Abrogation of IL-21 signaling in donor T cells maintains intrathymic ILC3s and ameliorates thymic GVHD in an IL-22–dependent fashion. (A-D) WT BALB/c recipients (H-2^d) transplanted with 5 × 10⁶ CD45.1 B6 TCD BM (H-2^b) cells and 1 × 10⁶ CD45.2 B6 T cells from either WT or Il21r^−/− B6 donors to induce GVHD. Thymus harvested on day 7 post-BMT. (A) Absolute number of intrathymic CD45⁺CD3^–CD8^–CD4⁺IL7R⁺RORγt⁺ ILC3s (n = 10 per group). (B) Absolute amount of thymic IL-22 measured by enzyme-linked immunosorbent assay (ELISA) (n = 9 per group). (C) ILC3s were isolated from thymus and incubated for 4 hours in the presence of brefeldin A and then examined for intracellular expression of IL-22 (n = 10 per group). (D) Absolute amount of thymic IL-23 measured by ELISA (n = 9 per group). (E-H) WT BALB/c recipients were transplanted with CD45.1 B6 TCD BM cells and WT or Il21r^−/− CD45.2 B6 donor T cells as above; thymus was harvested 21 days post-BMT. (E) Total thymus cellularity (n = 40-44 per group). (F) Absolute number of CD45^–EpCAM⁺MHCII⁺Ly51^hiUEA-1^lo cTECs and CD45^–EpCAM⁺MHCII⁺Ly51^loUEA-1^hi mTECs (n = 12-14 per group). (G) Proportion of thymocyte subsets by flow cytometry gated on CD45⁺ cells. (H) Absolute number of BM-derived (H-2^b+CD45.1⁺) CD4⁺CD8⁺ DP thymocytes (n = 9 per group). (I-J) B6→BALB/c BMT with WT B6 marrow, WT or Il21r^−/− B6 donor T cells, and WT or Il22^−/− BALB/c recipients (n = 10 per group). (I) Total thymus cellularity and (J) absolute number of CD4⁺CD8⁺ DP thymocytes at 21 days post-BMT. (K) Total thymic cellularity and absolute number of CD4⁺CD8⁺ DP thymocytes 21 days after B6→LP BMT (H-2^b→H-2^b) with WT B6 TCD BM and either WT or Il21r^−/− B6 T cells (n = 17 per group). Bar graphs represent mean ± SEM of at least 2 independent experiments. *P < .05; **P < .01; ***P < .001.