The Transcription Factor TCF-1 Initiates the Differentiation of TFH Cells During Acute Viral Infection
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ARTICLES The transcription factor TCF-1 initiates the differentiation of TFH cells during acute viral infection Lifan Xu1,4, Yi Cao1,4, Zhunyi Xie1,4, Qizhao Huang1, Qiang Bai1, Xia Yang1, Ran He1, Yaxing Hao1, Haoqiang Wang1, Tingting Zhao1, Zhonglei Fan2,3, Aijian Qin2,3, Jianqiang Ye2,3, Xinyuan Zhou1,5, Lilin Ye1,5 & Yuzhang Wu1,5 Induction of the transcriptional repressor Bcl-6 in CD4+ T cells is critical for the differentiation of follicular helper T cells (TFH cells), which are essential for B cell–mediated immunity. In contrast, the transcription factor Blimp1 (encoded by Prdm1) inhibits TFH differentiation by antagonizing Bcl-6. Here we found that the transcription factor TCF-1 was essential for both the initiation of TFH differentiation and the effector function of differentiated TFH cells during acute viral infection. Mechanistically, TCF-1 bound directly to the Bcl6 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. Thus, 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. Most antimicrobial vaccines licensed for human use elicit their effects been reported to bind to the Bcl6 locus and activate its expression18. by inducing the production of memory B cells and long-lived plasma The transcription factor Blimp1 (encoded by Prdm1) binds to the Bcl6 cells1, both of which are generally differentiated from germinal center promoter and strongly represses Bcl6 transcription14. Furthermore, (GC) B cells in secondary lymphoid tissues2. Follicular helper T cells Bcl-6 inhibits its own transcription14. Despite such advances in under- + (TFH cells), a unique subset of CD4 helper T cells, are specialized to standing, whether and how other factors are involved in the regulation facilitate the establishment of a GC reaction and the selection of GC of Bcl-6 expression in activated CD4+ T cells has remained unclear. Nature America, Inc. All rights reserved. Inc. Nature America, B cells bearing high-affinity antigen receptors for final differentiation The transcription factor TCF-1 (encoded by Tcf7) is a downstream 5 3 into memory B cells and long-lived plasma cells . TFH cells express effector of the canonical Wnt signaling pathway and is critical for abundant chemokine receptor CXCR5 to enable homing to B cell T cell development19,20. The role of TCF-1 in T cell–mediated © 201 follicles4–6, where they engage cognate B cells and provide essential immune responses is also emerging. In a Listeria infection model, help by secreting the cytokines interleukin 21 (IL-21) and IL-4 and TCF-1 has been shown to favor the formation of memory CD8+ T cells expressing CD40L (the ligand for the costimulatory receptor CD40) by inducing expression of the transcription factor Eomes21,22. TCF-1 7,8 and the costimulatory receptor ICOS . promotes commitment to the T helper type 2 (TH2) fate but inhibits 8 TFH differentiation is a multistage, multifactorial process . The T helper type 1 (TH1) differentiation by inducing expression of the transcriptional repressor Bcl-6, identified as a ‘master regulator’ transcription factor GATA-3 while repressing interferon-γ (IFN-γ) 9–11 23 of TFH differentiation , emerges on early TFH-like cells together production . In addition, TCF-1 reportedly reduces the inflamma- 12 with CXCR5 at day 2 or 3 after infection or immunization . Next, tory effects of the TH17 subset of helper T cells by dampening IL-17A + + 24 CXCR5 Bcl-6 TFH-like cells accumulate at the T cell–B cell border, expression . Despite these profound effects of TCF-1 on the regula- where they engage activated B cells. Subsequently, they migrate into tion of various T cell responses, its role in TFH differentiation has not B cell follicles and initiate GC reactions13, during which reinforced been determined. Bcl-6 expression drives the differentiation of fully functional TFH The transcriptional signatures of TFH cells overlap considerably 14 + 12 cells . Given the central role of Bcl-6 in TFH differentiation, it is with those of CD8 memory precursor cells . Indeed, both subsets critical to understand how Bcl-6 expression is regulated in activated have high expression of TCF-1 mRNA during acute infection with CD4+ T cells. Several axes of cytokines and members of the STAT lymphocytic choriomeningitis virus (LCMV)12. TCF-1 regulates family of transcription factors, including IL-6–STAT1, IL-12–STAT4 the formation of CD8+ memory cells mainly by inducing Eomes and IL-21–STAT3 (refs. 15–17), have been linked to the induction expression, which leads to upregulation of expression of the IL-2Rβ of Bcl-6 expression. In addition, the transcription factor Batf has cytokine receptor chain21. However, both Eomes and IL-2Rβ seem to 1Institute of Immunology, Third Military Medical University, Chongqing, China. 