Deletion of IL-21 signaling increases the number of donor graft–derived Tregs in the thymus. (A-E) WT BALB/c (H-2^d) recipients were transplanted with 5 × 10⁶ TCD BM cells from CD45.1⁺ B6 (H-2^b) mice and 1 × 10⁶ T cells derived from either WT (n = 10 recipients) or Il21r^−/− (n = 10 recipients) CD45.2⁺ B6 mice to induce GVHD. (A) Concatenated flow cytometry plots showing transplanted donor graft–derived T cells on day 7 post-BMT. (B) Proportion of transplanted donor T cells of all thymic T cells on day 7 post-BMT. (C) Absolute number of transplanted donor graft–derived T cells in the thymus on day 7 post-BMT. (D) Absolute number of total CD3⁺CD8^–CD25⁺CD4⁺FoxP3⁺ cells in the thymus on day 21 post-BMT (n = 9 per group). (E) Proportion of host-derived, graft T-cell–derived, and marrow-derived Tregs of all Tregs in the thymus on day 21 post-BMT, showing transplant with WT T cells in the left column and with IL-21R knockout (KO) T cells in right column; also shown are representative flow cytometry plots of Tregs derived from the donor marrow or from the T cells present in the donor graft. (F) Absolute number of donor BM–derived (H-2^b+CD45.1⁺) or donor T-cell–derived (H-2^b+CD45.2⁺) Tregs 21 days post-BMT (n = 9 per group). Dot plots concatenated from 5 independent observations from 1 experiment and gated on donor-derived (H-2^b+) Tregs. Bar graphs represent mean ± SEM, combined from at least 2 independent experiments. *P < .05; **P < .01; ***P < .001.