neoTCR isolation
Each purified patient peptide–HLA protein was multimerized, fluorescently labelled and DNA barcoded. The barcode followed the structure adaptor-UMI-barcode-UMI-adaptor (Integrated DNA Technologies). In brief, biotinylated peptide–HLA proteins and biotinylated DNA barcodes were mixed at a 3:1 molar ratio and then complexed into fluorescently labelled multimers with PE-streptavidin or APC-streptavidin (Life Technologies) at 4:1 molar ratio of streptavidin to biotin. Peptide–HLA multimers for each patient were then pooled, concentrated and used to stain cells.neoTCRs were isolated from a patient’s own peripheral blood. In brief, PBMCs were enriched for CD8 T cells by negative selection (Miltenyi Biotec) with the addition of CD16 and CD56 markers (R&D Systems) to prevent the loss of activated CD8 T cells. Enriched CD8 T cells were then stained with the pooled library of neoantigen peptide–HLA multimers plus a panel of fluorescently labelled antibodies against cell surface markers, including CD39, CD103 and CD95 (see Supplementary Table 