2Ministry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, China. 3Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China. 4These authors contributed equally to this work. 5These authors jointly directed this work. Correspondence should be addressed to L.Y. ([email protected]), X.Z. ([email protected]) or Y.W. ([email protected]). Received 6 May; accepted 15 June; published online 27 July 2015; doi:10.1038/ni.3229 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES 25,26 be unnecessary for TFH differentiation . Thus, it is unclear whether Role of TCF-1 in TFH differentiation high TCF-1 expression in the TFH cell subset serves a function or To investigate whether a greater abundance of TCF-1 is essential for merely represents a nonfunctional marker of the TFH-differentiation TFH differentiation during acute viral infection, we crossed mice with pathway. In this study, we determined that TCF-1 directly bound loxP-flanked Tcf7 alleles (Tcf7fl/fl) to mice with transgenic expression to the Bcl6 promoter and Prdm1 5′ regulatory regions, which acti- of Cre recombinase from the T cell–specific Cd4 promoter (Cd4- vated Bcl-6 expression but repressed Blimp1 expression. In addition, Cre) to generate mice with conditional deletion of Tcf7 in CD4+ TCF-1 formed a complex with Bcl-6 and increased Bcl6 transcrip- T cells (Tcf7fl/flCd4-Cre mice; called ‘Tcf7−/− mice’ here). CD4+ −/− tion. Thus, our findings suggest that TCF-1 promotes TFH differ- T cells from Tcf7 mice had largely abrogated TCF-1 expression entiation through multiple regulatory mechanisms upstream of the (Supplementary Fig. 2a). At day 8 after infection with LCMV, we Bcl-6–Blimp1 axis. observed a similar frequency and number of gp66 tetramer–positive CD4+ T cells in Tcf7−/− mice and control mice (Tcf7fl/fl mice with- RESULTS out Cd4-Cre) (Supplementary Fig. 2b), indicative of normal clonal + −/− + Higher TCF-1 expression in TFH cells than in naive CD4 T cells expansion of virus-specific Tcf7 CD4 T cells. However, very few + lo + −/− Upon acute viral infection, virus-specific naive CD4 T cells generally tetramer-positive SLAM CXCR5 TFH cells were present in Tcf7 27,28 + differentiate into TH1 cells or TFH cells . To investigate the expres- mice, whereas approximately 35% of the tetramer-positive CD4 sion of TCF-1 during this bifurcated differentiation, we transferred T cells in the control mice were TFH cells (Fig. 2a). This phenotype naive SMARTA cells (which have transgenic expression of a T cell anti- was recapitulated for other TFH cell markers, including ICOS, Bcl-6 gen receptor specific for LCMV epitope of glycoprotein amino acids and PD-1 (Supplementary Fig. 2c). The number of TFH cells was 66–77 (gp66) presented by I-Ab) into wild-type C57BL/6J recipients, also much lower in Tcf7−/− mice than in control mice (Fig. 2b). We which we subsequently infected with the Armstrong strain of LCMV. observed a similar phenotype for bulk activated Foxp3− CD4+ T cells At day 2 after infection, we observed that TCF-1 expression was nearly in Tcf7−/− mice (Supplementary Fig. 2d). Consistent with their lower hi − −/− tenfold lower in TH1 cells (Tim3 CXCR5 ) but twofold higher in TFH abundance of TFH cells, Tcf7 mice had a much lower frequency and cells (Tim3loCXCR5+) relative to its expression in naive cells (Fig. 1a). number of GC B cells, as well as a much lower titer of LCMV-specific We also observed the higher expression of TCF-1 in TFH cells than in immunoglobulin G (IgG), relative to that of their control counterparts −/− TH1 cells at day 8 after infection (Fig. 1a). Furthermore, at day 2 after (data not shown). The reduction in the TFH cell population in Tcf7 infection, we observed substantial nuclear enrichment for TCF-1 in mice was probably not due to enhanced differentiation of follicular TFH cells, but this was minimal in TH1 cells (Fig. 1b). regulatory T cells (Supplementary Fig. 2e) or altered apoptosis or Next we investigated the correlation between TCF-1 expression and proliferation (Supplementary Fig. 2f,g). Indeed, TCF-1 deficiency TFH differentiation. At day 2 after infection, TCF-1 expression was influenced the programming of TFH differentiation, as indicated by −/− positively correlated with expression of the key TFH-differentiation lower expression of Bcl-6, ICOS and CXCR5 in Tcf7 mice than in molecules CXCR5 and Bcl-6 and was negatively correlated with control mice (Fig. 2c). −/− + expression of the TH1 cell–associated molecules CD25, Tim3 and In Tcf7 mice, CD8 T cells are also deficient in TCF-1 expression, granzyme B (Fig. 1c). We observed a similar phenomenon at day 8 which may affect antigen loads and then influence the differentia- 21 after infection (Supplementary Fig. 1).