CD8⁺ Tregs represent a large fraction of iTregs that are generated in the recipient during GVHD. (A) GFP⁺ Tregs (CD4⁺ and CD8⁺) were sorted from CD45.1⁺ FoxP3.GFP mice (H-2^b) and mixed with GFP⁻ Tconvs (CD4⁺ and CD8⁺) sorted from CD45.2⁺ FoxP3.GFP mice (H-2^b). The T cells were 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). (A) Representative plot examining CD45.1 positivity of donor-derived GFP⁺ Tregs (host cells are also gated out by CD45.1/CD45.2 double positivity) is shown for CD4⁺ (top panels) and CD8⁺ (bottom panels) Tregs in the spleen on day 8 after transplantation. (B) The percentage of CD4⁺ and CD8⁺ Tregs derived from Tconvs (CD45.1⁻) was determined in the spleen and inguinal and mesenteric LNs of mice (n = 3) on day 8 after transplantation. Results are expressed as mean ± SEM. (C) Irradiated CD45.2⁺ B6D2F1 (H-2^bxd) mice were injected with splenocytes and T cell–depleted BM from CD45.1⁺ FoxP3.GFP (H-2^b) mice. On day 8 after transplantation, CD45.1⁺ CD4⁺ T cells (right plot) and CD8⁺ T cells (left plot) were analyzed for expression of Helios. (D) GFP⁻ Tconvs (CD4⁺ and CD8⁺) were sorted from CD45.1⁺ FoxP3.GFP (H-2^b) mice, combined with RAG^−/− splenocytes (H-2^b) and T cell–depleted BM (H-2^b), and injected into irradiated B6D2F1 mice (H-2^bxd). A representative plot examining donor T cells (CD45.1⁺) is shown for CD8⁺ (left plot) and CD4⁺ (middle plot) T cells in the spleen on day 8 after transplantation. The right plot was gated on all GFP⁺ cells and shows the relative contribution of CD8⁺ and CD4⁺ Tregs to the iTreg pool. One representative of 3 independent experiments is shown. (E) CD45.1⁺ GFP⁻ CD4⁺ and CD8⁺ Tconvs were sorted from FoxP3.GFP (CD45.1⁺):WT and Scurfy (CD45.1⁺):WT mixed BM chimeras. 0.6 × 10⁶ CD4⁺ Tconvs (WT GFP⁻ or Scurfy) were mixed with 0.4 × 10⁶ CD8⁺ Tconvs (WT GFP⁻ or Scurfy) and used as effectors for GVHD induction in irradiated B6D2F1 mice. Survival was monitored over an 8-week period. Two independent experiments were combined with a total of n = 9 or 10 mice/group to generate the survival curve. *P < .05 by log-rank test.