tumor necrosis factor-alpha-stimulated NF-kappaB activation [15]. Our next step was to determine whether Foxp3 could also suppress the activation of NF-kappaB caused by a strong viral transactivator protein. HTLV-I encodes a multifunctional transactivator protein, Tax, capable of activating both the NF-kappaB and CREB pathways [24-29]. Since the HTLV-I Tax protein can function at multiple levels in both the cytoplasm and the nucleus to stimulate activation of NF-kappaB [28,29], we hypothesized that overexpression of Foxp3 may interfere with this process. However, since Tax-dependent HTLV-I gene expression is independent of NF-kappaB [18], we also hypothesized that Foxp3 would not affect Tax-dependent activation of the HTLV-I LTR. To test these hypotheses, we overexpressed HTLV-I Tax, full-length Foxp3, and/or DeltaFKH in HEK 293T cells cotransfected with an HTLV-I LTR or NF-kappaB reporter vector. As shown in Figure 4A, HTLV-I Tax strongly up-regulated NF-kappaB-dependent transcriptional activation (~60-fold). Interestingly, overexpression of Foxp3, but not DeltaFKH, suppressed Tax-mediated activation of NF-kappaB-dependent transcription. These observations further suggest that the carboxyl-terminal FKH domain is required for inhibiting activation of NF-kappaB in the presence of Tax in HEK 293T cells, strikingly similar to the requirements of Foxp3 inhibition of basal NF-kappaB activation shown in Figure 2B. Transactivation of the HTLV-I LTR was stimulated about 55-fold by overexpression of Tax (Figure 4B), while transfection of Foxp3 suppressed Tax-dependent HTLV-I LTR activation, although HTLV-I LTR activation in the presence or absence of Tax is independent of NF-kappaB or NF-AT (another transcriptional activator known to interact with Foxp3). Furthermore, overexpression of DeltaFKH also led to suppression of HTLV-I transactivation by Tax to a similar extent as full-length Foxp3 (Figure 4B). The suppressive effects shown in Figure 4A and 4B were not the result of Foxp3 down-regulating the expression of the transfected Tax plasmid as determined by real-time RT-PCR (Figure 4C). These results strongly suggest that Foxp3 interacts with transcriptional regulators in addition to NF-kappaB and NF-AT, and that the carboxyl-terminal FKH domain, and therefore localization to the nucleus, are not required for inhibition of Tax-mediated HTLV-I LTR activation (even in HEK 293T cells). \nTo determine whether Foxp3 inhibited the transactivation functions of Tax by directly associating with this viral protein, we generated an expression vector in which HTLV-I Tax was fused in-frame to the carboxyl terminus of the Gal4 DNA-binding domain (Gal4-BD). This Gal4-BD-Tax fusion protein activated transcription of a synthetic promoter containing five Gal4 binding sites, while Gal4-BD was insufficient to stimulate transcription by itself (Figure 4D). Transactivation of the Gal4-resposive promoter by Gal4-BD-Tax remained relatively unaffected by overexpression of either EGFP (control), Foxp3, or DeltaFKH, suggesting that Foxp3 does not repress Tax transactivation by directly interfacing with the HTLV-I Tax protein. To confirm that Foxp3 had a direct effect on HTLV-I replication, we transfected HEK 293T cells with a well-characterized HTLV-I infectious molecular clone (termed ACH) [30] in the presence of full-length Foxp3, DeltaFKH, or control vector. ACH has been previously shown to direct the expression of viral antigens, produce infectious virus, and transform CD4+ T cells both in vitro and in vivo [31,32]. After 24 h, the amount of viral antigen expression, in this case Tax mRNA, was detected by a sensitive real-time RT-PCR assay. As illustrated in Figure 5, the level of Tax mRNA synthesized from ACH was down-regulated in the presence of Foxp3 compared to the level produced in the presence of the control vector. DeltaFKH did not have a discernable affect on Tax expression. These data indicate that Foxp3 is capable of repressing the expression of Tax from an infectious HTLV-I molecular clone. \n" ], "offsets": [ [ 0, 4256 ] ] } ]

[ { "id": "PMC-1447668-06-Results-04_T1", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 40, 43 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 62, 67 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 135, 140 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T4", "type": "Protein", "text": [ "tumor necrosis factor-alpha" ], "offsets": [ [ 210, 237 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 315, 320 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T6", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 475, 478 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T7", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 565, 568 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T8", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 732, 737 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T9", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 786, 789 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T10", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 883, 888 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T11", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 906, 909 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T12", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1000, 1003 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1017, 1022 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T14", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 1031, 1039 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T15", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1151, 1154 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T16", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1269, 1274 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T17", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 1284, 1292 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T18", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1305, 1308 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T19", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1510, 1513 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T20", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1575, 1580 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T21", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1726, 1729 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T22", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1765, 1770 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T23", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1782, 1785 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T24", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1880, 1883 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T25", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1979, 1984 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T26", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 2018, 2026 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T27", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 2080, 2083 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T28", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2119, 2124 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T29", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2211, 2216 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T30", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 2267, 2270 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T31", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2362, 2367 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T32", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 2567, 2570 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T33", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2650, 2655 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T34", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 2699, 2702 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T35", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 2802, 2805 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T36", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 2857, 2861 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T37", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 2882, 2886 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T38", "type": "Protein", "text": [ "Gal4-BD-Tax" ], "offsets": [ [ 2897, 2908 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T39", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 3014, 3018 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T40", "type": "Protein", "text": [ "Gal4-BD-Tax" ], "offsets": [ [ 3139, 3150 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T41", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3226, 3231 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T42", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 3236, 3244 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T43", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3262, 3267 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T44", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 3285, 3288 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T45", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 3345, 3348 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T46", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3374, 3379 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T47", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3562, 3567 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T48", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 3569, 3577 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T49", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 3712, 3715 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T50", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 3825, 3828 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T51", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 3928, 3931 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T52", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3996, 4001 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T53", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 4072, 4080 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T54", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 4118, 4121 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T55", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 4159, 4164 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T56", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 4208, 4211 ] ], "normalized": [] }, { "id": "PMC-1447668-06-Results-04_T68", "type": "Entity", "text": [ "nucleus" ], "offsets": [ [ 2523, 2530 ] ], "normalized": [] } ]

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"overexpression" ], "offsets": [ [ 1251, 1265 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T16" } ] }, { "id": "PMC-1447668-06-Results-04_E6", "type": "Gene_expression", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 1251, 1265 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T17" } ] }, { "id": "PMC-1447668-06-Results-04_E7", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 1708, 1722 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T21" } ] }, { "id": "PMC-1447668-06-Results-04_E8", "type": "Gene_expression", "trigger": { "text": [ "transfection" ], "offsets": [ [ 1749, 1761 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T22" } ] }, { "id": "PMC-1447668-06-Results-04_E9", "type": "Binding", "trigger": { "text": [ "interact" ], "offsets": [ [ 1965, 1973 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T25" } ] }, { "id": "PMC-1447668-06-Results-04_E10", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 2000, 2014 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T26" } ] }, { "id": "PMC-1447668-06-Results-04_E11", "type": "Negative_regulation", "trigger": { "text": [ "down-regulating" ], "offsets": [ [ 2217, 2232 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_E12" }, { "role": "Cause", "ref_id": "PMC-1447668-06-Results-04_T29" } ] }, { "id": "PMC-1447668-06-Results-04_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2237, 2247 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T30" } ] }, { "id": "PMC-1447668-06-Results-04_E13", "type": "Binding", "trigger": { "text": [ "interacts" ], "offsets": [ [ 2368, 2377 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T31" } ] }, { "id": "PMC-1447668-06-Results-04_E14", "type": "Localization", "trigger": { "text": [ "localization" ], "offsets": [ [ 2503, 2515 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T31" }, { "role": "ToLoc", "ref_id": "PMC-1447668-06-Results-04_T68" } ] }, { "id": "PMC-1447668-06-Results-04_E15", "type": "Binding", "trigger": { "text": [ "associating" ], "offsets": [ [ 2715, 2726 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T33" }, { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T34" } ] }, { "id": "PMC-1447668-06-Results-04_E16", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 3185, 3199 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T41" } ] }, { "id": "PMC-1447668-06-Results-04_E17", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 3185, 3199 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T42" } ] }, { "id": "PMC-1447668-06-Results-04_E18", "type": "Negative_regulation", "trigger": { "text": [ "interfacing" ], "offsets": [ [ 3317, 3328 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T45" }, { "role": "Cause", "ref_id": "PMC-1447668-06-Results-04_T43" } ] }, { "id": "PMC-1447668-06-Results-04_E19", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 3800, 3810 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T50" } ] }, { "id": "PMC-1447668-06-Results-04_E20", "type": "Transcription", "trigger": { "text": [ "synthesized" ], "offsets": [ [ 3937, 3948 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T51" } ] }, { "id": "PMC-1447668-06-Results-04_E21", "type": "Negative_regulation", "trigger": { "text": [ "down-regulated" ], "offsets": [ [ 3962, 3976 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_E20" }, { "role": "Cause", "ref_id": "PMC-1447668-06-Results-04_T52" } ] }, { "id": "PMC-1447668-06-Results-04_E22", "type": "Transcription", "trigger": { "text": [ "produced" ], "offsets": [ [ 4024, 4032 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T51" } ] }, { "id": "PMC-1447668-06-Results-04_E23", "type": "Regulation", "trigger": { "text": [ "affect" ], "offsets": [ [ 4108, 4114 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_E24" }, { "role": "Cause", "ref_id": "PMC-1447668-06-Results-04_T53" } ] }, { "id": "PMC-1447668-06-Results-04_E24", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 4122, 4132 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T54" } ] }, { "id": "PMC-1447668-06-Results-04_E25", "type": "Negative_regulation", "trigger": { "text": [ "repressing" ], "offsets": [ [ 4179, 4189 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_E26" }, { "role": "Cause", "ref_id": "PMC-1447668-06-Results-04_T55" } ] }, { "id": "PMC-1447668-06-Results-04_E26", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 4194, 4204 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-06-Results-04_T56" } ] } ]

[]

[]

[ { "id": "PMC-1447668-07-Results-05__text", "type": "abstract", "text": [ "Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load\nSince Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients. We therefore quantitated the Foxp3 protein expression in CD4+CD25+ T cells by flow cytometry and the HTLV-I proviral load (a surrogate marker of viral replication) by real-time PCR from eight patients with HAM/TSP and eight asymptomatic carriers (ACs). The data from this analysis is summarized in Table 1. As expected, patients with HAM/TSP exhibited significantly higher proviral loads (indicated as HTLV-I proviral DNA copies/100 cells) (34.68 +/- 23.19) compared to ACs (4.75 +/- 5.47) (p = 0.0008). The percentage of Foxp3+ cells within the CD4+CD25+ T cell population was significantly greater in ACs (43.23 +/- 12.95) than in HAM/TSP patients (18.59 +/- 5.77) (p = 0.0033). These data suggest that high levels of Foxp3 protein expression are associated with reduced HTLV-I replication in vivo. They also support our recent reports that high proviral loads, which have been shown to correlate with high Tax mRNA in HTLV-I-infected patients, are associated with reduced Foxp3 expression [8,34]. \n" ], "offsets": [ [ 0, 1313 ] ] } ]

[ { "id": "PMC-1447668-07-Results-05_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 10, 15 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 85, 90 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T3", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 130, 133 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T4", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 134, 138 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 261, 266 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T6", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 341, 346 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T7", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 369, 372 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T8", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 373, 377 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T9", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 834, 839 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T10", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 858, 861 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T11", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 862, 866 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T12", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1032, 1037 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T13", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1221, 1224 ] ], "normalized": [] }, { "id": "PMC-1447668-07-Results-05_T14", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1287, 1292 ] ], "normalized": [] } ]

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[]

[]

[ { "id": "PMC-1447668-08-Results-06__text", "type": "abstract", "text": [ "CREB Is a Target for Transcriptional Repression by Foxp3\nAlthough Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D). These results led us to hypothesize that Foxp3 acts on HTLV-I gene expression by interacting with proteins important for driving HTLV-I LTR activity in vivo. Previous studies have demonstrated that the Tax-responsive elements within the HTLV-I LTR play a crucial role in driving Tax-mediated transactivation of the HTLV-I LTR [18]. The Tax-responsive elements have been shown to resemble CREB binding sites, bind CREB in vitro and in vivo, and facilitate HTLV-I LTR activation both in the presence and in the absence of Tax [35,36]. Ching and colleagues [18] demonstrated that addition of a dominant-negative CREB expression vector resulted in nearly complete inhibition of Tax-mediated activation of the HTLV-I LTR, while blocking NF-kappaB activation by addition of a dominant-negative IKKbeta expression vector had no effect on Tax transactivation of the HTLV-I LTR. Therefore, we hypothesized that Foxp3 may inhibit Tax transactivation of the HTLV-I LTR via disruption of the CREB signaling pathway. To test this possibility, HEK 293T cells were transfected with an HTLV-I LTR or synthetic CREB reporter vector along with a control expression vector (EGFP) or expression vectors encoding Foxp3 or DeltaFKH. As shown in Figure 6A, Foxp3 down-regulated basal activation of the HTLV-I LTR and transcription of a synthetic CREB reporter vector, suggesting that Foxp3 down-regulates HTLV-I LTR activation by targeting the CREB pathway. Deletion of the FKH domain of Foxp3 dampened the suppressive effect of Foxp3, but did not completely abrogate suppression, as is seen with NF-kappaB-responsive promoters in HEK 293T cells. Like NF-kappaB activation, CREB transcriptional activation was also suppressed by expression of Foxp3, and to a similar extent DeltaFKH, in healthy donor CD4+ T cells (Figure 6B). Similarly, Foxp3 and DeltaFKH also repressed basal HTLV-I LTR activation in primary human CD4+ T cells (Figure 6C). To our knowledge, this is the first evidence implicating CREB as a molecular target of Foxp3. As observed with NF-kappaB activation, DeltaFKH was a more potent inhibitor of CREB activation in CD4+ T cells than in HEK 293T cells, further indicating that a cell type-specific mechanism of action may govern the function of this Foxp3 mutant. \nTo determine whether Foxp3 functioned by directly signaling through CREB, we utilized expression vectors encoding CREB-1 or c-Jun (a member of the activator protein 1 family of transcription factors) fused in-frame to the Gal4-BD (Gal4-BD-CREB-1 and Gal4-BD-c-Jun). As shown in Figure 6D, activation of a Gal4-responsive reporter vector by Gal4-BD-CREB-1 was down-regulated by Foxp3 compared to control vector (EGFP), indicating that Foxp3 functions by directly or indirectly interacting with CREB-1. However, Foxp3 failed to markedly affect transcriptional activation of Gal4-BD-c-Jun (c-Jun has also been demonstrated to bind to the HTLV-I LTR) and Gal4-BD-Tax (see Figure 5). Importantly, the mechanism of Foxp3-mediated inhibition of CREB-dependent transcription was not due to a block in CREB-1 protein expression, as determined by Western blot analysis (Figure 6E). Although these results demonstrate that Foxp3 functions as a co-repressor of CREB activation (in addition to NF-kappaB and NF-AT), we were unable to detect a direct physical interaction between CREB-1 and Foxp3 by coimmunoprecipitation or mammalian two-hybrid analysis (unpublished data). Therefore, our data suggest that Foxp3 may interfere with CREB signaling at an upstream event, such as phosphorylation of CREB or recruitment/function of coactivator proteins CREB-binding protein (CBP)/p300. \n" ], "offsets": [ [ 0, 3945 ] ] } ]

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"normalized": [] }, { "id": "PMC-1447668-08-Results-06_T9", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 834, 837 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T10", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 988, 991 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T11", "type": "Protein", "text": [ "IKKbeta" ], "offsets": [ [ 1102, 1109 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T12", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1145, 1148 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1216, 1221 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T14", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1234, 1237 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T15", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1506, 1511 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T16", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 1515, 1523 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T17", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1548, 1553 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T18", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1675, 1680 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T19", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1779, 1784 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T20", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1820, 1825 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T21", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2034, 2039 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T22", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 2065, 2073 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T23", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 2092, 2095 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T24", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2129, 2134 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T25", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 2139, 2147 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T26", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 2208, 2211 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T27", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2321, 2326 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T28", "type": "Protein", "text": [ "DeltaFKH" ], "offsets": [ [ 2367, 2375 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T29", "type": "Protein", "text": [ "CREB" ], "offsets": [ [ 2407, 2411 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T30", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 2426, 2429 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T31", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2596, 2601 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T32", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 2689, 2695 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T33", "type": "Protein", "text": [ "c-Jun" ], "offsets": [ [ 2699, 2704 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T34", "type": "Protein", "text": [ "activator protein 1" ], "offsets": [ [ 2722, 2741 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T35", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 2797, 2801 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T36", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 2806, 2820 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T37", "type": "Protein", "text": [ "Gal4-BD-c-Jun" ], "offsets": [ [ 2825, 2838 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T38", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 2880, 2884 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T39", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 2915, 2929 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T40", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2952, 2957 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T41", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3009, 3014 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T42", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 3068, 3074 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T43", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3085, 3090 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T44", "type": "Protein", "text": [ "Gal4-BD-c-Jun" ], "offsets": [ [ 3147, 3160 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T45", "type": "Protein", "text": [ "c-Jun" ], "offsets": [ [ 3162, 3167 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T46", "type": "Protein", "text": [ "Gal4-BD-Tax" ], "offsets": [ [ 3226, 3237 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T47", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3284, 3289 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T48", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 3368, 3374 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T49", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3487, 3492 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T50", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 3641, 3647 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T51", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3652, 3657 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T52", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 3769, 3774 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T53", "type": "Protein", "text": [ "CREB-binding protein" ], "offsets": [ [ 3911, 3931 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T54", "type": "Protein", "text": [ "CBP" ], "offsets": [ [ 3933, 3936 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T55", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 3938, 3942 ] ], "normalized": [] }, { "id": "PMC-1447668-08-Results-06_T62", "type": "Entity", "text": [ "FKH domain" ], "offsets": [ [ 1765, 1775 ] ], "normalized": [] } ]

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"role": "Theme", "ref_id": "PMC-1447668-08-Results-06_T50" }, { "role": "Theme", "ref_id": "PMC-1447668-08-Results-06_T51" } ] }, { "id": "PMC-1447668-08-Results-06_E18", "type": "Negative_regulation", "trigger": { "text": [ "interfere" ], "offsets": [ [ 3779, 3788 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-08-Results-06_E19" }, { "role": "Cause", "ref_id": "PMC-1447668-08-Results-06_T52" } ] }, { "id": "PMC-1447668-08-Results-06_E20", "type": "Negative_regulation", "trigger": { "text": [ "interfere" ], "offsets": [ [ 3779, 3788 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-08-Results-06_E21" }, { "role": "Cause", "ref_id": "PMC-1447668-08-Results-06_T52" } ] }, { "id": "PMC-1447668-08-Results-06_E19", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 3866, 3877 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-08-Results-06_T53" } ] }, { "id": "PMC-1447668-08-Results-06_E21", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 3866, 3877 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-08-Results-06_T55" } ] } ]

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[ { "id": "PMC-1447668-09-Results-07__text", "type": "abstract", "text": [ "Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment\nStimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinase A, which permits binding and recruitment of coactivators CBP/p300 to the promoter [37,38]. Phosphorylation of serine 133 does not, however, affect the DNA-binding activity of CREB in most cases [39-41]. Addition of forskolin to HEK 293T cells stimulated activation of a CREB reporter vector about 65 fold (Figure 7A). Overexpression of Foxp3 was capable of down-regulating forskolin-induced CREB transcriptional activation. The functional interaction between Foxp3 and CREB did not affect the DNA-binding activity of CREB-1, but did show a modest decrease in activating transcription factor 2 (ATF-2) DNA-binding activity in the presence of forskolin as determined by transcription factor ELISA (Figure 7B). \nWhile Foxp3 has been shown to bind to and repress activation of both NF-kappaB and NF-AT, exactly how Foxp3 functions to bring about this affect has not been elucidated. To determine how Foxp3 blocks CREB-dependent transcription, we examined whether Foxp3 was capable of (1) disrupting the recruitment of coactivator proteins and/or (2) preventing phosphorylation of CREB at serine 133 (which is a prerequisite for coactivator recruitment). Since both of these events are required for CREB-dependent gene expression, we hypothesized that Foxp3 may affect CREB activation at both steps. To determine whether Foxp3 can disrupt the function/recruitment of the coactivator protein p300, we introduced a Gal4 reporter vector and a Gal4-BD-CREB-1 expression vector into HEK 293T cells in the absence or presence of Foxp3, p300, and/or control expression vectors (Figure 7C). As expected, p300 overexpression stimulated transcription of the Gal4-BD-CREB-1 fusion protein. Foxp3, again, repressed basal levels of Gal4-BD-CREB-1 activation by more than 2-fold, while effectively neutralizing Gal4-BD-CREB-1 activation in the presence of p300. We next analyzed the effect of Foxp3 on phosphorylation of CREB at serine 133 in forskolin-treated HEK 293T cells by Western blot analysis. Overexpression of Foxp3 failed to reduce the detectable levels of CREB phosphorylation using a phosphospecific antibody for CREB-1 (unpublished data). However, when we attempted to determine whether Foxp3 could physically interact with the coactivator protein p300, we found that p300 immunoprecipitated Foxp3 when both proteins were overexpressed in HEK 293T cells (Figure 7D). Collectively, these results suggest that Foxp3 antagonizes CREB-dependent gene expression by directly interacting with coactivator p300 and interfering with its function and/or recruitment to CREB-responsive promoter sequences. \n" ], "offsets": [ [ 0, 2913 ] ] } ]

[ { "id": "PMC-1447668-09-Results-07_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T2", "type": "Protein", "text": [ "CBP" ], "offsets": [ [ 379, 382 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T3", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 383, 387 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T4", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 658, 663 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 781, 786 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T6", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 839, 845 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T7", "type": "Protein", "text": [ "activating transcription factor 2" ], "offsets": [ [ 881, 914 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T8", "type": "Protein", "text": [ "ATF-2" ], "offsets": [ [ 916, 921 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T9", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1037, 1042 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T10", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1133, 1138 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T11", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1218, 1223 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T12", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1281, 1286 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1569, 1574 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T14", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1638, 1643 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T15", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 1708, 1712 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T16", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 1730, 1734 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T17", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 1757, 1771 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T18", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1840, 1845 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T19", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 1847, 1851 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T20", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 1913, 1917 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T21", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 1965, 1979 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T22", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1996, 2001 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T23", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 2036, 2050 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T24", "type": "Protein", "text": [ "Gal4-BD-CREB-1" ], "offsets": [ [ 2114, 2128 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T25", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 2159, 2163 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T26", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2196, 2201 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T27", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2323, 2328 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T28", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 2429, 2435 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T29", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2504, 2509 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T30", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 2565, 2569 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T31", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 2585, 2589 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T32", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2609, 2614 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T33", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 2725, 2730 ] ], "normalized": [] }, { "id": "PMC-1447668-09-Results-07_T34", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 2815, 2819 ] ], "normalized": [] } ]

