Figure 3.

Immunomodulatory activity of TAM in the RAG model. A and B, Mice bearing RAG tumors were injected intraperitoneally with anti-CSF1R mAb or control IgG2a at 300 μg/animal 1 day before allocation (i.e., day 0). On day 1, when TVs were approximately 101 mm³, anti-CSF1R mAb or control IgG2a at 300 μg/animal were administered twice weekly thereafter for 2 weeks. A, CD45⁺ TILs were isolated from tumor tissue, and populations of immune cells were analyzed by flow cytometry. The gating strategy is shown in Supplementary Fig. S3. Percentages of TAMs, CD8⁺ T cells in CD45⁺ TILs, and PD-1⁺ Tim3⁺ CD8⁺ T cells and PD-1⁺ CD25⁺ CD8⁺ T cells in CD8⁺ T cells. Percentages of CD4⁺ T cells in CD45⁺ TILs and Treg (CD25⁺ CD127⁻ CD4⁺ T cells) in CD4⁺ T cells. N = 8. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, unpaired t test between groups. B, Expression levels of the indicated genes determined through qRT-PCR analysis. Data were normalized by the Gapdh gene. Data are presented as means + SEM (N = 8). **, P < 0.01; ****, P < 0.0001, unpaired t test between groups. C and D, T-cell coculture assay with F4/80⁺ cells (TAMs) isolated from RAG tumors was performed at the indicated ratios (T cells: F4/80⁺ cells) as described in Materials and Methods. C, Histograms of CMFDA-labeled CD8⁺ T cells cocultured with F4/80⁺ cells. D, Proliferation of CD8⁺ T cells and CD4⁺ T cells cocultured with F4/80⁺ cells compared with T-cell single culture (T cell only). Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Dunnett multiple comparison test) versus T cell only group.