Figure 5. Fntb promotes the maintenance of eT_reg cells by mTORC1 and ICOS signaling.

(A) Gene set enrichment analysis (GSEA) of the hallmark mTORC1 pathway in cT_reg cells stimulated with anti-CD3/28 plus IL-2 in the presence of vehicle or FTI for 48 h. (B) Quantification of the relative mean fluorescence intensity (MFI) of phosphorylated S6 (p-S6) and p-4EBP1 by flow cytometry analysis in WT cT_reg cells stimulated with anti-CD3/28 plus IL-2 in the presence of FTI for 0 or 18 h. (C) Immunoblot analysis of p-S6 and p-4EBP1 in cT_reg cells stimulated as in (B) for the indicated times. (D) Quantification of relative MFI of p-S6 in cT_reg cells from WT or Foxp3^CreFntb^fl/fl mice stimulated as in (B). (E) Quantification of relative MFI of p-S6 in cT_reg cells from WT and Cd4^CreFntb^fl/fl mice stimulated as in (B). (F and G) Quantification of percentage of splenic CD44^hiCD62L^lo eT_reg cells (F) or Ki67⁺ T_reg cells percentage in WT, Cd4^CreFntb^fl/fl, Cd4^CreFntb^fl/flTsc1^fl/fl or Cd4^CreTsc1^fl/fl mice. (H) Flow cytometry analysis and quantification of ICOS expression on cT_reg cells isolated from indicated mice after IL-2 or anti-CD3/28 plus IL-2 stimulation for 3 days. (I) Quantification of ICOS expression on WT cT_reg cells stimulated as in (H) in the presence of vehicle or FTI. (J) Flow cytometry analysis and quantification of ICOS expression on WT Bcl2^lo and Bcl2^hi eT_reg-cell subsets from spleen. (K) Flow cytometry analysis (left) and quantification of frequency (middle) and number (right) of splenic Bcl2^lo eT_reg cells in indicated mice. (L) Flow cytometry analysis and quantification of Mcl1 (left) or Bim (right) expression (based on mean fluorescence intensity, MFI) in splenic Bcl2^hi and Bcl2^lo eT_reg cells from indicated mice. (M) Icos expression in cT_reg cells from indicated mice after stimulation as in (B). (N) Principal component analysis plot of nucleosome-free reads of cT_reg and eT_reg cells from WT or Foxp3^CreFntb^fl/fl mixed bone marrow chimeras. (O) Odds ratio/odds ratio plot of the predicted enriched motifs in Foxp3^CreFntb^fl/fl vs. WT eT_reg cells compared with Foxp3^CreFntb^fl/fl vs. WT cT_reg from mixed bone marrow chimeras. (P) Flow cytometry and quantification of splenic eT_reg cell percentage and number in retrogenic mice [WT or Cd4^CreFntb^fl/fl bone marrow cells were transduced with pMIG-GFP empty vector (pMIG) or pMIG-GFP-ICOS (ICOS) retroviral vector; see Methods].

Data are representative of two (C) or four (J, K), or compiled from two (B for p-4EBP1, L for Bim, M), three (B for p-S6, D, E, L for Mcl1, P), four (F, G, J, K), five (H) or six (I) independent experiments, with 9 (B for p-S6), 7 (L for Mcl1), 6 (B for p-4EBP1, I, L for Bim, P for WT pMIG and WT ICOS), 3 (E, M, P for Cd4^CreFntb^fl/fl pMIG), 4 (F, G, J, K, P for Cd4^CreFntb^fl/fl ICOS) or 5 (D, H) biological replicates per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant; two-tailed unpaired Student’s t-test (B, D, E, H–K, L–P) or one-way ANOVA (F, G). Data are mean ± s.e.m. Numbers indicate percentage of cells in gates. Numbers in histograms indicate MFI. Controls were normalized to 1 for each comparison (usually in black bars). See also Figure S5.