TCR sequencing from A*68:02-EIFDRYGEEV tetramer-positive T cells from T cell cultures derived from PBMCs
PBMCs from patient SJ1 were expanded according to Cimen Bozkus et al, STAR Protoc, 2021 in the presence a pool of fusion peptides predicted to bind HLA-A*68:02 (EVKEFLAKA, EIFDRYGEEV, EIFDRYGEEVKEFL, EIFDRYGEEVKEFLA, EVEKEFLAKAKEDFL, 1 uM each). After expansion, cells were stained with a fluorescently-labeled A*68:02-EIFDRYGEEV tetramer to label fusion-specific T cells. Live, CD3+, CD8+, tetramer+ cells were single-cell sorted into individual wells of a 384-well plate loaded with Superscript VILO master mix and snap frozen at -80 C . After reverse transcription, cDNA underwent two rounds of nested multiplex PCR amplification using a forward primer mix specific for human V-segments and reverse primers specific for human TRAC and TRBC segments to obtain TCR amplicons.