Fig. 5.

Intratumoral T cell infiltration and spatial redistribution after Ang2/VEGFA blockade in combination with agonist CD40 antibodies. (A–C) Flow cytometry analysis of tumor-infiltrating CD8⁺ T cells in (A) MC38 and (B) B16-OVA tumors, and (C) FoxP3⁺ Treg cells (Left) and CD8⁺/Treg ratio (Right) in MC38 tumors after treatment with the indicated antibodies. Each data point represents one mouse. (D) Flow cytometry analysis of adoptively transferred OVA-specific CD8⁺ T cells (CD8⁺CD45.2⁺CD45.1⁻) in tumors, TDLNs, and spleen of CD45.1⁺C57BL/6 mice treated as indicated. Each data point represents one mouse. (E and F) Quantification of perivascular CD8⁺ T cells in MC38 tumors. (E) Data indicate the percentage of CD8⁺ T cells located in the perivascular space (5 to 25 μm from the closest CD31⁺ blood vessel) in 2 to 3 tumors per treatment. (F) Representative images show anti-CD31 (magenta) and anti-CD8 (brown) immunostaining, and hematoxylin staining (light blue) of tumor sections. (Scale bar, 100 μm.) (G) Intratumoral distribution of CD8⁺ T cells in MC38 tumors. Representative images (Upper) from tumor periphery and tumor core are shown after anti-CD8 (brown) immunostaining and hematoxylin staining (light blue) of tumor sections. (Scale bar, 50 μm.) For each tumor, the number of CD8⁺ T cells in the tumor periphery and tumor core (>200 μm from tumor margin) were quantified (Bottom). For each tumor, at least 4 images from each compartment were analyzed and the counts averaged. Each data point represents one mouse. Data indicate mean values ± SEM. Statistical analyses by 1-way ANOVA with Tukey’s correction for multiple comparisons (black) or pairwise Student’s t test (red) unless otherwise indicated in Dataset S2. The number of mice employed in each experiment is reported in Dataset S2.