Figure 4 from Functionally Characterizing the Renal Cell Carcinoma Tumor-Immune Microenvironment via Patient-Derived Ex Vivo Models

Characterization of the effects of a strong T cell activator, ImmunoCult (anti-CD3/CD28/CD2), on the RCC TME. A, Volcano plot illustrating changes in cytokine secretion in response to anti-CD3/CD28/CD2. Each point represents a cytokine, and statistically significantly upregulated cytokines are annotated and highlighted in dark red. B and C, CyTOF analysis for comparison of granzyme B, TNF (TNF-α), CCL4, or INF-γ and TNF expression between control and anti-CD3/CD28/CD2 in CD8+ (B) and CD4+ (C) T cells, respectively. Gated populations depict the percentage of positive cells for each marker. D, Heatmap showing changes in the percentage of marker-positive cells between control and anti-CD3/CD28/CD2 conditions in CD8+ and CD4+ T cells. E, UMAP of CD8+ T cells highlighting a newly formed “activated CD8 cell” cluster in response to anti-CD3/CD28/CD2 treatment. Bar plot to the right shows the proportion this newly formed cluster occupies in CD8+ T cells of each of the eight patients in anti-CD3/CD28/CD2 treatment. F, UMAP of CD4+ T cells highlighting a newly formed “ImmunoCult CD4 cells” cluster in response to anti-CD3/CD28/CD2 treatment. Bar plot to the right shows the proportion this newly formed cluster occupies in CD4+ T cells of each of the eight patients in anti-CD3/CD28/CD2 treatment. G and H, Volcano plot illustrating changes in gene expression in response to anti-CD3/CD28/CD2 in CD8+ T cells (G) or CD4+ T cells (H). Each point represents a gene, the significantly upregulated genes are colored dark red, whereas downregulated genes are dark blue. Only significantly regulated genes are labeled [adjusted P value < 0.05 and abs(avg_log2FC) > 0.5]. I, Dot plot of selected immunology-related hallmark pathways, comparing anti-CD3/CD28/CD2 and control conditions. Genes were identified using the FindMarkers method and included if the P value was < 0.05. Circle size represents the logP value, whereas color indicates the percentage of genes in our dataset that are upregulated (+) (in red) or downregulated (−) (in blue). J, Volcano plot illustrating changes in cluster size in response to anti-CD3/CD28/CD2. Each point represents a T cell cluster (CD8+ T cells and CD4+ T cells). Cell populations that are enriched in anti-CD3/CD28/CD2 and statistically significant (adjusted P value < 0.05 and log2FC > 0) are highlighted in dark red, and cell populations that are diminished in anti-CD3/CD28/CD2 and statistically significant (adjusted P value < 0.05 and log2FC < 0) are colored in dark blue. FC, fold change. K, Signature score distributions for terminally exhausted, progenitor exhausted, and IFN CD8+ T cell signatures comparing control and anti-CD3/CD28/CD2 cells. Statistical significance of differential signature enrichment (P value) between subtypes was determined by two-sided Wilcoxon rank-sum test (****, P ≤ 0.0001).