Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development

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 centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--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.

Figure 1. Ascl2 is selectively expressed in both mouse and human Tfh cells

a., Three populations of CXCR5^hiBcl6-RFP^hi (red), CXCR5⁺Bcl6-RFP^lo (blue) and CXCR5⁻Bcl6-RFP⁻ (black) cells were sorted from dLNs in Bcl6-RFP mice immunized with KLH emulsified in CFA subcutaneously. b. Ascl2, CXCR5 and Bcl6 transcriptional expression in sorted cells. c. Ascl2 mRNA expression among in vivo-generated Tfh, naïve, Th0, Th1, Th2, and Th17 cells by quantitative RT-PCR. d. Flow cytometric analysis of human tonsil CD4⁺ T cells by CXCR5 and PD-1 staining. e. The expression of Ascl2, CXCR5, Bcl6, and Gata3 mRNA in sorted cells. f. CXCR5 expression in CD4⁺ T cells activated by anti-CD3/anti-CD28 in the presence of TWS119 (1μM) (an inhibitor of glycogen synthase kinase-3beta (Gsk-3beta)) for three days. All experiments were repeated at least three times with similar results. (b, c, and e) bar graph displayed the relative level of mRNA as mean ± SD, n = 3 per group, *P<0.05, **P<0.01, two-tailed t-test.

Figure 2. Ascl2 expression induces the Tfh program

a. Flow cytometry analysis of surface CXCR5 expression from empty-RV-GFP, Ascl2-RV-GFP, and Bcl6-RV-GFP retrovirus-infected T cells, respectively. b. Sorted GFP⁺ T cells were subject to the measurement of CXCR5 mRNA by quantitative RT-PCR. c. Measurement of genes expression including Cxcr5, Bcl6, Prdm1, Batf, Sh2d1a, Cd40lg, Icos, pdcd1, Btla, and Il21. d. Surface CXCR5 expression in Ascl2-RV-GFP-and vector virus-infected WT, Bcl6^−/−, and Batf^−/− T cells. e. CCR7, PSGL-1, CD25, and CD122 expression by flow cytometry analysis. (f–k) Ascl2-RV-GFP- or Empty-RV-GFP- transduced GFP⁺ OT-II cell were transferred into naïve TCRβ^−/− mice subsequently immunized with NP-OVA/CFA. f. At day 2 and Day 6, flow cytometry analysis of donor cells with staining CXCR5 and Bcl6, n = 4. g. Quantification of CXCR5⁺ and CXCR5⁺Bcl6⁺ donor-derived T cells. h. GC B cells (GL-7^hi Fas^hi) in recipient mice, n = 4. i. Quantification of GC B cells. j. At day 8, dLNs were collected and subject to histochemistry staining of GC center and donor T cells. Green, GFP; Red, PNA; Blue, anti-IgD; Scale bar, 100μm, dot graph represents donor cells in GC, displayed as mean ± SD, n = 10. k. Titers of NP-specific antibodies in serum from mice day 8 after immunization, n = 4. l. Distribution of Ascl2-RV-GFP- and vector-infected GFP⁺ OT-II donor cells in B220⁺ B cell follicles from dLNs in mice immunized with OVA/CFA for four days, scale bar, 100μm, dot graph represents distribution with a ratio of donor cells in B cell follicle versus T zone, displayed as mean ± SD. Empty-RV-GFP, n = 21; Ascl2-RV-GFP, n=15. All experiments were repeated at least three times with similar results. (b, c, g, i, and j–l) graph displayed as mean ± SD, two-tailed t-test, N.S, no significance.

