Spatial immune infiltration patterns persist in mixed-population tumors following anti–PD-1 blockade and CD40 agonist combination treatment. (A) Schematic of tumors derived from a 1:1 mixture of CIT6-YFP and CIT9-RFP cell lines treated with anti–PD-1 + CD40 agonist combination treatment or isotype control antibodies at days (D)0 and 3 and subject to spatial analysis of T cell infiltration at day 6. (B) Average tumor volume of each treatment arm. Red arrows under the x axis shows the timing of treatments. n = 19–23. (C–H) Flow cytometric analysis of T cell subsets in each region of isotype control-treated and combination therapy-treated tumors, showing Th1 cell frequency (C), Treg frequency (D), frequency of all CD4 T cell subsets (E), Th1-to-Treg ratio (F), frequency of IFNγ⁺ CD8 T cells (G), and IFNγ⁺ CD8 T cell–to-Treg ratio (H). n = 10–16. All experiments were performed once. Statistical significance was determined by two-way ANOVA with Tukey’s correction posttest (B) or Student’s t test (C–H). *P < 0.05, **P < 0.01, and ***P < 0.001.