TGFβR expression is required for the optimal generation of CD4⁺ and CD8⁺ iTregs during GVHD. YFP⁺ CD4⁺ and CD8⁺ naive (CD25⁻CD45RB^hi) T cells were FACS sorted from tamoxifen-treated TGFβR2^F/F/ROSA-YFP/Cre^T2 and control TGFβR2^+/+/ROSA-YFP/Cre^T2 mice. (A) The TGFβR expression level on YFP⁺ T cells from TGFβR2^F/F/ROSA-YFP/Cre^T2 (open histogram) and TGFβR2^+/+/ROSA-YFP/Cre^T2 (shaded histogram) was determined by flow cytometry. (B) The YFP⁺ Tconvs were treated with plate-bound anti-CD3 and anti-CD28 with or without IL-2 and/or TGFβ for 4 days. FoxP3 expression by CD4⁺ (top panels) and CD8⁺ (bottom panels) T cells in each of the conditions was determined by flow cytometry. (C) The sorted CD45.2⁺YFP⁺ Tconvs (H-2^b) were mixed with Thy1.1⁺ WT competitor CD25⁻CD45RB^hi 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⁺YFP⁺ and Thy1.1⁺ WT competitor CD8⁺ (A) and CD4⁺ (B) 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 = 8 mice/group). One representative of 3 independent experiments is shown.