| BackgroudEffective B cell responses to infectious diseases or immunization require the assistance of CD4+ helper T cells. A specialized subset of CD4+ T cells named T follicular helper(TFH) cells are required for providing this help to antigen-specific B cells. Without cognate TFH help, activated B cells are unable to generate and maintain the germinal center(GC)response that is required for efficient somatic hyper mutation of immunoglobulin genes and the selective processes that facilitate affinity maturation of antibodies.Further more, the germinal center reaction is the origin of longlived memory B cells and long- lived plasma cells that populate the periphery and bone marrow(respectively),and provide long-term antibody- mediated protection against(re)exposure to pathogens. Thus, TFH cells play a critical role in the generation of effective and long- lived humoral immune responses to antigens.In LCMV infection model, na?ve CD4+ T cells differentiate into TFH and TH1 cell. TFH cells interact with B cells to support antibody-affinity maturation and memory formation. Whereas TH1 cells promote macrophage function and provide help to CD8+ T cells. The differentiation of TFH cells in vivo can be divided into several stages that include initiation, maintenance and full polarization. While constituting distinct effector subsets, TFH and TH1 cells share a common transitional stage during early differentiation, and differentiation of each subset relies on carefully orchestrated transcriptional programs. Central to the transcriptional programs that define these subsets are the regulators Bcl-6 and Blimp1. Bcl-6 expression upregulates early after T cell activation and continued enhances during the maintenance and polarization phases of the TFH cell response.Regulation of Bcl-6 in CD4+ T cells is controlled by multiple layers of signaling pathways, some of which are shared with non-TFH cells or engage potent negative feedback loops. Several axes of cytokines and members of the STAT family of transcription factors, including IL-6–STAT1, IL-12–STAT4 and IL-21–STAT3, have been linked to the induction of Bcl-6 expression. In addition, the transcription factor Batf has been reported to bind to the Bcl-6 locus and activate its expression. The transcription factor Blimp1(encoded by Prdm1) binds to the Bcl-6 promoter and strongly represses Bcl-6 transcription. Furthermore, Bcl-6 inhibits its own transcription. Despite such advances in understanding, whether and how other factors are involved in the regulation of Bcl-6 expression in activated CD4+ T cells has remained unclear.The transcription factor TCF-1(encoded by Tcf7) is a downstream effector of the canonical Wnt signaling pathway and is critical for cell development. The role of TCF-1 in T cell–mediated immune responses is also emerging. In a Listeria infection model, TCF-1 has been shown to favor the formation of memory CD8+ T cells by inducing expression of the transcription factor Eomes. TCF-1 promotes commitment to the T helper type 2(TH2) fate but inhibits T helper type 1(TH1) differentiation by inducing expression of the transcription factor GATA-3 while repressing interferon-?(IFN-?) production. In addition, TCF-1 reportedly reduces the inflammatory effects of the TH17 subset of helper T cells by dampening IL-17 A expression. Despite these profound effects of TCF-1 on the regulation of various T cell responses, its role in TFH differentiation has not been determined.TFH differentiation was examined in mice with conditiona l deletion(c KO) of TCF-1 in the T cell compartment(Tcf7f/f x Cd4-Cre,Cd4-c KO). It’s found dramatically reduced numbers of TFH cells and impaired germinal centre formation in Cd4-c KO mice, as compared to wild-type(WT) mice. With a mouse model that enables inducible deletion of Tcf7 upon tamoxifen treatment(Tcf7f/f x Ert2-Cre), Tamoxifen treatment immediately preceding LCMV infection resulted in reduced numbers of TFH cells. By contrast, treatment from day 4 to day 7 post infection had negligible effects on TFH cell numbers. Taken together, these findings support an important role for TCF-1 in the early stages of TFH cell differentiation. Inducible deletion of TCF-1 in mature TFH cells did not affect cell numbers, but these cells functioned poorly in promoting the generation of GCs and plasma cells in response to LCMV challenge. Taken together, these findings support the notion that TCF-1 is plays a critical role in TFH cell differentiation and function.How does