Abstract
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.
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Acknowledgements
We thank the Institut Clinique de la Souris (part of the International Knockout Mouse Consortium) for permission to use the mouse with conditional knockout of Tcf7; H.H. Xue (University of Iowa) for Tcf7fl/fl mice and retroviral vectors; R. Ahmed (Emory University) for SMARTA mice and retroviral vectors; CapitalBio for performing microarray experiments and data analysis; and the core facility center of Third Military Medical University for cell sorting. Supported by the National Basic Research Program of China (973 Program, 2013CB531500, to L.Y. and X.Z.), the National Natural Science Foundation of China (31470870 to X.Z., 81471624 to L.Y. and 31000631 to Z.X.), the China1000 Talent Plan Program (L.Y.) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (A.Q.).
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L.X., Y.C., Z.X., Q.H., Q.B., X.Y., R.H., Y.H., H.W., T.Z., Z.F., A.Q. and J.Y. performed the experiments; Q.B. analyzed the microarray data by GSEA; X.Z. and L.Y. designed the study, analyzed the data and wrote the paper with L.X., Y.C. and Z.X.; and X.Z., L.Y. and Y.W. supervised the study.
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Integrated supplementary information
Supplementary Figure 1 TCF-1 expression positively correlates with signatures associated with the TFH lineage.
Flow cytometry analysis of TCF-1 expression and other markers related to TFH and TH1 differentiation in SMARTA cells of spleens from SMARTA-chimera mice at day 8 after LCMV infection. The numbers in quadrants indicate the percentage of cells in each. The data are representative of three independent experiments.
Supplementary Figure 2 TCF-1 is essential for TFH differentiation after acute viral infection.
(a) Expression of TCF-1 in CD4+ T cells in spleens from Tcf7−/− and control mice. (b) Flow cytometry of CD4+ T cells in spleens from Tcf7−/− and control mice at day 8 after LCMV infection (left) and quantification of GP66-tetramer-positive cells (right). The numbers above the outlined areas indicate the percentages of tetramer-positive cells (left). (c) Flow cytometry of tetramer-positive CD4+ T cells in spleens from Tcf7−/− and control mice as in b. The numbers above the outlined areas indicate the percentages of TFH cells. (d) Flow cytometry of Foxp3− CD4+ T cells in spleens from Tcf7−/− and control mice (top). The numbers above the outlined areas indicate the percentages of CD44hiCXCR5+ TFH cells. The graphs show the frequency (below, left) and number (below, right) of TFH cells. (e) Flow cytometry of Foxp3+ CD4+ T cells in spleens from Tcf7−/− and control mice (top). The numbers above the outlined areas indicate the percentages of Foxp3+CXCR5+ TFR cells. The graphs show the frequency (below, left) and number (below, right) of TFR cells. (f) Flow cytometry of TFH cells in spleens from Tcf7−/− and control mice as in d. The numbers in quadrants indicate the percentage of cells in each. (g) Quantification of BrdU incorporation (left) and Ki-67 expression (right) in TFH cells from spleens of Tcf7−/− and control mice as in d. (h) Setup of BM chimera experiments. Donor BM cells from Tcf7−/− (CD45.2) and WT (CD45.1) mice were mixed (4:6), and transferred to irradiated WT (CD45.1) mice. After reconstitution, the recipient mice were infected with LCMV. (i) Setup of retrovirus-mediated TCF-1 knockdown in SMARTA cells. Retrovirus-transduced SMARTA cells (CD45.1) were adoptively transferred into WT mice (CD45.2), followed by LCMV infection. (j) Flow cytometry analysis of TCF-1 expression in transduced (GFP+) and non-transduced (GFP−) SMARTA cells at day 8 after infection. (k) Flow cytometry of GFP− and GFP+ SMARTA cells at day 8 after infection. The numbers adjacent to the outlined areas indicate the percentage of corresponding population. NS, not significant; *P < 0.05 and ***P < 0.001 (unpaired two-tailed t-test). The data are representative of two (c–g,j,k) or three (a,b) independent experiments with at least three mice per group (error bars (a,b,d,e,g), SEM).