Figure 3. Ascl2-dependent transcriptional regulation of Tfh-related genes

a. Scatterplot of the average signal of Tfh versus non-Tfh cell, and Ascl2-RV-GFP⁺ versus Empty-RV-GFP⁺ T cell gene expression microarray data. The green line indicates gene expression change by factor of 2; data normalized from two replicates are shown; n = 2. Genes with the most transcriptional changes are listed. b. Venn diagram of genes regulated by Ascl2 and Tfh-related genes. c. Distribution of Ascl2 ChIP-Seq peaks in Ascl2-overexpressed CD4⁺ T cells. d. Venn diagram of genes regulated by Ascl2 and Ascl2-bound genes. e. Ascl2 binding site is identical to E-box binding motif. f. Ascl2 binding peaks located at gene loci including Cxcr5, Cxcr4, Ccr7, Selplg1, Il2ra, Il2rb, Ifng, Tbx21, Il2, and Rorc. g. Comparison of mouse Cxcr5 genomic sequence to dog, human, and rat. Red regions denote CNS region. h. Ascl2 binding sites at Cxcr5 locus in in vivo-generated Tfh cells by ChIP-qPCR (primers information were listed in Supplementary Table II). Green indicates the CNS region. Data are a representative of two independent experiments.

Figure 4. Loss of Ascl2 in peripheral CD4⁺ T cells inhibits Tfh differentiation

a. Tfh cells (CXCR5^hi Bcl6^hi) in mediastinal LNs from Ascl2^fl/fl/CD4-Cre and Ascl2^+/+/CD4-Cre mice that received a sublethal dose of A/PR/8 (H1N1) virus intranasal infection nine days before, n = 5. b. Flow cytometry analysis of donor cells or host cells in mice receiving Cre-RV-GFP retroviral infected Ascl2^fl/fl/OT-II or Ascl2^+/+/OT-II GFP positive cells and subcutaneous 5-day- immunization of OVA/CFA, respectively, n = 3. c. Flow cytometry analysis of donor-derived Tfh cells in mice that received Id3-RV-GFP or control viral vector transduced OT-II cells and subcutaneous OVA/CFA immunization, n = 3. d. Flow cytometry analysis of donor-derived Tfh cells in Rag^−/− mice receiving equal numbers of naïve Id3^−/− and WT CD4⁺ T cells and subcutaneous KLH/CFA immunization for seven days, n = 5. e. Analysis and quantification of Tfh cells in chimeric mice reconstituted with equal number of Ascl2^fl/fl/ERT2-Cre and Ascl2^+/+/ERT2-Cre bone marrow cells after 5-day-tamoxifen treatment and 7-day- KLH/CFA immunization, n = 4. All above data are a representative of three independent experiments; Bar graph displays as mean ± SD, two-tailed t-test, N.S, no significance.

Extended Data Figure 1. Ascl2 exhibits unique epigenetic regulation in Tfh cell, and its expression is dependent on Wnt signal

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 set were newly generated; the rest were derived from GEO database (GSE14254)). b. Flow cytometric analysis of Ascl2 expression in three populations of activated CD44⁺ CD4⁺ T cell in draining LNs from Bcl6-RFP mice: CXCR5^hiBcl6^hi (red), CXCR5⁺Bcl6^lo (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; WT and Stat5^−/− naïve CD4⁺ T cells were cultured under Th0 condition, or together with IL-6, respectively. Ascl2, Bcl6, and Batf transcriptional expression were 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 (1μM)- treated T cells. All experiments were repeated at least three times with similar results. Bar graph displayed the relative level of mRNA as mean ± SD, n = 3 per group, *P<0.05, **P<0.01, two-tailed t-test, N.S, no significance.

