IL-2 is required for optimal generation of CD4⁺ and CD8⁺ iTregs during GVHD. Irradiated B6D2F1 mice (H-2^bxd) were injected with splenocytes and T cell–depleted BM from CD45.1⁺ FoxP3.GFP mice (H-2^b) and injected with vehicle (PBS) or with IL-2 IC. (A) Representative plots demonstrating FoxP3 expression in donor CD4⁺ (top plots) and CD8⁺ (bottom plots) T cells from the spleen on day 8 after transplantation is shown. (B) The fraction of FoxP3⁺ cells of CD4⁺ (top graph) and CD8⁺ (bottom graph) T cells in the spleen (open bars), inguinal LNs (hatched bars), and mesenteric LNs (filled bars) was determined by flow cytometry on day 8 after transplantation. Results are expressed as means ± SEM (n = 3 mice/group). (C) Sorted CD45.2⁺CD45RB^hi Tconvs from B6 or CD25^−/− mice (H-2^b) were mixed with Thy1.1⁺ CD45RB^hi WT competitor Tconvs (H-2^b) and combined with RAG^−/− splenocytes (H-2^b) and T cell–depleted BM (H-2^b) and injected into irradiated CD45.1/CD45.2 heterozygous B6D2F1 mice (H-2^bxd). On day 8 after transplantation, FoxP3 expression by the CD45.2⁺ B6 or CD25^−/− and Thy1.1⁺ WT competitor T cells in the spleen (open bars), inguinal LNs (hatched bars), and mesenteric LNs (filled bars) was determined by flow cytometry. A Treg generation index was calculated as described in “Characterization of Treg populations during GVHD,” and the results are represented as the mean percentage of control ± SEM (n = 3 mice/group). One representative of 2 independent experiments is shown.