Migratory CD103⁺ DC1 from either LR or HR recipients were sorted as above, synchronized by starvation, and then cocultured with CD4⁺CD25^– T cells harvested from BABL/C naive mice. (A) On day 5, cells were harvested, and expression of FoxP3 in CD4⁺T cells was analyzed by flow cytometry. (B) iDC1 were treated with or without IFN-γ and were then synchronized by starvation and cultured with CD4⁺CD25^– T cells in SFM supplemented with latent-TGF-β1, IL-2, and with or without anti-αvβ8 blocking antibody. (B and C) On day 5, cells were harvested and the expression of FoxP3 in CD4⁺T cells was analyzed (B), and soluble active TGF-β1 in the supernatant was assessed (C). (D) LR graft recipient mice were treated with ADWA-11 antibody or IgG-control on the day of the transplantation and once a week. (E and F) Two weeks after transplantation, DLN CD4⁺ T cell intracellular expression of IFN-γ (E) and FoxP3 (F) was analyzed. (G) Additional mice were followed (n = 8/group) for 8 weeks through slit lamp examination to evaluate graft opacity, and graft survival was evaluated. (H) Syngeneic naive CD4⁺CD25^– T cells were transferred i.v. at the time of the transplantation into HR Rag^–/– recipient mice. Mice were treated with ADWA-11 antibody or IgG control on the day of the transplant and once a week. (I and J) On day 21 after transplantation, DLN were harvested, and expression of FoxP3 in CD4⁺ T cells (I) and their suppressive capacity in proliferation CFSE-labeled CD4⁺ T cell (J) was analyzed by flow cytometry. Tregs from the DLN of mice treated with IgG control were sorted by FACS and adopted into newly transplanted HR recipient mice, and control mice received PBS (n = 8/group). (K) Mice were followed for 8 weeks for evaluating graft opacity and graft survival. Results (B and C) are of 2 sets of triplicates. Plots represent the mean (± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (2-tailed t test [E, F, and J] or 1-way ANOVA [A, B, C, and I] with Bonferroni correction).