Response to Programmed Cell Death-1 Blockade in a Murine Melanoma Syngeneic Model Requires Costimulation, CD4, and CD8 T Cells

IFNγ modulates PD-L1 expression in MC38, YUMM2.1, and YUMM1.1. A, Western blot analysis of PD-L1. MC38, YUMM2.1, and YUMM1.1 cells were cultured with or without IFNγ for 24 hours. B, expression of PD-L1 by flow cytometry on MC38, YUMM2.1, and YUMM1.1 cells at baseline and after 24 hours of stimulation with IFNγ. C, chromosomal copy-number variation in MC38, YUMM2.1, and YUMM1.1 cell lines. Y-axis represents Log₂ depth ratio vs. matched normal.

Both CD8 and CD4 cells mediate response to PD-1 blockade in MC38 and YUMM2.1. Tumor growth curves of MC38 (A) and YUMM2.1 (B) after anti–PD-1 and either anti-CD8 (anti–PD-1aCD8), anti-CD4 (anti–PD-1aCD4), anti-CD8 + anti-CD4 (anti–PD-1aCD8/4) or isotype control; 4 mice in each group, mean ± SD. (*, P < 0.001 isotype control, anti–PD-1aCD8, anti–PD-1aCD4, anti–PD-1aCD8/4 versus anti–PD-1 in MC38, P < 0.001 isotype control, anti–PD-1aCD4, anti–PD-1aCD8/4 versus anti–PD-1 in YUMM2.1, unpaired t test, n = 4); *, P = 0.003 anti–PD-1aCD8 versus anti–PD-1, unpaired t test, n = 4. The arrow indicates the day treatment with anti–PD-1 or isotype control was started. This experiment was performed in triplicate. On days 3 (d3) and 10 (d10) after treatment with anti–PD-1 or isotype control was started, MC38 and YUMM2.1 tumors were isolated and stained with fluorescent-labeled antibodies, analyzed by FACS. C and D, percentage of CD3⁺CD8⁺ (CD8 T cells) and CD3⁺CD4⁺ (CD4 T cells) in MC38 (C) and YUMM2.1. (D), tumors are shown (mean ± SD). *, P = 0.03 anti–PD-1 d10 versus control d10 in MC38; P = 0.03 anti–PD-1 d10 versus control d10 in YUMM2.1 (unpaired t test, n = 4). Results were consistent in 6 replicate experiments. E and F, statistical analysis of the 2C total number of CD8 T cells per gram of tumor in MC38 (E) and (F) YUMM2.1 tumors. *, P = 0.05 anti–PD-1 d10 versus control d10 in MC38, P = 0.02 anti–PD-1 d10 versus control d10 in YUMM2.1, unpaired t test, n = 8). G, representative immunofluorescence of CD8 T cells stained in YUMM2.1 tumors and spleens d10 after treatment with anti–PD-1 or isotype control was started.

Wnt/β-catenin pathway is not involved in CD8 T-cell decrease or anti–PD-1 antitumor response in YUMM2.1 tumor model. A, Western blot analysis of β-catenin in YUMM2.1 cells transduced with shRNA without β-catenin (sh YUMM2.1) or with shβ-catenin (shβ-catenin YUMM2.1) and YUMM1.1 cells transduced with shRNA without β-catenin (sh YUMM1.1) or with shβ-catenin (shβ-catenin YUMM1.1). B, quantification of CD3⁺CD8⁺ (CD8 T cells). Tumor cells harvested on days 3 and 10 after anti–PD-1 or isotype control were counted and analyzed by flow cytometry for CD3/CD8 staining; 3 mice in each group (mean ± SD). *, P = 0.003 anti–PD-1 d10 versus control d10 in sh-control YUMM2.1 tumors. C, shβ-catenin YUMM2.1 tumors. *, P = 0.008 anti–PD-1 d10 versus control d10 in shβ-catenin YUMM2.1 tumors, unpaired t test, n = 4. (D ) in vivo sh and shβ-catenin YUMM2.1 and (E) sh and shβ-catenin YUMM1.1 tumor growth curves with 3 to 4 mice in each group (mean ± SD) after anti–PD-1 or isotype control.

Increased antigen-presenting DCs in anti–PD-1-treated YUMM2.1 tumors. A, tumor growth curves of CD28KO or C57BL/6 mice bearing YUMM2.1 treated with anti–PD-1 or isotype control. B, tumor growth curves of CD80/86KO or C57BL/6 mice bearing YUMM2.1 treated with anti–PD-1 or isotype control. Four mice in each group (mean ± SD). The arrow indicates the day treatment with anti–PD-1 or isotype control was initiated. C, on day 10 after starting treatment, MC38, YUMM2.1, and YUMM1.1 tumors were isolated and stained with fluorescent-labeled antibodies and analyzed by FACS, with 3 mice in each group (mean ± SD). B220⁻ and B220⁺ cells presented as percentage of CD11c⁺ cells. *, P = 0.04 anti–PD-1 versus isotype control, CD11c⁺B220⁻ cells in MC38 tumors, unpaired t test, n = 3. D, B220⁻CD8⁺ and B220⁻CD103⁺ presented as percentage of CD11c⁺ cells. E, in vivo YUMM2.1 growth curve after anti–PD-1 ± anti-CD103 or isotype control ± anti-CD103, 4 mice in each group (mean ± SD). The arrow indicates the day anti–PD-1 or isotype control treatment was started. F, CD11b⁺ and CD11b⁺MHC-II^high DCs presented as percentage of CD11c⁺ cells. *, P = 0.04 anti–PD-1 versus control, P = 0.01 anti–PD-1 versus control in YUMM2.1 tumors, unpaired t test, n = 3.

Modulation of the tumor microenvironment by anti–PD-1 in MC38, YUMM2.1, and YUMM1.1. On day 10 after anti–PD-1 or isotype control, MC38, YUMM2.1, and YUMM1.1 tumors were isolated and stained with fluorescent-labeled antibodies and analyzed by FACS, with 3 mice in each group (mean ± SD). A, analysis of TAMs (CD11b⁺F4/80⁺). B, TAMs MHC-II^high (M1 TAMs, CD11b⁺F4/80⁺MHC-II^high) and TAMs MHC-II^low (M2 TAMs, CD11b⁺F4/80⁺MHC-II^low). *, P = 0.04 anti–PD-1 d10 versus control d10 TAMs; P = 0.02 anti–PD-1 d10 versus control d10 TAMs MHC-II^high in YUMM2.1 tumors, unpaired t test, n = 3. C, MO-MDSC (CD11b⁺Ly6C^highLy6G^low) and PMN-MDSC (CD11b⁺Ly6C^lowLy6G^high) presented as percentage of CD11b⁺ cells. D, analysis of T_regs (CD4⁺CD25⁺FOXp3⁺). E, representative FACS plots in tumors.