Figure 6. Autophagy protects T_reg cell stability by restraining mTORC1-dependent c-Myc expression and function.

(a) Flow cytometry analyzing c-Myc in T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) activated with anti-CD3 and anti-CD28 for 4 h. Numbers above graphs indicate MFI of c-Myc. (b) Immunoblot analysis of c-Myc in resting, and anti-CD3 and anti-CD28 stimulated T_reg cells from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice. (c,d) Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice received mock or rapamycin treatment. T_reg cells were isolated and stimulated with anti-CD3 and anti-CD28 for 4 h for analysis of c-Myc expression (c). Numbers above bracketed lines indicate percent c-Myc⁺ cells (c). Heat maps of Myc target gene expression in non-treated Foxp3^CreAtg7^fl/fl versus Foxp3^CreAtg7^+/fl T_reg cells, and rapamycin-treated Foxp3^CreAtg7^fl/fl versus Foxp3^CreAtg7^+/fl T_reg cells (d). Red color denotes upregulated genes in Foxp3^CreAtg7^fl/fl T_reg cells, and blue color denotes downregulated genes in Foxp3^CreAtg7^fl/fl T_reg cells (d). (e) Measurement of ECAR in T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) stimulated with anti-CD3 and anti-CD28 for 4 h in the presence of DMSO or JQ-1. (f) 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 and anti-CD28, and IL-2 for 96 h in the presence of DMSO or JQ-1. Numbers above graphs indicate MFI of YFP-Foxp3. NS, not significant (P > 0.05); * P < 0.05 and ** P < 0.001 (one-way ANOVA in e). Data are representative of four (a,f), three (b,c) or two (e) experiments, or from one (d) experiments (mean ± s.e.m in e).