Figure 4. Combined DAS + VAC therapy promotes a broader therapeutic CD8⁺ T cell repertoire in the tumor-draining lymph node and the tumor microenvironment. M05 melanoma bearing mice were left untreated or were treated (starting on d10 post tumor cell s.c. injection) with either 0.1 mg/day dasatinib (DAS) orally administered for 7 consecutive days, or contralateral s.c. vaccination (VAC) consisting of 10⁶ OVA_257–264 peptide-pulsed dendritic cells (DCs) genetically modified to overexpress IL-12, or a combination of DAS + VAC. Tumors and tumor-draining lymph nodes (TDLN) were harvested on day 34 and CD8⁺ T cells were isolated via anti-CD8 antibody labeling and magnetic beads. CD8+ effector cells were stimulated for 48h with M05 (OVA⁺) melanoma cells, B16 (OVA^neg) melanoma cells, EL4 thymoma control cells or EL4 cells pre-pulsed (for 4h at 37 °C) with CD8⁺ T cell peptide epitopes derived from other tumor-associated antigens (at an effector-to-target cell ratio of 10:1) as indicated. Cell-free supernatants were then recovered from each culture well and analyzed for IFNγ content by specific ELISA. Data are reported as the mean ± SD from triplicate ELISA determinations. Representative data from 1 of 3 independent experiments is depicted, Statistical analyses were performed by Student’s t test or 1-way ANOVA; *P < 0.05 vs. untreated (t test), **P < 0.05 vs. all other groups (ANOVA).