Fig. 2: Sequential TCR repertoires evolve over time leading to mixed dysfunctional single T cell states.

a Workflow of sequential TCRβ detected from nine metastases: 4 on-treatment (M0-day 373, M1-day 799, M2-day 1687, M3-day 1687) at distinct time points and 5 parallel multiregion postmortem metastases. scRNA-seq and scTCR-seq analyzed in isolated T cells from peripheral blood (day 2031). aPD-L1, anti-programmed death-ligand 1 monoclonal antibody; aPD-1 anti-programmed cell death protein 1 monoclonal antibody, CR complete response, M metastasis, PR partial response, PD progressive disease, Rec recurrence, SD stable disease, SC single-cell, TCRα T cell receptor alpha chain, TCRβ T cell receptor beta chain, TLR7 Toll-like receptor 7. b TCRβ CDR3 repertoire joined network to elucidate subnetworks private to on-treatment sequential chest wall metastases (yellow) or to parallel multiregion metastases (red) or shared to both sets of metastases (purple). Insert on the bottom shows amino acid sequences from a parallel multiregion metastases-specific subnetwork. Each node’s size corresponds to the number of samples where the sequence has been detected. Edges were formed between nodes only when the edit distance between the two CDR3 sequences equaled 1. Source data are provided as a Source Data file. c Uniform manifold approximation and projection (UMAP) analysis that displays single-cell transcriptomic landscape of sorted CD3+CD19- single T cells. Single T cells are colored by expression cluster, based on gene expression difference, of 11 T cell subsets and functional states. Mean unique molecular identifier (UMI) counts per cell were 3726 with a median number of genes detected per cell of 1254 (98.92% CD3+ cells of all cells, 97.17% expressing CD3ε, 89.83% expressing CD3δ). Clusters with percentages above 2% are depicted. Source data are provided as a Source Data file. d Heatmap from the scRNA-seq showing 9 clusters of T cell subpopulations resolved by z-scored differential expression of curated T cell marker genes. Caption shows four subclusters integrated within cluster 6. The top markers that define each one of those clusters are highlighted in red. Cluster 9 was characterized by cells with few detected genes and a high fraction of mitochondrial counts indicative of damaged cells. Source data are provided as a Source Data file. e UMAP embedding single cells from peripheral blood showing TCR clonotypes classified as singletons or expanded (N = 5204 cells with TCR) were projected onto the UMAP of peripheral blood T cells. Source data are provided as a Source Data file. f UMAP embedding single T cells from peripheral blood (N = 1284) and barplot colored by TCR clonotypes found in on-treatment sequential metastases (yellow), parallel multiregion metastases (red) or present in both tumor sources (purple). Source data are provided as a Source Data file.