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}, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T4" } ] }, { "id": "PMC-1447668-09-Results-07_E6", "type": "Binding", "trigger": { "text": [ "interaction" ], "offsets": [ [ 761, 772 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T5" } ] }, { "id": "PMC-1447668-09-Results-07_E7", "type": "Regulation", "trigger": { "text": [ "affect" ], "offsets": [ [ 804, 810 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E8" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_E6" } ] }, { "id": "PMC-1447668-09-Results-07_E8", "type": "Binding", "trigger": { "text": [ "binding" ], "offsets": [ [ 819, 826 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T6" } ] }, { "id": "PMC-1447668-09-Results-07_E9", "type": "Negative_regulation", "trigger": { "text": [ "decrease" ], "offsets": [ [ 869, 877 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E10" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_T5" } ] }, { "id": "PMC-1447668-09-Results-07_E10", "type": "Binding", "trigger": { "text": [ "binding" ], "offsets": [ [ 927, 934 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T7" } ] }, { "id": "PMC-1447668-09-Results-07_E11", "type": "Binding", "trigger": { "text": [ "bind" ], "offsets": [ [ 1061, 1065 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T9" } ] }, { "id": "PMC-1447668-09-Results-07_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1772, 1782 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T17" } ] }, { "id": "PMC-1447668-09-Results-07_E13", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1868, 1878 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T18" } ] }, { "id": "PMC-1447668-09-Results-07_E14", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1868, 1878 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T19" } ] }, { "id": "PMC-1447668-09-Results-07_E15", "type": "Positive_regulation", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 1918, 1932 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T20" } ] }, { "id": "PMC-1447668-09-Results-07_E16", "type": "Positive_regulation", "trigger": { "text": [ "stimulated" ], "offsets": [ [ 1933, 1943 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E17" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_E15" } ] }, { "id": "PMC-1447668-09-Results-07_E17", "type": "Transcription", "trigger": { "text": [ "transcription" ], "offsets": [ [ 1944, 1957 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T21" } ] }, { "id": "PMC-1447668-09-Results-07_E18", "type": "Negative_regulation", "trigger": { "text": [ "repressed" ], "offsets": [ [ 2010, 2019 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E19" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_T22" } ] }, { "id": "PMC-1447668-09-Results-07_E19", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 2051, 2061 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T23" } ] }, { "id": "PMC-1447668-09-Results-07_E20", "type": "Negative_regulation", "trigger": { "text": [ "neutralizing" ], "offsets": [ [ 2101, 2113 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E21" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_T22" } ] }, { "id": "PMC-1447668-09-Results-07_E21", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 2129, 2139 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T24" } ] }, { "id": "PMC-1447668-09-Results-07_E22", "type": "Positive_regulation", "trigger": { "text": [ "Overexpression" ], "offsets": [ [ 2305, 2319 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T27" } ] }, { "id": "PMC-1447668-09-Results-07_E23", "type": "Binding", "trigger": { "text": [ "interact" ], "offsets": [ [ 2527, 2535 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T29" }, { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T30" } ] }, { "id": "PMC-1447668-09-Results-07_E24", "type": "Binding", "trigger": { "text": [ "immunoprecipitated" ], "offsets": [ [ 2590, 2608 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T31" }, { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T32" } ] }, { "id": "PMC-1447668-09-Results-07_E25", "type": "Positive_regulation", "trigger": { "text": [ "when" ], "offsets": [ [ 2615, 2619 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E24" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_E26" } ] }, { "id": "PMC-1447668-09-Results-07_E27", "type": "Positive_regulation", "trigger": { "text": [ "when" ], "offsets": [ [ 2615, 2619 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E24" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_E28" } ] }, { "id": "PMC-1447668-09-Results-07_E26", "type": "Gene_expression", "trigger": { "text": [ "overexpressed" ], "offsets": [ [ 2639, 2652 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T31" } ] }, { "id": "PMC-1447668-09-Results-07_E28", "type": "Gene_expression", "trigger": { "text": [ "overexpressed" ], "offsets": [ [ 2639, 2652 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T32" } ] }, { "id": "PMC-1447668-09-Results-07_E29", "type": "Binding", "trigger": { "text": [ "interacting" ], "offsets": [ [ 2786, 2797 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T33" }, { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T34" } ] }, { "id": "PMC-1447668-09-Results-07_E30", "type": "Negative_regulation", "trigger": { "text": [ "interfering" ], "offsets": [ [ 2824, 2835 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_E31" }, { "role": "Cause", "ref_id": "PMC-1447668-09-Results-07_E29" } ] }, { "id": "PMC-1447668-09-Results-07_E31", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 2861, 2872 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-09-Results-07_T34" } ] } ]

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[ { "id": "PMC-1447668-10-Discussion__text", "type": "abstract", "text": [ "Discussion\nIn the present study, we show that Foxp3 functions as a potent repressor of NF-kappaB- and CREB-dependent transcriptional activation. Furthermore, the carboxyl-terminal FKH domain appears to be dispensable for mediating these effects, at least in T cell populations. This observation may become important in light of recent reports suggesting that Foxp3 expression in thymic epithelial cells was crucial for directing development of T cells in the thymus [42]. Interestingly, the majority of the genetic mutations associated with IPEX, a severe autoimmune disorder caused by functional inactivation of Foxp3, map to the carboxyl-terminal FKH domain or the leucine zipper domain in the central region of the protein. Only one mutation associated with IPEX to date has been mapped to the amino-terminal proline-rich region [43]. It is possible that the FKH domain has a complex tertiary structure that is particularly sensitive to misfolding caused by genetic mutations and that an intact FKH domain is absolutely critical for promoting Foxp3 function in the nucleus, whereas the structure of the amino-terminal proline-rich region may tolerate certain mutations as long as the NF-kappaB/NF-AT binding motif remains unaltered. This motif may also include the zinc finger domain. A logical region that may be targeted by the amino-terminal proline-rich region of Foxp3 is the Rel homology domain found in both NF-kappaB and NF-AT family proteins. A region that may also be important with respect to Foxp3 function is the leucine zipper domain, as demonstrated by the number of mutations associated with IPEX that have been mapped in this region of Foxp3. The role of this domain in Foxp3 function remains uncharacterized, but may play a role in dimer formation as it does in other Foxp family members [44]. \nBecause the pathogenesis of a number of retroviral-induced immunologic disorders such as HIV-1/AIDS and HTLV-I/HAM/TSP have been associated with dysregulation of Foxp3 expression [8,45], we also examined the role of Foxp3 in retroviral gene expression. HIV-1 LTR activation in CD4+ T cells is critically dependent on two tandem NF-kappaB sites located between nucleotide positions -102 and -81 within the HIV-1 enhancer region, whereas HTLV-I LTR activation in the presence or absence of the HTLV-I-encoded transactivator protein Tax is independent of NF-kappaB [18]. To our knowledge for the first time, Foxp3 was shown to have a direct effect on HIV-1 LTR transcription. Deletion of the NF-kappaB sites within the HIV-1 enhancer region reduced the responsiveness of the HIV-1 LTR to Foxp3-mediated suppression. In addition, the FKH domain of Foxp3 was required for this inhibitory effect in HEK 293T cells, but not in Jurkat T cells, similar to Foxp3-mediated suppression of a synthetic NF-kappaB reporter. The direct effect of Foxp3 down-regulating HIV-1 gene expression correlates well with recently reported evidence indicating that higher regulatory activity of CD4+CD25+ T cells from HIV-1-infected patients was associated with lower HIV-1 viral loads in these patients [46]. \nFoxp3 also affected two well-known functions of HTLV-I Tax: transactivation of the NF-kappaB pathway and, most surprisingly, transactivation of the HTLV-I LTR. Transactivation of the HTLV-I LTR by Tax involves the interaction of ATF/CREB factors with Tax in the nucleus. Binding of Tax enhances ATF/CREB dimerization and promotes assembly of Tax-ATF/CREB complexes onto specific sequences in the viral promoter known as Tax-responsive elements. This series of steps allows Tax to recruit coactivator proteins CBP/p300 to the viral promoter and facilitate a high level of viral gene expression [24-26]. Transactivation of the NF-kappaB pathway by Tax was inhibited by overexpression of full-length Foxp3, but not DeltaFKH, as seen with basal activation of the HIV-1 LTR and a synthetic NF-kappaB reporter in HEK 293T cells. However, Tax-mediated transactivation of the HTLV-I LTR was inhibited by overexpression of both full-length Foxp3 as well as DeltaFKH in both HEK 293T cells and CD4+ T cells. We demonstrated that Foxp3 did not directly affect the functioning of Tax, but rather Foxp3 targeted the transcription factors required for Tax transactivation (i.e., NF-kappaB and a then-unknown cellular factor, which we identified in this study as CREB). The negative effect of Foxp3 on HTLV-I gene expression was confirmed utilizing an HTLV-I infectious molecular clone. \nImportantly, we demonstrated that HTLV-I-infected individuals with the highest levels of Foxp3 protein expression within the CD4+CD25+ T cells population exhibited lower proviral loads than did individuals with the lowest levels of Foxp3 protein expression. Previous studies have demonstrated that the HTLV-I proviral load directly correlates with HTLV-I Tax mRNA load, the frequency of immunopathogenic virus-specific CD8+ T cells, and disease severity in patients with HAM/TSP [47]. These results have important implications on the utility of Foxp3 in controlling viral gene expression and thus pathogenesis of HAM/TSP. Therefore, Foxp3 becomes an attractive target for the development of novel therapeutic applications directed at modulating the expression of this important regulatory protein, especially in light of recent observations that the expression of Foxp3 can also be down-regulated by HTLV-I Tax [8]. \nAs the activation of the HTLV-I LTR depends primarily on ATF/CREB proteins (whether in the presence or the absence of Tax), we investigated whether Foxp3 could interact with this additional cellular signaling pathway. While the DNA-binding activity of CREB is, in most cases, constitutive, the transactivation potential of CREB is regulated by the phosphorylation of CREB and recruitment of CBP/p300 [48]. Our data demonstrate that Foxp3 interferes with the latter of these two processes and that the recruitment of the coactivator protein p300, and resulting transcriptional activation are blocked by Foxp3. This may be the result of the physical interaction we detected between Foxp3 and p300. With respect to HTLV-I LTR activity, while full-length Foxp3 inhibited both basal and Tax-dependent transcription by ~50%, DeltaFKH appeared less effective in suppressing basal activation (~25% inhibition) compared to Tax-dependent activation (~50% inhibition). The effect of DeltaFKH on basal activation of the HTLV-I LTR in HEK 293T cells was very similar to that shown for a synthetic CREB reporter, suggesting that the FKH domain of Foxp3 is important at some level. As observed with NF-kappaB activation, the Foxp3 mutant lacking the FKH domain was a stronger inhibitor of CREB activation in CD4+ T cells than in HEK 293T epithelial cells. Therefore, it appears that in CD4+ T cells, the FKH domain is dispensable for the proper functioning of Foxp3 with respect to both NF-kappaB and CREB activation. \nIn summary, this is, to our knowledge, the first direct evidence implicating a role for the Treg-specific transcription factor Foxp3 in regulating retroviral gene expression. In addition, we identify the CREB pathway as a molecular target of Foxp3. Since CREB has been shown to regulate multiple genes involved in transcription (e.g., JunD, c-Fos, signal transducer of activated T cells 3 [STAT3]), cell cycle (e.g., p15INK4b, cyclin A, cyclin D1), and immune regulation (e.g., IL-2, IL-6, T-cell receptor alpha) (reviewed in [48]), the findings presented in this report broaden the potential range of signaling pathways under the control of the regulatory protein Foxp3. Our evidence stresses the importance of Foxp3 expression and Treg function in the development and maintenance of protective immunity against HIV-1 and HTLV-I. Based on recent findings, Foxp3 may limit HIV-1 and HTLV-I transcription by interfering with activation of NF-kappaB and CREB pathways. However, observing that this inhibitory effect is not absolute, a low level of viral gene expression may persist in CD4+ T cells (in particular regulatory T cells, which are known reservoirs of HIV-1 and HTLV-I) and result in the accumulation of viral proteins that either stimulate NF-kappaB and/or CREB activation or directly inhibit Foxp3 expression or function. The imbalance of NF-kappaB and CREB activation caused by these viral gene products may be a crucial step in the pathogenesis of virus-induced immunological disorders such as AIDS and HAM/TSP. Future studies will be directed at identifying and characterizing cellular proteins that interact with Foxp3 both in the nucleus and cytoplasm, in order to better address how Foxp3 functions to guide the development and function of regulatory T cells in health and disease. \n" ], "offsets": [ [ 0, 8694 ] ] } ]

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"PMC-1447668-10-Discussion_T31" } ] }, { "id": "PMC-1447668-10-Discussion_E17", "type": "Gene_expression", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 3996, 4010 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T33" } ] }, { "id": "PMC-1447668-10-Discussion_E18", "type": "Gene_expression", "trigger": { "text": [ "overexpression" ], "offsets": [ [ 3996, 4010 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T34" } ] }, { "id": "PMC-1447668-10-Discussion_E19", "type": "Positive_regulation", "trigger": { "text": [ "highest" ], "offsets": [ [ 4544, 4551 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E20" } ] }, { "id": "PMC-1447668-10-Discussion_E20", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 4576, 4586 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T41" } ] }, { "id": "PMC-1447668-10-Discussion_E21", "type": "Negative_regulation", "trigger": { "text": [ "lowest" ], "offsets": [ [ 4688, 4694 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E22" } ] }, { "id": "PMC-1447668-10-Discussion_E22", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 4719, 4729 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T44" } ] }, { "id": "PMC-1447668-10-Discussion_E23", "type": "Regulation", "trigger": { "text": [ "modulating" ], "offsets": [ [ 5207, 5217 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E24" } ] }, { "id": "PMC-1447668-10-Discussion_E24", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 5222, 5232 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T48" } ] }, { "id": "PMC-1447668-10-Discussion_E25", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 5323, 5333 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T49" } ] }, { "id": "PMC-1447668-10-Discussion_E26", "type": "Negative_regulation", "trigger": { "text": [ "down-regulated" ], "offsets": [ [ 5355, 5369 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E25" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_T50" } ] }, { "id": "PMC-1447668-10-Discussion_E27", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 5766, 5777 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T53" } ] }, { "id": "PMC-1447668-10-Discussion_E28", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 5766, 5777 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T54" } ] }, { "id": "PMC-1447668-10-Discussion_E29", "type": "Negative_regulation", "trigger": { "text": [ "interferes" ], "offsets": [ [ 5828, 5838 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E27" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_T55" } ] }, { "id": "PMC-1447668-10-Discussion_E30", "type": "Negative_regulation", "trigger": { "text": [ "interferes" ], "offsets": [ [ 5828, 5838 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E28" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_T55" } ] }, { "id": "PMC-1447668-10-Discussion_E31", "type": "Binding", "trigger": { "text": [ "recruitment" ], "offsets": [ [ 5891, 5902 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T56" } ] }, { "id": "PMC-1447668-10-Discussion_E32", "type": "Negative_regulation", "trigger": { "text": [ "blocked" ], "offsets": [ [ 5981, 5988 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E31" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_T57" } ] }, { "id": "PMC-1447668-10-Discussion_E33", "type": "Positive_regulation", "trigger": { "text": [ "result" ], "offsets": [ [ 6015, 6021 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E29" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_E34" } ] }, { "id": "PMC-1447668-10-Discussion_E35", "type": "Positive_regulation", "trigger": { "text": [ "result" ], "offsets": [ [ 6015, 6021 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E30" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_E34" } ] }, { "id": "PMC-1447668-10-Discussion_E36", "type": "Positive_regulation", "trigger": { "text": [ "result" ], "offsets": [ [ 6015, 6021 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E32" }, { "role": "Cause", "ref_id": "PMC-1447668-10-Discussion_E34" } ] }, { "id": "PMC-1447668-10-Discussion_E34", "type": "Binding", "trigger": { "text": [ "interaction" ], "offsets": [ [ 6038, 6049 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T58" }, { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T59" } ] }, { "id": "PMC-1447668-10-Discussion_E37", "type": "Negative_regulation", "trigger": { "text": [ "lacking" ], "offsets": [ [ 6613, 6620 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T66" }, { "role": "Site", "ref_id": "PMC-1447668-10-Discussion_T120" } ] }, { "id": "PMC-1447668-10-Discussion_E38", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T72" } ] }, { "id": "PMC-1447668-10-Discussion_E39", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T73" } ] }, { "id": "PMC-1447668-10-Discussion_E40", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T74" } ] }, { "id": "PMC-1447668-10-Discussion_E41", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T76" } ] }, { "id": "PMC-1447668-10-Discussion_E42", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T77" } ] }, { "id": "PMC-1447668-10-Discussion_E43", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T78" } ] }, { "id": "PMC-1447668-10-Discussion_E44", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T79" } ] }, { "id": "PMC-1447668-10-Discussion_E45", "type": "Regulation", "trigger": { "text": [ "regulate" ], "offsets": [ [ 7172, 7180 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T80" } ] }, { "id": "PMC-1447668-10-Discussion_E46", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 7612, 7622 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T82" } ] }, { "id": "PMC-1447668-10-Discussion_E47", "type": "Negative_regulation", "trigger": { "text": [ "inhibit" ], "offsets": [ [ 8189, 8196 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_E48" } ] }, { "id": "PMC-1447668-10-Discussion_E48", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 8203, 8213 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T85" } ] }, { "id": "PMC-1447668-10-Discussion_E49", "type": "Binding", "trigger": { "text": [ "interact" ], "offsets": [ [ 8508, 8516 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-10-Discussion_T86" } ] } ]

[ { "id": "PMC-1447668-10-Discussion_1", "entity_ids": [ "PMC-1447668-10-Discussion_T74", "PMC-1447668-10-Discussion_T75" ] } ]

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[ { "id": "PMC-1447668-11-Materials_and_Methods-01__text", "type": "abstract", "text": [ "Cell culture.\nHEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California, United States). Jurkat T cells and primary human CD4+ T cells were cultured in RPMI-1640 medium (Invitrogen). Media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mug/ml streptomycin (Cambrex, East Rutherford, New Jersey, United States), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, Georgia, United States). \n" ], "offsets": [ [ 0, 456 ] ] } ]

[ { "id": "PMC-1447668-11-Materials_and_Methods-01_T1", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 163, 166 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-12-Materials_and_Methods-02__text", "type": "abstract", "text": [ "Patients and cell preparation.\nPBMCs were prepared by centrifugation over Ficoll-Hypaque gradients (BioWhittaker, Walkersville, Maryland, United States) from eight HAM/TSP patients and eight ACs, and the cells were viably cryopreserved in liquid nitrogen until tested. HAM/TSP was diagnosed according to WHO guidelines [49]. HTLV-I seropositivity was determined by ELISA (Abbott Laboratories, Abbott Park, Illinois, United States), with confirmation by Western blot analysis (Genelabs Technologies, Redwood City, California, United States). Blood samples were obtained after informed consent as part of a clinical protocol reviewed and approved by the NIH institutional review panel. \n" ], "offsets": [ [ 0, 685 ] ] } ]

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[ { "id": "PMC-1447668-13-Materials_and_Methods-03__text", "type": "abstract", "text": [ "Plasmids.\nExpression vectors encoding human Foxp3 (pCMV-Foxp3-IRES-EGFP) and human Foxp3 lacking the forkhead (FKH) domain (pCMV-DeltaFKH-IRES-EGFP) were generous gifts from S. Ziegler (Benaroya Research Institute). pEGFP-C2 was provided by I. Lipinski (NIDDK/NIH). pcDNA3 was provided by K. T. Jeang (NIAID/NIH). pCMV4-Tax was a generous gift from W. Greene (University of California San Francisco). pGL4-luc2 and pGL4-TKhRluc2 were purchased from Promega (Madison, Wisconsin, United States). pUC18 was purchased from Stratagene (La Jolla, California, United States). HIV-1 wt LTR and HIV-1 Delta-kappaB LTR luciferase reporter vectors were constructed by cloning the XhoI/HindIII LTR fragments from pHIV-CAT and pDelta-kappaB-HIV-CAT (AIDS Research and Reference Reagent Program, NIAID/NIH) into the multiple cloning site of pGL4-luc2. NF-kappaB, HTLV-I LTR, and CREB luciferase reporter and pCMV-p300-HA expression vectors were generously provided by B. Wigdahl (Drexel University College of Medicine). HTLV-I pACH infectious molecular clone has been described previously [30]. pFR-luc, pFA-CMV, pFA2-CREB-1, and pFA2-c-Jun were purchased from Stratagene. pFA-Tax (encoding a fusion protein consisting of the Gal4 DNA-binding domain fused in-frame to HTLV-I Tax) was constructed by PCR amplification of HTLV-I Tax using pCMV4-Tax as a template and BamHI/BglII-tagged primers. The amplified insert was digested and ligated into the BamHI/BglII sites of pFA-CMV. Plasmid contents were confirmed by DNA sequencing. \n" ], "offsets": [ [ 0, 1516 ] ] } ]

[ { "id": "PMC-1447668-13-Materials_and_Methods-03_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 44, 49 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 56, 61 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 83, 88 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T4", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 320, 323 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T5", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 609, 619 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T6", "type": "Protein", "text": [ "CAT" ], "offsets": [ [ 706, 709 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T7", "type": "Protein", "text": [ "CAT" ], "offsets": [ [ 732, 735 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T8", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 870, 880 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T9", "type": "Protein", "text": [ "p300" ], "offsets": [ [ 899, 903 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T10", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 1104, 1110 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T11", "type": "Protein", "text": [ "c-Jun" ], "offsets": [ [ 1121, 1126 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T12", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1163, 1166 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T13", "type": "Protein", "text": [ "Gal4" ], "offsets": [ [ 1212, 1216 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T14", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1261, 1264 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T15", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1313, 1316 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T16", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 1329, 1332 ] ], "normalized": [] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_T19", "type": "Entity", "text": [ "forkhead (FKH) domain" ], "offsets": [ [ 101, 122 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-13-Materials_and_Methods-03_E1", "type": "Gene_expression", "trigger": { "text": [ "Expression" ], "offsets": [ [ 10, 20 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T1" } ] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_E2", "type": "Gene_expression", "trigger": { "text": [ "Expression" ], "offsets": [ [ 10, 20 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T3" } ] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_E3", "type": "Negative_regulation", "trigger": { "text": [ "lacking" ], "offsets": [ [ 89, 96 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T3" }, { "role": "Site", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T19" } ] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_E4", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 907, 917 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T8" } ] }, { "id": "PMC-1447668-13-Materials_and_Methods-03_E5", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 907, 917 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-13-Materials_and_Methods-03_T9" } ] } ]

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[ { "id": "PMC-1447668-14-Materials_and_Methods-04__text", "type": "abstract", "text": [ "Isolation of primary human CD4+ T cells.\nCD4+ T cells were isolated from cryopreserved healthy donor PBMCs by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's guidelines. Purity of negatively selected CD4+ T cells was consistently higher than 96% as determined by flow cytometry. \n" ], "offsets": [ [ 0, 364 ] ] } ]

