Supplementary Figure 1: Ablation of autophagy in Foxp3^CreAtg7^fl/fl mice and analysis of homeostasis of Foxp3^CreAtg7^fl/fl mice, Foxp3^CreAtg5^fl/fl mice and mixed BM chimeras.

(a) Immunoblot analysis of LC3-I and LC3-II in naïve CD4 and T_reg cells from Foxp3^Cre mice and in T_reg cells from Foxp3^CreAtg7^fl/fl mice. Cells were rested in complete medium for 1 h in the presence or absence of 100 nM Baf1A. (b) Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (n=4 per genotype) were inoculated with MC38 colon adenocarcinoma cells. Flow cytometry analyzing CD4 and CD8 expression (left), and frequency of CD4⁺ and CD8⁺ T cells (right) in tumor-infiltrating lymphocytes. Numbers adjacent to outlined areas indicate percent CD4⁺ and CD8⁺ cells (left). (c) Number of CD4⁺ and CD8⁺ T cells in the spleen and PLNs of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (n=5 mice per genotype). (d) Flow cytometry analyzing IL-4 expression in Foxp3⁻CD4⁺ and CD8⁺ T cells from the spleen of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice. Numbers adjacent to outlined areas indicate percent IL-4⁺ cells. (e) Flow cytometry analyzing Foxp3 expression in CD4⁺ T cells in the colon lamina propria of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (3-4 weeks old). Numbers adjacent to outlined areas indicate percent Foxp3⁺ cells. (f) Flow cytometry analyzing YFP-Foxp3 expression in CD45.2⁺CD4⁺ T cells in the spleen of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl chimeras. Numbers adjacent to outlined areas indicate percent YFP-Foxp3⁺ cells. (g,h) Flow cytometry analyzing the expression of CD62L and CD44 (g), and IFN-γ (h) in Foxp3⁻CD4⁺ and CD8⁺ T cells and in the spleen of Foxp3^CreAtg5^+/fl and Foxp3^CreAtg5^fl/fl mice. Numbers in quadrants indicate percent cells in each (g), and numbers adjacent to outlined areas indicate percent IFN-γ⁺ cells (h). (i) Flow cytometry analyzing Foxp3 expression in CD4⁺ T cells in the spleen and PLNs of Foxp3^CreAtg5^+/fl and Foxp3^CreAtg5^fl/fl mice. Numbers adjacent to outlined areas indicate percent Foxp3⁺ cells. NS, not significant (P > 0.05); * P < 0.05 and ** P < 0.001 (two-tail unpaired Student's t-test in b,c). Data are representative of three (a, g-i), two (b) or five (d-f) experiments, or pooled from three out of six (c) experiments (mean ± s.e.m in b,c).