Host DCs and TGF-β are required for CD8 T_reg conversion. (A) T cells from B6.FoxP3-GFP donors were labeled with Cell Trace violet dye and transplanted with BM into lethally irradiated B6.CD11c-DOGxDBA2.F1 recipients or B6D2F1 recipients. Recipients were treated with either DT or saline to deplete host DCs (160 ng/d, day −2 → day 3). At day 4 after transplantation, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ conversion and proliferation were analyzed in the spleen and mLN. Representative plots of the mLN are shown, with numbers quantitated and displayed as mean ± SEM. ** P < .01. Combined data from 2 experiments; n = 6 per group. (B) BM and T cells (either CD8⁺ only or CD4⁺ + CD8⁺) from B6.FoxP3-GFP doors were transplanted into lethally irradiated bm1 recipients. At day 12 after transplantation, CD8⁺FoxP3⁺ T_reg cells were analyzed in the spleen and mLN. Representative plots gated on total live cells are shown with combined data represented in the bar graphs displaying mean ± SEM; n = 3 per group. (C) B6D2F1 recipients received either irradiation at day −1 or NK1.1 at day −2 and splenocytes from B6.FoxP3-GFP donors at day 0. At day 5 after transplantation, CD8⁺FoxP3⁺ populations were analyzed in the spleen and mLN. Representative plots gated on total live cells are shown, and percent contribution displayed as mean ± SEM with 3 per group. (D) Lethally irradiated B6D2F1 recipients were transplanted with BM and T cells from B6.FoxP3-GFP donors. Recipients were treated with antibodies against either TGF-β or IL-10R. At day 4 after transplantation, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ conversion and proliferation were analyzed in the spleen and mLN. Representative plots gated on CD8⁺ T cells in the mLN are shown, with numbers quantitated and displayed as mean ± SEM. Combined data from 2 experiments; n = 6 per group. **P < .01, control antibody versus anti–TGF-β