Figure 2: Gene set enrichment analysis (GSEA) of genes associated with self-tolerance in tumor-specific CD8+ T cells. Expression data of TILN [4] were analyzed for genes associated with self-tolerance [6] by GSEA using parameters recommended for expression datasets that contain a sample with less than 7 replicates: dataset and gene sets were converted into gene symbols, redundant probe sets were collapsed using probe medians, a Signal2Noise metric was used for ranking genes, and the weighted enrichment statistic and 1000 gene set permutations were employed [7,8]. The 144 genes associated with self-tolerance were previously published in K-means clusters 9 and 13 [6]. We converted 119 of these mouse genes to human homologues in DAVID [18,19]; before comparing them to genes expressed by tumorspecific CD8+ T cells [4,7,8]. (A) The plot shows enrichment of genes associated with tolerance in TILN compared to tumor-specific CD8+ T cells in circulation. The Normalized Enrichment Score (1.64, green line) considers the ranked list of expression differences between tumor-specific CD8+ T cells from the tumor and periphery (red=increased in TILN, blue=decreased in TILN). Vertical black lines indicate where genes overexpressed by self-tolerant versus functional T cells [6] fall in the ranked list [4] and significantly cluster among genes most expressed by TILN (p-value <0.001 and FDR <0.001) [7,8]. (B) The 39 genes that comprise the leading edge of the Enrichment Score [7,8] are shown in a corresponding heat map. The color gradient matches the location of genes associated with self-tolerance among the ranked list of gene expression by tumor-specific CD8+ T cells (red=increased in TILN, blue=decreased in TILN). Genes denoted with an asterisk are associated with the cell cycle (p-values=1.47 × 10-2 - 6.37 × 10-11) through the use of QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN, Redwood City, CA, USA, www.qiagen.com/ingenuity).