(A) Representative flow plots of T-bet expression in islet-infiltrating CD5^hi and CD5^lo Tregs (CD4⁺CD3⁺Foxp3⁺). Fluorescence minus 1 (FMO; minus T-bet staining) was used to set T-bet gating. (B) T-bet expression in islet-infiltrating Tregs (CD4⁺CD3⁺Foxp3⁺) and Tconvs (CD4⁺CD3⁺Foxp3^–). An average of 8 mice from 2 experiments is shown. (C) Relative expression of IFNγ by islet infiltrating CD4⁺Foxp3^– Tconvs and CD4⁺Foxp3⁺ CD5^hi or CD5^lo Tregs after 5hr ex vivo stimulation with PMA + ionomycin. An average of 11 mice from 3 experiments is shown. (D) Cell surface expression of IFNγR1 on islet-infiltrating Tregs. An average of 8 mice from 2 experiments is shown. (E–G) Analysis of inguinal draining LNs from 12-4.1 transgenic mice 12 days after immunization with InsB9-23 (INS), InsB9-23(R22E), or HEL11-25 peptide emulsified in IFA. An average of 6–7 mice from 4 experiments is shown. (E) Frequency of Tregs (CD4⁺CD3⁺Foxp3⁺). (F) Frequency of T-bet–expressing Tregs. (G) Frequency of T-bet–expressing Tconvs. (H) TCR-mediated T-bet induction in vitro. T-bet expression was induced in CD4⁺CD3⁺Foxp3⁺ Tregs sorted from the spleens of 8- to 14-week-old NOD mice by stimulating the cells for 48 hours with 1,000 U IL-2, 1 μg/ml plate-bound anti-CD28, and serial dilutions of plate-bound anti-CD3. Each culture also received either 25 ng/ml IFN (+IFNγ) or 2 μg/ml anti-IFNγ (α-IFNγ). Analysis is gated on CD4⁺CD3⁺Foxp3⁺ T cells. An average of 3 mice from 1 representative experiment is shown. Significance was determined by 1-way ANOVA with Bonferroni’s multiple-comparisons test (B–G) and 1-tailed paired t test (H). The mean ± SEM is shown. (P > 0.05), *P < 0.05, **P < 0.005, ***P < 0.0005.