(A) Schematic illustration of procedures and timelines for assessing suppressive function of Tregs in Rag^–/– mice receiving porcine NICC transplantation. DEREG mice were given CTLA4-Fc/MR1 described in Figure 1A. On ≥100 days after transplant, the CD4⁺GFP⁺ Tregs (tolerant Tregs) and CD127^hi Tregs from tolerant mice, as well as control CD4⁺GFP⁺ Tregs from naive mice (naive Tregs), were sorted and collected. Rag^–/– mice that received porcine NICC grafts were adoptively transferred with the tolerant Tregs, CD127^hi Tregs, or naive Tregs at day 22 after transplant. The 3 Treg adoptive transfer Rag^–/– mice groups and a Rag^–/– group that did not receive Tregs (CD4⁺ T cells only) were challenged with Foxp3^– CD4⁺GFP^– T cells from Ly5.1Foxp3^GFP mice on day 45 after transplant at 1:3 ratio (Treg/CD4⁺ T cell). The samples of these 4 groups and Rag^–/– mice with grafts and without transferring/challenging cells (transplant only) ≥120 days after transplant were collected. (B) Representative insulin-stained IHC images of porcine NICC grafts (brown as positive insulin staining) in Rag^–/– mice: 5 experimental groups. Scale bar: 150 μm. (C) Serum porcine C-peptide measurement for Rag^–/– mice experimental groups, including transplant only (n = 11), CD4⁺ T cells only (n = 12), naive Tregs (n = 7), tolerant Tregs (n = 7), and CD127^hi Tregs (n = 6). (D) Real-time RT-PCR performing measurement of Il-10, Tgf-β, Ebi3, and Blimp1 on sorted splenic CD127^hiCD4⁺GFP⁺Foxp3⁺ Tregs from tolerant mice day 100 after transplantation (CD127^hi Tregs) (n = 4), sorted splenic CD4⁺GFP^– T cells (CD4⁺ T cells) (n = 6), and CD4⁺GFP⁺Foxp3⁺ Tregs from naive mice (naive Tregs) (n = 6). Kruskal-Wallis test was used for the comparisons of serum porcine-C-peptide in C. A 1-way ANOVA followed by Tukey’s multiple comparison was used in D. Data were collected from 5 independent experiments and shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.