Pe cy7 sa

For selected neopeptides, pMHC tetramers were generated for staining of neoepitope-specific T cells. Neopeptides were selected based on the observed NART responses from the DNA barcode-labelled multimer screening. Following the observed increase in NART responses at 3 weeks post-treatment, NART responses in 3-week post-treatment PBMC samples were interrogated wherever sufficient patient material remained, otherwise 9-week post-treatment samples were analyzed. Single-fluorochrome pMHC specificity tetramers using were generated as described in detail previously^{81 (link),82 (link)}, using a library of streptavidin (SA)-conjugated flourochromes consisting of PE-SA (BioLegend, cat. #405204), APC-SA (BioLegend, cat. #405207), BV421-SA (BD, cat. #563259), PE-Cy7-SA (BD, cat. #557598), BV605-SA (BD, cat. #563260), PE-CF594-SA (BD, cat. #562284), BV650-SA (BD, cat. #563855), BUV395-SA (BD, cat. #564176). Up to eight patient-specific pMHC tetramers per sample were investigated. PBMC samples were stained with respective library of pMHC tetramers and with an antibody mix consisting of CD8-BV480, dump channel antibodies and a dead cell marker, as above. Tetramer-specific T cells analyzed as lymphocytes, single, live, CD8⁺, FITC^‒ and tetramer⁺ cells. Due to staining strategy, tetramer⁺ cells were gated by being CD8⁺.