Figure 2

Gene expression profile and functional analysis of CD8 tumour tissue-infiltrating lymphocytes (TILs) based on the expression patterns of PD-1 and TIM-3. (a) Clustering analysis for genes distinguishing four subsets of CD8 TILs extracted from three patients with renal cell carcinoma. In total, 912 genes were significantly upregulated or downregulated (log₂ fold change > [1], false discovery rate [FDR] < 0.05) as indicated by at least one group comparison. (b) Biological processes (Gene Ontology [GO] terms) enriched in group A and group B. (c) Principal component (PC) analysis of four subsets of CD8 T cells based on the expression patterns of PD-1 and TIM-3. (d) Gene set enrichment analysis (GSEA) of published data sets of biological processes (GO terms) enriched in group A and group B¹⁴. Statistical significance was determined by permutation testing with normalized enrichment score (NES). (e) Volcano plot of up- or downregulated genes between Fr. I vs. Fr. IV, Fr. II vs. Fr. IV, and Fr. III vs. Fr. IV. Genes specific only to exhausted T cells (blue) and to both exhausted CD8 T cells and CD4 regulatory T cells (Tregs) (red)⁷ are shown, and bold characters indicate the genes commonly having significant differences between these three combinations. (f) Comparison of multiple cytokine productivity (IFNγ, TNFα, and IL-2) of CD8 TILs from 18 patients in Fr. II, Fr. III, and Fr. IV among three sample types. (g) Cytotoxic functionality producing GZMB and both GZMB and PRF1 were compared within three fractions by Kruskal-Wallis analysis, and then a comparison of each sample type was performed by Bonferroni-corrected Mann-Whitney U test. The central tendency of the box plot indicates the median of each group, and the upper and lower ranges of the box plot show the 25^th and 75^th percentiles of each data set, respectively. **P < 0.01, *P < 0.05.