Abstract
In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centres1,2. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells3 or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation4. Here we show that expression of achaete-scute homologue 2 (Ascl2)—a basic helix–loop–helix (bHLH) transcription factor5—is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4+ T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.
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Change history
26 March 2014
Changes were made to Fig. 2f, h, i, j, l, and to Extended Data Figs 3, 7 and 8. Minor edits were made to the main text, and a number of incorrect details in the legends of Fig. 2 and Extended Data Figs 8 and 9 have been fixed.
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Acknowledgements
We thank J. A. Whitsett and J. P. Bridges at the University of Cincinnati for their provision of Ascl2 conditional knockout mice, D. Yi for help with ChIP-seq and microarray analysis, R. Dalla-Favera for Bcl6−/− mice, H. Hu for histochemistry staining, and the Dong laboratory members for their help. This work was supported in part by a grant from the National Institutes of Health (NIH; AI106654 to C.D.), an intramural research program (NIDCR to W.C. and H.N.), an NIH Lymphoma SPORE (to X.L.), an Odyssey fellowship from the MD Anderson Cancer Center (to X.L. and B.Z.), Chinese Ministry of Science and Technology ‘973’ program grants (2014CB542501 and 2012CB910402), and a National Natural Science Foundation of China grant (81361120397 to H.Q.).
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Authors and Affiliations
Contributions
X.L. designed and performed the experiments, and wrote the manuscript; X.C. and H.Q. performed histochemistry in Fig. 2j, l and Extended Data Fig. 9a, b, and B.Z., A.W., X.W., R.I.N., F.C. and S.S.N. contributed to the performance of the other experiments; X.Y., P.C., Q.T. and L.W. performed microarray, ChIP product sequencing and data analysis; L.G.v.d.F., H.N., W.C. and H.C. provided important mouse strains; and C.D. designed and directed the research.
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Extended data figures and tables
Extended Data Figure 1 Ascl2 exhibits unique epigenetic regulation in TFH cells, and its expression is dependent on Wnt signalling.
a, Genome-wide histone modifications (H3K4me3, permissive marker; H3K27me3, suppressive marker) across Bcl6, Maf, Batf, Irf4 and Ascl2 loci in T-cell subsets (in vivo TFH and non-TFH data sets were newly generated; the rest were derived from the Gene Expression Omnibus database (accession GSE1425428). b, Flow cytometric analysis of Ascl2 expression in three populations of activated CD44+CD4+ T cells in dLNs from Bcl6-RFP mice: CXCR5hiBcl6hi (red), CXCR5+Bcl6lo (blue) and CXCR5−Bcl6− (black) cells. c, Quantitative RT–PCR measurement of Ascl2, Bcl6 and Batf expression in Bcl6-RV-GFP, Batf-RV-GFP and control vector-infected CD4+ T cells; wild-type (WT) and Stat5−/− naive CD4+ T cells were cultured under TH0 conditions, or together with IL-6, respectively. Ascl2, Bcl6 and Batf transcriptional expression was measured by qRT–PCR. d, Quantitative RT–PCR measurement of Ascl2 in CD4+ T cells cultured under indicated conditions. e, Quantitative RT–PCR measurement of Cxcr5 and Bcl6 in control or TWS119- (1 μM) treated T cells. All experiments were repeated at least three times with similar results. Bar graphs show the relative level of mRNA as mean ± s.d., n = 3 per group. *P < 0.05, **P < 0.01, two-tailed t-test. NS, not significant.
Extended Data Figure 2 Ascl2 regulates a selective subset of TFH-relevant genes.
