CD8⁺ Tregs contribute significantly to the total Treg pool during GVHD. Irradiated CD45.2⁺ B6D2F1 mice (H-2^bxd) were injected with splenocytes and T cell–depleted BM from CD45.1⁺ FoxP3.GFP mice (H-2^b). (A) Representative FACS plots of splenocytes harvested on day 8 after BMT are shown. The plots are gated on donor (CD45.1⁺) CD4⁺ (left plot), CD8⁺ (middle plot), and FoxP3⁺ T cells (right plot). The numbers in the plots represent the fraction of cells within that quadrant. (B) The average contribution of CD8⁺FoxP3⁺ cells to the total FoxP3⁺ pool on day 8 after BMT (n = 3) was compared with naive unirradiated untransplanted B6 mice (n = 3). (C) The fraction of donor T cells that were FoxP3⁺ of CD4⁺ (left) or CD8⁺ (middle) and the fraction of donor CD8⁺ of total donor FoxP3⁺ T cells (right) were analyzed at various times after transplantation. One representative of 3 independent experiments is shown.