T cell isolation and effector function analysis.

CD8⁺ T cell negative selection kits (Miltenyi Biotec) were used to isolate peripheral T cells from mouse spleens (39). To analyze tumor-infiltrating T cells, the tumors were first digested with collagenase IV (Sigma-Aldrich), and then the tumor-infiltrating leukocytes were isolated by centrifugation at 40%–70% Percoll (GE) gradient. To measure the effector function of CD8⁺ T cells, T cells were first incubated in the presence of 5 μg/mL BFA with 1 μM ionomycin and 50 ng/mL PMA for 5 hours, then stained with APC/Cy7 conjugated anti-CD8a antibody (BioLegend, then stained with APC/Cy7 CD8a antibody (BioLegend, catalog 100714 for mouse, and catalog 300926 for human). Cells were then fixed and permeabilized with 4% paraformaldehyde (PFA) and stained with APC anti-IFN-γ antibody (Biolegend, catalog 505810 for mouse, and catalog 502512 for human), PE anti-TNF-α antibody (Biolegend, catalog 506306 for mouse, and catalog 502909 for human), and PerCP/Cyanine5.5 anti-human/mouse Granzyme B (BioLegend, catalog 372212. Generally, in order to gate cytokines or granule-producing cells, unstimulated T cells or T cells stained by an isotype control antibody were used as negative controls. This gating strategy was applied in all the flow cytometry analyses unless otherwise indicated.