Figure 2. Anti–PD-1–resistant tumor models demonstrate reduced CD8⁺ T cell and effector functions compared with sensitive tumors.

(A) Three of the 344SQ^PD1R lines, 3 of the 344SQ^PD1S lines, and the 344SQ parental line were implanted into WT mice. After 3 weeks, tumors were excised, processed into single cells, and stained for multicolor flow cytometry analysis of immune cell subsets. The total intratumoral T cells were gated as CD3⁺ as a percentage of total CD45⁺ cells. Under total T cells, we then analyzed the CD8⁺ T cells for total amounts and effector memory (CD62L^–CD44⁺) or naive (CD62L⁺CD44^–) status. Individual models are denoted by different symbols and colors. n = 2–5 mice per model. *P < 0.05 by t test. (B) Two representative cell lines for both 344SQ^PD1S and 344SQ^PD1R were implanted into WT mice and tumors grown until endpoint (about 6–7 weeks). Tumors were collected and analyzed via IHC for CD8⁺ T cells. A representative image per model is depicted (left). All tumors per group were combined and graphed as total CD8⁺ T cells per field of view (FOV) (right). n = 2 mice per cell line, 3–6 images per mouse tumor. ****P < 0.0001 by t test. Scale bars: 50 μm; insets zoomed 200%. (C) The KP^IgG and KP^PDL1 tumors from Figure 1E were collected for IHC and analyzed for total CD8⁺ T cells (top) and granzyme B staining (bottom). n = 3 tumors per condition, 5–6 images per tumor. *P < 0.05, ****P < 0.0001 by 1-way ANOVA with multiple comparisons corrected. Scale bars: 50 μm; insets zoomed 200%. (D) 344SQ^PD1S1 and 344SQ^PD1R2 models were implanted into WT mice and then treated with either IgG control or anti–PD-1 antibody. After 2 weeks of treatment, tumors were excised and analyzed via multicolor flow as described in A. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.