[ { "id": "PMC-1447668-14-Materials_and_Methods-04_T1", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 27, 30 ] ], "normalized": [] }, { "id": "PMC-1447668-14-Materials_and_Methods-04_T2", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 41, 44 ] ], "normalized": [] }, { "id": "PMC-1447668-14-Materials_and_Methods-04_T3", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 138, 141 ] ], "normalized": [] }, { "id": "PMC-1447668-14-Materials_and_Methods-04_T4", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 284, 287 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-15-Materials_and_Methods-05__text", "type": "abstract", "text": [ "Transient expression and luciferase assays.\nHEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent (Roche, Basel, Switzerland). Jurkat T cells were plated at 1 x 106 cells/well in six-well culture plates the day of transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent. Primary human CD4+ T cells (2 x 106) were nucleofected with the specified plasmid DNA (5 mug total) using the Human T Cell Nucleofection Kit (Amaxa, Gaithersburg, Maryland, United States). Forskolin (10 muM; Calbiochem, San Diego, California, United States) was added in some experiments 20 h posttransfection. Cells were harvested 24 h posttransfection and luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, United States) according to manufacturer's guidelines. pGL4-TKhRluc2 was used as an internal control to normalize for transfection efficiency. Nucleofected CD4+ T cells were also monitored for transfection efficiency and cell viability 24 h posttransfection as follows. Transfection efficiency was routinely ~30% as determined by flow cytometric analysis of EGFP expression. Cell viability, determined by staining with 7-amino-actinomycin D (7-AAD; BD Biosciences), was routinely ~70%. Both transfection efficiency and cell viability in nucleofected CD4+ T cells was independent of the plasmids used. \n" ], "offsets": [ [ 0, 1656 ] ] } ]

[ { "id": "PMC-1447668-15-Materials_and_Methods-05_T1", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 25, 35 ] ], "normalized": [] }, { "id": "PMC-1447668-15-Materials_and_Methods-05_T2", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 526, 529 ] ], "normalized": [] }, { "id": "PMC-1447668-15-Materials_and_Methods-05_T3", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 870, 880 ] ], "normalized": [] }, { "id": "PMC-1447668-15-Materials_and_Methods-05_T4", "type": "Protein", "text": [ "luciferase" ], "offsets": [ [ 918, 928 ] ], "normalized": [] }, { "id": "PMC-1447668-15-Materials_and_Methods-05_T5", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1210, 1213 ] ], "normalized": [] }, { "id": "PMC-1447668-15-Materials_and_Methods-05_T6", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1604, 1607 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-16-Materials_and_Methods-06__text", "type": "abstract", "text": [ "Foxp3 and HTLV-I Tax expression analysis by real-time RT-PCR.\nReal-time RT-PCR analysis of Foxp3 and HTLV-I Tax expression was performed as previously described [8,47]. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to manufacturer's guidelines, and cDNA was synthesized by reverse transcription using TaqMan Gold RT-PCR Kit using random hexamer primers (Applied Biosystems, Foster City, California, United States). Foxp3 and HTLV-I Tax mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The normalized values in each sample were calculated as the relative quantity of Foxp3 or HTLV-I Tax mRNA expression divided by the relative quantity of HPRT mRNA expression. The values were calculated by the following formula: normalized Foxp3 or HTLV-I Tax expression = 2Ct value of HPRT - Ct value of Foxp3 or HTLV-I Tax. \n" ], "offsets": [ [ 0, 948 ] ] } ]

[ { "id": "PMC-1447668-16-Materials_and_Methods-06_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T2", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 17, 20 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 91, 96 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T4", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 108, 111 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T5", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 484, 489 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T6", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 501, 504 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T7", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 703, 708 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T8", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 719, 722 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T9", "type": "Protein", "text": [ "HPRT" ], "offsets": [ [ 775, 779 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T10", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 861, 866 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T11", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 877, 880 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T12", "type": "Protein", "text": [ "HPRT" ], "offsets": [ [ 907, 911 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T13", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 926, 931 ] ], "normalized": [] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_T14", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 942, 945 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-16-Materials_and_Methods-06_E1", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 21, 31 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T1" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 21, 31 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T2" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 112, 122 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T3" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E4", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 112, 122 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T4" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E5", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 505, 520 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T5" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E6", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 505, 520 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T6" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E7", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 723, 738 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T7" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E8", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 723, 738 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T8" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E9", "type": "Transcription", "trigger": { "text": [ "mRNA expression" ], "offsets": [ [ 780, 795 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T9" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E10", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 881, 891 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T10" } ] }, { "id": "PMC-1447668-16-Materials_and_Methods-06_E11", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 881, 891 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-16-Materials_and_Methods-06_T11" } ] } ]

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[ { "id": "PMC-1447668-17-Materials_and_Methods-07__text", "type": "abstract", "text": [ "Real-time PCR.\nReal-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described [47,50]. DNA was extracted from 1 x 106 cells using Puregene DNA Isolation Kit (Gentra, Minneapolis, Minnesota, United States), and 100 ng of the sample DNA solution was analyzed by this system. The HTLV-I proviral DNA load was calculated by the following formula: copy number of HTLV-I (pX) per 100 cells = (copy number of pX)/(copy number of beta-actin/2) x 100. \n" ], "offsets": [ [ 0, 472 ] ] } ]

[ { "id": "PMC-1447668-17-Materials_and_Methods-07_T1", "type": "Protein", "text": [ "Tax" ], "offsets": [ [ 49, 52 ] ], "normalized": [] }, { "id": "PMC-1447668-17-Materials_and_Methods-07_T2", "type": "Protein", "text": [ "beta-actin" ], "offsets": [ [ 450, 460 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-18-Materials_and_Methods-08__text", "type": "abstract", "text": [ "Western blot analysis.\nHEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 mug total) using FuGene 6 transfection reagent (Roche). Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Igepal (NP-40), 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1x Complete Mini Protease Inhibitor [Roche]). Protein concentration was determined by Lowry assay (Bio-Rad, Hercules, California, United States) and colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices, Sunnyvale, California, United States) at an absorbance of 750 nm. Size fractionation was performed on 20 mug of protein/sample by SDS-PAGE, and the protein was transferred to nitrocellulose or PVDF membranes and subjected to immunoblotting using the indicated antibodies. Foxp3 was detected using rabbit anti-human Foxp3 polyclonal antibody (ab4728 or ab10563; Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, Massachusetts, United States). NF-kappaB p65 and CREB-1 were detected using rabbit anti-human polyclonal (p65) or monoclonal antibody (CREB-1; 48H2) (Cell Signaling Technology). Beta-actin was detected using a mouse monoclonal antibody (AC-15; Sigma, St. Louis, Missouri, United States). For coimmunoprecipitation analysis, cell lysates were precleared with 30 mul of protein A/G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, California, United States) and then incubated with mouse monoclonal anti-HA antibody (6E2; 1:100; Cell Signaling Technology) and 30 mul of protein A/G plus-agarose beads overnight. The immunoprecipitates were washed four times with RIPA buffer, resuspended in SDS sample buffer, and heated at 95 degreesC for 5 min. Proteins were then treated as described for Western blot analysis. \n" ], "offsets": [ [ 0, 1982 ] ] } ]

[ { "id": "PMC-1447668-18-Materials_and_Methods-08_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 957, 962 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 1000, 1005 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T3", "type": "Protein", "text": [ "p65" ], "offsets": [ [ 1201, 1204 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T4", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 1209, 1215 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T5", "type": "Protein", "text": [ "p65" ], "offsets": [ [ 1266, 1269 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T6", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 1295, 1301 ] ], "normalized": [] }, { "id": "PMC-1447668-18-Materials_and_Methods-08_T7", "type": "Protein", "text": [ "Beta-actin" ], "offsets": [ [ 1338, 1348 ] ], "normalized": [] } ]

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[ { "id": "PMC-1447668-19-Materials_and_Methods-09__text", "type": "abstract", "text": [ "Transcription factor DNA-binding analysis (TF-ELISA).\nCREB-1 and ATF-2 DNA-binding activity was analyzed with the TransFactor Profiling (Inflammation 1) Kit (BD Biosciences) according to the manufacturer's protocol. Nuclear extracts were prepared from HEK 293T cells transfected with a control vector (EGFP) or Foxp3 expression vector (1,000 ng) in the presence or absence of forskolin (10 muM for 4 h) using the TransFactor Extraction Kit (BD Biosciences). Protein concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany). Nuclear extracts (20 mug) were incubated in preblocked wells containing plate-bound double-stranded oligonucleotides corresponding to an ATF/CREB consensus sequence (emTGACATCAem). Wells were washed, incubated with the appropriate primary antibody, washed, incubated with secondary antibody (HRP-labeled), washed again, and finally developed with TMB substrate. Colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices) at an absorbance of 655 nm. \n" ], "offsets": [ [ 0, 1027 ] ] } ]

[ { "id": "PMC-1447668-19-Materials_and_Methods-09_T1", "type": "Protein", "text": [ "CREB-1" ], "offsets": [ [ 54, 60 ] ], "normalized": [] }, { "id": "PMC-1447668-19-Materials_and_Methods-09_T2", "type": "Protein", "text": [ "ATF-2" ], "offsets": [ [ 65, 70 ] ], "normalized": [] }, { "id": "PMC-1447668-19-Materials_and_Methods-09_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 311, 316 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-19-Materials_and_Methods-09_E1", "type": "Binding", "trigger": { "text": [ "binding" ], "offsets": [ [ 75, 82 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-19-Materials_and_Methods-09_T1" } ] }, { "id": "PMC-1447668-19-Materials_and_Methods-09_E2", "type": "Binding", "trigger": { "text": [ "binding" ], "offsets": [ [ 75, 82 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-19-Materials_and_Methods-09_T2" } ] }, { "id": "PMC-1447668-19-Materials_and_Methods-09_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 317, 327 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-19-Materials_and_Methods-09_T3" } ] } ]

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[ { "id": "PMC-1447668-20-Materials_and_Methods-10__text", "type": "abstract", "text": [ "Flow cytometric analysis of Foxp3 protein expression.\nCryopreserved PBMCs from HAM/TSP patients or HTLV-I-infected ACs were thawed and washed with FACS buffer (1x PBS, 0.1% NaN3, 5% FBS). Cells (1.5 x 106) were fixed by sequential formaldehyde/methanol fixation as follows. Cells were carefully resuspended in FACS buffer and fixed with 100 mul of reagent A (Fix & Perm kit; Caltag Laboratories, Burlingame, California, United States) at room temperature for 3 min followed by 2 ml of 70% methanol for 5 min at 4 degreesC. Cells were washed twice and permeabilized with 100 mul of reagent B (Fix & Perm kit) and stained for intracellular Foxp3 with mouse anti-human Foxp3 monoclonal antibody (0.5 mug of ab22510; Abcam) or the appropriate isotype control for 30 min. Cells were washed twice and stained with Cy5-conjugated goat anti-mouse immunoglobulin F(ab')2 secondary antibody (Caltag Laboratories) for an additional 30 min. Cells were washed twice and stained for surface CD4 expression with PE-labeled anti-CD4 (BD) and CD25 expression with FITC-labeled anti-CD25 (BD). Cells were washed twice and analyzed on a FACSCalibur (BD). Data analysis was performed using FlowJo (Tree Star, Ashland, Oregon, United States). \n" ], "offsets": [ [ 0, 1223 ] ] } ]

[ { "id": "PMC-1447668-20-Materials_and_Methods-10_T1", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 28, 33 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T2", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 638, 643 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T3", "type": "Protein", "text": [ "Foxp3" ], "offsets": [ [ 666, 671 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T4", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 977, 980 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T5", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1013, 1016 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T6", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 1026, 1030 ] ], "normalized": [] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_T7", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 1065, 1069 ] ], "normalized": [] } ]

[ { "id": "PMC-1447668-20-Materials_and_Methods-10_E1", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 42, 52 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-20-Materials_and_Methods-10_T1" } ] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 981, 991 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-20-Materials_and_Methods-10_T4" } ] }, { "id": "PMC-1447668-20-Materials_and_Methods-10_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1031, 1041 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-1447668-20-Materials_and_Methods-10_T6" } ] } ]

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[ { "id": "PMC-1447668-21-Materials_and_Methods-11__text", "type": "abstract", "text": [ "Statistical analyses.\nThe Mann-Whitney U test was used to compare the data between patients with HAM/TSP and AC. \n" ], "offsets": [ [ 0, 114 ] ] } ]

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[ { "id": "PMC-2065877-00-TIAB__text", "type": "abstract", "text": [ "EBV Latent Membrane Protein 1 Activates Akt, NFkappaB, and Stat3 in B Cell Lymphomas \nLatent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas. \nAuthor Summary\nEpstein-Barr virus (EBV) is linked to the development of multiple cancers, including post-transplant lymphoma, Hodgkin disease, and nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is expressed in many EBV-associated cancers and is responsible for most of the altered cellular growth properties that are induced by EBV infection. This study reveals that LMP1 induces lymphomas in B-1a lymphocytes, a cell type that is susceptible to transformation in aged mice. The lymphomas require Akt, NFkappaB, and Stat3 signaling for enhanced growth and survival. The activation of the Stat3, Akt, and NFkappaB signaling pathways likely underlies the ability of LMP1 to promote malignant transformation. \n" ], "offsets": [ [ 0, 1696 ] ] } ]

[ { "id": "PMC-2065877-00-TIAB_T1", "type": "Protein", "text": [ "Latent Membrane Protein 1" ], "offsets": [ [ 4, 29 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T2", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 40, 43 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T3", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 59, 64 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T4", "type": "Protein", "text": [ "Latent membrane protein 1" ], "offsets": [ [ 86, 111 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 113, 117 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 193, 197 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T7", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 459, 462 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T8", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 478, 483 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T9", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 516, 520 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T10", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 607, 610 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T11", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 611, 616 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T12", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 693, 698 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T13", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 858, 861 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T14", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 877, 882 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T15", "type": "Protein", "text": [ "Latent membrane protein 1" ], "offsets": [ [ 1150, 1175 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1177, 1181 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T17", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1356, 1360 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T18", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1486, 1489 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T19", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 1505, 1510 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T20", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 1577, 1582 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T21", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1584, 1587 ] ], "normalized": [] }, { "id": "PMC-2065877-00-TIAB_T22", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1653, 1657 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-00-TIAB_E1", "type": "Positive_regulation", "trigger": { "text": [ "Activates" ], "offsets": [ [ 30, 39 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T2" }, { "role": "Cause", "ref_id": "PMC-2065877-00-TIAB_T1" } ] }, { "id": "PMC-2065877-00-TIAB_E2", "type": "Positive_regulation", "trigger": { "text": [ "Activates" ], "offsets": [ [ 30, 39 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T3" }, { "role": "Cause", "ref_id": "PMC-2065877-00-TIAB_T1" } ] }, { "id": "PMC-2065877-00-TIAB_E3", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 445, 455 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T7" } ] }, { "id": "PMC-2065877-00-TIAB_E4", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 445, 455 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T8" } ] }, { "id": "PMC-2065877-00-TIAB_E5", "type": "Regulation", "trigger": { "text": [ "deregulated" ], "offsets": [ [ 738, 749 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T10" } ] }, { "id": "PMC-2065877-00-TIAB_E6", "type": "Regulation", "trigger": { "text": [ "deregulated" ], "offsets": [ [ 738, 749 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T11" } ] }, { "id": "PMC-2065877-00-TIAB_E7", "type": "Regulation", "trigger": { "text": [ "deregulated" ], "offsets": [ [ 738, 749 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T12" } ] }, { "id": "PMC-2065877-00-TIAB_E8", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 1186, 1195 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-00-TIAB_T15" } ] } ]

[ { "id": "PMC-2065877-00-TIAB_1", "entity_ids": [ "PMC-2065877-00-TIAB_T15", "PMC-2065877-00-TIAB_T16" ] } ]

[]

[ { "id": "PMC-2065877-01-Introduction__text", "type": "abstract", "text": [ "Introduction\nEpstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1]. EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3]. In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer. \nMany of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1]. Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4-8]. In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9-11]. While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization. LMP1 and CD40 interact with the same tumor necrosis factor receptor-associated factors (TRAFs) leading to activation of NFkappaB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12-16]. Activation of NFkappaB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18]. Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19]. \nIn vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment. In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell-dependent antigens [20,21]. Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22]. Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively. However, LMP1 is unable to restore affinity maturation and GC formation [23]. \nSeveral EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24-26]. The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%-50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26]. The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFkappaB, with specific activation of the NFkappaB family member cRel [27]. \nIn this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined. To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice. Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29]. LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4. In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target. The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway. Inhibition of the PI3K/Akt, NFkappaB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival. These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFkappaB, and Stat3 signaling pathways are required for its growth stimulatory effects. \n" ], "offsets": [ [ 0, 4970 ] ] } ]

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[ { "id": "PMC-2065877-02-Results-01__text", "type": "abstract", "text": [ "High Levels of LMP1 Expression Correlates with the Development of Lymphoma\nLMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer. It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26]. To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting. LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A). To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining. Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B). LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1. Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining. This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1. This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1. \n" ], "offsets": [ [ 0, 1785 ] ] } ]

[ { "id": "PMC-2065877-02-Results-01_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 15, 19 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 75, 79 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T3", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 253, 257 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 384, 388 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T5", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 509, 513 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 602, 606 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T7", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 625, 629 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T8", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 684, 688 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T9", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 845, 849 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T10", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 923, 927 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T11", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1055, 1059 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T12", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1183, 1187 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T13", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1229, 1233 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T14", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1306, 1310 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T15", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1342, 1346 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1417, 1421 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T17", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1469, 1473 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T18", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1552, 1556 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T19", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1623, 1627 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T20", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1659, 1663 ] ], "normalized": [] }, { "id": "PMC-2065877-02-Results-01_T21", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1778, 1782 ] ], "normalized": [] } ]

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"PMC-2065877-02-Results-01_E10", "type": "Positive_regulation", "trigger": { "text": [ "stronger" ], "offsets": [ [ 704, 712 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E9" } ] }, { "id": "PMC-2065877-02-Results-01_E11", "type": "Positive_regulation", "trigger": { "text": [ "higher level" ], "offsets": [ [ 829, 841 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T9" } ] }, { "id": "PMC-2065877-02-Results-01_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 909, 919 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T10" } ] }, { "id": "PMC-2065877-02-Results-01_E13", "type": "Gene_expression", "trigger": { "text": [ "detected" ], "offsets": [ [ 1046, 1054 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T11" } ] }, { "id": "PMC-2065877-02-Results-01_E14", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1188, 1198 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T12" } ] }, { "id": "PMC-2065877-02-Results-01_E15", "type": "Positive_regulation", "trigger": { "text": [ "strong" ], "offsets": [ [ 1222, 1228 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T13" } ] }, { "id": "PMC-2065877-02-Results-01_E16", "type": "Positive_regulation", "trigger": { "text": [ "weakly" ], "offsets": [ [ 1295, 1301 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T14" } ] }, { "id": "PMC-2065877-02-Results-01_E17", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1347, 1357 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T15" } ] }, { "id": "PMC-2065877-02-Results-01_E18", "type": "Positive_regulation", "trigger": { "text": [ "abundantly" ], "offsets": [ [ 1367, 1377 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E17" } ] }, { "id": "PMC-2065877-02-Results-01_E19", "type": "Positive_regulation", "trigger": { "text": [ "intense" ], "offsets": [ [ 1409, 1416 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T16" } ] }, { "id": "PMC-2065877-02-Results-01_E20", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 1459, 1468 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T17" } ] }, { "id": "PMC-2065877-02-Results-01_E21", "type": "Positive_regulation", "trigger": { "text": [ "increase" ], "offsets": [ [ 1540, 1548 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E22" } ] }, { "id": "PMC-2065877-02-Results-01_E22", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1557, 1567 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T18" } ] }, { "id": "PMC-2065877-02-Results-01_E23", "type": "Gene_expression", "trigger": { "text": [ "expressing" ], "offsets": [ [ 1597, 1607 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T19" } ] }, { "id": "PMC-2065877-02-Results-01_E24", "type": "Positive_regulation", "trigger": { "text": [ "high levels" ], "offsets": [ [ 1608, 1619 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E23" } ] }, { "id": "PMC-2065877-02-Results-01_E25", "type": "Positive_regulation", "trigger": { "text": [ "high" ], "offsets": [ [ 1654, 1658 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E26" } ] }, { "id": "PMC-2065877-02-Results-01_E26", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1664, 1674 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T20" } ] }, { "id": "PMC-2065877-02-Results-01_E27", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 1758, 1767 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_E28" } ] }, { "id": "PMC-2065877-02-Results-01_E28", "type": "Gene_expression", "trigger": { "text": [ "levels" ], "offsets": [ [ 1768, 1774 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-02-Results-01_T21" } ] } ]

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[ { "id": "PMC-2065877-03-Results-02__text", "type": "abstract", "text": [ "LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion\nTo determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (kappa, lambda) were analyzed by flow cytometry. Similar numbers of naive (IgM+IgD+IgG-) splenic B cells with a strong bias towards kappa light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data). Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells. B-1 cells are further separated into CD5+ (B-1a) and CD5- (B-1b) subsets. To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5. All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells. Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A). These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30]. Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5-), indicating that LMP1 does not affect B cell differentiation (Figure 2B). \nDue to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a kappa or lambda light chain. Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both kappa and lambda light chains (Figure 2A). Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of kappa chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality. To further assess light chain expression, the passaged samples were tested by immunoblotting for kappa and lambda light chains (Figure 2C). Interestingly, very low levels of expression of both light chains were detected by flow cytometry. The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell. In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for kappa and lambda light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains. \nA previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for kappa and lambda light chains [31]. These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion. \n" ], "offsets": [ [ 0, 3115 ] ] } ]

[ { "id": "PMC-2065877-03-Results-02_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 79, 83 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T3", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 178, 182 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 448, 452 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 486, 490 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 614, 618 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T7", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 779, 782 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T8", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 795, 798 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T9", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 916, 919 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T10", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 945, 949 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T11", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 997, 1000 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T12", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1316, 1320 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T13", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 1412, 1416 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T14", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 1417, 1420 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T15", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 1452, 1456 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T16", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 1457, 1460 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T17", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1480, 1484 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T18", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1745, 1749 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T19", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1920, 1924 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T20", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2570, 2574 ] ], "normalized": [] }, { "id": "PMC-2065877-03-Results-02_T21", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2994, 2998 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-03-Results-02_E1", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 183, 193 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-03-Results-02_T3" } ] }, { "id": "PMC-2065877-03-Results-02_E2", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2980, 2990 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-03-Results-02_T21" } ] } ]

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[ { "id": "PMC-2065877-04-Results-03__text", "type": "abstract", "text": [ "LMP1 Promotes B Cell Survival and Proliferation In Vitro\nPrimary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32]. To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2'-deoxy-uridine (BrdU) incorporation for proliferation. In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A). In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4. Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A). The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma. As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18-20 mo. The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old. There was no correlation between age and MTS activity. \nEMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes. Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B). Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase. This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells. Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data). Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C). Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%). This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation. The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1). \n" ], "offsets": [ [ 0, 2930 ] ] } ]

