Fig. 2

Characterization of the TME of TAb2 and TCh3 tumors. Flow cytometry analysis was performed for spleen controls (n = 7), or tumor-infiltrating immune cells from TAb2 (n = 8) and TCh3 (n = 7) tumors for panel A-E and G. Spleens and tumors were harvested on day 20 post-injection. Spleens from TCh3 tumor-bearing mice were used as spleen controls. A Quantification of the percentage of CD11b⁺, CD4⁺, or CD8⁺ cells in CD45⁺ population of spleen controls, TAb2 and TCh3 tumors. Significance was calculated using Kruskal-Wallis test. B Representative flow plots of different MDSC populations (left panel) and macrophages (right panel). M-MDSCs are CD11b⁺Ly6C^highLy6G⁻, PMN-MDSCs are CD11b⁺Ly6C^lowLy6G⁺, and TAMs are CD11b⁺Ly6C⁻Ly6G⁻F4/80⁺. C Quantification of the percentages of MDSCs (left) and TAMs (right) in spleen, TAb2 and TCh3 tumors. P values are shown for Tukey’s multiple comparisons by two-way ANOVA (MDSCs) and one-way ANOVA (TAMs). D Representative flow plots for TAMs expressing CD86 and/or CD206. E Quantification of the percentages of M1 (CD86⁺CD206⁻) and M2 (CD86⁻CD206⁺) TAMs. P values are shown for Tukey’s multiple comparisons by two-way ANOVA. F Representative images of multispectral imaging (MSI) analysis of TAb2 (top) or TCh3 (bottom) tumors stained for Arginase 1 (red), F4/80 (green), Keratin 5 (cyan), and DAPI (blue). The white bars on the top left corners of the image indicate scale, 100 μm (left panel) and 20 μm (right panel). G Effector functions of CD8 TILs. The frequency of the CD8⁺ T cells producing single or double cytokines (IFNγ⁺, TNFα⁺, and IFNγ⁺TNFα⁺) in response to ex vivo stimulation. Significance was calculated using two-way ANOVA with Tukey’s multiple comparison test