a, Flow cytometry analysis of CXCR5 expression in CD4+ T cells transduced with vector control, Ascl2-RV-GFP, Bcl6-RV-GFP, Batf-RV-GFP and Maf-RV-GFP. Data are representative of two independent experiments. b, Transcriptional expression of Ccr7, Psgl1, Il2ra and Il2rb in Ascl2-RV-GFP- or control-vector-infected T cells was measured by quantitative RT–PCR. Data are representative of two independent experiments. Bar graphs show the relative level of mRNA as mean ± s.d., n = 3, two-tailed t-test. c, Quantitative RT–PCR measurement of gene expression including the TH1-related genes Tbx21 and Ifng, as well as the TH17-related genes Rorc and Il17a. Data are representative of three independent experiments. Bar graphs display the relative level of mRNA as mean ± s.d., n = 3, two-tailed t-test. d, Bcl6-RV-GFP- or control-viral-vector-infected GFP+ OT-II cells were adoptively transferred into naive congenic mice, followed by subcutaneous OVA/CFA immunization. At day 2 after immunization, flow cytometry analysis of donor-derived cells in dLNs was carried out with CXCR5 and PD1 staining. Data are representative of two independent experiments (n = 3). e, Quantification of donor-derived TFH cells. Bar graphs show mean ± s.d., n = 3, two-tailed t-test. NS, not significant. f, Vector-transduced GFP+CXCR5−CD4+ T (RV) cells, Ascl2-RV-GFP-infected GFP+CXCR5− (CXCR5−) and GFP+CXCR5+ (CXCR5+) CD4+T cells were sorted and subjected to microarray assay. g, Hierarchical clustering and principal component analysis (PCA) were applied on seven microarray data sets including RV, CXCR5− and CXCR5+, as well as TFH and non-TFH cells (derived from GSE40068)4. h, The clustered heatmap of ∼350 genes from RV, CXCR5−, CXCR5+, TFH and non-TFH cells. The colour coding applies to gene expression level (log2) with 0 as a median.
Extended Data Figure 3 Regulation of TH-cell differentiation by Ascl2.
a, Naive CD4+ T cells from C57BL6 mice were activated under neutral conditions and infected with Ascl2-RV-GFP or control vector (empty-RV-GFP) virus, followed by continuous culture under TH1, TH2, TH17, inducible (i)Treg and TFH-like conditions for 3–4 days. Quantification of signature genes by intracellular staining and real-time RT–PCR. b, c, Ascl2-RV-GFP- or control-vector-transduced T cells were cultured under TH2 conditions for 4 days. b, After re-stimulation with PMA and ionomycin for 5 h, TH2-related gene expression, including IL-4, IL-5 and IL-13, was measured by flow cytometric analysis. c, GFP+ T cells were sorted and re-stimulated by plate-bound anti-CD3, and transcriptional expression of IL-4, IL-5 and IL-13 was measured by quantitative RT–PCR; cytokines in supernatants from re-stimulation were subjected to ELISA analysis. d, e, Ascl2-RV-GFP- or control-vector-transduced OT-II cells were adoptively transferred into naive congenic mice, followed by subcutaneous OVA/CFA immunization for 7 days. d, After re-stimulation with OVA, flow cytometry analysis of donor-derived cells from dLNs was carried out with intracellular IL-4 and IL-21 staining. e, GFP+ donor-derived T cells were sorted from dLNs, re-stimulated with anti-CD3, and subjected to quantitative RT–PCR measurement of Il21 and Il4 mRNA expression. All data are representative of two independent experiments. a–c, e, Bar graphs show mean ± s.d., n = 3, two-tailed t-test. NS, not significant.
Extended Data Figure 4 CXCR5 expression is directly mediated by Ascl2.
a, b, Naive CD4+ T cells were pre-activated and transduced with empty-RV-GFP, Id3-RV-GFP, Ascl2-RV-GFP or Ascl2-RV-GFP, together with Id3-RV-GFP retrovirus. a, Flow cytometry analysis of CXCR5 expression in retrovirus-infected T cells. Data are representative of two independent experiments. b, GFP+ cells were sorted from Ascl2-RV-GFP or Ascl2-RV-GFP plus Id3-RV-GFP retrovirus-infected T cells, and subjected to Ascl2-binding analysis of the Cxcr5 gene locus using a ChIP assay. Primer information is listed in Supplementary Table 2. Data are representative of two independent experiments. c, Luciferase reporter assay of enhancer activity for the Ascl2-bound region of the Cxcr5 locus. CNS-containing PGL3 plasmid was transfected with either empty-RV-GFP or Ascl2-RV-GFP into the EL4 T-cell line. Bar graph shows mean ± s.d., n = 3. **P < 0.01, two-tailed t-test. d, Map of Cxcr5 gene locus and Ascl2-binding peaks at the Cxcr5 locus. Arrows indicate the Ascl2-responsive CNS region.
