Supplementary Figure 4: Characterization of mice with T_reg cells expressing an active form of STAT5.

(a) The frequencies of STAT5b-CA-expressing CD4⁺Foxp3⁺ cells among total CD4⁺Foxp3⁺ cells in the spleen of Foxp3^Cre-ERT2Rosa26^Stat5bCA mice after a single tamoxifen gavage on day 0. (b) The frequencies of STAT5b-CA-expressing CD4⁺Foxp3⁺ cells among total CD4⁺Foxp3⁺ cells in the indicated organs of Foxp3^Cre-ERT2Rosa26^Stat5bCA mice three months after a single tamoxifen gavage. (c) TCR Vβ usage of the T_reg cells in various tissues 2 months after tamoxifen gavage. MLNs, mesenteric lymph nodes; PPs, Peyer’s patches. (d) Changes in body weights after tamoxifen gavage. 4-month-old Foxp3^Cre-ERT2 (black, n=7) and Foxp3^Cre-ERT2Rosa26^Stat5bCA (blue, n=7) mice were gavaged with tamoxifen and body weights were monitored the following 4 months. (e) Serum chemistry profiles 4.5 months after tamoxifen gavage. (f–h) General characterization of T_reg cells of Foxp3^Cre-ERT2 (black) and Foxp3^Cre-ERT2Rosa26^Stat5bCA (blue) mice three months after a single tamoxifen gavage. (f) The expression levels of indicated molecules on CD4⁺Foxp3⁺ cells in the indicated organs. (g) The frequencies of Foxp3⁺ cells among CD3⁺CD4⁺ cells (upper graph) and the expression levels of Foxp3 in the CD3⁺CD4⁺Foxp3⁺ cells (lower graph) in the indicated organs. (h) The frequencies of Foxp3⁺ cells among CD3⁺CD8⁺ cells in the indicated organs. (i) In vitro suppressor activity of T_reg cells. T_reg cells were isolated from Foxp3^Cre-ERT2 (control) and Foxp3^Cre-ERT2Rosa26^Stat5bCA (STAT5b-CA) mice and co-cultured with T naive cells (responder cells). The proliferative activities of T_reg and responder cells were determined by flow cytometry based on the dilution of CellTrace Violet (CTV) fluorescence intensity. Typical dye dilution patterns of T naive cells at a 4:1 responder vs. T_reg cell ratio are shown in the left two panels. Summary graphs showing the proliferation of co-cultured responder T cells (Foxp3⁻) and T_reg cells (Foxp3⁺) are shown in the right two panels. CTV MFI of cells inversely correlates with cell division. Cells were analyzed by flow cytometry (a–c, f–i). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed unpaired Student’s t test). Data are from one experiment representative of two (c, d, i) or three or more (a, b, f–h) independent experiments with similar results with three or more biological replicates per group in each experiment. Each dot represents a single mouse (b, e–h). mean ± s.e.m. (a, b, d–i).