Immunosuppressive effect of BRAF^V600Ein B cells in vitro. (A) OSUCLL cells with doxorubicin-induced BRAF^V600E expression were incubated 1:1 with normal human CD3 T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with antibodies to CD3 and CD28. After a 4-day incubation, proliferation of CD4⁺ and CD8⁺ T cells was determined by CFSE flow cytometry. Proliferation caused dilution of the CFSE and is evidenced by the appearance of lower-intensity peaks. Data are representative of 3 separate experiments using T cells from different donors. Statistical analysis of division indices from the 3 independent experiments are included. (B) The experiment from panel A was repeated with or without dabrafenib (2 µM). Data are representative of 3 similar experiments using T cells from different donors. Statistical analysis of division indices from 3 independent experiments are included. (C) The experiment in panel A was repeated with the different cell types in the same or in opposite chambers of 0.4 µm transwell plates. In this experiment, CD4 and CD8 T cells were seeded separately. Data are representative of 3 separate experiments using T cells from different donors. Statistical analysis of division indices from 3 independent experiments are included. Vertical bars represent mean values, and error bars represent standard error of the mean. Stim., stimulated; Unstim., unstimulated.