Extended Data Figure 2. Ascl2 regulates a selective subset of Tfh-relevant genes

a. Flow cytometry analysis of CXCR5 expression in CD4⁺ T cell transduced with vector control, Ascl2-RV-GFP, Bcl6-RV-GFP, Batf-RV-GFP and Maf-RV-GFP. Data are a representative of two independent experiments. b. Transcriptional expression of Ccr7, Psgl1, Il2ra, and Il2rb in Ascl2-RV-GFP- or control Vector-infected T cells were measured by quantitative RT-PCR. Data are a representative of two independent experiments. Bar graph displayed the relative level of mRNA as mean ± SD, n = 3, two-tailed t-test. c. Quantitative RT-PCR measurement of genes expression including Th1-related Tbx21 and Ifng, as well as Th17-related Rorc and Il17a. Data are a representative of three independent experiments. Bar graph displayed the relative level of mRNA as mean ± SD, n = 3, two-tailed t-test. d. Bcl6-RV-GFP- or control viral vector-infected GFP⁺ OT-II cells were adoptively transferred into naïve congenic mice, respectively, followed with subcutaneous OVA/CFA immunization. At day 2 after immunization, flow cytometry analysis of donor-derived cells in dLNS with CXCR5 and PD1 staining. Data are a representative of two independent experiments, n = 3. e. Quantification of donor-derived Tfh cells. Bar graph displayed as mean ± SD, n = 3, two-tailed t-test, N.S, no significance. f. Vector-transduced GFP⁺CXCR5⁻ CD4⁺ T (RV) cell, Ascl2-RV-GFP-infected GFP⁺CXCR5⁻ (CXCR5⁻) and GFP⁺CXCR5⁺ (CXCR5⁺) CD4⁺T cell were sorted and subject to microarray assay. g. Hierarchic clustering and principal component analysis (PCA) were applied on seven microarray dataset including RV, CXCR5⁻, CXCR5⁺ as well as Tfh and non-Tfh cells (derived from GSE40068 ). h. The clusterd heatmap of ~350 genes from RV, CXCR5⁻, CXCR5⁺, Tfh, Non-Tfh cells. The color-coding applies to gene expression level (log2) with 0 as a median.

Extended Data Figure 3. Regulation of Th cell differentiation by Ascl2

a. Naïve CD4⁺ T cells from C57BL6 mice were activated under neutral condition and infected with Ascl2-RV-GFP or control vector (empty-RV-GFP) virus, followed with continuous culture under Th1, Th2, Th17, iTreg, and Tfh-like condition for 3–4 days, respectively. 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 condition for 4 days. b. After re-stimulation with PMA and ionomycin for 5h, Th2-related genes including IL-4, IL-5, and IL-13 expression were measured by flow cytometric analysis. c. GFP⁺ T cells were sorted and re-stimulated by plate-bound anti-CD3, transcriptional expression of IL-4, IL-5, and IL-13 were measured by quantitative RT-PCR; cytokines in supernatants of re-stimulation were subject to ELISA analysis. (d–e). Ascl2-RV-GFP- or control vector- transduced OT-II cells were adoptively transferred into naïve congenic mice, respectively, followed with subcutaneous OVA/CFA immunization for seven days. d. After restimulation with OVA, flow cytometry analysis of donor-derived cells from dLNs with intracellular IL-4 and IL-21 staining. e. GFP⁺ donor-derived T cells were sorted from dLNs, re-stimulated with anti-CD3, and subject to quantitative RT-PCR measurement of IL-21 and IL-4 mRNA expression. All data are a representative of two independent experiments. (a–c, and e) bar graph displayed as mean ± SD, n = 3, two-tailed t-test, N.S, no significance.

Extended Data Figure 4. CXCR5 expression is directly mediated by Ascl2

Naïve 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, respectively. a. Flow cytometry analysis of CXCR5 expression in retroviral virus-infected T cells. Data are a 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 subject to Ascl2 binding analysis on Cxcr5 gene locus via ChIP assay. Primer information was listed in Supplementary Table II. Data are a representative of two independent experiments. c. Luciferase reporter assay of enhancer activity for Ascl2-bound region at Cxcr5 locus. Conserved noncoding sequence (CNS)-containing PGL3 plasmid was transfected with either empty-RV-GFP or Ascl2-RV-GFP into EL4 T cell line. Bar graph displayed as mean ± SD, n = 3, **P<0.01, two-tailed t-test. d. Map of Cxcr5 gene locus and Ascl2 binding peaks at Cxcr5 locus. Arrow indicates the Ascl2-responsive CNS region.

