CD4⁺and CD8⁺T cells isolated from aGVHD and clGVHD skin lesions differ in their cytokine profiles. Cells isolated from collagenase-digested skin biopsies of aGVHD (n = 3) or clGVHD (n = 2) patients as well as of HCs (n = 5) were stimulated for 4 hours with phorbolmyristate acetate/ionomycin. Samples that had been incubated with brefeldin A only (data not shown) served as negative controls. Thereafter, cells were stained for CD3, CD4, CD8, IFN-γ, IL-4, IL-17, and IL-22 and analyzed with 7-color flow cytometry; gates were set based on isotype-matched controls. (A) FACS gating strategy. (B) CD4⁺ T cells in aGVHD skin produce higher amounts of IL-22 than in clGVHD. Data are given as percentages ± SD of CD4⁺ and CD8⁺ T cells that stained positive for IL-22, IL-17/IL-22, IL17, IL-4, or IFN-ү. (C) Representative FACS plots of cytokine expression (IFN-γ, IL-4, IL-17, and IL-22) detected in CD4⁺ and CD8⁺ T cells by intracellular flow cytometry. *P < .05, **P < .01.