Reduced intratumoral macrophages correlate with loss of tumor protection. (A) 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 three independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, ****P < 0.0001). (B) The density of CD11b⁺ F4/80⁺ TAMs is depicted as an absolute number of cells per mg of the spleen (upper panel) and a total number of CD11b⁺ F4/80⁺ TAMs in tumor-draining lymph nodes (lower panel) on day 16 after tumor challenge. Data were pooled from three independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, *P < 0.05). (C) The density of CD11c⁺ F4/80⁻ DCs was depicted as an absolute number of cells per mg of tumor and spleen and a total number of CD11c⁺ F4/80⁻ DCs in tumor-draining lymph nodes on day 16 after tumor challenge. Data were pooled from three independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, ns, not significant). (D and E) The density of neutrophils is depicted as an absolute number of cells per mg of tumor on day 16 after tumor challenge (D) and (E) the MFI of various surface receptors on the surface of neutrophils. Data were pooled from three independent experiments (n = 3 mice per group; one-way ANOVA, post hoc, ns, not significant *P < 0.05). (F) Mice were i.d. challenged on the right flank with B16-F10 tumor cells. Subsequently, i.p. injections of 100 µg anti-CTLA-4, combined with intradermal vaccination on the left flank with irradiated 10⁶ IVAX or irradiated parental B16 tumor cells, were administered on days 3, 6, 9, and 12. For specific cell depletion groups, as indicated, 500 µg of anti-Ly6G, anti-CSF1R, or isotype control antibodies were coadministered with the combination of IVAX plus anti-CTLA-4 on days 3, 6, 9, and 12. The data represent three independent experiments (n = 5 mice per group). Error bars indicate means ± SEM. Irradiated parental B16 tumor cells (Vac) were used as a control for the IVAX group.