CD8⁺FoxP3⁺ differentiate after allogeneic transplantation from CD8⁺FoxP3⁻ in the mLN. (A) After lethal irradiation at day −1, BM and T cells from B6.FoxP3-GFP donors were transplanted into B6D2F1, BALB/c, C3H.SW, or C57Bl6 recipients. At day 12 after transplantation, CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ were analyzed in the spleen, pLN, and mLN. Representative plots of total live cells in the pLN are shown. Experiment was performed 3 times (n = 10, 7, 7, 6). (B) After lethal irradiation at day −1, BM and T cells from B6.FoxP3-GFP donors were transplanted into B6D2F1 recipients. Spleens were stained for intracellular FoxP3 at day 5 after transplantation. (C) After lethal irradiation at day −1, BM and T cells were transplanted from PTPrc^a donors at day 0. Sorted cell populations (CD3⁺, CD4⁺FoxP3⁻, CD4⁺FoxP3⁺, or CD8⁺FoxP3⁻) from B6.FoxP3-GFP donors were also transplanted with the donor graft. At day 12 after transplantation, CD8⁺FoxP3⁺ conversion was analyzed in the spleen and pLN. Representative plots of total live cells from peripheral LN are shown from 2 experiments; n = 6 per group. (D) Recipients were transplanted and analyzed as in panel C; however, CD8⁺FoxP3⁻ and CD4⁺FoxP3⁻ were matched to that within the whole CD3⁺ graft. Experiment was performed twice with results displayed as mean ± SEM; n = 6 per group. (E) At day 0, BM and T cells from B6.FoxP3-GFP donors were transplanted into lethally irradiated B6D2F1 recipients. At time points shown, spleen, and mLN were analyzed for CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺cells. Combined data from 2 experiments; n = 6 per group. (F) After lethal irradiation on day −1, recipients were transplanted with the following grafts: (1) WT CD4⁺ T cells and WT CD8⁺ T cells, (2) WT CD4⁺ T cells and B6.FoxP3-luc⁺ CD8⁺ T cells, (3) B6.FoxP3-luc⁺ CD4⁺ T cells and WT CD8⁺ T cells, or (4) B6.FoxP3-luc⁺ CD4⁺ T cells and B6.FoxP3-luc⁺ CD8⁺ T cells. Recipients were culled at day 4 after transplantation, 5 minutes after luciferin injection, and the spleen, inguinal LN, liver, thymus, lung, and gut removed and imaged individually. Representative images from 2 experiments are shown (n = 6 per group). (G) Mice were transplanted as in panel E; however, donor T cells were labeled with Cell Trace violet proliferation dye and mLN analyzed for CD8 T_reg conversion. Representative plots gated on live donor CD8⁺ T cells are shown, with numbers in graph showing mean ± SEM.