(A) Flow cytometric analysis of IL-17A–producing CD45.2⁺CD4⁺ Treg cells from cLP of recipient mice as in Figure 4D, 6 weeks after transfer, followed by restimulation with PMA/ionomycin for 6 hours. (B) Flow cytometric analysis of YFP and RFP expression in CD4⁺ T cells (upper panel) or Foxp3 expression in CD4⁺RFP⁺ T cells (lower panel) in Foxp3^YFPCreR26^RFPTsc1^+/+ and Foxp3^YFPCreR26^RFPTsc1^f/f mice. (C) Immunoblot analysis of TSC1 in sorted CD4⁺RFP^–YFP^–, CD4⁺RFP⁺YFP^–, and CD4⁺RFP⁺YFP⁺ T cells from Foxp3^YFPCreR26^RFPTsc1^+/+ and Foxp3^YFPCreR26^RFPTsc1^f/f mice. (D and E) Sorted CD4⁺RFP⁺YFP⁺ Treg cells (D, right panel) or CD4⁺RFP⁺YFP^– ex-Treg cells (D, left panel), or CD4⁺CD62L⁺RFP^–YFP^– naive T cells (E) from Foxp3^YFPCreR26^RFPTsc1^+/+ and Foxp3^YFPCreR26^RFPTsc1^f/f mice were stimulated with anti-CD3/CD28 for 36 hours. Cytokine production was measured by Bio-Plex multicytokine assay. (F) Sorted CD4⁺RFP⁺YFP⁺ Treg cells were stimulated with anti-CD3/CD28, IL-2, and the indicated cytokines for 48 hours. Cytokine production was measured by Bio-Plex multicytokine assay. (G and H) Rag1^–/– mice were given sorted CD4⁺RFP⁺YFP⁺ (CD45.2⁺) Treg cells from Foxp3^YFPCreR26^RFPTsc1^+/+ or Foxp3^YFPCreR26^RFPTsc1^f/f mice, together with CD4⁺CD45RB^hi (CD45.1⁺) T cells. Flow cytometric analysis of RFP and YFP expression (G) or cytokine production (H) in CD45.2⁺CD4⁺RFP⁺ T cells of the recipient mice 6 weeks after transfer. Data are representative of (A, B, G, and H) or compiled from (D–F) two to five independent experiments. Error bars indicate the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed, unpaired Student’s t test.