Figure 1. T_reg cells have active autophagy and require Atg7 for mediating tumor immune tolerance and self-tolerance.

(a) Representative images (scale bars, 5 μm) (left) and quantification of percentages of cells with GFP-LC3⁺ puncta (right) in peripheral naïve CD4⁺ cells and T_reg cells purified from GFP-LC3 mice (n=3 mice). (b–d) Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (n=4 mice per genotype) were inoculated with MC38 colon adenocarcinoma cells, and tumor growth (b) was measured. Flow cytometry analyzing IFN-γ expression in Foxp3⁻CD4⁺ and CD8⁺ T cells (c, left), frequency of IFN-γ⁺ cells (c, right) and Foxp3 expression in CD4⁺ T cells (d) in tumor-infiltrating lymphocytes. Numbers in quadrants indicate percent cells in each throughout, and numbers adjacent to outlined areas indicate percent Foxp3⁺ cells (d). (e–g) Analysis of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (10–12 weeks old) under steady state. Total cellularity of spleen and PLNs (n=8 mice per genotype) (e). Flow cytometry analyzing the expression of CD62L and CD44 (f, left) and IFN-γ and IL-17 (g, left), and frequency of CD44^hiCD62L^lo cells (n=6 mice per genotype) (f, right) and IFN-γ⁺ or IL-17⁺ cells (n=6 mice per genotype) (g, right) in splenic Foxp3⁻CD4⁺ and CD8⁺ T cells. Numbers adjacent to outlined areas indicate percent IFN-γ⁺ or IL-17⁺ cells (g, left). (h) Hematoxylin and eosin staining of colon, esophagus, liver, lung, pancreas, prostate gland, salivary gland, and skin from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (19–23 weeks old). Magnification and scale bars: ×40 and 100 μm (colon and skin), ×20 and 200 μm (esophagus, liver, pancreas, prostate gland and salivary gland) and ×10 and 200 μm (lung). (i) Flow cytometry analyzing YFP-Foxp3 expression in CD4⁺ T cells (left), and frequency and number of YFP-Foxp3⁺ cells (right) in the thymus, spleen, PLNs and mesenteric lymph nodes (MLNs) of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (10–12 weeks old) (n=7 mice per genotype). Numbers adjacent to outlined areas indicate percent YFP-Foxp3⁺ cells (g, left). NS, not significant (P > 0.05); * P < 0.05 and **P < 0.001 (two-tail unpaired Student’s t-test in a-c,e–g,i). Data are representative of two (a–d,h) experiments, or pooled from five out of six (e), four out of six (f,g) or three out of six (i) experiments (mean ± s.e.m in a–c,e–g,i).