Expression levels of noncognate peptide/MHC-I complexes dictate the antigenicity of B16 cells. (A) Expression levels of MHC-I (H-2Kb and H-2Db, top panels) and H-2Kb/Ova₂₅₇ complexes (bottom panel) on a stable B16 clone expressing single-chain H-2Kb/Ova₂₅₇ (B16-scKb^Ova) compared with B16 and IFNγ-treated B16 (100 U/mL, 24 hours) cells measured by flow cytometry. Levels of H-2Kb/Ova₂₅₇ complexes were measured using antibody 25-D1.16.⁵³  (B) Antigenicity of B16-scKb^Ova was evaluated with freshly isolated CD8 T cells from Trp1₄₅₅TriVax- and Trp2₁₈₀TriVax-immunized WT mice using cytokine release ELISA. IFNγ-treated (100 U/mL, 24 hours) and nontreated parental B16 cells were included for comparison. Cultures consisted of 3 × 10⁵ CD8 T cells coincubated with 1 × 10⁵ stimulator cells for 40 hours before removing culture supernatants for cytokine measurements. Results represent the average values of IFNγ (columns) and SD (error bars) from triplicate wells. These experiments were repeated twice with similar results.