Skin affected by cutaneous GVHD is enriched in Th1 but not Th17 cells. Punch biopsies from skin affected by aGVHD collected at the time of diagnosis (n = 7), psoriasis (n = 3), and healthy controls (n = 10) were cultured on 3-dimensional matrices in the presence of T-cell growth factors. After 3 weeks, suspension cells were restimulated with phorbol myristate acetate and ionomycin and gated CD4⁺ T cells were analyzed for the proportion of (A) IL-17 (GVHD⁺, 0.44% ± 0.18%; psoriasis, 24.6% ± 9.46%; healthy donors, 12.24% ± 1.60%), (B) IL-22 (GHVD⁺, 2.72% ± 0.61% psoriasis 24.05% ± 1.74%; healthy donors, 22.4% ± 3.82%), and (F) IFN-γ–producing cells (GVHD⁺, 57.36% ± 10.75%; healthy donors, 21.06% ± 4.63%). Unstimulated samples were analyzed for expression of (C) CCR4 (GVHD⁺, 14.88% ± 5.44%; healthy donors, 61.80% ± 6.73%) and (D) CCR6 (GVHD⁺, 3.4% ± 1.46%; healthy donors, 71.97% ± 8.87%). (G) Expression of cutaneous lymphocyte-associated antigen on circulating CD4⁺ T cells was measured in patients with cutaneous GVHD (14.69% ± 2.99%, n = 8) and compared with time-matched transplanted patients without GVHD (6.62% ± 1.43%, n = 9). (E) CD3 staining was used to identify T-cell infiltrates, and IL-17/IL-22 staining was used to identify cytokine expression patterns in the skin of patients with aGVHD (n = 6) and psoriasis (n = 1). The sections were examined using an Olympus BX61 microscope equipped with a 20×/0.75 objective lens and images were taken with an Olympus DP71 digital camera and acquired with DP controller 3.1.1.267 Olympus software. *P < .05. **P < .0001. Error bars represent SEM.