Combining anti–PD-L1 with anti-CD40 significantly impairs tumor growth and increases survival in the Mycn-high nGEMM model. (A) Mycn-nGEMM-high cells were transplanted in the renal capsule (RC) of C57BL/6J hosts on day 0. After BLI imaging and randomization, treatments started on day 7 with 200 µg of anti–PD-L1 (or corresponding IgG), CD40 agonist, or a combination of both injected i.p. (IP) every 3 days for a total of 5 doses. (B) Kaplan–Meier curves for C57BL/6J mice, transplanted with Mycn-nGEMM-high cells in the renal capsule on day 0, and treated with indicated antibodies (n = 10–12 mice per group). (C) Pictures of three tumors harvested at day 21 (5 treatment doses) for indicated treatment group. (D) Tumor volumes were measured for tumors harvested at day 21 for each treatment group. Indicated values are mean of n = 3 tumors per group ± SD. (E) Proportion of CD45⁺ cells in tumors from each treatment group as a percentage of total cells in the tumors (mean values of n = 3 tumors) ± SD. (F) Proportion of GFP⁺H-2Kd/H2Dd⁺ cells in tumors from each treatment group as a percentage of total cells in the tumors (mean values of n = 3 tumors) ± SD. (G) Proportion of CD38⁺ macrophages as a percentage of total cells in the tumors (mean values for n = 3 tumors) ± SD. (H) Proportion of CD11c⁺ DCs as a percentage of total cells in the tumors (mean values for n = 3 tumors) ± SD. (I) Proportion of CD4⁺ T cells as a percentage of total cells in the tumors (mean values for n = 3 tumors) ± SD. (J) Proportion of CD8⁺ T cells as a percentage of total cells in the tumors (mean values for n = 3 tumors) ± SD. (K) Proportion of B220⁺ B cells as a percentage of total cells in the tumors (mean values for n = 3 tumors) ± SD. (L) Cartoon summarizing effect of anti–PD-L1 and CD40 agonist therapies on the TME and growth of Mycn-nGEMM tumors. Statistical significance was determined by ordinary one-way ANOVA test (D–K), and Kaplan–Meier survival analyses were performed by log-rank test (B). *P < 0.05, **P < 0.01, ***P < 0.001, and ns = no significance.