IL-6 trans signaling regulates Th17 and Th22 differentiation. (A-J) Donor BALB/c CD4⁺ T-cell cytokine expression was assessed at day 7 after transplantation in recipient B6 wild-type (B6.WT) and B6.sgp130Fc (IL-6 trans signaling inhibited) mice. (A) High-dimensional analysis of cytokine expression from donor CD4⁺ T cells isolated from the mLNs using t-distributed stochastic neighbor embedding (6 parameters: IFNγ, TNF, GM-CSF, IL-10, IL-17A, and IL-22; concatenated from 1 experiment, where n = 6 mice per group). (B) Representative contour plots of IL-22 and IL-17A expression in CD4⁺ T cells and IFNγ, IL-22, and IL-10 in IL-17A⁻ CD4⁺ T cells from the mLNs (concatenated from 4-5 mice per group). (C) Proportion and total numbers of Th17 cells (CD4⁺IL-17A⁺) either not expressing IL-22 (IL-22⁻Th17) or expressing IL-22 (IL-22⁺Th17) and Th22 cells (CD4⁺IL-17A⁻IL-22⁺) in the spleen, liver, mLNs, and peripheral LNs (pLNs) of B6.WT and sgp130Fc mice (n = 10-22 mice per group from 2-4 separate experiments). (D-E) Frequency and total numbers of Th1 (CD4⁺IFNγ⁺IL-17A⁻IL-22⁻) and Th22 (CD4⁺IL-17A⁻IL-22⁺) cells expressing IFNγ (D) and GM-CSF (E) as a proportion of total CD4⁺ T cells (n = 10-11 mice per group from 2-3 separate experiments). (F) Frequency and total number of CD4⁺ T cells expressing IL-10 in multiple organs (10-22 mice per group from 2-4 separate experiments). (G) Percentage and total number of type-1 T regulatory cells (Tregs) (Tr1, CD4⁺IFNγ⁺IL-10⁺) and FoxP3⁺ Tregs expressing IL-10 (CD4⁺FoxP3⁺IL-10⁺) in the spleen and FoxP3⁺ Tregs (CD4⁺FoxP3⁺) as a percentage of total CD4⁺ T cells in the spleen (n = 6-13 mice per group from 1-2 experiments). (H) Peripheral blood serum cytokine levels of IL-6, IL-17A, IL-17F, IFNγ, TNF, and GM-CSF at day 4 and day 7 after transplantation (n = 16-28 mice per group from 4 experiments). Data presented as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001.