Supplementary Figure 3 TCF-1 is required for TFH differentiation after infection with influenza A virus.
(a) Flow cytometry of Foxp3− CD4+ T cells in mediastinal lymph nodes of BM chimeras as in Supplementary Fig. 2 h at day 8 after influenza A virus infection, showing the CXCR5+ TFH population (left) and a summary of the results (right). The numbers adjacent to the outlined areas indicate the percentages of TFH cells. (b) Quantification of MFIs of Bcl-6, ICOS and CXCR5 on TFH cells as in a. *P < 0.05 and **P < 0.01 (paired two-tailed t-test). The data are representative of two independent experiments with four mice per group (error bars, SEM).
Supplementary Figure 4 Ectopic expression of TCF-1 enhances TFH differentiation.
(a) Flow cytometry analysis of GFP− (non-transduced) and GFP+ (overexpressing p33 or p45) SMARTA cells in spleens at day 8 after LCMV infection. The numbers adjacent to the outlined areas indicate the percentages of SLAMloCXCR5+ TFH cells. (b) Summary of TFH frequency and quantification of MFIs of CXCR5, ICOS and Bcl-6 on TFH cells expressing p33 or p45. (c) Experimental setup. Retroviruses overexpressing p33 were introduced into CD45.1+ SMARTA cells, and the cells were transferred into WT recipients (CD45.2), which were subsequently infected with LCMV. At day 8 after infection, transduced GFP+ SMARTA TFH cells were sorted and transferred into day 1-infected recipients, followed by analysis at day 5 after cell transfer. (d) The graphs show a summary of the frequency and number of PNAhiFAShi GC B cells (top) gated on the B220+CD19+ population and CD138hiB220lo plasma cells (below) in spleens at day 5 after cell transfer as in c. *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired (d) or paired (b) two-tailed t-test).The presented data are representative of two (d) or three (a,b) independent experiments with at least three (a,b) or four (d) mice per group (error bar (d), SEM).
Supplementary Figure 5 Altered gene expression in TCF-1-deficient TFH cells.
(a) Sorting strategy. (b–e) WT and Tcf7−/− TFH and TH1 cells were sorted on day 8 after LCMV infection, followed by mRNA extraction and RT-qPCR analysis. NS, not significant; *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two-tailed t-test).The data are representative of two (b–e) independent experiments with two replicates from three mice per group (error bars (b–e), SEM).
Supplementary Figure 6 Alignment of the putative TCF-1-binding sites in the Bcl6 promoter and Prdm1 5′ regulatory regions.
The conserved TCF-1-binding motifs “CAAAG” (or “CTTTG” on the reverse strand) are highlighted in red, and their locations relative to the transcriptional starting site (TSS) of Bcl6 or Prdm1 are marked.
Supplementary Figure 7 TCF-1 physically interacts with Bcl-6 protein.
Immunoprecipitation of lysates of activated CD4+ T cells from infected mice at day 8 after infection with a Bcl-6 antibody or control IgG and immunoblot analysis with Bcl-6 and TCF-1 antibodies. The presented data are representative of two independent experiments.
Supplementary Figure 8 ICAT inhibits TFH differentiation.
Flow cytometry analysis of GFP− (non-transduced) and GFP+ (overexpressing ICAT) SMARTA cells in spleens at day 3 after LCMV infection. The numbers adjacent to the outlined areas indicate the percentages of Tim3loCXCR5+ TFH cells (left). The graph shows a summary of results (right). ***P < 0.001 (paired two-tailed t-test).The presented data are representative of three independent experiments with at least five mice per group.
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Xu, L., Cao, Y., Xie, Z. et al. The transcription factor TCF-1 initiates the differentiation of TFH cells during acute viral infection. Nat Immunol 16, 991–999 (2015). https://doi.org/10.1038/ni.3229
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DOI: https://doi.org/10.1038/ni.3229
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