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: γδ thymocytes from Rag1Cre-Nfatc1fl/fl mice exhibit an NKT γδ phenotype(A) Differently expressed genes from freshly isolated Rag1Cre-Nfatc1fl/fl and Nfatc1fl/fl γδ thymocytes.(B) Cell type identification analysis of RNA-seq data of Nfatc1fl/fl and RagCre-Nfatc1fl/fl γδ thymocytes shows the dominance of NKT cell phenotype.(C) Flow cytometry of γδ thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice upon staining with anti-CD4 and anti-CD8. Right, percentages of CD4+ γδ thymocytes.(D) Flow cytometry of thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice gated for CD4+ TCRδ+ cells upon staining with anti-NK1.1. Right, percentages of CD4+ TCRδ+ thymocytes expressing the NK1.1 surface marker.(E) Flow cytometry of total thymocytes from Nfatc1fl/fland RagCre-Nfatc1fl/fl mice gated for CD4+ TCRδ+ cells upon staining with anti-Vγ1.1 and anti-Vδ6.3. Right, percentages of CD4+ TCRδ+ thymocytes expressing the Vγ1.1+ and Vδ6.3+ gene segments.(F) Number of γδ thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice expressing the transcription factor PLZF analyzed by flow cytometry (left) and percentages of γδ thymocytes expressing PLZF (right).(G) (Above) Flow cytometry analysis of PLZF+ γδ thymocytes from WT (CD45.1+CD45.2+) and Rag1Cre-Nfatc1f/fl (CD45.2+) mouse bone marrow chimera. Total thymocytes are gated for TCRδ and TCRβ expression. The percentages of γδ PLZF+ thymocytes is calculated from the WT and the Rag1Cre-Nfatc1f/fl γδ thymocyte compartments. Data are representative of at least two independent experiments with four mice from every genotype and are shown as mean ± SEM. The statistical significance was determined by unpaired student’s t-tests. ∗∗p value <0.005, ∗∗∗p value <0.001, ∗∗∗∗p value <0.000. See also Figure S2.