LEF-1 and TCF-1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6

Abstract

Follicular helper T cells (T(FH) cells) are specialized effector CD4(+) T cells that help B cells develop germinal centers (GCs) and memory. However, the transcription factors that regulate the differentiation of T(FH) cells remain incompletely understood. Here we report that selective loss of Lef1 or Tcf7 (which encode the transcription factor LEF-1 or TCF-1, respectively) resulted in T(FH) cell defects, while deletion of both Lef1 and Tcf7 severely impaired the differentiation of T(FH) cells and the formation of GCs. Forced expression of LEF-1 enhanced T(FH) differentiation. LEF-1 and TCF-1 coordinated such differentiation by two general mechanisms. First, they established the responsiveness of naive CD4(+) T cells to T(FH) cell signals. Second, they promoted early T(FH) differentiation via the multipronged approach of sustaining expression of the cytokine receptors IL-6Rα and gp130, enhancing expression of the costimulatory receptor ICOS and promoting expression of the transcriptional repressor Bcl6.

Figure 1. Lef1 expression is associated with T_FH cells and regulates early T_FH differentiation

(a) RNA-seq analysis of early T_FH (IL-2Rα⁻Blimp1-YFP⁻) versus T_H1 (IL-2Rα⁺Blimp1-YFP⁺) CD45.1⁺ Blimp1-YFP SMARTA cells isolated from B6 mice 3 d after SMARTA cell transfer and LCMV infection (left panels), and that of T_H1 (CXCR5⁻), T_FH (PD-1^loCXCR5⁺), and GC T_FH (PD-1^hiCXCR5⁺) sorted 8 d after LCMV from CD45.2⁺ B6 mice (right panels). Heatmaps of selected genes of interest are shown. (b) Scatter plot of genes showing ≥ |1.5 fold| differential expression in early T_FH in comparison to T_H1 cells. Select genes of interest are marked. (c) Immunoblot of LEF-1 (two isoforms) and β-actin from shCtrl⁺ and shLef1⁺ SMARTA cells. (d-f) Analysis of shCtrl⁺ or shLef1⁺ CD45.1⁺ SMARTA cells (Ametrine⁺CD45.1⁺CD4⁺CD19⁻), three days after transfer of shRNA-RV-infected SMARTA cells into B6 mice and LCMV infection. (d) shRNA⁺ SMARTA cell frequency among total CD4⁺ T cells. (e-f) Phenotyping of shCtrl⁺ and shLef1⁺ SMARTA cells. (e) Bcl6⁺CXCR5⁺ T_FH cells. (f) IL-2Rα⁻CXCR5⁺ T_FH cells. Quantitation shown as % of SMARTA cells (mean ± s.e.m.). Data are a composite of two independent experiments (n = 7 per group). * P < 0.05, ** P < 0.001 (Student’s t-test).

Figure 2. LEF-1-dependent T_FH differentiation supports germinal center responses

(a-e) Frequencies and phenotypes of shCtrl⁺ or shLef1⁺ CD45.1⁺ SMARTA cells assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice and LCMV infection. (a) Abundance of shRNA⁺ SMARTA cells (Ametrine⁺CD45.1⁺CD4⁺CD19⁻) among total CD4⁺ T cells. (b) Frequency of SLAM^loCXCR5⁺ T_FH cells. Quantitation shown as % of SMARTA cells. (c) Expression of CXCR5 on shCtrl⁺ (blue) and shLef1⁺ (red) SMARTA cells. (d-e) Frequencies of shCtrl⁺ and shLef1⁺ SMARTA GC T_FH cells among SMARTA cells phenotyped as PSGL-1^loCXCR5⁺(d) and Bcl6^hiCXCR5⁺ (e). (f) Abundance of GC B cells (Bcl6⁺CD19⁺) among total B cells. Data are representative of two independent experiments (n = 4–5 per group, mean ± s.e.m.). * P < 0.05 (Student’s t-test).

Figure 3. Genetic ablation of LEF-1 impairs GC T_FH differentiation

(a-c) Flow cytometry of T_FH and GC T_FH cells in spleens of Lef1^−/− mice and littermate controls infected with vaccinia virus for 8 days. Plots are gated on CD44^hiCD62L^loGFP⁺CD4⁺ T cells. (a) SLAM^loCXCR5⁺ T_FH cells. (b-c) Abundance of GC T_FH cells phenotyped as Bcl6⁺CXCR5⁺ (b) and PD-1^hiCXCR5⁺(c). Cumulative data from four independent experiments are shown (mean ± s.d.). * P < 0.01, ** P < 0.001 (Student’s t-test).

Figure 4. Both TCF-1 and LEF-1 contribute to regulation of T_FH differentiation and B cell responses

(a) Flow cytometry of Tcf7-GFP expression in SMARTA CD4⁺ T cells (CD45.2⁺CD4⁺) at 8 d after Tcf7^GFP/+ SMARTA cell transfer into CD45.1⁺ recipients and LCMV infection. Gated populations of T_H1 (CXCR5⁻SLAM^hi) and T_FH (CXCR5⁺SLAM^lo) cells were analyzed for Tcf7-GFP expression. Numbers indicate percent Tcf7-GFP⁺ cells. Data are representative of ≥ 2 experiments. (b-f) Flow cytometry of Tcf7^−/−Lef1^−/−Tcf7^−/−, and littermate controls 8 d after infection i.v. with vaccinia virus. (b-d) Abundance of SLAM^loCXCR5⁺ T_FH cells (b), Bcl6⁺CXCR5⁺ GC T_FH cells (c), and PD-1^hiCXCR5⁺ GC T_FH cells (d) gated on CD44^hiCD62L⁻GFP⁺CD4⁺ T cells in spleen. (e-f) Abundance of GL7⁺Fas⁺ GC B cells (e) and IgD^loCD138⁺ plasma cells (f) in the same animals (mean ± s.d.). Cumulative data from ≥ 3 experiments are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).

