T-cell proliferation induced by TCR engagement, but not IL-2, is markedly inhibited by dasatinib. (A) PBMCs were loaded with CFSE and then stimulated with OKT3 (100 ng/mL; top panels) or PHA (0.5%; bottom panels) in the presence or absence of dasatinib (10 nM). After 5 days, T lymphocytes were analyzed by flow cytometry for proliferation based on CFSE dilution using WinMDI 2.8 and ModFit LT. OKT3 or PHA stimulation alone is shown by the gray-shaded area, whereas OKT3 or PHA in the presence of dasatinib is indicated by the red line, which overlapped the untreated control. (B) PBT expanded in IL-2 for 7 days were rested overnight, loaded with CFSE, and then stimulated with IL-2 or IL-2 + OKT3 in the presence or absence of dasatinib (10 nM). After 5 days, T lymphocytes were analyzed by flow cytometry for proliferation. Gray, unstimulated; blue, IL-2 alone; green, IL-2 + OKT3; red, IL-2 + OKT3 + dasatinib. (C) PBMCs were loaded with CFSE and then stimulated with OKT3 (100 ng/mL). Dasatinib (10 nM) was added at time zero (ii), after 24 hours (iii), after 48 hours (iv), or after 72 hours (v) before analysis at 120 hours. The percentage of proliferating T cells is indicated in each panel. (D) PBMCs were stimulated with PHA or CD3/CD28 beads for 72 hours and then stained with propidium iodide. DNA content was then measured by flow cytometry.