Cytokine production in TILs from CT26 tumor-bearing mice. TILs were harvested from CT26 tumor-bearing mice and stimulated with PMA and ionomycin before intracytoplasmic cytokine staining. (A) Expression of cytokine in Tim-3⁻PD-1⁻, Tim-3⁻PD-1⁺, and Tim-3⁺PD-1⁺ CD8⁺ TILs. Data shown are representative of five independent analyses. FMO, fluorescence minus one (anti-cytokine antibody). (B) Frequency of Tim-3⁻PD-1⁺ and Tim-3⁺PD-1⁺ cells among CD8⁺ cytokine-producing and -nonproducing TILs (n = 5). The horizontal bars indicate means. *, P < 0.0001; **, P = 0.0261, unpaired Student’s t test.