Intrathymic ILCs are eliminated and their production of IL-22 is reduced in mice with GVHD. (A-G) BALB/c recipients (H-2^d) were transplanted with 5 × 10⁶ TCD BM cells with or without 1 × 10⁶ T cells from B6 donors (H-2^b) to induce GVHD and were analyzed on day 7 after transplant (n = 7-10 mice per group). (A) Total number and (B) frequency of cTECs, mTECs, and DP thymocytes. (C) Back-gating analysis of thymic IL-22⁺ cells after TCD allo-BMT; thymic harvest and incubation in vitro with brefeldin A for 5 hours, followed by intracellular and surface antibody staining. Representative flow cytometry plots were concatenated from 5 independent observations with isotype antibody (RORγt, IL-7Rα) or CD45^– (CD4, CCR6, CD8, CD3, NKp46) negative controls or whole thymus positive controls (CD45). (D) Total number of intrathymic CD45⁺CD3^–CD8^–CD4⁺IL7R⁺RORγt⁺ ILC3s in BMT recipients or normal untransplanted controls. (E-F) Thymocytes were harvested and incubated for 5 hours with brefeldin A. Intracellular IL-22 was assessed by flow cytometry, demonstrating the frequency (E) and absolute number (F) of IL-22⁺ ILC3s in BMT recipients or normal untransplanted controls. (G) Absolute levels of intrathymic IL-23 on day 7 after BMT in BALB/c recipients or normal untransplanted controls. (H) B6 (CD45.2) recipients were transplanted with 10 000 Lineage^–Sca1⁺ckit⁺ BM cells from congenic CD45.1⁺ donors, and BM, spleen, and thymus were analyzed on day 4 after transplant for ILC3 chimerism and absolute numbers of donor and host ILC3s (n = 5-15 mice per group). (I) Absolute number of host-derived (CD45.2⁺H-2^d+) and donor BM–derived (CD45.1⁺H-2^b+) thymic ILCs 7 days after B6→BALB/c transplant (performed as in panels A-G), gated on CD45⁺CD3^–CD8^–CD4⁺ cells (n = 9-10 per group). Bar graphs represent mean ± SEM of at least 2 independent experiments.*P < .05; **P < .01; ***P < .001. FSC, forward scatter; ns, not significant.