[ { "id": "PMC-2065877-04-Results-03_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T2", "type": "Protein", "text": [ "CD40" ], "offsets": [ [ 107, 111 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T3", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 146, 149 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 179, 183 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T5", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 291, 294 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T6", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 568, 571 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T7", "type": "Protein", "text": [ "CD40" ], "offsets": [ [ 589, 593 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T8", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 629, 633 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T9", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 694, 697 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T10", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 743, 746 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T11", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 762, 766 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T12", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 848, 851 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T13", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 869, 873 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T14", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 1323, 1327 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T15", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1379, 1383 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1448, 1452 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T17", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1527, 1531 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T18", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1616, 1619 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T19", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1685, 1688 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T20", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1761, 1765 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T21", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1811, 1814 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T22", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1910, 1913 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T23", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1976, 1980 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T24", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2031, 2035 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T25", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2130, 2134 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T26", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2345, 2349 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T27", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2477, 2481 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T28", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2613, 2617 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T29", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2625, 2629 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T30", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2652, 2656 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T31", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2671, 2675 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T32", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2715, 2719 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T33", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 2828, 2831 ] ], "normalized": [] }, { "id": "PMC-2065877-04-Results-03_T34", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 2857, 2860 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-05-Results-04__text", "type": "abstract", "text": [ "Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling\nTo investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA). IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46mu (Figure 4A). However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A). Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B). In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells. However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C). \nAlthough wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest. The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D). However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E). In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling. \n" ], "offsets": [ [ 0, 1616 ] ] } ]

[ { "id": "PMC-2065877-05-Results-04_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 14, 18 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T2", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 60, 63 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T3", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 68, 73 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T4", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 107, 110 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T5", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 146, 149 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T6", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 162, 165 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T7", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 229, 232 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T8", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 351, 354 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T9", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 391, 395 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T10", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 466, 470 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T11", "type": "Protein", "text": [ "GAPDH" ], "offsets": [ [ 527, 532 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T12", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 537, 540 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T13", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 600, 605 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T14", "type": "Protein", "text": [ "pStat6" ], "offsets": [ [ 607, 613 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T15", "type": "Protein", "text": [ "IL4 receptor" ], "offsets": [ [ 632, 644 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 688, 692 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T17", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 796, 800 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T18", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 907, 910 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T19", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 919, 924 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T20", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1023, 1026 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T21", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1267, 1270 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T22", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1336, 1339 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T23", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1398, 1401 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T24", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1437, 1441 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T25", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1511, 1515 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T26", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 1564, 1567 ] ], "normalized": [] }, { "id": "PMC-2065877-05-Results-04_T27", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 1598, 1603 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-05-Results-04_1", "entity_ids": [ "PMC-2065877-05-Results-04_T13", "PMC-2065877-05-Results-04_T14" ] } ]

[]

[ { "id": "PMC-2065877-06-Results-05__text", "type": "abstract", "text": [ "LMP1 Upregulates IL10 and Constitutively Activates Stat3\nTo identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNgamma. Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32. None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967. Transcription of IL10, IL15, and IFNgamma were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46mu (Figure 5A). There was no significant difference in the expression of IL15 and IFNgamma between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNgamma is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes. Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A). Production of IL15 and IFNgamma has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34]. However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35-38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined. Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated alpha and beta isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B). However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation. This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive. Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A). This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1. Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3. This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C). Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C). This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction. The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C). \nNuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice. However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D). The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development. \n" ], "offsets": [ [ 0, 3755 ] ] } ]

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"normalized": [] }, { "id": "PMC-2065877-06-Results-05_T9", "type": "Protein", "text": [ "IL15" ], "offsets": [ [ 291, 295 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T10", "type": "Protein", "text": [ "IL9" ], "offsets": [ [ 297, 300 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T11", "type": "Protein", "text": [ "IL2" ], "offsets": [ [ 302, 305 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T12", "type": "Protein", "text": [ "IL6" ], "offsets": [ [ 307, 310 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T13", "type": "Protein", "text": [ "IFNgamma" ], "offsets": [ [ 316, 324 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T14", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 432, 435 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T15", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 525, 528 ] ], "normalized": [] }, { "id": "PMC-2065877-06-Results-05_T16", "type": "Protein", "text": [ "IL10" ], 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"ref_id": "PMC-2065877-06-Results-05_E42" }, { "role": "Cause", "ref_id": "PMC-2065877-06-Results-05_T46" } ] }, { "id": "PMC-2065877-06-Results-05_E44", "type": "Negative_regulation", "trigger": { "text": [ "Neutralizing" ], "offsets": [ [ 2423, 2435 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T47" } ] }, { "id": "PMC-2065877-06-Results-05_E45", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 2576, 2586 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T48" } ] }, { "id": "PMC-2065877-06-Results-05_E46", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 2726, 2735 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T50" } ] }, { "id": "PMC-2065877-06-Results-05_E47", "type": "Positive_regulation", "trigger": { "text": [ "enhanced" ], "offsets": [ [ 2848, 2856 ] ] }, "arguments": [ { "role": "Theme", "ref_id": 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"PMC-2065877-06-Results-05_T57" } ] }, { "id": "PMC-2065877-06-Results-05_E52", "type": "Localization", "trigger": { "text": [ "translocation" ], "offsets": [ [ 3280, 3293 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T60" }, { "role": "ToLoc", "ref_id": "PMC-2065877-06-Results-05_T104" } ] }, { "id": "PMC-2065877-06-Results-05_E53", "type": "Positive_regulation", "trigger": { "text": [ "consequence" ], "offsets": [ [ 3309, 3320 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_E52" }, { "role": "Cause", "ref_id": "PMC-2065877-06-Results-05_E54" } ] }, { "id": "PMC-2065877-06-Results-05_E54", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 3324, 3334 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T60" } ] }, { "id": "PMC-2065877-06-Results-05_E55", "type": "Localization", "trigger": { "text": [ "detected" ], "offsets": [ [ 3363, 3371 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T61" }, { "role": "ToLoc", "ref_id": "PMC-2065877-06-Results-05_T108" } ] }, { "id": "PMC-2065877-06-Results-05_E56", "type": "Localization", "trigger": { "text": [ "detectable" ], "offsets": [ [ 3471, 3481 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T62" }, { "role": "ToLoc", "ref_id": "PMC-2065877-06-Results-05_T110" } ] }, { "id": "PMC-2065877-06-Results-05_E57", "type": "Localization", "trigger": { "text": [ "detected" ], "offsets": [ [ 3538, 3546 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T62" }, { "role": "ToLoc", "ref_id": "PMC-2065877-06-Results-05_T110" } ] }, { "id": "PMC-2065877-06-Results-05_E58", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 3625, 3635 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-06-Results-05_T65" } ] } ]

[]

[]

[ { "id": "PMC-2065877-07-Results-06__text", "type": "abstract", "text": [ "LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway\nLMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5]. Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40]. To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels. The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A). This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells. Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40]. In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3. This further suggests that additional pathways are involved in the regulation of GSK3. \nTo identify other potential Akt targets, immunoblot analysis for p-mTOR was performed. Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B). Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42]. Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation. Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels. Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development. However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways. \nThe Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44]. LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45]. To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C). Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C). These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27. \n" ], "offsets": [ [ 0, 3505 ] ] } ]

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"normalized": [] }, { "id": "PMC-2065877-07-Results-06_T9", "type": "Protein", "text": [ "pAkt" ], "offsets": [ [ 346, 350 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T10", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 421, 425 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T11", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 450, 454 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T12", "type": "Protein", "text": [ "pAkt" ], "offsets": [ [ 498, 502 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T13", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 579, 583 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T14", "type": "Protein", "text": [ "pAkt" ], "offsets": [ [ 647, 651 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T15", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 664, 667 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T16", "type": "Protein", "text": [ "glycogen synthase kinase 3" ], "offsets": [ [ 675, 701 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T17", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 703, 707 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T18", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 778, 782 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T19", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 917, 921 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T20", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 951, 954 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T21", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 962, 966 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T22", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1035, 1038 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T23", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 1066, 1070 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T24", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1178, 1181 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T25", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 1221, 1225 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T26", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 1308, 1312 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T27", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1343, 1346 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T28", "type": "Protein", "text": [ "p-mTOR" ], "offsets": [ [ 1380, 1386 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T29", "type": "Protein", "text": [ "p-mTOR" ], "offsets": [ [ 1412, 1418 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T30", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1440, 1444 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T31", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 1535, 1539 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T32", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1548, 1551 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T33", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 1624, 1627 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T34", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 1895, 1900 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T35", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 1931, 1936 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T36", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 1976, 1981 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T37", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 2017, 2022 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T38", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 2072, 2077 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T39", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2125, 2129 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T40", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 2251, 2254 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T41", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 2306, 2309 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T42", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 2336, 2341 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T43", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2357, 2361 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T44", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 2382, 2385 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T45", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 2419, 2424 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T46", "type": "Protein", "text": [ "p27" ], "offsets": [ 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"Protein", "text": [ "Cdk2" ], "offsets": [ [ 2926, 2930 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T55", "type": "Protein", "text": [ "p27" ], "offsets": [ [ 2936, 2939 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T56", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 2966, 2970 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T57", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 2995, 2999 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T58", "type": "Protein", "text": [ "pRb" ], "offsets": [ [ 3042, 3045 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T59", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 3086, 3088 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T60", "type": "Protein", "text": [ "Cdk2" ], "offsets": [ [ 3100, 3104 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T61", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 3200, 3204 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T62", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 3258, 3260 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T63", "type": "Protein", "text": [ "Cdk2" ], "offsets": [ [ 3293, 3297 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T64", "type": "Protein", "text": [ "p27" ], "offsets": [ [ 3323, 3326 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T65", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 3369, 3371 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T66", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 3398, 3402 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T67", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 3479, 3484 ] ], "normalized": [] }, { "id": "PMC-2065877-07-Results-06_T68", "type": "Protein", "text": [ "p27" ], "offsets": [ [ 3499, 3502 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-08-Results-07__text", "type": "abstract", "text": [ "LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFkappaB, and Stat3 Pathways\nTo explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFkappaB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay. As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors. However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11-7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7). Triciribine inhibits the activation of Akt and at 20 muM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46]. The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 muM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7). The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IkappaBalpha (Figure 8). Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 muM. Phosphorylated Stat3 and IkappaBalpha were still present at 25 muM. These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells. \nInhibition of NFkappaB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18]. BAY11-7085, an inhibitor of NFkappaB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 muM, and at 5 muM the cells were completely nonviable (Figure 7). This is well within the reported IC50 of 10 muM. Phosphorylated Akt, Stat3, and total IkappaBalpha were still present up to treatment with 15 muM and then were no longer detected (Figure 8). This finding suggests that inhibition of NFkappaB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3. \nCucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2. It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47]. Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 muM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47]. Phosphorylated Akt, Stat3, and total IkappaBalpha were not detectable past 1 muM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8). A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IkappaBalpha were also not affected (Figure 8). These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFkappaB signaling. \nLMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity. However, effects of SB202190 were only apparent at high doses (>10 muM), much higher than the reported IC50 of 0.35 muM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7). Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11-7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFkappaB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7). \n" ], "offsets": [ [ 0, 4416 ] ] } ]

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[ { "id": "PMC-2065877-09-Discussion__text", "type": "abstract", "text": [ "Discussion\nThis study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis. LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development. Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas. Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation. Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis. Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20]. It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion. The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas. Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation. These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51]. This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53]. In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice. In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54]. \nB-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood. B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell-independent responses to common microbial antigens. Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29]. Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30]. The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells. The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth. These same pathways can also become sporadically activated in aged mice and also result in lymphoma development. This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55-58]. Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development. \nThe lymphomas were marked by the upregulation of IL10, constitutive activation of Stat3 signaling, and a requirement for activation of Akt, NFkappaB, and Stat3 pathways (Figures 5 and 7). Induction of IL10 is associated with the transformation of B-1 lymphomas in mice [59,60] and is frequently associated with EBV-positive B cell malignancies acting as a B cell growth factor [35-38]. In addition, LMP1 has been shown to stimulate IL10 expression in Burkitt lymphoma cell lines [61,62]. This suggests that although Stat3 is constitutively activated in the lymphoma cells, the induction of IL10 may further enhance Stat3 activation or may contribute to other IL10-responsive signaling pathways. \nLMP1 activates both the canonical and non-canonical pathways of NFkappaB signaling [14,63-65], and inhibition of NFkappaB blocked the survival of LMP1 transgenic lymphocytes and LMP1-positive and -negative lymphoma cells. NFkappaB and PI3K signaling are crucial for CD40-induced proliferation, and mice deficient for cRel or the p85 regulatory subunit of PI3K are unresponsive to mitogenic stimuli, including CD40 ligation [66-68]. We have previously shown that cRel is specifically activated in both wild-type and LMP1 transgenic lymphomas, suggesting that activation of cRel is associated with B cell transformation [27]. Our observations suggest that similar to CD40-induced proliferation, LMP1 induces proliferation through PI3K-mediated activation of Akt and activation of NFkappaB components such as cRel. CD40 also induces downregulation of the cell cycle inhibitor p27 through a PI3K-dependent manner, and the LMP1 lymphoma cells also had decreased levels of p27 with phosphorylation of Rb and increased Cdk2 (Figure 6C) [66]. Although LMP1 has been shown to deregulate the Rb pathway in epithelial cells [69], to our knowledge this is the first demonstration of this property in B lymphocytes. \nThe requirement for Akt activation was confirmed by the striking inhibition of lymphoma viability by triciribine, an Akt inhibitor. However, the activated pAkt did not lead to phosphorylation and inactivation of the downstream target GSK3 (Figure 6A). This effect has also been described in EBV-positive HD biopsies [40]. In contrast, rapamycin, U0126, and SB202190 did not affect the survival of LMP1 transgenic lymphocytes or the wild-type and LMP1 transgenic lymphoma cells (Figure 7A, 7C, and 7E). This lack of effect by rapamycin confirmed the absence of activated p-mTOR levels (Figure 6B). These findings suggest that other Akt targets contribute to malignant progression. One key target is likely the inhibition of the Fox01 transcription factors. Repression of the pro-apoptotic transcription factor FoxO1 in a PI3K-dependent manner can inhibit expression of bcl6, a transcription factor necessary for GC formation [49,70]. It has been shown that overstimulation of CD40 signaling with agonistic antibodies inhibits GC formation [71]. Similarly, due to mimicry of CD40 signaling, transgenic LMP1 mice are also defective in GC formation [23,49]. The constitutive signaling by LMP1 likely blocks GC formation through downregulation of bcl6. Interestingly, clinical studies indicate that expression of LMP1 and bcl6 are mutually exclusive in non-HD and classical HD [72,73]. Thus, the LMP1 transgenic lymphomas mirror aspects of EBV-induced HD. Although the activation of Akt and the lack of Fox01 in the lymphoma cells suggest that LMP1 affects bcl6 and GC formation through this pathway, regulation of other Forkhead targets involved in cell cycle progression, such as p27 and CyclinD2, likely contribute to malignant transformation. Indeed, loss of FoxO1 expression in lymphoma cells correlated with a loss of p27 (Figure 6B and 6C). CyclinD2 has also been shown to be upregulated by LMP1 through release of FoxO1-mediated repression [70]. \nIn summary, in this transgenic model of lymphomagenesis, LMP1 promotes malignancy in B-1a cells, a population that is predisposed to clonal expansion with age. The malignant lymphocytes were distinguished by constitutively active Stat3 signaling, decreased p27, and activated Akt and NFkappaB pathways, properties that are associated with promoting the growth and survival of B lymphocytes. Importantly, Akt, NFkappaB, and Stat3 pathways were critically required for the growth and survival of malignant lymphocytes as well as healthy LMP1 transgenic lymphocytes. The growth of EBV-transformed lymphocytes also requires activation of NFkappaB, and these studies provide insight into how LMP1 contributes to EBV-associated transformation. The transgenic lymphomas mirror multiple aspects of EBV-induced tumors and suggest that in vivo these properties of LMP1 are major factors in the development of cancer. \n" ], "offsets": [ [ 0, 8435 ] ] } ]

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] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E31" } ] }, { "id": "PMC-2065877-09-Discussion_E31", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 5601, 5611 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T58" } ] }, { "id": "PMC-2065877-09-Discussion_E32", "type": "Negative_regulation", "trigger": { "text": [ "inhibitor" ], "offsets": [ [ 5698, 5707 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T59" } ] }, { "id": "PMC-2065877-09-Discussion_E33", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 5722, 5731 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T60" } ] }, { "id": "PMC-2065877-09-Discussion_E34", "type": "Positive_regulation", "trigger": { "text": [ "lead" ], "offsets": [ [ 5745, 5749 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E35" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E33" } ] }, { "id": "PMC-2065877-09-Discussion_E36", "type": "Positive_regulation", "trigger": { "text": [ "lead" ], "offsets": [ [ 5745, 5749 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E37" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E33" } ] }, { "id": "PMC-2065877-09-Discussion_E35", "type": "Phosphorylation", "trigger": { "text": [ "phosphorylation" ], "offsets": [ [ 5753, 5768 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T61" } ] }, { "id": "PMC-2065877-09-Discussion_E37", "type": "Negative_regulation", "trigger": { "text": [ "inactivation" ], "offsets": [ [ 5773, 5785 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T61" } ] }, { "id": "PMC-2065877-09-Discussion_E38", "type": "Negative_regulation", "trigger": { "text": [ "absence" ], "offsets": [ [ 6126, 6133 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E39" } ] }, { "id": "PMC-2065877-09-Discussion_E39", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 6137, 6146 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T64" } ] }, { "id": "PMC-2065877-09-Discussion_E40", "type": "Regulation", "trigger": { "text": [ "target" ], "offsets": [ [ 6265, 6271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E41" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_T65" } ] }, { "id": "PMC-2065877-09-Discussion_E41", "type": "Negative_regulation", "trigger": { "text": [ "inhibition" ], "offsets": [ [ 6286, 6296 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T66" } ] }, { "id": "PMC-2065877-09-Discussion_E42", "type": "Negative_regulation", "trigger": { "text": [ "Repression" ], "offsets": [ [ 6333, 6343 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T67" } ] }, { "id": "PMC-2065877-09-Discussion_E43", "type": "Negative_regulation", "trigger": { "text": [ "inhibit" ], "offsets": [ [ 6423, 6430 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E44" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E42" } ] }, { "id": "PMC-2065877-09-Discussion_E44", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 6431, 6441 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T68" } ] }, { "id": "PMC-2065877-09-Discussion_E45", "type": "Negative_regulation", "trigger": { "text": [ "downregulation" ], "offsets": [ [ 6801, 6815 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T73" } ] }, { "id": "PMC-2065877-09-Discussion_E46", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 6871, 6881 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T74" } ] }, { "id": "PMC-2065877-09-Discussion_E47", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 6871, 6881 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T75" } ] }, { "id": "PMC-2065877-09-Discussion_E48", "type": "Negative_regulation", "trigger": { "text": [ "exclusive" ], "offsets": [ [ 6912, 6921 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E46" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E46" } ] }, { "id": "PMC-2065877-09-Discussion_E49", "type": "Negative_regulation", "trigger": { "text": [ "exclusive" ], "offsets": [ [ 6912, 6921 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E46" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E47" } ] }, { "id": "PMC-2065877-09-Discussion_E50", "type": "Negative_regulation", "trigger": { "text": [ "exclusive" ], "offsets": [ [ 6912, 6921 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E47" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E46" } ] }, { "id": "PMC-2065877-09-Discussion_E51", "type": "Negative_regulation", "trigger": { "text": [ "exclusive" ], "offsets": [ [ 6912, 6921 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E47" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E47" } ] }, { "id": "PMC-2065877-09-Discussion_E52", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 7041, 7051 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T77" } ] }, { "id": "PMC-2065877-09-Discussion_E53", "type": "Negative_regulation", "trigger": { "text": [ "lack" ], "offsets": [ [ 7067, 7071 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T78" } ] }, { "id": "PMC-2065877-09-Discussion_E54", "type": "Regulation", "trigger": { "text": [ "affects" ], "offsets": [ [ 7121, 7128 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T80" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E55" } ] }, { "id": "PMC-2065877-09-Discussion_E55", "type": "Regulation", "trigger": { "text": [ "pathway" ], "offsets": [ [ 7164, 7171 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T78" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_T79" } ] }, { "id": "PMC-2065877-09-Discussion_E56", "type": "Regulation", "trigger": { "text": [ "regulation" ], "offsets": [ [ 7173, 7183 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T81" } ] }, { "id": "PMC-2065877-09-Discussion_E57", "type": "Regulation", "trigger": { "text": [ "regulation" ], "offsets": [ [ 7173, 7183 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T82" } ] }, { "id": "PMC-2065877-09-Discussion_E58", "type": "Negative_regulation", "trigger": { "text": [ "loss" ], "offsets": [ [ 7327, 7331 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E59" } ] }, { "id": "PMC-2065877-09-Discussion_E59", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 7341, 7351 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T83" } ] }, { "id": "PMC-2065877-09-Discussion_E60", "type": "Positive_regulation", "trigger": { "text": [ "correlated" ], "offsets": [ [ 7370, 7380 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_E61" }, { "role": "Cause", "ref_id": "PMC-2065877-09-Discussion_E58" } ] }, { "id": "PMC-2065877-09-Discussion_E61", "type": "Negative_regulation", "trigger": { "text": [ "loss" ], "offsets": [ [ 7388, 7392 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T84" } ] }, { "id": "PMC-2065877-09-Discussion_E62", "type": "Positive_regulation", "trigger": { "text": [ "upregulated" ], "offsets": [ [ 7455, 7466 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T85" } ] }, { "id": "PMC-2065877-09-Discussion_E63", "type": "Negative_regulation", "trigger": { "text": [ "decreased" ], "offsets": [ [ 7774, 7783 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-09-Discussion_T90" } ] } ]

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[ { "id": "PMC-2065877-10-Materials_and_Methods-01__text", "type": "abstract", "text": [ "Transgenic mice.\nGeneration of LMP1 mice under the Ig heavy chain promoter and enhancer have been described previously and were maintained as heterozygotes on a Balbc background [26]. LMP1 mice were genotyped by Southern blot and PCR analysis of tail DNA as described previously [26]. Spleen and liver sections were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-mum sections were stained with hematoxylin and eosin for histopathological analysis. Lymphomas were passaged by intraperitoneal injection of 1 x 108 splenocytes into SCID mice and sacrificed upon development of an extended abdomen. Animals were housed in the Association for Assessment and Accreditation for Animal Care-approved animal facility at the University of North Carolina at Chapel Hill. All protocols were approved by the Institutional Animal Care and Use Committee. \n" ], "offsets": [ [ 0, 855 ] ] } ]

[ { "id": "PMC-2065877-10-Materials_and_Methods-01_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 31, 35 ] ], "normalized": [] }, { "id": "PMC-2065877-10-Materials_and_Methods-01_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 184, 188 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-11-Materials_and_Methods-02__text", "type": "abstract", "text": [ "Isolation and growth of B cells.\nSplenocytes were prepared by homogenizing spleen tissue with two frosted slides and debris was filtered through a 100-mum cell strainer. Erythrocytes were lysed using 0.8% ammonium chloride solution (StemCell Technologies) for 10 min on ice and washed twice with PBS. B cells were isolated using CD19-MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and grown in Iscove's medium supplemented with heat-inactivated 10% fetal bovine serum and antibiotic/antimycotic (GIBCO). Splenocytes isolated from SCID-passaged lymphomas consisted of 80%-90% B cells as determined by flow cytometry, and were hence not further purified with CD19-MACS beads. Splenocytes were seeded at 1.25 x 106 cells/ml and where applicable, recombinant mouse IL4 was added at 100 ng/ml, recombinant mouse IL10 at 10 ng/ml, and rat IgG1 anti-mouse IL10 and rat IgG1 isotype control at 10 mug/ml (R&D Systems). For BrdU incorporation assays, splenocytes were pulsed for 24 h with 10 muM BrdU 1 d post-harvest. \n" ], "offsets": [ [ 0, 1038 ] ] } ]

