Combination therapy reduces the suppressive efficacies of TAMs. TAMs were isolated from tumors of indicated treatment groups and control as described in the Materials and methods. These macrophages were then incubated with naïve untouched total T cells isolated from the spleen with simultaneous in vitro stimulation with anti-CD3 (1.25 µg/ml) and anti-CD28 (1.25 µg/ml) antibodies for 48 h. Cells were stained with indicated antibodies. (A and B) Representative flow cytometry plots (A) and bar graphs (B) of IFN-γ in CD8 T cells. (C) MFI of CD206 expression on macrophages isolated from tumors of indicated treatment groups. (D) Frequencies of CD206⁺ macrophages in TAMs isolated from tumors of indicated treatment groups. Data represent two independent experiments (n = 5 mice per group; one-way ANOVA, post hoc, ns, not significant *P < 0.05; ***P < 0.001, ****P < 0.0001). Error bars indicate means ± SEM. Irradiated parental B16 tumor cells (Vac) were used as a control for the IVAX group.