TCF-1 promote TFH cell differentiation? Our findings brings insight into the the mechanism of TCF-1 regulates TFH cell differentiation and function.Main contents1. Screening candidate genes by Microarray.To elucidate the molecular mechanisms by which TCF-1 regulates TFH differentiation, we sorted TFH and TH1 cells from wild-type and TCF-1-deficient mice at day 8 after infection with LCMV. We extract RNA of the sorted cells and perform microarray analysis. By gene cluster analysis with microarray data, we found TCF-1 influenced transcriptional profiles only in TFH cells. The gene pattern of TCF-1-deficient TFH cells shows great different from wild-type TFH cells. We identified 569 genes that were downregulated and 513 genes that were upregulated in TCF-1-deficient TFH cells relative to their expression in wild-type TFH cells. We selected genes from published data sets(GEO accession codes GSE21379 and GSE21381) that are upregulated and downregulated in TFH cells relative to their expression in non- TFH cells, for gene-set–enrichment analysis(GSEA) with our data. The gene set linked to the TH1 lineage(downregulated in TFH cells) was ‘concentrated’ in Tcf7-/- TFH cells. We found TCF-1 seriously impacts Bcl6 and Prdm1 expression.2. Direct regulation of Bcl6 and Prdm1 by TCF-1We identified a single peak and two peaks that showed enrichment at the Bcl6 locus and Prdm1 locus, respectively, through the use of the UCSC Genome Browser website(data not shown). We screened two conserved consensus TCF-1-binding sequences in the Bcl6 promoter region and three in the 5’regulatory region of Prdm1. Using Ch IP followed by quantitative PCR, we observed enrichment for the binding of TCF-1 to the Bcl6 promoter and the 5’regulatory region of Prdm1 relative to the binding of isotype- matched control Ig G to each locus in sorted TFH cells, but not in TH1 cells, obtained from mice at day 8 after infection with LCMV.Using Promega Dual Luciferase Assay system, we demonstrated that 293 T cells transfected with plasmid containing Bcl6 promoter with mutated TCF-1-binding motif showed much lower relative luciferase activity than cells with wild-type Bcl6 promoter. The relative luciferase activity in 293 T cells transfected with mutated Prdm1 regulator increased compared with WT control. Subsequently, we performed invivo reporter assays using a self- inactivating retroviral system. The results further confirmed that TCF-1 promoted Bcl6 expression but repressed Prdm1 expression via direct DNA-binding activity.3. Protein-protein interaction be tween TCF-1 and Bcl-6We also observed binding of TCF-1 to Bcl-6 in a co-immunoprecipitation assay of 293 T human embryonic kidney cells co-transfected with plasmids expressing Bcl-6 and the p33 or p45 isoform of TCF-1.The interaction between TCF-1 and Bcl-6 was further confirmed by endogenous co-immunoprecipitation with lysates of activated CD4+ T cells. We overexpressed p45, Bcl-6 and stabilized ?-catenin in 293 T, the result of co-immunoprecipitation assay suggested ?-catenin disrupted the p45–Bcl-6 interaction. In contrast to p45, transcriptional corepressors TLE4 enhanced formation of the p33–Bcl-6 complex. Using invivo reporter plasmid containing Mutated Bcl6 promoter which can be directly binding by Bcl-6 and cannot be regulated by TCF-1, We demonstrated the P33-Bcl-6 interaction potentially favored the transcription of Bcl-6 during TFH differentiation.In the end, we co-transduced TCF-1 knockdown vector and Bcl-6 promoter-Thy-1.1 reporter vectors into activated SMARTA cells. In the presense sh-TCF-1, frequece of Thy1.1+ cells which indicated Bcl-6 promoter expression dropped by one half. We also found that TFH cell deficiency that was mediated by knockdown of TCF-1 could be completely rescued by overexpression of Bcl-6.ConclusionsIn summary, this stud y revealed the mechanism of TCF-1 regulates TFH cell differentiation and function. We demonstrated TCF-1 directly regulating the transcription of Bcl6 and Prdm1. TCF-1 bound directly to the Bcl-6 promoter and Prdm1 5’regulatory regions, which promoted Bcl-6 expression but repressed Blimp1 expression. TCF-1-null TFH cells upregulated genes associated with non-TFH cell lineages. The p33 isoform of TCF-1 formed a complex with Bcl-6 and promoted Bcl-6 transcription.TCF-1 functions as an important hub upstream of the Bcl-6–Blimp1 axis to initiate and secure the differentiation of TFH cells during acute viral infection. |
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