Dasatinib inhibits the effects of CD40 ligation but does not restrict the expression of accessory molecules on CLL cells. (A) PBMCs from patients with CLL were first cultured on a layer of CD40L expressing fibroblasts with or without dasatinib (1 μM/mL), imatinib (1 μM/mL), ibrutinib (1 μM/mL), or Bay 11-7082 (0.1 μM/mL) for 2 days, after which the CLL cells were thoroughly washed to remove the inhibitors. The untreated and treated CLL cells were resuspended in fresh media and cultured in a 1:1 ratio with autologous PBMCs for 2 days with soluble stimulatory CD3/CD28 antibodies after which expression of CD25 was measured on T cells (HD, n = 4; CLL, n = 7). In a similar experiment, expression of (B) MHC-I, MHC-II, CD54, CD58, CD70, CD80, CD86, 4-1BBL, OX40L, and CD95 were measured on CLL cells after incubation with 1 μM dasatinib for 2 days using the 3T40 system (CLL, n = 8). (C) Before culturing CD40-stimulated CLL cells with autologous T cells, CLL cells were preincubated for 1 hour with CD54 and CD58 blocking antibodies after which T cells were stimulated for 2 days and analyzed for expression of CD25 (HD, n = 4; CLL, n = 2-3). P values were calculated using a 1-way ANOVA for panels A-C. Data are presented as mean ± SEM; ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. MFI, mean fluorescence intensity; Stim, stimulated.