Identification of individual potentially tumor-reactive T cells

The characterization based on OR and STARTRAC indexes highlighted certain meta-clusters which enriched potentially tumor-reactive T cells (pTRTs). In addition, we also identified individual cells with hallmarks of tumor-reactive T cells. First, with "REACTOME_TCR_SIGNALING" from MSigDB and common proliferation markers as genesets, GSEA implemented in R package clusterProfiler was applied to the z-score expression profiles of mini-clusters to obtain the normalized enrichment scores (NES) and p-values. Mini-clusters with NES > 0 and p-value < 0.05 were considered as having high TCR signaling or proliferation, and tumor infiltrated T cells in those mini-clusters were used as “baits”. Second, the expanded clonotypes to which the baits belong were considered as potentially tumor-reactive clonotypes. At last, all cells of those potentially tumor-reactive clonotypes were considered as pTRTs, including both cells that exhibited signature of TCR signaling or proliferation and cells that did not. Although whether those cells were truly tumor-reactive required further experimental validation, it was helpful for the characterization of meta-clusters to analyze the distribution patterns of those pTRTs.