Extended Data Figure 5. Coordinated function of Ascl2 and Batf/IRF4 in regulating Tfh-related genes

(ChIP-Seq data of Maf, Batf, and IRF4 were derived from GSE40918 ) 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, Prmd1, 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 co-localization 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 were performed on Ascl2-overexpressed T cell cultured under Th0 condition. ChIP-Seq assay of Maf, Batf and IRF4 were performed on Th0 cells by Dan R. Littman’s group, derived from GSE40918 .

Extended Data Figure 6. Loss of Ascl2 in CD4⁺ T cells leads to impairment of germinal center responses during Influenza virus infection

Control and Ascl2^fl/fl/CD4-Cre mice were infected intranasally with influenza virus A/PR8. a. Body weight of control and Ascl2^fl/fl/CD4-Cre mice were monitored daily after infection. b. Mice were sacrificed at day9 after infection, viral titer in the lungs were assessed by measurement of active Hemagglutinin (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 center B cells (GL-7^hi Fas^hi) in lung dLNs from influenza-infected control and Ascl2^fl/fl/CD4-Cre mice. e. Frequencies of Tfh cells in spleens from influenza-infected control and Ascl2^fl/fl/CD4-Cre mice; f. Frequencies of GC B cells in spleens from influenza-infected control and Ascl2^fl/fl/CD4-Cre mice; g. After 9 day post infection (dpi), measurement of CD4⁺/CD8⁺ T cell ratio in lung, bronchoalveolar lavage fluid (BALF), spleen and dLNs from control and Ascl2^fl/fl/CD4-Cre mice. h. Flow cytometry analysis of Granzyme B and IFNγ production from both CD4⁺ and CD8⁺ T cell in dLNs. (i–j). Mixed chimeric mice were reconstituted with both Ascl2^+/+/ETR2-Cre and Ascl2^fl/fl/ETR2-Cre bone marrow cells at a ratio of 1:1. Eight weeks later, chimeric mice were administered 200μl tamoxifen (5mg/ml) in sunflower seed oil i.p every other day for total 5 days, and then followed with influenza virus A/PR8 infection. At 9 dpi, measurement of CD4⁺/CD8⁺ T cell ratio and Tfh cell generation in dLNs (i) and spleens (j). All the Data above are a representative of three independent experiments. Graph displayed as mean ± SD, n = 5 per group, *P<0.05, **P<0.01, (a, c) two way ANOVA, (b, e, f, and g–j) two-tailed t-test, N.S, no significance.

Extended Data Figure 7. In the absence of Ascl2, bHLH family member E47 may play a redundant role in Tfh cell differentiation

a. Quantitative RT-PCR measurement of E47 expression in naïve CD4⁺ T cell from Ascl2^fl/fl/CD4-Cre and litter mate control mice; Tfh and non-Tfh were obtained from dLNs of Ascl2^fl/fl/CD4-Cre and littermate control mice immunization with KLH in CFA, the expression of E47 was measured by real time RT-PCR. b. Flow cytometry analysis of CXCR5 expression in T cell 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 Ascl2^fl/fl/CD4-Cre (CD45.2⁺) mice and mixed at a ratio of 1:1 or 0:1 before transferred into irradiated Rag1^−/− recipient mice (8×10⁶ cell/mouse). Eight weeks later, mice were either utilized 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 cells maturation in thymus of mixed chimeric mice containing both Ascl2^+/+/CD4-Cre and Ascl2^fl/fl/CD4-Cre bone marrow cells. d. Flow cytometry analysis of T cells maturation in thymus of chimeric mice containing only Ascl2^fl/fl/CD4-Cre bone marrow cells. e. Seven days after immunization, Tfh cells in dLNs of mixed chimeric mice (Ascl2^+/+/CD4-Cre and Ascl2^fl/fl/CD4-Cre) were measured by flow cytometry. f. Flow cytometry analysis of Tfh cells in dLNs of chimeric mice (Ascl2^fl/fl/CD4-Cre). g. The percentages of both CD4⁺ T cells and B220⁺ B cells in dLNs of mixed chimeric mice (Ascl2^+/+/CD4-Cre and Ascl2^fl/fl/CD4-Cre). All the Data above are a representative of two independent experiments. Graph displayed as mean ± SD, n = 3 per group, two-tailed t-test, N.S, no significance.

