T-cell receptor beta CDR3 sequencing of tumor-associated antigen and minor histocompatibility antigen specific T cells
T-cell clones directed against the HLA*02:01-restricted minor histocompatibility antigen HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. T-cell clones directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, Proteinase-3-VLQ, PRAME-VLD and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors.Sequencing of the CDR3 region of the T-cell receptor beta (TRB) chain (including the variable chain, diversity chain and joining chain) was performed on selected T-cell clones using ARTISAN PCR adapted for TCR. mRNA was purified from T-cell clones using the Dynabead mRNA DIRECT Kit and mixed with TRBC1 and TRBC2 constant region-specific primers and anchored template-switching oligonucleotides. TRB cDNA was synthesized using SMARTscribe reverse transcriptase and amplified by Phusion Flash using a forward anchor-specific primer and a nested reverse primer specific for the constant region of TRB: 5'-TGT GGG AGA TCT CTG CTT CTG-3'. Sanger sequencing was performed using the same reverse primer at the Leiden Genome Technology Center.