Figure 6. Irf6 promotes susceptibility to T cell killing by enhancing TNF-induced apoptosis.

a, Normalized viability of 4662 parental EV, Esc EV, and Esc Irf6 tumors with varying concentrations of TNF-α in the presence of IFN-γ (0.2 μg/ml) plus cycloheximide (1 μg/ml) for 48 h. IC₅₀ values are 0.03472 ug/ml for parental EV, 0.6494 ug/ml for Esc Irf6, and not determined for Esc EV tumors. b, Mean fluorescence intensities (MFIs) of active caspase-3 in 4662 parental EV, Esc EV, and Esc Irf6 tumors treated with or without TNF-α (0.5 μg/ml) plus IFN-γ in the presence of cycloheximide for 48 h by flow cytometry. c, The percentages of cleaved caspase-3 among s.c. implanted YFP⁺ 4662 parental EV, Esc EV, and Esc Irf6 tumors with or without immunotherapy by IF staining. Tumors were prepared a week after treatment. d, Normalized viability of 4662 Esc EV and Esc Irf6 tumors, treated with vehicle or z-VAD (20 μM), in response to TNF-α plus IFN-γ in the presence of cycloheximide for 48 h. e, Left, immunoblots of IRF6 and TNF-related cell death mediators in 4662 Esc EV and Esc Irf6 tumors with or without ablation of each gene. Right, normalized viability of 4662 Esc EV and Esc Irf6 tumors with or without indicated gene ablation in response to TNF-α plus IFN-γ in the presence of cycloheximide for 48 h. f, OVA-tdTomato⁺ 4662 Esc EV and Esc Irf6tumors with or without indicated gene ablation were used as target cells for OT-I co-culture. g, OVA-tdTomato⁺ 4662 Esc EV and Esc Irf6 tumors were co-cultured with or without activated OT-I and TNF-α neutralizing Ab (5 μg/ml) for 2 d. 7-AAD expression on tumors was measured by flow cytometry. Data represent two independent experiments.