Differential effects of dasatinib on T cells and DCs. (A) A robust in vitro priming system for assessment of dasatinib-mediated effects. The protocol has been validated previously²²  and is described in the “Methods” section. (B) The direct effects of dasatinib were assessed by adding a single dose of dasatinib (50 nM) at various time points after the initial coincubation of T cells and DCs. On day 10, individual wells were evaluated for MHC-multimer⁺ CD8⁺ T cells (left); this fraction was multiplied by the total number of cells per well to obtain the absolute number of antigen-specific T cells per well (right; 7 parallel wells per group, bars represent the mean with standard deviation). (C) T-cell priming with dasatinib-treated (50 nM) DCs (day −1) and addition of dasatinib (50 nM) at the time of priming resulted in complete blockade of antigen-specific T-cell proliferation. (D) T-cell priming with dasatinib-treated (50 nM) DCs (day −1), without addition to the T-cell culture, increased the percentage of MHC-multimer⁺ cells compared with matured DCs without dasatinib pretreatment. Before coincubation with T cells, the DCs were extensively washed to remove residual dasatinib. (E) Comparison of dasatinib-treated DCs and DCs matured with LPS/IFNγ only. Differences in the percentage of MHC-multimer⁺ cells and the absolute yield of antigen-specific T cells were highly significant (Student t test) on days 7 and 10 of culture (evaluation of 7 parallel wells, technical replicates). (F) Primed T cells (gated on viable CD8⁺ MHC-multimer⁺ cells) expressed higher levels of CD28 and CD62L on day 10 of culture, when naïve T cells had been primed with dasatinib-pretreated DCs as opposed to DC matured without dasatinib. Each of the results is representative of at least 3 separate experiments.