Extended Data Figure 5 Coordinated function of Ascl2 and Batf/IRF4 in regulating TFH-related genes.
a–g, ChIP-Seq data for Maf, Batf and IRF4 were derived from GSE40918 (ref. 25). a, Venn diagram of ChIP-seq peaks from Ascl2 and Maf. b, Distribution of ChIP-seq peaks by Ascl2, Batf and IRF4 on gene loci including Bcl6, Prdm1, Ascl2 and Maf. c, Venn diagram of ChIP-seq peaks from Ascl2, Batf and IRF4. d, Distribution of ChIP-seq peaks by Ascl2, Batf and IRF4 on gene loci including Cxcr5, Cxcr4, Ccr7, Selplg1, Il2, Il2ra and Il2rb. Blue frame represents the colocalization of peaks. e, Distribution of ChIP-seq peaks by Ascl2, Batf and IRF4 on TH1-related Tbx21 and Ifng gene loci. f, Distribution of ChIP-seq peaks by Ascl2, Batf and IRF4 on TH2-related Gata3, Il4, Il13 and Il5 gene loci. g, Distribution of ChIP-seq peaks by Ascl2, Batf and IRF4 on TH17-related Rorc, Il17a, Il17f and Il21 gene loci. ChIP-seq assay of Ascl2 was performed on Ascl2-overexpressing T cells cultured under TH0 conditions. ChIP-seq assays of Maf, Batf and IRF4 were performed on TH0 cells by D. R. Littman’s group, derived from GSE40918 (ref. 25).
Extended Data Figure 6 Loss of Ascl2 in CD4+ T cells leads to impairment of germinal centre responses during influenza virus infection.
a–j, Control and Ascl2fl/fl/CD4-Cre mice were infected intranasally with influenza virus A/PR8. a, The body weight of control and Ascl2fl/fl/CD4-Cre mice was monitored daily after infection. b, Mice were killed at day 9 after infection, and viral titre in the lungs was assessed by measurement of active HA gene expression with quantitative RT–PCR. c, Virus-specific total IgGs in the sera were measured by ELISA. d, Flow cytometry analysis of germinal centre B cells (GL7hiFAShi) in lung dLNs from influenza-infected control and Ascl2fl/fl/CD4-Cre mice. e, Frequencies of TFH cells in spleens from influenza-infected control and Ascl2fl/fl/CD4-Cre mice. f, Frequencies of germinal centre B cells in spleens from influenza-infected control and Ascl2fl/fl/CD4-Cre mice. g, After 9 d.p.i., the CD4+/CD8+ T-cell ratio was measured in the lung, BALF, spleen and dLNs from control and Ascl2fl/fl/CD4-Cre mice. h, Flow cytometry analysis of granzyme B and IFN-γ production from both CD4+ and CD8+ T cells in dLNs. i, j, Mixed chimaeric mice were reconstituted with both Ascl2+/+/ETR2-Cre and Ascl2fl/fl/ETR2-Cre bone marrow cells at a ratio of 1:1. Eight weeks later, chimaeric mice were administered 200 μl tamoxifen (5 mg ml−1) in sunflower seed oil i.p. every other day for a total of 5 days, and this was followed by influenza virus A/PR8 infection. At 9 d.p.i.,the CD4+/CD8+ T-cell ratio and TFH-cell generation was measured in dLNs (i) and spleens (j). All data are representative of three independent experiments. Graphs display mean ± s.d., n = 5 per group. *P < 0.05, **P < 0.01, two-way ANOVA (a, c), two-tailed t-test (b, e, f, g–j). NS, not significant.
Extended Data Figure 7 In the absence of Ascl2, bHLH family member E47 may have a redundant role in TFH-cell differentiation.
a, Quantitative RT–PCR measurement of E47 expression in naive CD4+ T cells from Ascl2fl/fl/CD4-Cre and littermate control mice; TFH and non-TFH cells were obtained from dLNs of Ascl2fl/fl/CD4-Cre and littermate control mice immunized with KLH in CFA, and the expression of E47 was measured by real-time RT–PCR. b, Flow cytometry analysis of CXCR5 expression in T cells infected with E47-RV-CFP or control vector retrovirus. c–g, T-cell-depleted bone marrow cells were obtained from Ascl2+/+/CD4-Cre (CD45.1+ CD45.2+) and Ascl2fl/fl/CD4-Cre (CD45.2+) mice and mixed at a ratio of 1:1 or 0:1 before being transferred into irradiated Rag1−/− recipient mice (8 × 106 cells per mouse). c–g, Eight weeks later, mice were either used for measurement of thymic T-cell maturation (c, d), or immunized with KLH in CFA for monitoring peripheral TFH-cell differentiation (e–g). c, Flow cytometry analysis of T-cell maturation in thymus of mixed chimaeric mice containing both Ascl2+/+/CD4-Cre and Ascl2fl/fl/CD4-Cre bone marrow cells. d, Flow cytometry analysis of T-cell maturation in thymus of chimaeric mice containing only Ascl2fl/fl/CD4-Cre bone marrow cells. e, Seven days after immunization, TFH cells in dLNs of mixed chimaeric mice (Ascl2+/+/CD4-Cre and Ascl2fl/fl/CD4-Cre) were measured by flow cytometry. f, Flow cytometry analysis of TFH cells in dLNs of chimaeric mice (Ascl2fl/fl/CD4-Cre). g, The percentages of both CD4+ T cells and B220+ B cells in dLNs of mixed chimaeric mice (Ascl2+/+/CD4-Cre and Ascl2fl/fl/CD4-Cre). All data above are representative of two independent experiments. Graphs show mean ± s.d., n = 3 per group, two-tailed t-test. NS, not significant.
