Figure 8: The proinflammatory cytokine TNF-α is required for T-cell-mediated death upon SMC and immune checkpoint inhibitor treatment.

(a) Isolated CD8 T-cells derived from the spleen and lymph nodes from mice previously cured of intracranial CT-2A tumours were cocultured with CT-2A cells in the presence of vehicle or 5 μM LCL161 and 20 μg ml⁻¹ isotype-matched IgG or α-PD-1 for 24 h. Viable T-cells were processed for flow cytometry using the following antibodies: CD3 (APC-Cy7), CD8 (BV711), GrzB (AF647) and TNF-α (PE). Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min–max range of the values. Significance was compared with vehicle and IgG-treated mice as assessed by ANOVA with Dunnett’s multiple comparison test. *P<0.05; **P<0.01; ***P<0.001. n=5 for each treatment group. (b) CD8⁺ T-cells were cocultured with mKate2-tagged CT-2A cells (CT-2A-mKate2) for 72 h in the presence of vehicle or 5 μM LCL161 and 20 μg ml⁻¹ of control IgG, α-PD-1 or α-TNF-α. Enumeration of mKate2-positive cells was acquired using the Incucyte Zoom software. Crosses depict mean, solid horizontal lines depict median, boxes depict 25th to 75th percentile, and whiskers depict min–max range of the values. Significance was compared with vehicle and IgG-treated mice as assessed by ANOVA with Dunnett’s multiple comparison test. **P<0.01; ***P<0.001. n=5 for each treatment group. Scale bar, 100 μm. (c) Mice bearing intracranial CT-2A tumours were treated at the indicated post-implantation day with vehicle or 75 mg kg⁻¹ LCL161 (oral) or i.p. with the relevant isotype IgG control or 500 μg α-TNF-α or 250 μg α-PD-1. Data represent the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison: **P<0.01. Numbers in parentheses represent number of mice per group.