(A) Illustration of the Treg suppression assay performed by coculturing naive CTV-labeled CD4⁺ Tconv cells with Tregs from B6 FOXP3-GFP mice treated with control IgG or mIL-2 (0.5 mg/kg). (B) Percentages of proliferating CD4⁺ Tconv cells after coculturing with different ratios of Tregs (n = 3/group; representative data are from 3 independent experiments). (C) Illustration of the experimental design to assess the functional surface markers and inhibitory cytokines of splenic Tregs in which B6 FOXP3-GFP mice were treated with control IgG or mIL-2 at 0.5 mg/kg subcutaneously on days 0 and 3, and splenocytes were harvested on day 4. Flow cytometric analyses of (D) total Tregs, (E) CTLA-4⁺, (F) ICOS⁺, and (G) LAG3⁺ Tregs. Flow cytometric analyses of (H) IL-10⁺ and (I) LAP⁺ (TGF-β⁺) Tregs after in vitro stimulation with a PMA/ionomycin/brefeldin cocktail for 6 hours (n = 4/group, data are representative of 2 independent experiments). (J) Illustration of B6.mOVA to WT B6 skin transplantation, in which the recipient mice were treated with subcutaneous control IgG or mIL-2 (0.5 mg/kg) with or without anti–CTLA-4 antibodies (combination of 2 antibodies from the clones of UC10-4F10-11 and 9H10; 200 μg/mouse for each clone, intraperitoneally) twice a week starting on day 0 (n = 3–12/group, data were pooled from 4 different experiments). (K) Kaplan-Meier curves of the graft survivals. (L) Flow cytometric analysis of peripheral blood Tregs from the recipient mice. (M) Plasma anti-OVA antibody levels as determined by ELISA on days 0 and 21 (n = 3–4/group). Graphs show the mean ± SD. A 2-tailed t test was used for 2-group comparisons (B and D–I) and 1-way (M) and 2-way (L) ANOVA with Tukey’s multiple-comparison test for 3 or more group comparisons as appropriate. The long-rank test was used for graft survival comparisons (K). CTV, CellTrace Violet.