Cytokine release and CTLA-4 expression of expanded allo-specific Tregs. (A) Cytoplasmic staining of cytokines produced by expanded allo-specific CFSE⁻ Tregs and non-Tregs. Flow cytometric contour plots show the relative number of cytokine-producing CD4⁺ T cells. (B) Cytokine secretion of expanded allo-specific Tregs. Supernatants were removed after incubation of expanded Tregs and non-Tregs with PMA and ionomycin in serum-free medium. Each bar represent mean ± SD of cytokine levels of 4 individual experiments. (C) TGF-β/IFN-γ cytokine ratio of Tregs and non-Tregs. *P < .05; **P < .01.