Macrophage depletion is associated with decreased TIL functions. (A) Cumulative frequency of tumor-infiltrating IFN-γ⁺ CD8 T cells, TNF-α⁺ CD8 T cells, and IFN-γ⁺ TNF-α⁺ CD8 T cells from two independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, *P < 0.05). (B) Cumulative frequency of tumor-infiltrating IFN-γ⁺ CD4 Teff cells, TNF-α⁺ CD4 Teff cells, and IFN-γ⁺ TNF-α⁺ CD4 Teff cells from two independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, *P < 0.05). (C) Cumulative frequency of tumor-infiltrating granzyme B⁺ and IFN-γ⁺ granzyme B⁺ CD8 T cells from two independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, *P < 0.05). (D and E) Recruitment and polarization of M1-like macrophages depend on IFN-γ. (D) The density of CD11b⁺ F4/80⁺ TAMs is depicted as an absolute number of cells per mg of tumor on day 16 after tumor challenge. Data were pooled from two representative experiments out of three independent experiments (n = 5 mice per group; one-way ANOVA, post hoc, **P < 0.01; ***P < 0.001) (E) Frequency (left panel) of MHC II⁺ TAMs and mean fluorescent intensity (right panel) of MHC II molecule expressed on the surface of TAMs. Data were pooled from two representative experiments out of three independent experiments (n = 5 mice per group; one-way ANOVA, post hoc, *P < 0.05; **P < 0.01). Error bars represent means ± SEM. Irradiated parental B16 tumor cells (Vac) were used as a control for the IVAX group.