Fig 2: TCF1 and LEF1 Redundantly Control Tfr Generation and Germinal Center B Cell Responses(A) The expression of Bcl6 (left) and Cxcr5 (right) detected by RNA-seq in the three Tregs.(B) GSEA analysis revealing that Bcl6 gene signature was enriched in TCF1+ aTregs not TCF1- eTregs. NES, normalized enrichment score.(C) Gene ontology (GO) annotations of the over-represented genes in TCF1+ aTregs identified by k-means clustering analysis (see Figure S7A for detail).(D) Flow cytometric analysis of Bcl6 and CXCR5 in the three Treg subsets.(E) Superimposing Tfr cells onto the bulk Treg plot (TCF1 versus CD62L). Inset numbers show the percentages of Tfr cells fell into each quadrant.(F) Flow cytometric analysis of Foxp3 expression in CD4+ Bcl6+ CXCR5+ T cells from spleen of indicated mice (left). The percentages and numbers of Tfr and Tfh cells (right).(G) Flow cytometric analysis of the expression of Bcl6 and FAS expression in CD4- B220+ cells in the spleen of indicated mice.(H) Immunofluorescence staining of splenic frozen sections from Tcf7wt/wtLef1wt/wt; Foxp3Cre (WT) and Tcf7fl/flLef1fl/fl;Foxp3Cre (dKO) mice. Statistical data show the intensities of GL7 staining in each individual B220+ follicles normalized by the area of B cell follicles.(I) Immunofluorescence staining of samples as in (H) with Foxp3, GL7, and CD4. Arrows in the top panel (WT) depicting Tfr cells. The numbers of Foxp3+ T cells within each individual GCs was counted and then normalized by the size of the GC area.(J) ChIP-PCR analysis showing enriched binding of TCF1 to Bcl6 (upstream -500) in WT Tregs. Splenic CD4+ T cells from Tcf7fl/flCD4Cre mice were used as negative control to show the background of TCF1 binding.The data are representative of n = 2 replicates for each Treg subsets (A–C); n = 3 independent experiments (D, E, and J) or n = 2 experiments with n = 3–4 mice in each group (F–I). The statistic calculation in (B) was false discovery rate (FDR) q value. Shown are mean ± SEM. The p values in (A, D, and F–I) were calculated using the two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars, 100 µm (H) and 50 µm (I).See also Figure S7 and Tables S4 and S5.

Fig 3: TCF1 and LEF1 Are Required for Treg’s Competitive Fitness by Regulating STAT5 Expression and Activation(A) The percentages of CD25 expression in TCF1+ and TCF1- Tregs.(B) In vitro pSTAT5 assays in TCF1 + and TCF1- Tregs, stimulated with IL-2 at various concentrations.(C) GSEA analysis revealing that IL-2/STAT5 signaling was significantly under-represented in dKO Tregs. NES, normalized enrichment score.(D) In vitro pSTAT5 assays in dKO and WT Tregs, stimulated with IL-2 at various concentrations. (left) The percentages of pSTAT5; (right) the MFI of pSTAT5 (gated on pSTAT5+).(E) Ex vivo analysis of total STAT5 protein and pSTAT5 in dKO and WT Tregs. Representative FACS plots (top). Percentages of total STAT5 protein and pSTAT5, and the ratios of pSTAT5/total STAT5, respectively (bottom).(F) ChIP-PCR analysis showing enriched binding of TCF1 to Stat5b promoter.(G) In vivo Treg survival assays. Splenic CD4+ T cells were isolated from WT (Foxp3Cre) or dKO mice, i.p. transferred into Tcra-/- mice and analyzed 5 weeks later. FACS plots and statistical data show the percentages of Tregs reconstituted from transferred donor cells.(H) In vivo Treg survival assays under competitive conditions. Splenic CD4+ T cells were isolated from CD45.1 WT (Foxp3Cre), CD45.2 WT (Foxp3Cre), or CD45.2 dKO mice, mixed at a ratio of 3:1 between CD45.1 versus CD45.2 donor cells, i.p. transferred into Tcra-/- mice and analyzed 5 weeks later. FACS plots and statistical data show the percentages of Tregs reconstituted from each type of donor cells in spleen, pLNs, and mLNs.(I) Schematic diagram showing that splenic CD4+ T cells from WT or dKO mice were MACS enriched and transduced with retroviruses expressing Tcf7, or caSTAT5, i.p. transferred into Tcra-/- mice and analyzed 3 weeks later (top). Representative FACS plots and statistical data showing the reconstitution of Tregs from different inputs as described in the inset legend (middle). The MFIs of TCF1 and LEF1 in Tregs from the conditions as described in the inset legend (bottom). The data are representative of n = 3 (A, B, D, and E) and n = 2 (F, G, and I) independent experiments and one experiment with n = 2 replicates for each Treg subsets (C) or one experiment with n = 3 mice in each group (H). n = 4–6 mice in each group. Shown are mean ± SEM. The p values were calculated using two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. RV, retrovirus; Ctl, control; wk, week.See also Figure S6.

Fig 4: TCF1 and LEF1 Are Dispensable for Treg’s Suppressive Capacity(A) Schematic diagram of experimental settings.(B) Representative histograms of cell trace violet (CTV) dilution in indicated groups (left) and statistical data of Treg-mediated suppression on Tresp cells (right).(C) Representative histograms of CD80 expression in DCs in indicated groups (top) and statistical data of Treg-mediated suppression on CD80 expression at indicated ratios (bottom).(D) Representative histograms of CD86 expression in DCs in indicated groups (top) and statistical data of Treg-mediated suppression on CD86 expression at indicated ratios (bottom).(E) Representative histograms of CTLA4 expression in DCs in WT (left) and dKO Tregs and statistical data (right).The data are representative of n = 2 independent experiments with pooled n = 3 mice in each group. Shown are mean ± SEM. *p < 0.05; **p < 0.01; n.s., non-significant (two-tailed unpaired Student’s t test).See also Figure S4.

Fig 5: Gradient Expression of TCF1 and LEF1 Distinguishes Peripheral Treg into Distinct Subsets(A) Flow cytometric analysis of the expression of CD44, CD62L, and TCF1 in Tregs from the spleens of 6- to 8-week old B6 mice.(B) Histograms summarize the MFIs of TCF1, CD62L, and CD44 in the R1 (blue), R2 (green), and R3 (red) subsets, as depicted in (A).(C) The proportions of the R1, R2, and R3 Treg subsets in the spleen, pLNs, and mLNs.(D) Flow cytometric analysis of TCF1 expression in splenic Tregs of adult (11 weeks old) and aged (55 weeks old) mice.(E) Flow cytometric analysis of the expression of LEF1 and CD62L in cells prepared as in (A).The data are representative of n = 3 independent experiments with n = 3–4 mice in each group. Shown are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., non-significant (two-tailed unpaired Student’s t test). wk, week; Spl, spleen; pLN, peripheral (cervical, axillary, brachial, and inguinal) lymph nodes; mLN, mesenteric lymph nodes.See also Figure S1.