[ { "id": "PMC-2065877-11-Materials_and_Methods-02_T1", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 329, 333 ] ], "normalized": [] }, { "id": "PMC-2065877-11-Materials_and_Methods-02_T2", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 684, 688 ] ], "normalized": [] }, { "id": "PMC-2065877-11-Materials_and_Methods-02_T3", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 788, 791 ] ], "normalized": [] }, { "id": "PMC-2065877-11-Materials_and_Methods-02_T4", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 834, 838 ] ], "normalized": [] }, { "id": "PMC-2065877-11-Materials_and_Methods-02_T5", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 876, 880 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-12-Materials_and_Methods-03__text", "type": "abstract", "text": [ "MTS assay.\nThe 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions. For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4. Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 x 106 cells/ml at 100 mul per well. MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks. For neutralization assays, splenocytes were seeded at 5 x 106 cells/ml at 100 mul per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures. Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding. For inhibitor studies, splenocytes were seeded at 1 x 107 cells/ml at 100 mul per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures. The inhibitors BAY11-7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences. For non-malignant splenocyte cultures, B cell activation was induced with 10 mug/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch). Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding. \n" ], "offsets": [ [ 0, 1423 ] ] } ]

[ { "id": "PMC-2065877-12-Materials_and_Methods-03_T1", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 302, 305 ] ], "normalized": [] }, { "id": "PMC-2065877-12-Materials_and_Methods-03_T2", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 389, 392 ] ], "normalized": [] }, { "id": "PMC-2065877-12-Materials_and_Methods-03_T3", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 719, 722 ] ], "normalized": [] }, { "id": "PMC-2065877-12-Materials_and_Methods-03_T4", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 744, 747 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-13-Materials_and_Methods-04__text", "type": "abstract", "text": [ "Immunohistochemistry.\nParaffin-embedded spleen sections were deparaffinized in Histoclear (National Diagnostics) and rehydrated in graded ethanol. Sections were antigen retrieved by microwaving in citrate buffer (pH 6.0) for 15 min (LMP1 staining) and 10 min (pStat3 staining). For LMP1 staining, sections were blocked with 1% BSA and 0.1% cold fish skin gelatin followed with streptavidin/biotin block (Vector Labs). Rat IgG anti-LMP1 (clones 8G3 and 1G6, Ascenion) were used at 1:10 dilution from tissue culture supernatants, followed with 8 mug/ml biotinylated mouse F(ab') anti-rat IgG (H+L) pre-adsorbed to mouse serum (Jackson ImmunoResearch) and 2 mug/ml streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResearch). Stains were developed with BCIP/NBT and counterstained in Nuclear Fast Red (Dako). Phospho-Stat3 was detected with 4 mug/ml of pStat3 antibody (Tyr705, Cell Signaling) and detected with anti-rabbit Poly-HRP IHC detection kit (Chemicon). \n" ], "offsets": [ [ 0, 970 ] ] } ]

[ { "id": "PMC-2065877-13-Materials_and_Methods-04_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 233, 237 ] ], "normalized": [] }, { "id": "PMC-2065877-13-Materials_and_Methods-04_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 282, 286 ] ], "normalized": [] }, { "id": "PMC-2065877-13-Materials_and_Methods-04_T3", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 431, 435 ] ], "normalized": [] }, { "id": "PMC-2065877-13-Materials_and_Methods-04_T4", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 823, 828 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-14-Materials_and_Methods-05__text", "type": "abstract", "text": [ "Flow cytometry.\nOne million splenocytes were stained with the appropriate primary antibody unconjugated or conjugated to FITC, PE, or APC diluted in stain buffer (PBS with 3% FBS). For BrdU detection, the FITC-BrdU flow kit was used as instructed by the manufacturer (BD Bioscience). Briefly, cells were exposed to EMA (Molecular Probes) for exclusion of dead cells, stained for surface antigens, fixed in paraformaldehyde, and permeabilized with saponin. To expose BrdU epitopes, cells were treated with Dnase and stained with FITC-conjugated anti-BrdU antibody. Flow cytometry was performed on FACScalibur using the CellQuest program (Becton Dickinson). Further analysis was conducted on the Summit v4.2 program (Dako). \n" ], "offsets": [ [ 0, 723 ] ] } ]

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[ { "id": "PMC-2065877-15-Materials_and_Methods-06__text", "type": "abstract", "text": [ "Rnase protection assay.\nTotal RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen). The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences). Briefly, 50 ng of mCK-1 probe set was labeled with [alpha-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion). The labeled probe was quantitated using a scintillation counter, and 6 x 105 cherenkov cpm was used to hybridize to 4 mug of total RNA using the RPA kit (BD Biosciences). Samples were denatured at 90 degreesC and hybridized at 56 degreesC overnight. Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol. Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics). Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare). \n" ], "offsets": [ [ 0, 1085 ] ] } ]

[ { "id": "PMC-2065877-15-Materials_and_Methods-06_T1", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 52, 56 ] ], "normalized": [] }, { "id": "PMC-2065877-15-Materials_and_Methods-06_T2", "type": "Protein", "text": [ "Rnase A" ], "offsets": [ [ 817, 824 ] ], "normalized": [] }, { "id": "PMC-2065877-15-Materials_and_Methods-06_T3", "type": "Protein", "text": [ "T1" ], "offsets": [ [ 829, 831 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-16-Materials_and_Methods-07__text", "type": "abstract", "text": [ "PCR analysis of kappa chain rearrangement.\nDNA was isolated from splenocytes using the Dneasy Tissue Kit (Qiagen), with Rnase treatment. PCR reactions contained 100 ng of genomic DNA, 0.2 muM each primer, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (NEB) performed in 1X ThermoPol buffer (NEB). Primers used were Vkappacon and Jkappa5-1degrees and have been described previously [74]. PCR conditions were 94 degreesC for 2 min, 40 cycles of 94 degreesC for 30 s, 63 degreesC for 90 s, and 72 degreesC for 1 min, followed by 1 cycle of 72 degreesC for 5 min. \n" ], "offsets": [ [ 0, 558 ] ] } ]

[ { "id": "PMC-2065877-16-Materials_and_Methods-07_T1", "type": "Protein", "text": [ "Taq DNA polymerase" ], "offsets": [ [ 229, 247 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-17-Materials_and_Methods-08__text", "type": "abstract", "text": [ "Immunoblot analysis.\nWhole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma). Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 degreesC and the supernatants were collected for further analysis. Protein concentrations were determined with the Bio-Rad DC protein assay system. Lysates were boiled in the presence of 2.5% beta-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-mum Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit. Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase-tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce). \n" ], "offsets": [ [ 0, 955 ] ] } ]

[ { "id": "PMC-2065877-17-Materials_and_Methods-08_T1", "type": "Protein", "text": [ "peroxidase" ], "offsets": [ [ 822, 832 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-18-Materials_and_Methods-09__text", "type": "abstract", "text": [ "Antibodies.\nFITC-conjugated goat anti-mouse IgM, rat IgG2akappa anti-mouse IgD (clone 11-26), rat IgG1kappa anti-mouse kappa (clone 187.1), rat IgG2bkappa anti-mouse lambda (clone JC5-1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech. APC-conjugated rat IgG2akappa anti-mouse CD19 (clone 6D5), PE-conjugated mouse IgG2akappa anti-LMP1 (clone S12), un-conjugated mouse IgG2a anti-Rb (clone 2), and anti-Cdk2 (clone 55) were purchased from BD Bioscience. PE-conjugated rat IgG2akappa anti-mouse CD5 (clone 53-7.3) was purchased from eBioscience. Rat anti-LMP1 (clones 8G3, 1G6, 7E10, and 7G8) was purchased from Ascenion. Rabbit anti-pAkt (Ser473), anti-pGSK3alpha/beta (Ser21/9), anti-pStat3 (Tyr705), anti-pStat6 (Tyr641), anti-pmTOR (Ser2448), anti-Stat6, anti-Akt, anti-FoxO1, and anti-p27 were purchased from Cell Signaling. Rabbit anti-Stat3 (H-190), anti-IkappaBalpha (C-21), and goat anti-beta actin (I-19) were purchased from Santa Cruz Biotechnology. Mouse IgG1kappa anti-GSK3 was purchased from Upstate Biotechnology. Rabbit anti-pRb (Thr373) was purchased from EMD Biosciences. \n" ], "offsets": [ [ 0, 1173 ] ] } ]

[ { "id": "PMC-2065877-18-Materials_and_Methods-09_T1", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 360, 364 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 414, 418 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T3", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 463, 465 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T4", "type": "Protein", "text": [ "Cdk2" ], "offsets": [ [ 486, 490 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T5", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 577, 580 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 637, 641 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T7", "type": "Protein", "text": [ "pAkt" ], "offsets": [ [ 716, 720 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T8", "type": "Protein", "text": [ "pGSK3alpha" ], "offsets": [ [ 736, 746 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T9", "type": "Protein", "text": [ "beta" ], "offsets": [ [ 747, 751 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T10", "type": "Protein", "text": [ "pStat3" ], "offsets": [ [ 768, 774 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T11", "type": "Protein", "text": [ "pStat6" ], "offsets": [ [ 790, 796 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T12", "type": "Protein", "text": [ "pmTOR" ], "offsets": [ [ 812, 817 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T13", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 834, 839 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T14", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 846, 849 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T15", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 856, 861 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T16", "type": "Protein", "text": [ "p27" ], "offsets": [ [ 872, 875 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T17", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 924, 929 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T18", "type": "Protein", "text": [ "IkappaBalpha" ], "offsets": [ [ 944, 956 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T19", "type": "Protein", "text": [ "beta actin" ], "offsets": [ [ 979, 989 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T20", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 1064, 1068 ] ], "normalized": [] }, { "id": "PMC-2065877-18-Materials_and_Methods-09_T21", "type": "Protein", "text": [ "pRb" ], "offsets": [ [ 1123, 1126 ] ], "normalized": [] } ]

[]

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[ { "id": "PMC-2065877-19-Supporting_Information-01__text", "type": "abstract", "text": [ "Accession Numbers\nThe GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the EBV genome sequence is AJ507799. The gene identifier for LMP1 is BNLF1. \n" ], "offsets": [ [ 0, 167 ] ] } ]

[ { "id": "PMC-2065877-19-Supporting_Information-01_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 151, 155 ] ], "normalized": [] }, { "id": "PMC-2065877-19-Supporting_Information-01_T2", "type": "Protein", "text": [ "BNLF1" ], "offsets": [ [ 159, 164 ] ], "normalized": [] } ]

[]

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[]

[ { "id": "PMC-2065877-20-caption-01__text", "type": "abstract", "text": [ "High LMP1 Expression Correlates with the Development of Lymphoma\nLMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice. \n(A) Lymphomas are identified with a number (1-7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control. \n(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 mum. \n" ], "offsets": [ [ 0, 545 ] ] } ]

[ { "id": "PMC-2065877-20-caption-01_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 5, 9 ] ], "normalized": [] }, { "id": "PMC-2065877-20-caption-01_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 65, 69 ] ], "normalized": [] }, { "id": "PMC-2065877-20-caption-01_T3", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 133, 137 ] ], "normalized": [] }, { "id": "PMC-2065877-20-caption-01_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 219, 223 ] ], "normalized": [] }, { "id": "PMC-2065877-20-caption-01_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 312, 316 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-20-caption-01_E1", "type": "Positive_regulation", "trigger": { "text": [ "High" ], "offsets": [ [ 0, 4 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-20-caption-01_E2" } ] }, { "id": "PMC-2065877-20-caption-01_E2", "type": "Gene_expression", "trigger": { "text": [ "Expression" ], "offsets": [ [ 10, 20 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-20-caption-01_T1" } ] }, { "id": "PMC-2065877-20-caption-01_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 70, 80 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-20-caption-01_T2" } ] } ]

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[ { "id": "PMC-2065877-21-caption-02__text", "type": "abstract", "text": [ "LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion\n(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan-B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (kappa and lambda) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for kappa and lambda light chains. \n(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5-). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results. \n(C) Immunoblot analysis for kappa and lambda light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control. \n" ], "offsets": [ [ 0, 999 ] ] } ]

[ { "id": "PMC-2065877-21-caption-02_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 119, 123 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T3", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 164, 168 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T4", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 182, 185 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 310, 314 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 379, 383 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T7", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 582, 586 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T8", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 620, 624 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T9", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 625, 628 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T10", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 655, 659 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T11", "type": "Protein", "text": [ "CD5" ], "offsets": [ [ 660, 663 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T12", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 797, 801 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T13", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 911, 915 ] ], "normalized": [] }, { "id": "PMC-2065877-21-caption-02_T14", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 939, 943 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-22-caption-03__text", "type": "abstract", "text": [ "LMP1 Promotes B Cell Survival and Proliferation In Vitro\n(A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean +/- SEM of triplicate samples averaged from multiple mice where \"n\" the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas. \n(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d. \n(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown. \n" ], "offsets": [ [ 0, 908 ] ] } ]

[ { "id": "PMC-2065877-22-caption-03_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T2", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 98, 102 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T3", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 201, 204 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 404, 408 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 448, 452 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T6", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 497, 501 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T7", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 533, 537 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T8", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 642, 645 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T9", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 716, 720 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T10", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 772, 775 ] ], "normalized": [] }, { "id": "PMC-2065877-22-caption-03_T11", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 830, 834 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-23-caption-04__text", "type": "abstract", "text": [ "Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas \n" ], "offsets": [ [ 0, 74 ] ] } ]

[ { "id": "PMC-2065877-23-caption-04_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 47, 51 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-23-caption-04_E1", "type": "Gene_expression", "trigger": { "text": [ "Transgenic" ], "offsets": [ [ 52, 62 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-23-caption-04_T1" } ] } ]

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[ { "id": "PMC-2065877-24-caption-05__text", "type": "abstract", "text": [ "Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture\n(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes. The L32 and GAPDH housekeeping genes were used as a loading control. Arrow indicates the position of the protected probe. \n(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out. \n(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations. Shown are the results from LMP1 transgenic lymphoma 3. The results are the mean +/- SEM of triplicate samples from a single representative experiment that was repeated twice with similar results. \n" ], "offsets": [ [ 0, 1038 ] ] } ]

[ { "id": "PMC-2065877-24-caption-05_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 14, 18 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T2", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 70, 73 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T3", "type": "Protein", "text": [ "Stat6" ], "offsets": [ [ 74, 79 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T4", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 132, 135 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T5", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 164, 168 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T6", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 183, 187 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T7", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 216, 219 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T8", "type": "Protein", "text": [ "GAPDH" ], "offsets": [ [ 224, 229 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T9", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 375, 379 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T10", "type": "Protein", "text": [ "pStat6" ], "offsets": [ [ 410, 416 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T11", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 442, 446 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T12", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 525, 528 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T13", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 697, 701 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T14", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 742, 745 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T15", "type": "Protein", "text": [ "IL4" ], "offsets": [ [ 774, 777 ] ], "normalized": [] }, { "id": "PMC-2065877-24-caption-05_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 868, 872 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-24-caption-05_E1", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 400, 409 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-24-caption-05_T10" } ] } ]

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[ { "id": "PMC-2065877-25-caption-06__text", "type": "abstract", "text": [ "LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3\n(A) Relative expression of IL10, IL15, and IFNgamma mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay. Mouse lymphoma cell lines 967 and K46mu were used as controls. Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32. The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967. \n(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1. Arrows indicate the positions of the alpha and beta isoforms of Stat3. Actin was used as a loading control. \n(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice. Scale bar, 20 mum. \n" ], "offsets": [ [ 0, 1040 ] ] } ]

[ { "id": "PMC-2065877-25-caption-06_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T2", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 17, 21 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T3", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 62, 67 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T4", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 95, 99 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T5", "type": "Protein", "text": [ "IL15" ], "offsets": [ [ 101, 105 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T6", "type": "Protein", "text": [ "IFNgamma" ], "offsets": [ [ 111, 119 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T7", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 135, 139 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T8", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 160, 164 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T9", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 393, 396 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T10", "type": "Protein", "text": [ "L32" ], "offsets": [ [ 411, 414 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T11", "type": "Protein", "text": [ "pStat3" ], "offsets": [ [ 517, 523 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T12", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 545, 549 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T13", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 564, 568 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T14", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 655, 659 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T15", "type": "Protein", "text": [ "IL10" ], "offsets": [ [ 688, 692 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T16", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 773, 777 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T17", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 865, 870 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T18", "type": "Protein", "text": [ "pStat3" ], "offsets": [ [ 966, 972 ] ], "normalized": [] }, { "id": "PMC-2065877-25-caption-06_T19", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 998, 1002 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-25-caption-06_E1", "type": "Positive_regulation", "trigger": { "text": [ "Upregulates" ], "offsets": [ [ 5, 16 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_E2" }, { "role": "Cause", "ref_id": "PMC-2065877-25-caption-06_T1" } ] }, { "id": "PMC-2065877-25-caption-06_E2", "type": "Gene_expression", "trigger": { "text": [ "Expression" ], "offsets": [ [ 22, 32 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T2" } ] }, { "id": "PMC-2065877-25-caption-06_E3", "type": "Positive_regulation", "trigger": { "text": [ "Activates" ], "offsets": [ [ 52, 61 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T3" }, { "role": "Cause", "ref_id": "PMC-2065877-25-caption-06_T1" } ] }, { "id": "PMC-2065877-25-caption-06_E4", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 81, 91 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T4" } ] }, { "id": "PMC-2065877-25-caption-06_E5", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 81, 91 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T5" } ] }, { "id": "PMC-2065877-25-caption-06_E6", "type": "Transcription", "trigger": { "text": [ "expression" ], "offsets": [ [ 81, 91 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T6" } ] }, { "id": "PMC-2065877-25-caption-06_E7", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 507, 516 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T11" } ] }, { "id": "PMC-2065877-25-caption-06_E8", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 948, 957 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-25-caption-06_T18" } ] } ]

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[ { "id": "PMC-2065877-26-caption-07__text", "type": "abstract", "text": [ "LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway\n(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3alpha/beta, and (B) activated p-mTOR, and total levels of FoxO1. Arrows indicate the positions of alpha and beta isoforms of GSK3. The white line indicates that intervening lanes have been spliced out. \n(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27. Actin was used as a loading control. \n" ], "offsets": [ [ 0, 673 ] ] } ]

[ { "id": "PMC-2065877-26-caption-07_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 0, 4 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T2", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 15, 18 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T3", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 49, 51 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T4", "type": "Protein", "text": [ "CD19" ], "offsets": [ [ 122, 126 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T5", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 156, 160 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T6", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 181, 184 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T7", "type": "Protein", "text": [ "pAkt" ], "offsets": [ [ 222, 226 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T8", "type": "Protein", "text": [ "pGSK3alpha" ], "offsets": [ [ 273, 283 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T9", "type": "Protein", "text": [ "beta" ], "offsets": [ [ 284, 288 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T10", "type": "Protein", "text": [ "p-mTOR" ], "offsets": [ [ 308, 314 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T11", "type": "Protein", "text": [ "FoxO1" ], "offsets": [ [ 336, 341 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T12", "type": "Protein", "text": [ "GSK3" ], "offsets": [ [ 403, 407 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T13", "type": "Protein", "text": [ "Rb" ], "offsets": [ [ 544, 546 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T14", "type": "Protein", "text": [ "pRb" ], "offsets": [ [ 578, 581 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T15", "type": "Protein", "text": [ "Cdk2" ], "offsets": [ [ 603, 607 ] ], "normalized": [] }, { "id": "PMC-2065877-26-caption-07_T16", "type": "Protein", "text": [ "p27" ], "offsets": [ [ 630, 633 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-26-caption-07_E1", "type": "Regulation", "trigger": { "text": [ "targets" ], "offsets": [ [ 242, 249 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T8" } ] }, { "id": "PMC-2065877-26-caption-07_E2", "type": "Regulation", "trigger": { "text": [ "targets" ], "offsets": [ [ 242, 249 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T9" } ] }, { "id": "PMC-2065877-26-caption-07_E3", "type": "Positive_regulation", "trigger": { "text": [ "inactivated" ], "offsets": [ [ 261, 272 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T8" } ] }, { "id": "PMC-2065877-26-caption-07_E4", "type": "Positive_regulation", "trigger": { "text": [ "inactivated" ], "offsets": [ [ 261, 272 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T9" } ] }, { "id": "PMC-2065877-26-caption-07_E5", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 298, 307 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T10" } ] }, { "id": "PMC-2065877-26-caption-07_E6", "type": "Positive_regulation", "trigger": { "text": [ "activated" ], "offsets": [ [ 568, 577 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-26-caption-07_T14" } ] } ]

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[ { "id": "PMC-2065877-27-caption-08__text", "type": "abstract", "text": [ "Akt, NFkappaB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells\nMTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes. Splenocytes were cultured with or without inhibitors of NFkappaB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations. The results are the mean +/- SEM of triplicate samples. Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed. This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results. \n" ], "offsets": [ [ 0, 722 ] ] } ]

[ { "id": "PMC-2065877-27-caption-08_T1", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 0, 3 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T2", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 19, 24 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T3", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 150, 154 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T4", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 190, 194 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T5", "type": "Protein", "text": [ "mTOR" ], "offsets": [ [ 293, 297 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T6", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 311, 314 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T7", "type": "Protein", "text": [ "MEK1" ], "offsets": [ [ 330, 334 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T8", "type": "Protein", "text": [ "2" ], "offsets": [ [ 335, 336 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T9", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 365, 370 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T10", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 548, 552 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T11", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 605, 609 ] ], "normalized": [] }, { "id": "PMC-2065877-27-caption-08_T12", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 668, 672 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-27-caption-08_E1", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 261, 271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-27-caption-08_T5" } ] }, { "id": "PMC-2065877-27-caption-08_E2", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 261, 271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-27-caption-08_T6" } ] }, { "id": "PMC-2065877-27-caption-08_E3", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 261, 271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-27-caption-08_T7" } ] }, { "id": "PMC-2065877-27-caption-08_E4", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 261, 271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-27-caption-08_T8" } ] }, { "id": "PMC-2065877-27-caption-08_E5", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 261, 271 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-27-caption-08_T9" } ] } ]

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[ { "id": "PMC-2065877-28-caption-09__text", "type": "abstract", "text": [ "Analysis of Akt, NFkappaB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells\n(A-D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFkappaB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFkappaB inhibitor, BAY11-7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations. Arrows indicate the positions of alpha and beta isoforms of Stat3. Actin was used as a loading control. \n" ], "offsets": [ [ 0, 507 ] ] } ]

