Fig. 2. The phenotype of CD8⁺ T cells and decreased resistance to anti-PD-1 Ab in CCL5−/− mice.

a Representative histological staining (left) and analysis (right) of CD8 in frozen section from the tumor of CCL5+/+ or CCL5−/− mice. b Representative flow cytometry plot (left) and analysis (right) of CD8⁺ T cells percentage in TILs of control group CCL5+/+ or CCL5−/− mice. n = 5 mice per group. c The tumor growth curves of CT26^shNTC and CT26^shCCL5 tumor cells were injected into the back of female WT or KO mice treated with CD8 T-cell-depleting antibody. Tumor growth was monitored every 2–3 days. d Quantitation of liver metastasis determined by counting of foci per liver lobe. Each group contained five mice. e Representative flow cytometry plot (left) and analysis (right) of effector CD8⁺ T cells percentage among CD8⁺ cells in TILs of CCL5+/+ or CCL5−/− mice. Bar chart shows the FACS quantification of effector (CD8⁺CD44^hiCD62L^low), memory (CD8⁺CD44^hiCD62L^hi), and naïve (CD8⁺CD44^lowCD62L^low) T-cell subpopulations. f FACS quantification of the percentage of CD69⁺, LAG3⁺, TIM3⁺, ICOS⁺, PD-1⁺ cells among CD8⁺ T cells in TILs of CCL5+/+ or CCL5−/− mice. g RT-PCR quantification of relative PD-1 mRNA expression in CD8⁺ T cells in TILs. h FACS quantification of the percentage of PD-L1⁺ cells in TILs. i The tumor growth curves of CT26^shNTC and CT26^shCCL5 tumor cells were injected into the back of female WT or KO mice. Mice received anti-PD-1 monoclonal antibody i.p. on days 3, 6, 9, 12 post tumor injection. Tumor growth was monitored every 2–3 days. j Quantitation of liver metastasis determined by counting of foci per liver lobe after tumor burden of 40 days. Each group contained five mice. *P < 0.05, **P < 0.05, ***P < 0.001. Data are represented as mean ± SEM; n = 5 mice per group