IFNγ-treated H-2Kb-loss tumor variants are recognized by H-2Db–restricted CD8 T cells. (A) Expression levels of MHC-I and PD-L1 on H-2Kb–loss variants (B16^Kb−, B16F1^Kb−, and JB/RH^Kb−). The cells were incubated (IFNγ) or not (No Tx) with 100 U/mL of IFNγ for 24 hours, and stained with antibodies as indicated, followed by flow cytometric analysis. (B) CD8 T cells, which were freshly isolated from mice vaccinated with Trp1₄₅₅TriVax (H-2Db–restricted), were evaluated for their capacity to recognize B16, 2 B16 H-2Kb-loss variants (B16^Kb− and B16F1^Kb−), and an H-2Kb⁻ chemically induced melanoma (JB/RH^Kb−) using a cytokine-release ELISA assay. The cells were either incubated with IFNγ (100 U/mL, 24 hours) or not (No Tx). Cultures consisted of 3 × 10⁵ CD8 T cells coincubated with stimulator cells (3:1 ratio) 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. Numbers above columns represent the percentage response of the IFNγ-treated cells compared with that of the nontreated group.