T-cell proliferation and cytokine production induced by TCR/CD3 engagement plus CD28 costimulation is potently inhibited by dasatinib. PBMCs were loaded with CFSE and then stimulated with anti-CD3/CD28 beads in the presence or absence of 10 nM dasatinib. (A) Microscopic analysis of PBMC cultures at 72 hours. (B) CFSE staining was analyzed by flow cytometry at 120 hours. Numbers on plots are the percentage of proliferating T cells. (C) Cytokine production was analyzed in supernatants at 120 hours. (D) Peripheral blood T cells were stimulated with plate-bound anti-CD3 plus soluble anti-CD28 antibody for 3 days in the presence of various concentrations of dasatinib or cyclosporin A, or in the presence of DMSO vehicle alone. T-cell proliferation was measured by [³H] thymidine incorporation. Assays were performed in triplicate, and the percentage inhibition relative to controls treated with DMSO vehicle was calculated.