Figure 3.

Combination 3M‐052/PD‐1 blockade increases primary tumor immunogenicity and adaptive immune responses. BALB/c mice were injected with 1 × 10⁵ 4T1.2 cells into the 4th mammary fat pad, and palpable tumors were i.t. injected with vehicle or 3M‐052 on day 7 post‐inoculation. Mice were treated with four doses of isotype or anti‐PD1 mAb in a neoadjuvant setting before primary tumor resection on day 14. Primary tumors were assessed for (a) number of CD206⁺ F4/80⁺ cells; (b) number of TCRβ⁺CD8⁺ cells; (c) ratio of TCRβ⁺CD8⁺: FoxP3⁺CD25⁺ TCRβ⁺CD4⁺ cells; (d) MHC‐I (H2‐K^d) and (e) MHC‐II expression of F4/80⁺ cells; MHC‐I expression of (f) CD11b⁺ DCs and (g) isolated tumor cells (mCherry⁺); number of (h) CD69⁺ TCRβ⁺CD4⁺ cells, (i) CD69⁺ TCRβ⁺CD8⁺ cells and (j) IFN‐γ⁺TCRβ⁺CD8⁺ cells; and (k) ratio of tumor: TCRβ⁺CD8⁺ cells. (l) TCRβ⁺CD8⁺ cells from day 18 peripheral blood (PB) was assessed for specificity against 4T1.2 cells denoted by IFN‐γ production. Experiments were independently repeated twice, and representative data are shown. MFI: mean fluorescence intensity. All experiments n = 7 mice/group except (c, k) n = 8 or 9 mice/group, (l) n = 10 mice/group. Statistical analysis was performed by one‐way ANOVA with post hoc Tukey's multiple comparison test. Error bars are SEM. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.