[ { "id": "PMC-2065877-28-caption-09_T1", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 12, 15 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T2", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 31, 36 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T3", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 150, 154 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T4", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 180, 183 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T5", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 199, 204 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T6", "type": "Protein", "text": [ "Akt" ], "offsets": [ [ 243, 246 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T7", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 318, 323 ] ], "normalized": [] }, { "id": "PMC-2065877-28-caption-09_T8", "type": "Protein", "text": [ "Stat3" ], "offsets": [ [ 462, 467 ] ], "normalized": [] } ]

[ { "id": "PMC-2065877-28-caption-09_E1", "type": "Negative_regulation", "trigger": { "text": [ "inhibitor" ], "offsets": [ [ 247, 256 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-28-caption-09_T6" } ] }, { "id": "PMC-2065877-28-caption-09_E2", "type": "Negative_regulation", "trigger": { "text": [ "inhibitors" ], "offsets": [ [ 324, 334 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2065877-28-caption-09_T7" } ] } ]

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[ { "id": "PMC-2065877-29-caption-10__text", "type": "abstract", "text": [ "Lymphomas Are Clonal by kappa Light Chain Rearrangement\nPCR analysis of total spleen genomic DNA for the mouse kappa locus. Primers were designed to amplify all V-J rearrangements, including VJ1, VJ2, VJ4, and VJ5. Specific PCR products for the four rearrangements corresponding to the calculated sizes are indicated with arrows. Bands of other sizes are due to non-specific products. If a tumor was present in the background of normal spleen cells, a single band appears brighter than all the other specific bands. Wild-type and LMP1 transgenic lymphocytes are positive for all four V-J rearrangements and serve as positive controls. Clonality is determined by the appearance of a predominant band at the correct size for a single V-J rearrangement. \n(739 KB PDF) \n" ], "offsets": [ [ 0, 766 ] ] } ]

[ { "id": "PMC-2065877-29-caption-10_T1", "type": "Protein", "text": [ "LMP1" ], "offsets": [ [ 530, 534 ] ], "normalized": [] } ]

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[ { "id": "PMC-2065877-30-caption-11__text", "type": "abstract", "text": [ "Click here for additional data file. \n" ], "offsets": [ [ 0, 38 ] ] } ]

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[ { "id": "PMC-2626671-00-TIAB__text", "type": "abstract", "text": [ "Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs \nActivation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs. \n" ], "offsets": [ [ 0, 1136 ] ] } ]

[ { "id": "PMC-2626671-00-TIAB_T1", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T2", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 114, 117 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T3", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 328, 336 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T4", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 501, 506 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T5", "type": "Protein", "text": [ "Eomesodermin" ], "offsets": [ [ 529, 541 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T6", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 543, 548 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T7", "type": "Protein", "text": [ "interferon (IFN) gamma" ], "offsets": [ [ 622, 644 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T8", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 744, 749 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T9", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 774, 779 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T10", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 790, 795 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T11", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 884, 893 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T12", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 895, 903 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T13", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 909, 919 ] ], "normalized": [] }, { "id": "PMC-2626671-00-TIAB_T14", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1002, 1007 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-00-TIAB_1", "entity_ids": [ "PMC-2626671-00-TIAB_T5", "PMC-2626671-00-TIAB_T6" ] } ]

[]

[ { "id": "PMC-2626671-01-INTRODUCTION__text", "type": "abstract", "text": [ "INTRODUCTION\nNaive CD8+ T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-gamma and TNF upon restimulation through the TCR (1, 2). In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3, 4). In this study, we have used in vitro systems such as those developed to study CD4+ T cell differentiation to define the molecular basis of effector CTL differentiation (5, 6). \nThe T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7). In uninfected mice, compound deletion of the Tbx21 (encoding T-bet) and eomesodermin genes is associated with a selective loss of CD8+ T cells with an IL-2Rbeta-high, memory phenotype (8). Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9). Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+ T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes. \nRunx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10-13). Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+ single-positive (SP) thymocytes but not in CD4+ SP cells (14). Although Runx3 is not expressed in naive CD4+ T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of the Il4 and Ifng cytokine genes (15, 16). In contrast, all three Runx proteins are expressed in mature CD8+ T cells (10, 12), and Runx3-deficient CD8+ T cells show reduced cytolytic activity (12, 13). We therefore tested whether Runx3 influenced cytolytic T cell differentiation. \nIn this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function. T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-gamma expression. Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at the Prf1 locus; in contrast, perforin expression is unaffected in T-bet-deficient cells. T cells lacking Runx3 show decreased expression of IFN-gamma and granzyme B, and Runx3 also binds the promoter regions of the Ifng and Gzmb genes. Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator of Gzmb expression but synergizes with T-bet and Eomes, respectively, to promote transcription of the Ifng and Prf1 genes. \n" ], "offsets": [ [ 0, 3160 ] ] } ]

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"T-bet" ], "offsets": [ [ 661, 666 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T9", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 793, 798 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T10", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 809, 814 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T11", "type": "Protein", "text": [ "eomesodermin" ], "offsets": [ [ 820, 832 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T12", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 878, 881 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T13", "type": "Protein", "text": [ "IL-2Rbeta" ], "offsets": [ [ 899, 908 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T14", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 961, 966 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T15", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 971, 976 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T16", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1183, 1188 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T17", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1193, 1198 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T18", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1379, 1382 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T19", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1612, 1615 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T20", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1616, 1619 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T21", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1646, 1651 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T22", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1656, 1664 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T23", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1723, 1726 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T24", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1771, 1774 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T25", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1800, 1805 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T26", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1832, 1835 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T27", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1914, 1919 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T28", "type": "Protein", "text": [ "Il4" ], "offsets": [ [ 2002, 2005 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T29", "type": "Protein", "text": [ "Ifng" ], "offsets": [ [ 2010, 2014 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T30", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 2101, 2104 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T31", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2128, 2133 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T32", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 2144, 2147 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T33", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2227, 2232 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T34", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2308, 2313 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T35", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 2391, 2396 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T36", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 2512, 2517 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T37", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 2571, 2580 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T38", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2593, 2598 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T39", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 2615, 2620 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T40", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 2625, 2633 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T41", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 2655, 2660 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T42", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2665, 2670 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T43", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 2683, 2687 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T44", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 2708, 2716 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T45", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 2745, 2750 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T46", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2784, 2789 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T47", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 2819, 2828 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T48", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 2833, 2843 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T49", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 2849, 2854 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T50", "type": "Protein", "text": [ "Ifng" ], "offsets": [ [ 2894, 2898 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T51", "type": "Protein", "text": [ "Gzmb" ], "offsets": [ [ 2903, 2907 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T52", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 3007, 3012 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T53", "type": "Protein", "text": [ "Gzmb" ], "offsets": [ [ 3039, 3043 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T54", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 3075, 3080 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T55", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 3085, 3090 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T56", "type": "Protein", "text": [ "Ifng" ], "offsets": [ [ 3138, 3142 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T57", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 3147, 3151 ] ], "normalized": [] }, { "id": "PMC-2626671-01-INTRODUCTION_T82", "type": "Entity", "text": [ "promoter regions" ], "offsets": [ [ 2870, 2886 ] ], "normalized": [] } ]

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], "offsets": [ [ 942, 951 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-01-INTRODUCTION_T14" } ] }, { "id": "PMC-2626671-01-INTRODUCTION_E6", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 942, 951 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-01-INTRODUCTION_T15" } ] }, { "id": "PMC-2626671-01-INTRODUCTION_E7", "type": "Binding", "trigger": { "text": [ "targeting" ], "offsets": [ [ 1443, 1452 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-01-INTRODUCTION_T16" } ] }, { "id": "PMC-2626671-01-INTRODUCTION_E8", "type": "Binding", "trigger": { "text": [ "targeting" ], "offsets": [ [ 1443, 1452 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-01-INTRODUCTION_T17" } ] }, { "id": "PMC-2626671-01-INTRODUCTION_E9", "type": "Transcription", "trigger": { "text": [ "expressed" ], "offsets": [ [ 1674, 1683 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-01-INTRODUCTION_T21" } ] 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[]

[ { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01__text", "type": "abstract", "text": [ "A cell culture system to monitor effector CTL differentiation\nWe used a simple cell culture system to examine the kinetics of effector gene expression during CD8+ T cell differentiation. Naive CD8+ T cells from P14 TCR transgenic mice were activated for 2 d with anti-CD3 and anti-CD28 or with splenic APCs in the presence of Gp33 peptide, and were cultured in media containing 100 U/ml of recombinant human IL-2 (rhIL-2). We used TCR transgenic mice for these experiments because they provide a reliable source of CD8+ T cells that are truly naive; however, we chose not to stimulate cells with antigen in most experiments so as to avoid contamination with proteins and nucleic acids derived from APCs. There were only minor differences in gene expression during differentiation induced by antigen/APC versus anti-CD3/anti-CD28, and the major conclusions presented in this report are the same for both activating conditions. \nUnder our culture conditions, activated CD8+ T cells expanded exponentially and accumulated for >8 d. We limited our analysis to the first 6-8 d after activation, a period that coincides with clonal expansion of CD8+ T cells after activation in vivo. \n" ], "offsets": [ [ 0, 1179 ] ] } ]

[ { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 158, 161 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T2", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 193, 196 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T3", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 268, 271 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T4", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 281, 285 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T5", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 408, 412 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T6", "type": "Protein", "text": [ "rhIL-2" ], "offsets": [ [ 414, 420 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T7", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 515, 518 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T8", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 815, 818 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T9", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 824, 828 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T10", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 967, 970 ] ], "normalized": [] }, { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T11", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1139, 1142 ] ], "normalized": [] } ]

[]

[ { "id": "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_1", "entity_ids": [ "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T5", "PMC-2626671-02-RESULTS_AND_DISCUSSION-01_T6" ] } ]

[]

[ { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02__text", "type": "abstract", "text": [ "Distinct expression kinetics of perforin and granzyme B during CTL development in culture\nOur experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A-D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A-D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-gamma and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A). \nWe evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F). By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay. Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19). Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E). Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F). \nThese results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2. Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated. \n" ], "offsets": [ [ 0, 2355 ] ] } ]

[ { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T1", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 32, 40 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T2", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 45, 55 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T3", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 152, 160 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T4", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 162, 172 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T5", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 205, 208 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T6", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 284, 292 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T7", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 309, 317 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T8", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 386, 394 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T9", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 396, 400 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T10", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 582, 592 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T11", "type": "Protein", "text": [ "Gzmb" ], "offsets": [ [ 594, 598 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T12", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 773, 783 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T13", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 928, 937 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T14", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 942, 945 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T15", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1522, 1530 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T16", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 2017, 2027 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T17", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 2115, 2123 ] ], "normalized": [] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T18", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 2217, 2221 ] ], "normalized": [] } ]

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"PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T10" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E13", "type": "Positive_regulation", "trigger": { "text": [ "up-regulated" ], "offsets": [ [ 663, 675 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T10" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E14", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 707, 716 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T10" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E15", "type": "Positive_regulation", "trigger": { "text": [ "remained high" ], "offsets": [ [ 819, 832 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T12" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E16", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 904, 913 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T13" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E17", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 904, 913 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T14" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E18", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 992, 1001 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E16" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E19", "type": "Positive_regulation", "trigger": { "text": [ "increased" ], "offsets": [ [ 992, 1001 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E17" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E20", "type": "Gene_expression", "trigger": { "text": [ "express" ], "offsets": [ [ 1482, 1489 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T15" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E21", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2028, 2038 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T16" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E22", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2124, 2134 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T17" } ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E23", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 2162, 2169 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_E22" } ] } ]

[ { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_1", "entity_ids": [ "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T8", "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T9" ] }, { "id": "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_2", "entity_ids": [ "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T10", "PMC-2626671-03-RESULTS_AND_DISCUSSION-02_T11" ] } ]

[]

[ { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03__text", "type": "abstract", "text": [ "Distinct kinetics of T-bet and Eomes expression during CTL differentiation\nThe T-box transcription factors T-bet and Eomes have been linked to the regulation of genes encoding effector cytokines (e.g., Ifng) and genes important for cytolytic function (e.g., Prf1 and GzmB) (20). We investigated the kinetics of expression of these transcription factors in our in vitro cultures (Fig. 1, A-D). T-bet mRNA and protein were not detectable in naive CD8+ T cells, but were strongly induced upon TCR priming (day 2) and remained expressed through day 6 of differentiation (Fig. 1, A and C; quantified in Fig. 1, B and D). In contrast, Eomes expression was low or undetectable at both the mRNA and protein levels in naive CD8+ T cells, and TCR priming in culture had only a modest effect on its expression at day 2 (Fig. 1, A and C). Strong induction of Eomes mRNA and protein was only observed at day 4 and later (Fig. 1, A and C). T-bet mRNA expression slightly preceded the expression of GzmB mRNA; similarly, Eomes mRNA and protein were expressed approximately1 d ahead of the reexpression of perforin mRNA and protein, respectively (Fig. 1, B and D). \nThis detailed kinetic analysis suggested that, under our culture conditions, T-bet and Eomes contribute to distinct aspects of gene transcription during CTL differentiation. T-bet is required early for IFN-gamma production, and our data suggested that Eomes might not function during this early period but rather might contribute later to the control of perforin expression. Our data seemed most consistent with a model in which TCR signals induce T-bet, which in turn induces IFN-gamma (17) and possibly granzyme B; subsequently, Eomes is induced during the period of clonal expansion in IL-2 and activates perforin expression. \n" ], "offsets": [ [ 0, 1780 ] ] } ]

[ { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T1", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 21, 26 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T2", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 31, 36 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T3", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 107, 112 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T4", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 117, 122 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T5", "type": "Protein", "text": [ "Ifng" ], "offsets": [ [ 202, 206 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T6", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 258, 262 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T7", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 267, 271 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T8", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 393, 398 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T9", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 445, 448 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T10", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 629, 634 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T11", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 715, 718 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T12", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 847, 852 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T13", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 926, 931 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T14", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 984, 988 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T15", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1006, 1011 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T16", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1090, 1098 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T17", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1227, 1232 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T18", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1237, 1242 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T19", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1324, 1329 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T20", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1352, 1361 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T21", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1402, 1407 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T22", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1504, 1512 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T23", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1598, 1603 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T24", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1627, 1636 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T25", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 1655, 1665 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T26", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1681, 1686 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T27", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 1739, 1743 ] ], "normalized": [] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T28", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1758, 1766 ] ], "normalized": [] } ]

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"regulation" ], "offsets": [ [ 147, 157 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T7" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E12", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 311, 321 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T3" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E13", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 311, 321 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T4" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E14", "type": "Gene_expression", "trigger": { "text": [ "detectable" ], "offsets": [ [ 425, 435 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T8" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E15", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 477, 484 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T8" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E16", "type": "Positive_regulation", "trigger": { "text": [ "remained" ], "offsets": [ [ 514, 522 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E17" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E17", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 523, 532 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T8" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E18", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 635, 645 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T10" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E19", "type": "Positive_regulation", "trigger": { "text": [ "low or undetectable" ], "offsets": [ [ 650, 669 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E18" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E20", "type": "Regulation", "trigger": { "text": [ "effect" ], "offsets": [ [ 774, 780 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E21" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E21", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 788, 798 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T10" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E22", "type": "Positive_regulation", "trigger": { "text": [ "induction" ], "offsets": [ [ 834, 843 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T12" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E23", "type": 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"trigger": { "text": [ "control" ], "offsets": [ [ 1493, 1500 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E31" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E31", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1513, 1523 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T22" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E32", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 1591, 1597 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T23" } ] }, { "id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_E33", "type": "Positive_regulation", "trigger": { "text": [ "induces" ], "offsets": [ [ 1619, 1626 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T24" }, { "role": "Cause", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T23" } 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"Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1767, 1777 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-04-RESULTS_AND_DISCUSSION-03_T28" } ] } ]

[]

[]

[ { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04__text", "type": "abstract", "text": [ "Perforin and granzyme B expression are not appreciably regulated by T-bet\nTo test the model outlined in the previous paragraph directly, we compared the expression of IFN-gamma, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice. As expected (17, 21), naive Tbx21-/- CD8+ T cells produced IFN-gamma poorly upon activation (Fig. 2 A). Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A). This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1). In contrast, T-bet-deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4). We consistently observed a modest reduction in GzmB mRNA in T-bet-deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C). \nTo examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21-/- mice with retroviruses containing internal ribosome entry site (IRES)-GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions. Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet-deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6). As expected, Eo-VP16 also rescued the early defect in IFN-gamma production observed in T-bet-deficient CD8+ T cells (Fig. 2 D). However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet-deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16. \n" ], "offsets": [ [ 0, 1774 ] ] } ]

[ { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T1", "type": "Protein", "text": [ "Perforin" ], "offsets": [ [ 0, 8 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T2", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 13, 23 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T3", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 68, 73 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T4", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 167, 176 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T5", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 178, 186 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T6", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 192, 202 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T7", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 206, 209 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T8", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 231, 236 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T9", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 238, 243 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T10", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 289, 294 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T11", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 298, 301 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T12", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 320, 329 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T13", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 401, 406 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T14", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 455, 458 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T15", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 591, 596 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T16", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 669, 674 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T17", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 730, 738 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T18", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 837, 841 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T19", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 850, 855 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T20", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 966, 976 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T21", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1021, 1026 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T22", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1048, 1051 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T23", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 1073, 1078 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T24", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 1152, 1155 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T25", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1228, 1233 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T26", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1235, 1242 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T27", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1305, 1312 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T28", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 1332, 1335 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T29", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1358, 1366 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T30", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1393, 1398 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T31", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1409, 1412 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T32", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1469, 1476 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T33", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1510, 1519 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T34", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1543, 1548 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T35", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1559, 1562 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T36", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1593, 1600 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T37", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 1616, 1620 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T38", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1653, 1658 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T39", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1694, 1699 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T40", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 1714, 1718 ] ], "normalized": [] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T41", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1764, 1771 ] ], "normalized": [] } ]

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"arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_E20" }, { "role": "Cause", "ref_id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T27" } ] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_E20", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1367, 1377 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T29" } ] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_E21", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 1399, 1408 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T30" } ] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_E22", "type": "Negative_regulation", "trigger": { "text": [ "rescued" ], "offsets": [ [ 1482, 1489 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_E23" }, { "role": "Cause", "ref_id": 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[ { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_1", "entity_ids": [ "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T8", "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T9" ] }, { "id": "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_2", "entity_ids": [ "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T25", "PMC-2626671-05-RESULTS_AND_DISCUSSION-04_T26" ] } ]

[]

[ { "id": "PMC-2626671-06-RESULTS_AND_DISCUSSION-05__text", "type": "abstract", "text": [ "Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3-/- (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3-/- CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-gamma (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3-/- T cells at day 6 of culture (Fig. 3, A and B). Runx3-/- T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-gamma, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C). \nWe previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-gamma and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet-deficient T cells led to an increase in both perforin and IFN-gamma expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-gamma, and granzyme B. \nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near -1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the -1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17). \nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3-/- mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3-/- CD8+CD4- SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3-/- CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3-/- CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-gamma (Fig. 4, A and B). In addition, Runx3-/- T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3-/- cells, even though it restored the capacity to induce IFN-gamma expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes. \nAs expected from their defect in perforin and granzyme B expression, Runx3-/- CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3-/- CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3-/- mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3-/- CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas-Fas ligand pathway. \n" ], "offsets": [ [ 0, 4900 ] ] } ]

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[]

[]

[ { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06__text", "type": "abstract", "text": [ "Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation\nCollectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C). Runx3 is present in naive CD8+ T cells before activation (12). It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-gamma. Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression. Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3. \nSurprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-gamma at day 4 (Fig. S2). For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3-/- cells (Fig. 4 A). Runx3 continues to be required for IFN-gamma expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A). \nAn unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C). T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-gamma upon restimulation, but its function is less important at later times. Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-gamma in restimulated CTLs (8). Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type. \nSurprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C). Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively. T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet-/- or Runx3-/- cells. Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression. \nCollectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rbeta signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C). Our data recall the \"feed-forward\" interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-gamma, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes. Whole-genome experiments in these and other systems will be required to establish whether cooperation between T-box and Runx family transcription factors is a general feature of cellular differentiation programs. \n" ], "offsets": [ [ 0, 3729 ] ] } ]

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"normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T8", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 431, 439 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T9", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 445, 454 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T10", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 456, 461 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T11", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 642, 647 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T12", "type": "Protein", "text": [ "Runx1" ], "offsets": [ [ 652, 657 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T13", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 690, 695 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T14", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 712, 717 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T15", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 759, 762 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T16", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 764, 768 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T17", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 774, 783 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T18", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 808, 811 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T19", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 816, 820 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T20", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 842, 847 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T21", "type": "Protein", "text": 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"type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1255, 1258 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T29", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1391, 1396 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T30", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1436, 1439 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T31", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1475, 1484 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T32", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1556, 1561 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T33", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1613, 1618 ] ], "normalized": [] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T34", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1639, 1644 ] ], "normalized": [] }, { "id": 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"role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T27" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E43", "type": "Positive_regulation", "trigger": { "text": [ "needed" ], "offsets": [ [ 1400, 1406 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E44" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T29" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E44", "type": "Gene_expression", "trigger": { "text": [ "produce" ], "offsets": [ [ 1467, 1474 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T31" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E45", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 1572, 1579 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T32" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E46", "type": "Regulation", "trigger": { "text": [ "functions downstream" ], "offsets": [ [ 1589, 1609 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T32" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T33" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E47", "type": "Positive_regulation", "trigger": { "text": [ "promoting" ], "offsets": [ [ 1648, 1657 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E48" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T32" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E49", "type": "Positive_regulation", "trigger": { "text": [ "promoting" ], "offsets": [ [ 1648, 1657 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E48" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T34" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E48", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1668, 1678 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T35" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E50", "type": "Positive_regulation", "trigger": { "text": [ "contribute" ], "offsets": [ [ 1747, 1757 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E51" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T36" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E52", "type": "Positive_regulation", "trigger": { "text": [ "contribute" ], "offsets": [ [ 1747, 1757 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E51" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T37" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E51", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1770, 1780 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T38" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E53", "type": "Positive_regulation", "trigger": { "text": [ "induces" ], "offsets": [ [ 1812, 1819 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T40" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T39" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E54", "type": "Positive_regulation", "trigger": { "text": [ "induces" ], "offsets": [ [ 1812, 1819 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T40" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T41" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E55", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2057, 2063 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T44" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T42" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E56", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2057, 2063 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T44" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T43" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E57", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2057, 2063 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T45" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T42" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E58", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2057, 2063 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T45" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T43" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E59", "type": "Regulation", "trigger": { "text": [ "regulating" ], "offsets": [ [ 2166, 2176 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E60" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T46" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E61", "type": "Regulation", "trigger": { "text": [ "regulating" ], "offsets": [ [ 2166, 2176 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E62" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T47" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E60", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2201, 2211 ] ] }, "arguments": [ { "role": "Theme", "ref_id": 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"role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E66" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E63" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E67", "type": "Positive_regulation", "trigger": { "text": [ "preceded" ], "offsets": [ [ 2285, 2293 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E68" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E64" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E66", "type": "Positive_regulation", "trigger": { "text": [ "up-regulation" ], "offsets": [ [ 2294, 2307 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T52" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E68", "type": "Positive_regulation", "trigger": { "text": [ "up-regulation" ], "offsets": [ [ 2294, 2307 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T53" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E69", "type": "Regulation", "trigger": { "text": [ "had no role" ], "offsets": [ [ 2363, 2374 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E70" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T54" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E70", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2387, 2397 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T55" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E71", "type": "Regulation", "trigger": { "text": [ "affect" ], "offsets": [ [ 2448, 2454 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E72" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T56" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E72", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2466, 2476 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T57" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E73", "type": "Gene_expression", "trigger": { "text": [ "expressed" ], "offsets": [ [ 2482, 2491 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T56" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E74", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 2550, 2559 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T60" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E75", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 2673, 2682 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T61" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E76", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 2776, 2785 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T63" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E77", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2855, 2861 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E78" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T65" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E79", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 2855, 2861 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E78" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T66" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E78", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 2871, 2881 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T67" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E80", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 3000, 3007 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T69" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E81", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 3000, 3007 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T70" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E82", "type": "Regulation", "trigger": { "text": [ "interaction" ], "offsets": [ [ 3161, 3172 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T72" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T72" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E83", "type": "Regulation", "trigger": { "text": [ "interaction" ], "offsets": [ [ 3161, 3172 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T72" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T73" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E84", "type": "Regulation", "trigger": { "text": [ "interaction" ], "offsets": [ [ 3161, 3172 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T73" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T72" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E85", "type": "Regulation", "trigger": { "text": [ "interaction" ], "offsets": [ [ 3161, 3172 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T73" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T73" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E86", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 3326, 3333 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T75" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T76" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E87", "type": "Positive_regulation", "trigger": { "text": [ "induces" ], "offsets": [ [ 3376, 3383 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T77" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T75" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E88", "type": "Positive_regulation", "trigger": { "text": [ "required" ], "offsets": [ [ 3458, 3466 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E89" }, { "role": "Cause", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T79" } ] }, { "id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_E89", "type": "Positive_regulation", "trigger": { "text": [ "induce" ], "offsets": [ [ 3470, 3476 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-07-RESULTS_AND_DISCUSSION-06_T80" } ] } ]

