Autoimmunity is characterized by T_H1- and T_H17-mediated proinflammatory cytokine production that is suppressed by CD4⁺CD25⁺Foxp3⁺ T cells. Lethally irradiated Balb/c mice received transplants of TCD BM (10 × 10⁶) plus 3 × 10⁵ B6 spleen cells. At 20 days after transplantation, spleen cells (adjusted to yield a T-cell dose of 0.5 × 10⁶ cells) were transplanted into B6 Rag mice alone (n = 7) or together with 0.5 × 10⁶ purified CD4⁺CD25⁺ T cells from B6.PL animals (n = 7). (A) B6 Rag mice that received GVHD spleen cells alone or GVHD spleen cells plus Tregs were bled 42 to 83 days after BMT, and serum was analyzed for presence of IL-1β, TNF-α, G-CSF, IL-6, IFN-γ, and IL-17. Data are presented as the mean plus or minus SEM and are cumulative results from 2 experiments. IFN-γ levels were undetectable in 4 of 6 mice that received Tregs. (B) CD4⁺ T cells were purified from the spleens of B6 Rag mice that received GVHD spleen cells only (■) or together with Tregs (□) 42 to 83 days after transplantation. Cells were then restimulated with PMA and ionomycin in the presence of GolgiStop and then intracellularly stained with IFN-γ, IL-17, or IL-4–specific antibodies. Dot plots show the percentage of CD4⁺ T cells that stained positive for each of the cytokines. (C,D) The average absolute number of IFN-γ and IL-17–positive CD4⁺ T cells from pooled spleen cell suspensions obtained from mice (n = 3/group) that received transplants of primary GVHD spleen cells alone (■) or together with an equivalent number of Tregs (□). Data are from 1 of 2 experiments that yielded similar results. (E) Lymphocytes were isolated from the colon lamina propria or livers of B6 Rag mice (n = 5) 72 days after transplantation with primary GVHD spleen cells. Dot plots show the percentage of CD4⁺ T cells that were positive for IL-17 and/or IFN-γ. Data are from 1 of 2 experiments that yielded similar results.