A cold tumor population drives resistance of mixed-population tumors to anti–PD-1 blocking and CD40 agonistic antibody combination treatment. (A) Dosing schedule of anti–PD-1 and CD40 agonist treatment. (B) Tumor growth curves of CIT6-YFP and CIT9-RFP tumors showing tumor volume of individual tumors and average volume of isotype control and anti–PD-1 + CD40 agonist combination treatment arms. Red arrows under the x axis shows the timing of treatments (day [D]0 and 3), fraction shown on the top right corner of individual tumor volume plots shows the number of mice whose tumors completely regressed after treatment. n = 8–11. (C) Kaplan–Meier curves comparing the survival of mice from each treatment arm. Statistical test was performed using log-rank test. (D) Frequency of tumor-infiltrating T cells after treatment in CIT6-YFP and CIT9-RFP tumors. n = 7–11. (E and F) Tumor growth and survival of mice harboring tumors derived from a 1:1 mixture of CIT6-YFP and CIT9-RFP cell lines. n = 18–23. (G) Frequency of tumor-infiltrating T cells after treatment in 1:1 mixture-derived tumors. n = 18–23. All experiments were performed once. Statistical significance was determined by two-way ANOVA (B and E) or Student’s t test (D and G), and Kaplan–Meier survival analyses were performed by log-rank (Mantel–Cox) test (C and F). *P < 0.05 and **P < 0.01.