Extended Data Figure 8 Loss of Bcl6 in CD4+ T cells does not affect early TFH-cell homing ability in vivo.
Equal amounts of Cre-RV-GFP-transduced wild-type (WT)/OT-II, Bcl6−/−/OT-II and Ascl2 fl/fl/OT-II cells were transferred into congenic mice, and this was followed by subcutaneous OVA/CFA immunization. a, At day 3 after immunization, flow cytometry analysis of donor-derived TFH-cell generation was carried out with CXCR5 and PD1 staining. b, Quantification of donor-derived CXCR5+ T cells. c, At day 8 after immunization, donor-derived TFH-cell generation was examined using CXCR5 and PD1 staining. d, Quantification of donor-derived CXCR5+PD1+ TFH cells. e, At day 3 after immunization, dLNs were isolated and subjected to histochemical staining of B-cell follicles and donor T cells. Green, GFP; red, B220; scale bar, 100 μm, n = 4. All data are representative of two independent experiments. Graphs display mean ± s.d., n = 4, two-tailed t-test, NS, not significant.
Extended Data Figure 9 Ectopic expression of Id3 inhibits TFH-cell generation in vivo.
a–c, Naive OT-II CD4+ T cells were activated and transduced with Id3-RV-GFP or control viral vector (empty-RV-GFP) for 3 days. GFP+ T cells were then sorted and transferred into naive congenic mice that were subsequently immunized with OVA/CFA. a, At day 4 after immunization with OVA/Alum/LPS, immunohistochemical staining of section slides of dLNs was carried out. Red, IgD+ B cells; green, GFP+ donor-derived OT-II cells. Data are representative of two independent experiments, n = 6. Scale bar, 100 µm. b, Quantification of GFP+ OT-II cell distributions in dLNs. Data are representative of two independent experiments. Dot graph shows mean ± s.d., n = 17, two-tailed t-test. c, Optical density (OD) values of OVA-specific antibodies in serum from mice on day 7 after immunization with OVA/CFA, measured by threefold serial dilution in OVA- (100 μg ml−1) coated plates. Data are representative of two independent experiments. Graphs show mean ± s.d., n = 6. *P < 0.05, **P < 0.01, one-way ANOVA. d, Naive CD4+ OT-II cells were pre-activated and co-transduced with empty-RV-GFP/empty-RV-hCD2, empty-RV-GFP/Bcl6-RV-hCD2, Id3-RV-GFP/Bcl6-RV-hCD2, or Id3-RV-GFP/empty-RV-hCD2. Sorted hCD2+GFP+ OT-II cells were transferred into congenic mice, followed by subcutaneous OVA/CFA immunization for 7 days. Measurement of donor-derived TFH cells was carried out with CXCR5 and PD1 staining. Data are representative of two independent experiments. Bar graphs show mean ± s.d., n = 3, two-tailed t-test.
Extended Data Figure 10 Schematic model of the sequential roles of Ascl2/Id3 and Bcl6/Blimp1 during TFH-cell differentiation.
Ascl2 expression plus simultaneous Id3 reduction in activated CD4+ T cells orchestrates T cells to migrate towards B-cell follicles and initiate the TFH program by inducing expression of the chemokine receptors CXCR5 and CXCR4, and suppressing expression of CCR7 and PSGL1 and the IL-2 signal pathway, as well as TH1 and TH17 differentiation. Upon interacting with cognate B cells at the T–B border, CXCR5+ T cells begin to increase Bcl6 expression, which eventually facilitates TFH maturation in B follicles and germinal centre formation.
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Liu, X., Chen, X., Zhong, B. et al. Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development. Nature 507, 513–518 (2014). https://doi.org/10.1038/nature12910
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DOI: https://doi.org/10.1038/nature12910
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