Extended Data Figure 8. Loss of Bcl6 in CD4⁺ T cells does not affect early Tfh cell homing ability in vivo

Equal amount of retrovirus Cre-RV-GFP-transduced WT/OT-II, Bcl6^−/−/OT-II and Ascl2 ^fl/fl/OT-II cells were transferred into congenic mice, respectively, and followed with subcutaneous OVA/CFA immunization. a. At day 3 post immunization, flow cytometry analysis of donor-derived Tfh cell generation with CXCR5 and PD1 staining. b. Quantification of donor-derived CXCR5⁺ T cell. c. At day 8 post immunization, examination of donor-derived Tfh cell generation with CXCR5 and PD1 staining. d. Quantification of donor-derived CXCR5⁺ PD1⁺ Tfh cells. e. At day 3 post immunization, dLNs were isolated and subject to histochemistry staining of B cell follicles and donor T cells. Green, GFP; Red, anti-B220; Scale bar, 100μm, n=8. All the Data above are a representative of two independent experiments. Graph displayed as mean ± SD, n = 4, two-tailed t-test, N.S, no significance.

Extended Data Figure 9. Ectopic expression of Id3 inhibits Tfh cell generation in vivo

(a–c) Naïve OT-II CD4⁺ T cells were activated and transduced with Id3-RV-GFP or control viral vector (empty-RV-GFP) for three days. GFP⁺ T cell were then sorted and transferred into naïve congenic mice subsequently immunized with OVA/CFA. a. At day3 post immunization, immunohistochemistry staining of section slide of draining lymph nodes. Red, B220⁺ B cells; Green, GFP⁺ donor derived OT-II cells. Data are a representative of two independent experiments, n = 6. b. Quantification of GFP⁺ OT-II cell distributions in dLNs. Data are a representative of two independent experiments; Dot graph displayed as mean ± SD, n=17, two-tailed t-test. c. OD values of OVA-specific antibodies in serum from mice day 7 after immunization, measured by 3 fold serial dilution in OVA (100μg/ml)-coated plates. Data are a representative of two independent experiments; Graph displayed as mean ± SD, n = 6, *P<0.05, **P<0.01, one way ANOVA. d. Naïve 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, respectively. Sorted hCD2⁺GFP⁺ OT-II cells were transferred into congenic mice, followed with subcutaneous OVA/CFA immunization for seven days. Measurement of donor-derived Tfh cells with CXCR5 and PD1 staining. Data are representative of two independent experiments; Bar graph displayed as mean ± SD, n = 3, two-tailed t-test.

Extended Data Figure 10. The schematic model on the sequential roles of Ascl2/Id3 and Bcl6/Blimp1 during Tfh differentiation

Ascl2 expression plus simultaneous Id3 reduction in activated CD4⁺ T cells orchestrates T cells to migrate toward B cell follicles and initiate Tfh program by inducing chemokine receptor CXCR5, CXCR4 expression, and suppressing CCR7, PSGL1, IL-2 signal pathway, as well as Th1, Th17 differentiation. Upon interacting with cognate B cells at T-B border, CXCR5+ T cells begin to increase Bcl6 expression, which eventually facilitates Tfh maturation in B follicles and GC formation.