PD-1 and Tim-3 expression in TILs. BALB/c mice were implanted with CT26 colon adenocarcinoma or 4T1 mammary adenocarcinoma. C57BL/6 mice were implanted with B16F10 melanoma. TILs were harvested and stained with 7AAD to exclude dead cells and antibodies against CD8, CD4, Tim-3, and PD-1. (A) Expression of Tim-3 and PD-1 on gated CD4⁺ and CD8⁺ TILs from a BALB/c mouse bearing CT26 tumor. FMO, fluorescence minus one controls for Tim-3 and PD-1 staining. Data shown are representative of more than five independent analyses. (B) Frequency of CD8⁺ cells in TILs expressing Tim-3 and PD-1 from tumor-bearing mice. The horizontal bars indicate means. *, P < 0.001; **, P < 0.05, one-way ANOVA followed by Tukey’s multiple comparison test. CT26 (n = 5), 4T1 (n = 6), and B16 (n = 9). (C) Frequency of CD8⁺Tim-3⁺ cells in spleens of tumor-bearing mice compared with spleens of naive tumor-free mice. The horizontal bars indicate means. *, P < 0.001, one-way ANOVA, Tukey’s multiple comparison test; **, P = 0.0188, unpaired Student’s t test. BALB/c, n = 11; CT26, n = 8; 4T1, n = 7; C57BL/6, n = 5; B16, n = 10.

CD44 and CD62L expression in Tim-3– and PD-1–expressing TILs. TILs were harvested from CT26 tumor-bearing mice and stained with 7AAD to exclude dead cells and antibodies against CD8, CD44, CD62L, Tim-3, and PD-1. (A) Representative staining on CD8⁺ Tim-3⁻PD-1⁻, Tim-3⁻PD-1⁺, and Tim-3⁺PD-1⁺ TILs is shown. FMO, controls for CD44 and CD62L staining are shown. Data are representative of three independent analyses. (B) Summary data showing the frequency of effector/memory (CD44^hiCD62L^low) and central memory (CD44^hiCD62L^hi) and CD44^int cells within the CD8⁺ Tim-3⁻PD-1⁻, Tim-3⁻PD-1⁺, and Tim-3⁺PD-1⁺ TILs. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA and Tukey’s multiple comparison test. n = 3. Error bars represent SEM.

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.

Proliferation and cell cycle entry in TILs from CT26 tumor-bearing mice. TILs were harvested from CT26 tumor-bearing mice and stimulated with 1 µg/ml of anti-CD3 before staining with antibodies against CD8, Tim-3, PD-1, and Ki-67 and TO-PRO-3–iodide. (A) Expression of Ki-67 and TO-PRO-3 staining in CD8⁺ TILs showing the different phases of the cell cycle: G0, G1, and S→M. Data shown are representative of six independent analyses. (B) Ratio of Tim-3⁺PD-1⁺ to Tim-3⁻PD-1⁺ TILs (n = 6) in different phases of cell cycle. *, P < 0.05, one-way ANOVA and Tukey’s multiple comparison test. n = 6. Error bars represent SEM.

Effect of targeting the Tim-3 and PD-1 signaling pathways on tumor growth. (A) 5 × 10⁵ CT26 cells were implanted into wild-type BALB/c mice. Mice were then treated with anti-Tim-3, anti-PD-L1, anti-Tim-3 + anti-PD-L1, or control immunoglobulins (RatIgG₁ + RatIgG_2b). Error bars represent SEM. Two independent experiments are shown. Left, control (n = 5), anti–Tim-3 (n = 5), anti–PD-L1 (n = 6), and anti–Tim-3 + anti–PD-L1 (n = 5). Right, control (n = 4), anti–Tim-3 (n = 5), anti–PD-L1 (n = 4), and anti–Tim-3 + anti–PD-L1 (n = 3). (B) Pooled data from the experiments shown in A. Error bars represent SEM. Left, *, P < 0.01 compared with control or anti–Tim-3 group. Right, *, P < 0.01 compared with control group and P < 0.05 compared with anti–Tim-3 group, one-way ANOVA and Tukey’s multiple comparison test.

Blockade of the Tim-3 and PD-1 signaling pathways restores IFN-γ production. TILs were harvested from CT26 tumor-bearing mice and cultured in vitro in the presence of soluble anti-CD3 and anti–Tim-3, anti–PD-L1, anti–Tim-3 plus anti–PD-L1, or control immunoglobulins. After 96 h, culture supernatant was collected and IFN-γ measured by cytometric bead array. Data are expressed as the difference in cytokine production over that observed in cultures with control immunoglobulins. Data shown are from three independent TILs samples from two independent experiments.