Qualitative effects of dasatinib-treated DCs on T-cell priming (day 10 of culture). (A) Dasatinib does not improve the priming capacity of immature DCs. Shown are T-cell responses with differentially treated immature DCs. (B) Effects after dasatinib treatment are not the result of contaminating bystander cells. Immature DCs were harvested, stained with APC-labeled anti-CD11c-antibodies, and purified with anti-APC-beads (>95% purity) and only then matured. (C) Dasatinib-associated effects are mediated in trans. Melan-A peptide–pulsed DCs were either matured with LPS/IFNγ alone or coincubated with dasatinib. Melan-A-pulsed, LPS/IFNγ–matured DCs were mixed with LPS/IFNγ–matured DCs pulsed with a mock peptide (STEAP1(292.2L)) or with dasatinib/LPS/IFNγ–matured DCs pulsed with the mock peptide at a 1:1 ratio. (D) T cells primed with dasatinib-pretreated DCs have higher functional avidity. Responses to titrated peptide onto CD14⁺ monocytes (IFNγ⁺ response normalized for MHC-multimer⁺ cells). (E) Avidity testing using Melan-A+ melanoma cells (FM55, HLA-A2+, Melan-A+) at a 2:1 Effector:Target ratio (5-hour stimulation). Zebra plots show the MHC-multimer staining of the unstimulated cell lines. Pseudocolor plots show the stimulated cells gated on live, CD8⁺ T cells (in brackets: IFNγ⁺TNFα⁺ T cells normalized for the MHC-multimer⁺cells). First row: T cells primed with DCs matured with LPS/IFNγ (left) vs DCs matured with dasatinib/LPS/IFNγ. Middle row: Identical maturation conditions plus 5 μg/mL of neutralizing IL-12 antibody at the time of priming. Last row: DCs matured with IL1β, TNFα, and PgE₂ in the absence or presence of dasatinib were used for priming. Results are representative for at least 3 independent experiments.