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[ { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01__text", "type": "abstract", "text": [ "Antibodies and reagents.\nThe following antibodies used for intracellular or surface stains were obtained from eBioscience: anti-IL-2, anti-IFN-gamma, anti-TNF, anti-granzyme B, anti-CD8, anti-CD25, and anti-CD44. Anti-CD69 was purchased from BD. For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory. The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti-Pol-II (Santa Cruz Biotechnology, Inc.). The T-bet antibody was provided by L. Glimcher (Harvard School of Public Health, Boston, MA). \nThe following reagents were used for the experiments presented in this report: Annexin V-FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems). The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO. \n" ], "offsets": [ [ 0, 1015 ] ] } ]

[ { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T1", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 128, 132 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T2", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 139, 148 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T3", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 155, 158 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T4", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 165, 175 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T5", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 182, 185 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T6", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 192, 196 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T7", "type": "Protein", "text": [ "CD44" ], "offsets": [ [ 207, 211 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T8", "type": "Protein", "text": [ "CD69" ], "offsets": [ [ 218, 222 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T9", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 277, 282 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T10", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 329, 334 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T11", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 442, 450 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T12", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 465, 470 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T13", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 534, 539 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T14", "type": "Protein", "text": [ "Annexin V" ], "offsets": [ [ 704, 713 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T15", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 749, 752 ] ], "normalized": [] }, { "id": "PMC-2626671-08-MATERIALS_AND_METHODS-01_T16", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 790, 793 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02__text", "type": "abstract", "text": [ "Isolation and culture of primary CD8+ T cells.\nCD8+ T cells from 4-8-wk-old Tcra-/- x P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21-/- (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells. CD8+ T cells from Runx3-/- mice on the ICR background were purified by positive selection (Miltenyi Biotec). All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees. For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 mug/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 mug/ml goat anti-hamster IgG. After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 x 105 cells/ml in media supplemented with 100 U/ml rhIL-2. Every 24 h, viable cells were counted and readjusted to 5 x 105 cells/ml with fresh media containing the corresponding amount of rhIL-2. \n" ], "offsets": [ [ 0, 1107 ] ] } ]

[ { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 47, 50 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T2", "type": "Protein", "text": [ "Tcra" ], "offsets": [ [ 76, 80 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T3", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 132, 137 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T4", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 275, 278 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T5", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 293, 298 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T6", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 616, 619 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T7", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 715, 718 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T8", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 741, 745 ] ], "normalized": [] }, { "id": "PMC-2626671-09-MATERIALS_AND_METHODS-02_T9", "type": "Protein", "text": [ "rhIL-2" ], "offsets": [ [ 1098, 1104 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03__text", "type": "abstract", "text": [ "Isolation of CD8+ T cells from Runx3-/- mice.\nRunx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13). We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4- SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads. This yielded a Runx3 KO SP \"enriched\" population that contained 75% CD8+CD4- cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1). The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2. As previously reported, TCR-induced proliferation of Runx3-/- CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13). However, the Runx3-/- cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1). As expected from their ability to up-regulate CD25, Runx3-/- CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1). Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3-/- CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not. The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells. \n" ], "offsets": [ [ 0, 1740 ] ] } ]

[ { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T1", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 31, 36 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T2", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 46, 51 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T3", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 86, 89 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T4", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 188, 191 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T5", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 206, 211 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T6", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 225, 228 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T7", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 229, 232 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T8", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 240, 243 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T9", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 244, 247 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T10", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 283, 286 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T11", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 318, 323 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T12", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 371, 374 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T13", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 375, 378 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T14", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 437, 440 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T15", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 441, 444 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T16", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 499, 502 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T17", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 509, 513 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T18", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 613, 617 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T19", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 672, 677 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T20", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 681, 684 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T21", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 733, 736 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T22", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 781, 786 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T23", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 894, 898 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T24", "type": "Protein", "text": [ "CD69" ], "offsets": [ [ 903, 907 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T25", "type": "Protein", "text": [ "CD25" ], "offsets": [ [ 965, 969 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T26", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 971, 976 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T27", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 980, 983 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T28", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 1006, 1010 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T29", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1204, 1207 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T30", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1517, 1522 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T31", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1526, 1529 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T32", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1559, 1562 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T33", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1648, 1653 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T34", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1719, 1724 ] ], "normalized": [] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T35", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1728, 1731 ] ], "normalized": [] } ]

[ { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E1", "type": "Negative_regulation", "trigger": { "text": [ "deficient" ], "offsets": [ [ 52, 61 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T2" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E2", "type": "Negative_regulation", "trigger": { "text": [ "silence" ], "offsets": [ [ 78, 85 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E3" }, { "role": "Cause", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E1" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E3", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 90, 100 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T3" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E4", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 737, 747 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T21" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E5", "type": "Positive_regulation", "trigger": { "text": [ "up-regulation" ], "offsets": [ [ 877, 890 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T23" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E6", "type": "Positive_regulation", "trigger": { "text": [ "up-regulation" ], "offsets": [ [ 877, 890 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T24" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E7", "type": "Positive_regulation", "trigger": { "text": [ "up-regulate" ], "offsets": [ [ 953, 964 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T25" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E8", "type": "Negative_regulation", "trigger": { "text": [ "silenced" ], "offsets": [ [ 1195, 1203 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E9" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E9", "type": "Gene_expression", "trigger": { "text": [ "expression" ], "offsets": [ [ 1208, 1218 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T29" } ] }, { "id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_E10", "type": "Gene_expression", "trigger": { "text": [ "coexpress" ], "offsets": [ [ 1549, 1558 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2626671-10-MATERIALS_AND_METHODS-03_T32" } ] } ]

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[ { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04__text", "type": "abstract", "text": [ "FACS-based cytotoxicity assay.\nTo measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 muM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates. After the coincubation period, cells were stained with Annexin V-FITC and anti-CD8-allophycocyanin. Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined. \n" ], "offsets": [ [ 0, 550 ] ] } ]

[ { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 165, 168 ] ], "normalized": [] }, { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04_T2", "type": "Protein", "text": [ "Annexin V" ], "offsets": [ [ 323, 332 ] ], "normalized": [] }, { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04_T3", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 347, 350 ] ], "normalized": [] }, { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04_T4", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 454, 457 ] ], "normalized": [] }, { "id": "PMC-2626671-11-MATERIALS_AND_METHODS-04_T5", "type": "Protein", "text": [ "Annexin V" ], "offsets": [ [ 509, 518 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-12-MATERIALS_AND_METHODS-05__text", "type": "abstract", "text": [ "Cytokine and surface marker staining.\nTo assess cytokine production, cells were restimulated with 10 nM PMA + 1 muM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28). To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1x PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD). \n" ], "offsets": [ [ 0, 650 ] ] } ]

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[ { "id": "PMC-2626671-13-MATERIALS_AND_METHODS-06__text", "type": "abstract", "text": [ "Retroviral transduction of primary CD8+ T cells.\nFor transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g. At approximately42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 mug/ml polybrene containing fresh plus concentrated virus. The plates were centrifuged at 700 g for 1 h at RT before returning to 37degreesC for an additional 5 h. Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L. Reiner (University of Pennsylvania, Philadelphia, PA) (8). \n" ], "offsets": [ [ 0, 723 ] ] } ]

[ { "id": "PMC-2626671-13-MATERIALS_AND_METHODS-06_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 283, 286 ] ], "normalized": [] }, { "id": "PMC-2626671-13-MATERIALS_AND_METHODS-06_T2", "type": "Protein", "text": [ "Eomes-VP16" ], "offsets": [ [ 597, 607 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-14-MATERIALS_AND_METHODS-07__text", "type": "abstract", "text": [ "ChIP and real-time PCR analysis.\n20 x 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT. Fixation was stopped with 120 mM glycine on ice for 5 min. Fixed cells were washed 2x with cold PBS, 1x with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1x with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). After washes, cell pellets were resuspended at 40 x 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5-1-kb DNA fragments. After preclearing the sheared chromatin with protein A-sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 x 106 cell equivalents: 2.5 mug anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4degreesC; protein A-sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2x with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1x with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1x with TE buffer. After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3). Both input and ChIP chromatin were then treated with RNase A (5 mug total) for 1 h at 37degreesC, followed by the addition of proteinase K (100 mug total) and overnight incubation at 65degreesC to reverse cross-linking. DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-mul volume. For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 mul ChIP DNA was used per PCR reaction (performed in duplicates). Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences. Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets. The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP. \n" ], "offsets": [ [ 0, 2579 ] ] } ]

[ { "id": "PMC-2626671-14-MATERIALS_AND_METHODS-07_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 42, 45 ] ], "normalized": [] }, { "id": "PMC-2626671-14-MATERIALS_AND_METHODS-07_T2", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1067, 1072 ] ], "normalized": [] }, { "id": "PMC-2626671-14-MATERIALS_AND_METHODS-07_T3", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1094, 1099 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-15-MATERIALS_AND_METHODS-08__text", "type": "abstract", "text": [ "Northern and Western blot analyses.\nRNA isolation and Northern blot analysis was performed as previously described (29). In brief, 10 mug of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane. Membranes were hybridized with 1 ng/ml alpha-[32P]dCTP-labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.). All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis. Band intensities were acquired by phosphorimaging analysis. \nFor Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 mul per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors. Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10-30 mug of total protein was loaded per well). Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane. \n" ], "offsets": [ [ 0, 1418 ] ] } ]

[ { "id": "PMC-2626671-15-MATERIALS_AND_METHODS-08_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 782, 785 ] ], "normalized": [] }, { "id": "PMC-2626671-15-MATERIALS_AND_METHODS-08_T2", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 875, 879 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-16-MATERIALS_AND_METHODS-09__text", "type": "abstract", "text": [ "Online supplemental material.\nFig. S1 shows the characterization of peripheral CD8+ T cells from Runx3-/- mice. Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture. Primer sequences used for ChIP experiments are shown in Table S1. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1. \n" ], "offsets": [ [ 0, 375 ] ] } ]

[ { "id": "PMC-2626671-16-MATERIALS_AND_METHODS-09_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 79, 82 ] ], "normalized": [] }, { "id": "PMC-2626671-16-MATERIALS_AND_METHODS-09_T2", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 97, 102 ] ], "normalized": [] }, { "id": "PMC-2626671-16-MATERIALS_AND_METHODS-09_T3", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 157, 162 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-17-Supplementary_Material__text", "type": "abstract", "text": [ "Supplementary Material\n[Supplemental Material Index]\n" ], "offsets": [ [ 0, 53 ] ] } ]

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[ { "id": "PMC-2626671-18-caption-01__text", "type": "abstract", "text": [ "Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-gamma, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (-) or 1 (+) muM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin-granzyme B) pathway. Data are representative of at least five (A-E) or three (F) independent experiments. \n" ], "offsets": [ [ 0, 1305 ] ] } ]

[ { "id": "PMC-2626671-18-caption-01_T1", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 35, 38 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T2", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 80, 84 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T3", "type": "Protein", "text": [ "Gzmb" ], "offsets": [ [ 86, 90 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T4", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 92, 97 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T5", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 99, 104 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T6", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 111, 116 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T7", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 156, 159 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T8", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 419, 422 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T9", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 593, 603 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T10", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 605, 614 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T11", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 620, 623 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T12", "type": "Protein", "text": [ "Granzyme B" ], "offsets": [ [ 625, 635 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T13", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 819, 822 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T14", "type": "Protein", "text": [ "Annexin V" ], "offsets": [ [ 944, 953 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T15", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 987, 990 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T16", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1189, 1197 ] ], "normalized": [] }, { "id": "PMC-2626671-18-caption-01_T17", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 1198, 1208 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-18-caption-01_1", "entity_ids": [ "PMC-2626671-18-caption-01_T4", "PMC-2626671-18-caption-01_T5" ] } ]

[]

[ { "id": "PMC-2626671-19-caption-02__text", "type": "abstract", "text": [ "Regulation of perforin, granzyme B, and IFN-gamma expression by T-bet and Eomes in differentiating CTLs. (A) IFN-gamma expression by WT (Tbx21+/+) and T-bet-deficient (Tbx21-/-) T cells. Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-gamma expression was assessed by intracellular staining. Numbers show the percentage of IFN-gamma+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet-deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP). Total cellular RNA was analyzed on day 6 of culture. The frequency of transduced cells in the cultures was equivalent for both constructs (approximately65-70% GFP+ cells; not depicted). (C) Granzyme B and IFN-gamma expression by Tbx21+/+ and Tbx21-/- T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-gamma production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin. Numbers show the percentage of GFP+ IFN-gamma+ cells. Results are representative of three (A and C) or two (B and D) independent experiments. \n" ], "offsets": [ [ 0, 1288 ] ] } ]

[ { "id": "PMC-2626671-19-caption-02_T1", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 14, 22 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T2", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 24, 34 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T3", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 40, 49 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T4", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 64, 69 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T5", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 74, 79 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T6", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 109, 118 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T7", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 137, 142 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T8", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 151, 156 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T9", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 168, 173 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T10", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 193, 196 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T11", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 307, 316 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T12", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 399, 408 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T13", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 447, 451 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T14", "type": "Protein", "text": [ "GzmB" ], "offsets": [ [ 456, 460 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T15", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 486, 491 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T16", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 502, 505 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T17", "type": "Protein", "text": [ "Eomes-VP16" ], "offsets": [ [ 603, 613 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T18", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 615, 622 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T19", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 641, 644 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T20", "type": "Protein", "text": [ "Granzyme B" ], "offsets": [ [ 851, 861 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T21", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 866, 875 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T22", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 890, 895 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T23", "type": "Protein", "text": [ "Tbx21" ], "offsets": [ [ 903, 908 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T24", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 987, 996 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T25", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 1033, 1040 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T26", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 1053, 1056 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T27", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 1176, 1179 ] ], "normalized": [] }, { "id": "PMC-2626671-19-caption-02_T28", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1181, 1190 ] ], "normalized": [] } ]

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[ { "id": "PMC-2626671-19-caption-02_1", "entity_ids": [ "PMC-2626671-19-caption-02_T17", "PMC-2626671-19-caption-02_T18" ] } ]

[]

[ { "id": "PMC-2626671-20-caption-03__text", "type": "abstract", "text": [ "Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. beta-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3-/- CD8+ T cells differentiated for 6 d. beta-Actin was used as a loading control. (C) Expression of granzyme B, IFN-gamma, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A-C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the -1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP. \n" ], "offsets": [ [ 0, 1140 ] ] } ]

[ { "id": "PMC-2626671-20-caption-03_T1", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 13, 18 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T2", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 76, 81 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T3", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 83, 88 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T4", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 90, 95 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T5", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 101, 109 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T6", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 124, 129 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T7", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 140, 145 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T8", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 149, 152 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T9", "type": "Protein", "text": [ "beta-Actin" ], "offsets": [ [ 189, 199 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T10", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 261, 265 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T11", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 285, 290 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T12", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 301, 306 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T13", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 310, 313 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T14", "type": "Protein", "text": [ "beta-Actin" ], "offsets": [ [ 347, 357 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T15", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 407, 417 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T16", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 419, 428 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T17", "type": "Protein", "text": [ "TNF" ], "offsets": [ [ 430, 433 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T18", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 439, 443 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T19", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 477, 482 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T20", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 493, 498 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T21", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 502, 505 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T22", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 579, 589 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T23", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 601, 604 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T24", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 722, 727 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T25", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 732, 737 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T26", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 745, 749 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T27", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 986, 989 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T28", "type": "Protein", "text": [ "Prf1" ], "offsets": [ [ 1076, 1080 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T29", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1099, 1104 ] ], "normalized": [] }, { "id": "PMC-2626671-20-caption-03_T30", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1127, 1132 ] ], "normalized": [] } ]

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[]

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[ { "id": "PMC-2626671-21-caption-04__text", "type": "abstract", "text": [ "Runx3 controls Eomes, perforin, granzyme B, and IFN-gamma expression in effector CTLs. Runx3+/+ or Runx3-/- CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (approximately75-90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-gamma after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-gamma expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-gamma expression. Eomes may participate in sustaining late IFN-gamma expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression. \n" ], "offsets": [ [ 0, 1695 ] ] } ]

[ { "id": "PMC-2626671-21-caption-04_T1", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T2", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 15, 20 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T3", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 22, 30 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T4", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 32, 42 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T5", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 48, 57 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T6", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 87, 92 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T7", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 99, 104 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T8", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 108, 111 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T9", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 191, 194 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T10", "type": "Protein", "text": [ "Eomes-VP16" ], "offsets": [ [ 227, 237 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T11", "type": "Protein", "text": [ "Eo-VP16" ], "offsets": [ [ 239, 246 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T12", "type": "Protein", "text": [ "Myc" ], "offsets": [ [ 251, 254 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T13", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 255, 260 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T14", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 262, 267 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T15", "type": "Protein", "text": [ "Eomes-VP16" ], "offsets": [ [ 506, 516 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T16", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 545, 550 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T17", "type": "Protein", "text": [ "VP16" ], "offsets": [ [ 640, 644 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T18", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 687, 697 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T19", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 702, 711 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T20", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 934, 944 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T21", "type": "Protein", "text": [ "GFP" ], "offsets": [ [ 1052, 1055 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T22", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1195, 1200 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T23", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1220, 1225 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T24", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1279, 1288 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T25", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1301, 1306 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T26", "type": "Protein", "text": [ "CD8" ], "offsets": [ [ 1327, 1330 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T27", "type": "Protein", "text": [ "Runx1" ], "offsets": [ [ 1354, 1359 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T28", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1372, 1377 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T29", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1379, 1387 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T30", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 1389, 1399 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T31", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1405, 1414 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T32", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1427, 1432 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T33", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1468, 1477 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T34", "type": "Protein", "text": [ "Runx3" ], "offsets": [ [ 1498, 1503 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T35", "type": "Protein", "text": [ "Eomes" ], "offsets": [ [ 1508, 1513 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T36", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1523, 1528 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T37", "type": "Protein", "text": [ "perforin" ], "offsets": [ [ 1556, 1564 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T38", "type": "Protein", "text": [ "T-bet" ], "offsets": [ [ 1625, 1630 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T39", "type": "Protein", "text": [ "Gzmb" ], "offsets": [ [ 1645, 1649 ] ], "normalized": [] }, { "id": "PMC-2626671-21-caption-04_T40", "type": "Protein", "text": [ "granzyme B" ], "offsets": [ [ 1663, 1673 ] ], "normalized": [] } ]

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"PMC-2626671-21-caption-04_T40" } ] } ]

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[]

[ { "id": "PMC-2626671-22-caption-05__text", "type": "abstract", "text": [ "[Supplemental Material Index]\n" ], "offsets": [ [ 0, 30 ] ] } ]

[]

[]

[]

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[ { "id": "PMC-2791889-00-TIAB__text", "type": "abstract", "text": [ "Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose \nSummary\nCD4+ Tcells producing interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) are reported in chronic infections. However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined. We showed that development of IL-10-producing Th1 cells required high Tcell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation. Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells. Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 wasa common requirement for production of IL-10 by all Th cell subsets. IL-10 expression also correlated with c-maf expression. Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production. \n" ], "offsets": [ [ 0, 1235 ] ] } ]

[ { "id": "PMC-2791889-00-TIAB_T1", "type": "Protein", "text": [ "Interleukin-10" ], "offsets": [ [ 0, 14 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T2", "type": "Protein", "text": [ "Interleukin-12" ], "offsets": [ [ 48, 62 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T3", "type": "Protein", "text": [ "STAT4" ], "offsets": [ [ 71, 76 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T4", "type": "Protein", "text": [ "ERK" ], "offsets": [ [ 102, 105 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T5", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 158, 161 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T6", "type": "Protein", "text": [ "interleukin-10" ], "offsets": [ [ 180, 194 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T7", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 196, 201 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T8", "type": "Protein", "text": [ "interferon-gamma" ], "offsets": [ [ 207, 223 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T9", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 225, 234 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T10", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 324, 329 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T11", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 408, 413 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T12", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 489, 493 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T13", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 498, 502 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T14", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 536, 541 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T15", "type": "Protein", "text": [ "STAT4" ], "offsets": [ [ 550, 555 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T16", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 629, 634 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T17", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 674, 679 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T18", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 768, 773 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T19", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 911, 915 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T20", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 920, 924 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T21", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 967, 972 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T22", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 997, 1002 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T23", "type": "Protein", "text": [ "c-maf" ], "offsets": [ [ 1035, 1040 ] ], "normalized": [] }, { "id": "PMC-2791889-00-TIAB_T24", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1216, 1221 ] ], "normalized": [] } ]