Figure 5. Enhanced Lef1 expression leads to augmented T_FH differentiation

(a) Expression of LEF-1 in GFP-RV⁺ (blue) and Lef1-RV⁺ (red) SMARTA cells assessed by flow cytometry. (b-g) Frequencies and phenotypes of GFP-RV⁺ or Lef1-RV⁺ SMARTA cells (CD45.1⁺CD4⁺CD19⁻) assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice (CD45.2⁺) and LCMV infection. (b) Abundance of RV⁺ SMARTA cell (GFP⁺CD45.1⁺CD4⁺CD19⁻) among total CD4⁺ T cells. (c) Abundance of SLAM^loCXCR5⁺ T_FH cells among RV⁺ SMARTA cells. (d-e) Expression of canonical T_FH markers CXCR5 (d) and PD-1 (e) on CXCR5⁻ T_H1 and CXCR5⁺ T_FH cells by Lef1-RV⁺ and GFP-RV⁺ cells, normalized to the mean MFI per group (mean ± s.e.m.). (f-g) Abundance of GC T_FH cells phenotyped as PSGL-1^loCXCR5⁺(f) and PD-1^hiCXCR5⁺(e) of RV⁺ SMARTA cells. Data are a composite of two independent experiments (n = 9 per group). * P < 0.01, ** P < 0.001 (Student’s t-test).

Figure 6. LEF-1 regulates expression of IL-6 receptor chains and ICOS

(a-b) RNA-seq analysis of GFP-RV⁺ and Lef1-RV⁺ SMARTA cells (CD45.1⁺CD4⁺CD19⁻) sorted into CXCR5⁺ T_FH or CXCR5⁻ T_H1 populations isolated from B6 mice 4 d after SMARTA cell transfer and LCMV infection. (a) GSEA enrichment analysis showing enrichment of T_FH and GC T_FH gene signatures in Lef1-RV⁺ T_H1 cells compared to GFP-RV⁺ T_H1 cells. (b) Heat map of selected genes upregulated in Lef1-RV⁺ T_H1 cells compared to GFP-RV⁺ T_H1 cells. (c-h) Flow cytometry of GFP-RV⁺ or Lef1-RV⁺ SMARTA cells (CD45.1⁺CD4⁺CD19) assessed at 3 d after SMARTA cell transfer into B6 mice (CD45.2⁺) and LCMV infection. (c-d) Expression of IL6Rα(c) and gp130 (d) on RV⁺ SMARTA cells. (e-f) Comparative expression of IL-6Rα (e) and gp130 (f) GFP-RV⁺ (red) and Lef1-RV⁺ (blue) T_H1 and T_FH cells. (g-h) Abundance of ICOS on total RV⁺ SMARTA cells (g), CXCR5⁺ T_FH and CXCR5⁻ T_H1 cell subpopulations (h) in the same animal (mean ± s.e.m.). Data are a composite of three independent experiments (n = 10–14 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).

Figure 7. LEF-1 and TCF-1-dependent transcriptional regulation of T_FH-related genes

(a) RNA-seq analysis of PD-1^hiCXCR5⁺ GC T_FH cells sorted from spleens of Lef1^−/−Tcf7^−/− and littermate controls 8 d after vaccinia virus infection. Green lines mark mean gene expression of ≥ |1.5 fold| differences. Select genes of interest are marked. (b) Heatmap of selected differentially regulated genes between control and Lef1^−/−Tcf7^−/− GC T_FH cells. (c-e) Flow cytometry of Lef1^−/−Tcf7^−/− and littermate controls 8 d after infection i.v. with vaccinia virus. CXCR5⁺ T_FH and CXCR5⁻ T_H1 cells were analyzed for gp130 (c), ICOS (d), and IL-6Rα (e) expression (mean ± s.d.). Bar graphs are normalized to the mean MFI on control T_FH cells. Data are a composite of 4 independent experiments (n = 5–9 per group). (f) Gene expression of Ascl2 and Prdm1 was determined by quantitative RT-PCR in CXCR5⁻ T_H1, PD-1^loCXCR5⁺ T_FH, and PD-1^hiCXCR5⁺ GC T_FH cells sorted from Lef1^−/−Tcf7^−/− and littermate controls 8 d after infection i.v. with vaccinia virus. Data are from 2 experiments with each sample measured in duplicate and normalized to control T_FH cells. ND, not reliably detected. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).

Figure 8. TCF-1 binds to key T_FH-associated genes in T_FH cells

(a-d) ChIP assays using anti-TCF-1 antibody or control IgG were performed on naive control CD4⁺ T cells (CD44^loCD62L⁺CD4⁺); naive Tcf7^−/− CD4⁺ T cells (GFP⁺CD44^loCD62L⁺CD4⁺); WT T_FH cells (CXCR5⁺CD44^hiCD62L⁻CD4⁺); and WT T_H1 cells (CXCR5⁻CD44^hiCD62L⁻CD4⁺). T_FH and T_H1 cells were sorted from B6 mice 8 d after vaccinia virus infection. Quantitation of enriched TCF-1 binding was done at the positive control Axin2 gene (a), the TSS of the Il6ra and Il6st genes (b), the TSS and a −2.8 kb upstream regulatory region of the Bcl6 gene (c), and the TSS of Ascl2 and intron 3 of Prdm1 (d), and data are means ± s.d. from 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t-test).