IL-6 classical signaling in donor T cells drives Th22- and Th17-dependent GVHD. (A-L) Lethally irradiated (1300 cGy) B6D2F1 mice received BM and T cells or TCD BM allografts from control mice (CD4Cre⁻ × IL-6R^fl) or mice with T cells deficient in IL-6 classical signaling (CD4Cre⁺ × IL-6R^fl). (B) Deletion of IL-6R on Cre⁺ recipients was confirmed by flow cytometric analysis of mIL-6R expression on peripheral blood CD4⁺ T cells before transplantation. (C) Functional confirmation was performed by stimulation of CD4⁺ T cells (in peripheral whole blood) with IL-6 (to induce classical signaling) or hyper–IL-6 (H-IL-6; to induce trans signaling) and upregulation of phosphorylated STAT3 (pSTAT3) expression measured by flow cytometry. (D) Survival indices by Kaplan-Meier analyses and weekly clinical GVHD scores of recipients after transplantation (BM and T-cell grafts, n = 12 mice per group; TCD, n = 6 mice per group; from 2 experiments). (E) Survival indices of recipient mice receiving grafts containing BM and the listed combination of sorted CD4⁺ (CD90.2⁺CD4⁺γδTCR⁻7AAD⁻) or CD8⁺ (CD90.2⁺CD4⁻γδTCR⁻7AAD⁻) T cells from CD4Cre⁻ × IL-6R^fl (CD4 wild-type [CD4.WT] or CD8.WT) or CD4Cre⁺ × IL-6R^fl mice (T-cell grafts, n = 24 mice per group; TCD grafts, n = 8 mice per group; from 4 experiments). (F-I) CD4⁺ T-cell cytokine expression was assessed at day 7 after transplantation after stimulation. (F-G) Representative contour plots (mLNs; concatenated from 5 mice per group) (F) and frequencies of IFNγ, TNF, and GM-CSF expressing in CD4⁺ T cells isolated from the spleen and mLNs (n = 10 mice per group from 2 experiments) (G). (H-I) Representative flow cytometry plots of IL-22 and IL-17A in CD4⁺ T cells isolated from the mLNs (concatenated from 5 mice per group) (H) and frequencies and total numbers of Th22 (CD4⁺IL-22⁺IL-17⁻), IL-22⁺Th17 (CD4⁺IL-22⁺IL-17⁺), and IL-22⁻Th17 (CD4⁺IL-22⁻IL-17⁺) in the spleen, liver, mLNs, and peripheral LNs (pLNs) (n = 10 mice per group from 2 experiments) (I). (J) Peripheral blood serum levels of IL-17A at day 4 and day 7 after transplantation. (K-M) Frequency and total numbers of Tbet⁺ and RORγt⁺ in CD4⁺ T cells (K), Tr1 cells (CD4⁺IFNγ⁺IL-10⁺EOMES⁺) (L),⁷³  and Tregs (CD4⁺FoxP3⁺) in the spleen at day 7 after transplantation (M) (n = 10 mice per group from 2 experiments). Data presented as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001. TBI, total-body irradiation.