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"ref_id": "PMC-2791889-00-TIAB_T11" } ] }, { "id": "PMC-2791889-00-TIAB_E10", "type": "Phosphorylation", "trigger": { "text": [ "phosphorylation" ], "offsets": [ [ 515, 530 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_T12" } ] }, { "id": "PMC-2791889-00-TIAB_E11", "type": "Phosphorylation", "trigger": { "text": [ "phosphorylation" ], "offsets": [ [ 515, 530 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_T13" } ] }, { "id": "PMC-2791889-00-TIAB_E12", "type": "Positive_regulation", "trigger": { "text": [ "induced" ], "offsets": [ [ 542, 549 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_E13" }, { "role": "Cause", "ref_id": "PMC-2791889-00-TIAB_T14" } ] }, { "id": "PMC-2791889-00-TIAB_E13", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 577, 587 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_T15" } ] }, { "id": "PMC-2791889-00-TIAB_E14", "type": 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"PMC-2791889-00-TIAB_T19" } ] }, { "id": "PMC-2791889-00-TIAB_E19", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 897, 907 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_T20" } ] }, { "id": "PMC-2791889-00-TIAB_E20", "type": "Positive_regulation", "trigger": { "text": [ "requirement" ], "offsets": [ [ 937, 948 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_E21" }, { "role": "Cause", "ref_id": "PMC-2791889-00-TIAB_E18" } ] }, { "id": "PMC-2791889-00-TIAB_E22", "type": "Positive_regulation", "trigger": { "text": [ "requirement" ], "offsets": [ [ 937, 948 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_E21" }, { "role": "Cause", "ref_id": "PMC-2791889-00-TIAB_E19" } ] }, { "id": "PMC-2791889-00-TIAB_E21", "type": "Gene_expression", "trigger": { "text": [ "production" ], "offsets": [ [ 953, 963 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-00-TIAB_T21" } ] }, { 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[]

[ { "id": "PMC-2791889-01-Introduction__text", "type": "abstract", "text": [ "Introduction\nInterleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties that inhibits macrophage and dendritic cell (DC) function (Moore etal., 2001). IL-10 limits the immune and inflammatory responses to pathogens and gut flora and prevents damage to the host (Moore etal., 2001; O'Garra and Vieira, 2004), but when dysregulated may result in chronic infection (Brooks etal., 2006; Ejrnaes etal., 2006; Moore etal., 2001). IL-10 is expressed by T helper 2 (Th2) cells, B cells, DCs, and macrophages (Moore etal., 2001), and also by Th1 cells (Anderson etal., 2007; Assenmacher etal., 1994; Del Prete etal., 1993; Gerosa etal., 1996; Jankovic etal., 2007; Pohl-Koppe etal., 1998) and (reviewed in O'Garra and Vieira, 2007; Trinchieri, 2007), certain regulatory (Treg) Tcells (Moore etal., 2001; O'Garra and Vieira, 2004; Roncarolo etal., 2006), and Th17 cells (Awasthi etal., 2007; Fitzgerald etal., 2007; McGeachy etal., 2007; Stumhofer etal., 2007). \nIn vitro human CD4+ and CD8+ Tcell clones, or mouse CD4+ Tcells that produce both interferon-gamma (IFN-gamma) and IL-10, can be differentiated by Tcell receptor (TCR)-stimulation in the presence of IL-12 (Chang etal., 2007; Gerosa etal., 1996; Jeannin etal., 1996; Meyaard etal., 1996; Windhagen etal., 1996). Furthermore, Th1 cell clones coproducing IFN-gamma and IL-10 have been isolated from bronchoalveolar lavage (BAL) of active pulmonary tuberculosis (TB) patients (Gerosa etal., 1999). IL-10 production by Th1 cells was also reported in animals infected with Toxoplasma gondii (Jankovic etal., 2002; Shaw etal., 2006) or with Leishmania major (Anderson etal., 2007) and shown to be required for regulation of the immune response in these infections (Anderson etal., 2007; Jankovic etal., 2007). The relative amounts of IL-10 and IFN-gamma produced by Th1 cells may influence the balance between clearance and persistent infection with certain pathogens (Moore etal., 2001; Trinchieri, 2007), thus determining whether chronic infection or immunopathology ensues. \nTh1, Th2, and Th17 cell responses differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Ivanov etal., 2007; Stockinger and Veldhoen, 2007). Th1 cell development requires signal transducer and activator of transcription (STAT)1activation, induced by type I IFN or IFN-gamma, the transcription factor T-box 21 (T-bet), and IL-12-induced STAT4 signaling, which can couple with IL-18-induced IRAK and NF-kappaB transcription factors to drive the high amounts of IFN-gamma required to eradicate intracellular pathogens (Glimcher and Murphy, 2000). Th2 cell development, with expression of IL-4, IL-5, and IL-13, requires IL-4, STAT6, and the transcription factor GATA binding protein (GATA)-3 (Glimcher and Murphy, 2000). The development of Th17 cells requires IL-6, TGF-beta, and the STAT3-dependent expression of the transcription factor RORgammat (Ivanov etal., 2007; Stockinger and Veldhoen, 2007). \nTh1 and Th2 cell responses can also be induced by varying the dose of antigen presented to the naive Tcell by the antigen-presenting cell (APC). Whereas high doses of antigen, with sustained TCR signaling and extracellular-signal regulated (ERK) mitogen-activated protein kinase (MAPK) phosphorylation, result in Th1 cells producing IFN-gamma via an IL-12-independent mechanism, low doses of antigen, with transient ERK1 and ERK2 activation, favor Th2 responses and IL-4 secretion (Constant etal., 1995; Hosken etal., 1995; Jorritsma etal., 2003; Yamane etal., 2005). \nBecause Th1 and Th2 cells cross regulate each other's development and function and can suppress Th17 cell responses, and all differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Stockinger and Veldhoen, 2007), IL-10 produced by all these Th cells may thus act as a feedback regulator to control the pathology associated with an overexuberant, albeit efficacious, inflammatory response. Whether IL-10 production by these different Th cell subsets is induced by independent and/or common mechanisms is unknown. \nHere, we showed that invitro differentiation of IL-10-producing Th1 cells from naive CD4+ Tcells required IL-12-induced STAT4 signaling, strong TCR activation (high antigen dose), and sustained ERK1 and ERK2 phosphorylation. Furthermore, we showed that activation of ERK1 and ERK2 is a requirement for production of IL-10 by Th1, Th2, and Th17 cell subsets. This common but highly regulated pathway for IL-10 induction and maintenance ensures its function as a feedback loop to control damage to the host and also allows a protective response to ensue as opposed to chronic infection. \n" ], "offsets": [ [ 0, 4651 ] ] } ]

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[]

[ { "id": "PMC-2791889-02-Results-01__text", "type": "abstract", "text": [ "IL-12 and High Doses of Antigen Induce the Development of Th1 Cells Producing IL-10\nTo study the differentiation of Th1 cells coproducing IFN-gamma and IL-10, we cultured purified TCR-transgenic DO11.10 naive CD4+ Tcells with purified DCs as APCs and increasing doses of ovalbumin peptide 323-339 (OVA). Culture with high doses of antigen for 7 days gave rise to Th1 cells expressing IFN-gamma upon restimulation (Constant etal., 1995; Hosken etal., 1995), but not IL-10 (FigureS1A available online). Culture with low antigen doses under the same conditions led to the differentiation of Th2 cells, which expressed both IL-4 and IL-10 upon restimulation (FigureS1A). Culture of naive CD4+ Tcells in an APC-free system by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of IL-12 resulted in IFN-gamma-producing Th1 cells, a proportion of which coproduced IL-10, as did Th2 cells resulting from culture in IL-4 (FigureS1B). \nTo investigate whether the lack of IL-10 produced by Th1 cells resulted from inhibition of IL-10 production by DCs and high antigen dose or alternatively required IL-12, we cultured naive CD4+ Tcells with increasing doses of antigen presented by DC in the presence of IL-12. At low doses of antigen, IL-12 abrogated the development of Th2 cells and induced IFN-gamma expression but only low levels of IL-10 expression, suggesting that IL-12 per se was not sufficient to induce significant IL-10 production in Th1 cells (Figure1A). Strikingly, as the antigen dose was increased, Th1 populations driven with IL-12 now contained higher numbers of IL-10-producing cells (Figure1A) and produced more IL-10 protein upon restimulation (Figures 1A and 1B). Thus, the development of Th1 cells producing IL-10 required both IL-12 and high doses of antigen. Th1 cells differentiated to produce large amounts of IL-10 and IFN-gamma and lost theircapacity to produce IL-2 (Figure1B). Because the presence of IL-12 reduced the proliferation of CD4+ Tcells at both high andlow antigen doses (Table S1), and only the former showed IL-10 production, the development of high IL-10-producing cellsis most likely not related to limited IL-2. Naive CD4+ Tcells from DO11.10/recombination-activating gene 1 (Rag1)-deficient animals, cultured with high doses of antigen in the presence of IL-12, also resulted in IL-10 expression by Th1 cells, showing that this expression was not dependent on the presence of effector or memory Tcells or Treg cells (FigureS2). Although it has been suggested that TGF-beta can induce IL-10 in CD4+ Tcells (Kitani etal., 2003; Schiott etal., 2000), we found that in developing Th1 and Th2 cells this was not the case (data not shown). In fact, neutralization of TGF-beta led to increased IL-10 production by both Tcell subsets (FigureS3). Thus, the development of IL-10-producing Th1 cells only depended on the presence of IL-12 together with high antigen dose and not on other soluble factors such as IL-2 or TGF-beta or on the presence of other Tcell types. \n" ], "offsets": [ [ 0, 3013 ] ] } ]

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[ 384, 393 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T9", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 465, 470 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T10", "type": "Protein", "text": [ "IL-4" ], "offsets": [ [ 620, 624 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T11", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 629, 634 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T12", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 684, 687 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T13", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 743, 746 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T14", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 756, 760 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T15", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 791, 796 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T16", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 809, 818 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T17", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 873, 878 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T18", "type": "Protein", "text": [ "IL-4" ], "offsets": [ [ 923, 927 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T19", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 977, 982 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T20", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1033, 1038 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T21", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1105, 1110 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T22", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1130, 1133 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T23", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1210, 1215 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T24", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1242, 1247 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T25", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1299, 1308 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T26", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1343, 1348 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T27", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1377, 1382 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T28", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1431, 1436 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T29", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1548, 1553 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T30", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1586, 1591 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T31", "type": "Protein", "text": [ "IL-10" ], 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"PMC-2791889-02-Results-01_T39", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2057, 2062 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T40", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2099, 2104 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T41", "type": "Protein", "text": [ "IL-2" ], "offsets": [ [ 2158, 2162 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T42", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 2170, 2173 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T43", "type": "Protein", "text": [ "recombination-activating gene 1" ], "offsets": [ [ 2195, 2226 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T44", "type": "Protein", "text": [ "Rag1" ], "offsets": [ [ 2228, 2232 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T45", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 2308, 2313 ] ], "normalized": [] }, { "id": "PMC-2791889-02-Results-01_T46", "type": "Protein", 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[]

[ { "id": "PMC-2791889-03-Results-02__text", "type": "abstract", "text": [ "IL-10 Production by Th1 Cells Is Dependent on STAT4 but Not on STAT6, IFN-gamma, or IL-4 Signaling\nTo further elucidate the mechanisms required for the development of Th1 cells producing IL-10, we investigated the role of STAT4, one of the signaling pathways activated by IL-12 (Murphy etal., 2000). Naive CD4+ D011.10 Tcells deficient in STAT4 (Ouyang etal., 1998) were cultured in the presence of IL-12 and OVA. Again, IL-10-producing Th1 cells were differentiated at the high antigen dose in the presence of IL-12 in DO11.10 Tcells (Figure2A). In contrast, in the absence of STAT4, the percentage of cells expressing IFN-gamma was dramatically diminished as expected and resulted in an increase in the percentage of cells expressing IL-4, but not IL-10 (Figure2A), suggesting that STAT4 contributes to IL-10 expression by Th1 cells. \nBecause IL-10 expression is associated with an IL-4-induced Th2 cell phenotype, we investigated whether the differentiation of the IL-10-producing Th1 cells depended on signaling through the IL-4 receptor via STAT6 activation (Glimcher and Murphy, 2000; Murphy etal., 2000). The absence of STAT6 did not impair the differentiation of IL-10-producing Th1 cells in the presence of IL-12 and OVA (Figure2A). In fact, a higher percentage of STAT6-deficient cells compared with WT cells produced both IL-10 and IFN-gamma (Figure2A), which may be the result of the loss of Th2 cell control over a Th1 cell response. As expected, lack of STAT6 abrogated both IL-4 and IL-10 production by Tcells developed with IL-4 or with low antigen dose (Figures 2B and 2C). However, in the absence of STAT4 signaling, IL-10 and IL-4 production by Th2 cells was if anything increased (Figures 2B and 2C). Thus, in contrast to what was observed under Th1 conditions, IL-10 expression by Th2 cells depended on STAT6, but not on STAT4, signaling (Figures 2B and 2C). \nTo investigate whether the inability of STAT4-deficient Tcells to produce IL-10 might be due to the absence of IFN-gamma, as suggested before (Shaw etal., 2006), we differentiated DO11.10 or DO11.10 IFN-gamma-deficient naive CD4+ Tcells in the presence of IL-12 and increasing doses of OVA. The secretion of IL-10 as induced by high antigen dose, and IL-12 was not affected by an absence of IFN-gamma (Figure2D), showing that the expression ofIL-10 by Th1 cells is independent of IFN-gamma. In the absence of IFN-gamma, we observed an increase in the secreted IL-4 as expected (data not shown). \nWe also tested for any potential role of IL-4 in the development of Th1 cells producing IL-10 by culturing DO11.10 or DO11.10 IL-4-deficient naive CD4+ Tcells with IL-12 and increasing doses of antigen. As observed in the absence of STAT6 (Figure2A), IL-4 deficiency had no effect on the development of Th1 cells producing IL-10 (Figure2E), but compromised the development of Th2 cells producing IL-10 (Figure2F). Thus, our data suggested that IL-10 production by Th1 or Th2 cells was dependent on the specific signaling pathways required for their differentiation, given that STAT4 is required for the induction of IL-10 production by Th1 cells and STAT6 for Th2 cells. \n" ], "offsets": [ [ 0, 3149 ] ] } ]

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[]

[ { "id": "PMC-2791889-04-Results-03__text", "type": "abstract", "text": [ "High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells\nTo address the mechanisms regulating IL-10 production by Th1 cells invivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS). Tcells were recovered from the inguinal lymph nodes 3 days after priming and restimulated invitro with OVA peptide for 48 hr. This invivo immunization induced IL-10 and IFN-gamma production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure3A) and with higher doses of OVA (3 muM versus 1 muM, data not shown). To test the role of STAT4 and STAT6 signaling in the invivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before. In vivo expression of both IL-10 and IFN-gamma was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the invitro system. Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient Tcells (Figures 3B and 3C). \n" ], "offsets": [ [ 0, 1370 ] ] } ]

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[ { "id": "PMC-2791889-05-Results-04__text", "type": "abstract", "text": [ "IL-10 Production Is Maintained by High TCR Signal Strength and IL-12\nWe next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells. For this, CD4+ Tcells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A-4D). High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells. Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D). However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure4E). Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells. \nTo determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ Tcells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure4F). Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10. Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-gamma but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (FigureS4). Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells. \n" ], "offsets": [ [ 0, 2166 ] ] } ]

[ { "id": "PMC-2791889-05-Results-04_T1", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T2", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 63, 68 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T3", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 158, 163 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T4", "type": "Protein", "text": [ "STAT4" ], "offsets": [ [ 172, 177 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T5", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 208, 213 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T6", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 238, 241 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T7", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 348, 353 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T8", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 404, 409 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T9", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 439, 444 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T10", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 540, 545 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T11", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 642, 647 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T12", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 678, 683 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T13", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 771, 776 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T14", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 781, 786 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T15", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 827, 832 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T16", "type": "Protein", "text": [ "IL-12p40" ], "offsets": [ [ 885, 893 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T17", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 944, 949 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T18", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1037, 1042 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T19", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1109, 1114 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T20", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1154, 1157 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T21", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1217, 1222 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T22", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1362, 1367 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T23", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1427, 1432 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T24", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1472, 1477 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T25", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1508, 1513 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T26", "type": "Protein", "text": [ "OVA" ], "offsets": [ [ 1548, 1551 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T27", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1616, 1621 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T28", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1699, 1704 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T29", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1744, 1749 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T30", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 1768, 1771 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T31", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1825, 1830 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T32", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1897, 1906 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T33", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1918, 1923 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T34", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1962, 1967 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T35", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 2000, 2005 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T36", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 2083, 2088 ] ], "normalized": [] }, { "id": "PMC-2791889-05-Results-04_T37", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2134, 2139 ] ], "normalized": [] } ]

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[ { "id": "PMC-2791889-06-Results-05__text", "type": "abstract", "text": [ "IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation\nOur data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12. Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ Tcells (data not shown) as previously demonstrated (Jorritsma etal., 2003) but also in CD4+ Tcells restimulated with the same high and low antigen doses (Figure5A). Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure5B). \nWe then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure5C), a compound that blocks downstream ERK activation. To ensure that only Tcell signaling was being affected by U0126, we used an APC-free system in which the Tcells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-gamma after 1 week of culture (Figure5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain etal., 2007; Mody etal., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain etal., 2007; Mody etal., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure5D and FigureS5A). Upon addition of inhibitors to othersignaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3beta inhibitor, CT99021 (Bain etal., 2007), no effect on IL-10 production was observed (FigureS5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3beta pathways. \n" ], "offsets": [ [ 0, 2497 ] ] } ]

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"normalized": [] }, { "id": "PMC-2791889-06-Results-05_T9", "type": "Protein", "text": [ "CD4" ], "offsets": [ [ 494, 497 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T10", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 613, 617 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T11", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 622, 626 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T12", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 785, 789 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T13", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 794, 798 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T14", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 829, 834 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T15", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 953, 957 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T16", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 962, 966 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T17", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1012, 1017 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T18", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 1284, 1287 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T19", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1313, 1318 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T20", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 1390, 1395 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T21", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1446, 1451 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T22", "type": "Protein", "text": [ "IFN-gamma" ], "offsets": [ [ 1456, 1465 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T23", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 1564, 1569 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T24", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 1591, 1594 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T25", "type": "Protein", "text": [ "MEK5" ], "offsets": [ [ 1634, 1638 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T26", "type": "Protein", "text": [ "ERK5" ], "offsets": [ [ 1663, 1667 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T27", "type": "Protein", "text": [ "MEK1" ], "offsets": [ [ 1684, 1688 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T28", "type": "Protein", "text": [ "MEK2" ], "offsets": [ [ 1694, 1698 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T29", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 1723, 1727 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T30", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 1732, 1736 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T31", "type": "Protein", "text": [ "MEK1" ], "offsets": [ [ 1830, 1834 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T32", "type": "Protein", "text": [ "MEK2" ], "offsets": [ [ 1839, 1843 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T33", "type": "Protein", "text": [ "MEK1" ], "offsets": [ [ 1902, 1906 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T34", "type": "Protein", "text": [ "MEK2" ], "offsets": [ [ 1911, 1915 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T35", "type": "Protein", "text": [ "MEK5" ], "offsets": [ [ 1924, 1928 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T36", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2007, 2012 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T37", "type": "Protein", "text": [ "p38 MAPK" ], "offsets": [ [ 2159, 2167 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T38", "type": "Protein", "text": [ "GSK3beta" ], "offsets": [ [ 2196, 2204 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T39", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2257, 2262 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T40", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 2329, 2334 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T41", "type": "Protein", "text": [ "IL-12" ], "offsets": [ [ 2396, 2401 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T42", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 2411, 2415 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T43", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 2420, 2424 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T44", "type": "Protein", "text": [ "p38" ], "offsets": [ [ 2466, 2469 ] ], "normalized": [] }, { "id": "PMC-2791889-06-Results-05_T45", "type": "Protein", "text": [ "GSK3beta" ], "offsets": [ [ 2477, 2485 ] ], "normalized": [] } ]

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[]

[]

[ { "id": "PMC-2791889-07-Results-06__text", "type": "abstract", "text": [ "IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation\nTo address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker etal., 2006; Veldhoen etal., 2009; Veldhoen etal., 2006), in the presence or absence of the MEK inhibitor (PD184352). We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure5D and FigureS5B). In contrast, inhibitors of p38 MAPK or of GSK-3beta activation did not affect the expression of IL-10 by these subsets (FigureS5B). Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells. \n" ], "offsets": [ [ 0, 948 ] ] } ]

[ { "id": "PMC-2791889-07-Results-06_T1", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 0, 5 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T2", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 53, 57 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T3", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 62, 66 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T4", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 97, 102 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T5", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 158, 162 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T6", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 167, 171 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T7", "type": "Protein", "text": [ "CD3" ], "offsets": [ [ 224, 227 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T8", "type": "Protein", "text": [ "CD28" ], "offsets": [ [ 237, 241 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T9", "type": "Protein", "text": [ "MEK" ], "offsets": [ [ 368, 371 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T10", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 409, 413 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T11", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 418, 422 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T12", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 480, 485 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T13", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 523, 528 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T14", "type": "Protein", "text": [ "MEK" ], "offsets": [ [ 613, 616 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T15", "type": "Protein", "text": [ "p38 MAPK" ], "offsets": [ [ 691, 699 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T16", "type": "Protein", "text": [ "GSK-3beta" ], "offsets": [ [ 706, 715 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T17", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 760, 765 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T18", "type": "Protein", "text": [ "ERK1" ], "offsets": [ [ 814, 818 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T19", "type": "Protein", "text": [ "ERK2" ], "offsets": [ [ 823, 827 ] ], "normalized": [] }, { "id": "PMC-2791889-07-Results-06_T20", "type": "Protein", "text": [ "IL-10" ], "offsets": [ [ 901, 906 ] ], "normalized": [] } ]

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}, { "id": "PMC-2791889-07-Results-06_E5", "type": "Positive_regulation", "trigger": { "text": [ "Activation" ], "offsets": [ [ 67, 77 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T3" } ] }, { "id": "PMC-2791889-07-Results-06_E6", "type": "Gene_expression", "trigger": { "text": [ "production" ], "offsets": [ [ 103, 113 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T4" } ] }, { "id": "PMC-2791889-07-Results-06_E7", "type": "Positive_regulation", "trigger": { "text": [ "dependent" ], "offsets": [ [ 145, 154 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_E6" }, { "role": "Cause", "ref_id": "PMC-2791889-07-Results-06_E8" } ] }, { "id": "PMC-2791889-07-Results-06_E9", "type": "Positive_regulation", "trigger": { "text": [ "dependent" ], "offsets": [ [ 145, 154 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_E6" }, { "role": "Cause", "ref_id": "PMC-2791889-07-Results-06_E10" } ] }, { "id": "PMC-2791889-07-Results-06_E8", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 172, 182 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T5" } ] }, { "id": "PMC-2791889-07-Results-06_E10", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 172, 182 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T6" } ] }, { "id": "PMC-2791889-07-Results-06_E11", "type": "Negative_regulation", "trigger": { "text": [ "inhibitor" ], "offsets": [ [ 372, 381 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T9" } ] }, { "id": "PMC-2791889-07-Results-06_E12", "type": "Positive_regulation", "trigger": { "text": [ "activation" ], "offsets": [ [ 423, 433 ] ] }, "arguments": [ { "role": "Theme", "ref_id": "PMC-2791889-07-Results-06_T10" } ] }, { "id": "PMC-2791889-07-Results-06_E13", "type": 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