Fig. 3.

Efficacy of α-PD-1 therapy of tongue-implanted mEER tumors is enhanced by combination treatment with α-CTLA-4 but not α-Lag3. Mice were challenged with mEER tumor cells (4 × 10⁴) in the tongue and treated with antibodies targeting individual checkpoint receptors PD-1, CTLA-4, or Lag3 or using combinations of α-PD-1 and α-CTLA-4 or α-PD-1 and α-Lag-3 antibodies. The percentages of mice surviving in the different groups are shown (a). Statistical significance was calculated using Log-rank (Mantel-Cox) test. The significant difference for each treatment group compared to untreated control group is indicated by colored stars and between groups is shown on the legend; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Tongue tumor volume was measured by MRI (T2-weighted sagittal image) at day 19 after tumor implantation and representative data is shown for one mouse in each group (b) along with group means ± SD (n = 4–16 mice/group) (c). **p < 0.01, ****p < 0.0001, one-way ANOVA. Flow cytometry analyses of TIL isolated at day 15 from tongue tumor-bearing mice subjected to different treatments showing frequencies of total CD8⁺ T cells, Granzyme B expressing CD8⁺ T cells (d), CD4⁺FoxP3⁺ Treg, CD11b⁺Gr-1⁺ MDSC (e) as well as ratios of GrnzB⁺CD8⁺ T cells to Treg and to MDSC (f). Data shown are mean + SD from two experiments (except for anti-Lag3 group) with individual data points representing pooled TILs of 2–3 tumors. Statistical significance was calculated using one-way ANOVA with Turkey post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001