Up-regulation of CD8 coreceptors after in vitro culture restores the capacity of T cells to recognize B16 cells expressing high levels of noncognate MHC-I. Tissue-culture CD8 T cells were produced by placing the purified CD8 T cells from Trp1₄₅₅TriVax-immunized mice in medium containing 50 U/mL IL-2 and 20 ng/mL IL-7 for 7 days. (A) Comparison of the levels of CD8α expression between freshly isolated and cultured CD8 T cells from Trp1₄₅₅TriVax-immunized mice compared with naive CD8 T cells from nonvaccinated mice. MFI, mean fluorescence intensity of CD8α. Right panel shows histograms gating on the Trp1₄₅₅ tetramer-positive populations. (B) Antigen-induced IFNγ production of cultured and freshly isolated CD8 T cells from Trp1₄₅₅TriVax-immunized mice evaluated by ELISA. CD8 T cells were evaluated for their capacity to recognize B16 treated or not with IFNγ (100 U/mL, 24 hours) and B16-scKb^Ova. CD8 T cells (3 × 10⁵) were incubated with tumor cells (1 × 10⁵) for 40 hours, and supernatants were removed for cytokine measurements. Supernatants from T cells without tumor cells (T cell alone) were included as controls. Results represent the average amounts of IFNγ and SD (error bars) from triplicate cultures. These experiments were repeated twice with similar results.