Immune cell infiltration and function in CIT6-YFP, CIT9-RFP, and mixed-population tumors and in tumors treated with PD-1 blockade + CD40 agonist combination therapy. (A) Expression of MHC-II (I-A/I-E) on tumor cells in CIT6-YFP and CIT9-RFP tumors (n = 6). (B) Frequency of CD11b⁺ cells (n = 6–7). (C) Frequency of CD69⁻ CD25⁺ and PD-1⁺–activated T cells in in vitro co-culture assay of CD8 T cells and macrophages sorted from CIT6-YFP, CIT9-RFP, and mixed-population tumors (n = 3). (D) Frequency of Teff subsets broken down by subset to show statistical test results (n = 3). (E) Frequency of neutrophils, monocytes, and DCs (n = 6–7). All data in A–E are a representation of two independent experiments. (F–H) CIT6-YFP + CIT9-RFP mixed-population tumors were microdissected into YFP, mixed, and RFP regions and analyzed for immune infiltration by flow cytometry. (F) Frequency of total CD45⁺ immune cells in each region (n = 8–9). (G) Frequency of total CD3⁺ T cells, CD4 T cells, and CD8 T cells presented as % live cells. (H) Frequency of total macrophages presented as % live cells. Data in G and H are a representation of two independent experiments with ≥6 mice per experiment. (I–K) B cells and main myeloid cell populations in (I) CIT6-YFP tumors (n = 7–8), (J) CIT9-RFP tumors (n = 9–11), and (K) CIT6-YFP + CIT9-RFP tumors (n = 18–23). Experiments in I–K were performed once. (L) Total CD4 T cell (CD45⁺ CD3⁺ CD4⁺) and total CD8 T cell (CD45⁺ CD3⁺ CD8⁺) frequencies in each region of isotype control-treated and combination therapy-treated tumors analyzed at day 6 after treatment initiation (n = 10–16). Experiment was performed once. Statistical significance was determined by Student’s t test (A and G–L), one-way (B and E), or two-way ANOVA (C, D, and F) with Tukey’s correction. *P < 0.05, **P < 0.01, and ***P < 0.001.