Figure 2. Atg7 contributes to T_reg cell survival and lineage stability.

(a) Flow cytometry analyzing active caspase-3 expression (left), and frequency of caspase-3⁺ cells (right) in T_reg cells from the spleen of Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (n=5 mice per genotype). Numbers adjacent to outlined areas indicate percent caspase-3⁺ cells (left). (b,c) Flow cytometry analyzing active caspase-3 expression and 7-AAD staining (b) and Bim expression (c) in T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) stimulated with anti-CD3, anti-CD28, and IL-2 for overnight. Numbers above bracketed lines indicate percent caspase-3⁺ or 7-AAD⁺ cells (b), and numbers above graph indicate mean fluorescence intensity (MFI) of Bim (c). (d) Flow cytometry analyzing the expression of IFN-γ and IL-17 (left), and frequency of IFN-γ⁺ cells and IL-17⁺ cells (right) in splenic T_reg cells from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice (n=5 mice per genotype). Numbers in quadrants indicate percent cells in each throughout. (e,f) T_reg cells (sorted from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) were transferred into Rag1^−/− mice. Flow cytometry analyzing CellTrace dilution (e, left) and the expression of Foxp3 (e, left) and IFN-γ and IL-17 (f, left), and frequency of Foxp3⁻ cells (e, right) (n=3 mice for Atg7^+/fl and n=4 mice for Atg7^fl/fl) and IFN-γ⁺ cells and IL-17⁺ cells (f, right) (n=6 mice for Atg7^+/fl and n=7 mice for Atg7^fl/fl) in divided CellTrace-labeled donor cells (gated on CD4⁺TCRβ⁺). Numbers adjacent to outlined areas indicate percent CellTrace^lo cells (e, left), and numbers above bracketed lines indicate percent Foxp3⁻ cells (e, left). (g) Flow cytometry analyzing YFP-Foxp3 expression in divided T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) that were activated in vitro with anti-CD3, anti-CD28 and IL-2 for 96 h. Numbers above graphs indicate MFI of YFP-Foxp3. NS, not significant (P > 0.05); * P < 0.05 and ** P < 0.001 (two-tail unpaired Student’s t-test in a,d–f). Data are pooled from two out of five (a), four out of ten (d) or two out of two (f) experiments, or representative of three (b,c), ten (e) or five (g) experiments (mean ± s.e.m in a,d–f).