Figure 6. COX-2/PGE2 signaling inhibition in combination with αPD-1 treatment causes a phenotypic shift in tumor-infiltrating CD8+ T cells.
(A) Frequency of tumor-infiltrating immune cells out of CD45+ cells in melanoma tumors at day 7 post-treatment, as measured by CyTOF (n=5 per treatment group). P-value by two-way ANOVA. (B) Hierarchical clustering and heatmap representing the mean intensity expression of select surface and intracellular markers associated with intratumoral CD8+ T functional states measured by CyTOF. (C) Uniform Manifold Approximation and Projection (UMAP) plots displaying the relative abundance of different CD8+ T cell clusters across treatment groups as defined by unsupervised self-organizing map clustering (FLowSOM). (D) Treatment-based comparison of the relative abundance of Cluster 6 within CD8+ T cells, which is characterized by high expression of TOX. (E) Treatment-based comparison of the relative abundance of Cluster 1 within CD45+ T cells, which is characterized by high expression of CD62L and the stem-like marker TCF1. (F) Bar plots of the median intensity (MI) of PD-1, CD25 and T-bet expression in non-Treg CD4+ T cells. (G) Bar plots of the frequency of CD44+ IFN-γ+ cells within CD8+ or CD4+ T cells. (H) Heatmap representing log2 fold change in expression of cytokines, chemokines and T cell-surface markers and (I) Paired dot plots representing levels of IL-2, IFN-γ and CXCL10 48h following anti-CD3 and/or CXB treatment of PDTFs from four independent tumors (averaged from 1-2 repeat cultures). (J) Representative FACS plots and paired dot plots of OX40 (% of CD4) or CD137 (% of CD8) expression 48h following anti-CD3 and/or CXB treatment of PDTFs from four independent tumors (averaged from 1-2 repeat cultures). P-value by one-way ANOVA (D, E, F, G, I, J). Data representative of 2 independent experiments with n=5 mice per group (A-G) or of 2 independent PDTF cultures (H-J). *p<0.05, **p<0.01.