5 race

The generation of CD4⁺ T cell clones was described previously.^{35 (link)} Briefly, Patient EB97 was vaccinated with 300 μg MAGE-A3 protein mixed with an immunological adjuvant AS-15 (GlaxoSmithKline), and later a set of MAGE-A3 peptides at sites close to the protein/adjuvant injection site. Patient R12 received dendritic cells pulsed with a pool of MAGE-A3 peptides. Peripheral blood mononuclear cells (PBMC) from vaccinated patients were labeled with DP4/MAGE-A3 tetramers, and single CD4⁺CD8⁻ tetramer⁺ cells were sorted and stimulated with irradiated DP4⁺ feeder cells pulsed with MAGE-A3.DP4 peptide KKLLTQHFVQENYLEY. TCR sequences were obtained from DP4/MAGE-A3 tetramer⁺ clones by following the manufacturer’s protocol for 5′ RACE (rapid amplification of cDNA ends) (Clontech, CA), using a TCRα constant region reverse primer (5′-CAC TGT TGC TCT TGA AGT CC-3′) and a TCRβ constant region reverse primer (5′-CAG GCA GTA TCT GGA GTC ATT GAG-3′).

V-CDR3 regions of TCRA and TCRB mRNA from T-cell clones were sequenced using 5′ RACE (Clontech, Mountain View, CA) (36 (link)). TCR expression plasmid was created by placing TCRA/TCRB V-CDR3 (Genbank KT955849/KT955850) into pMP71flex (34 (link)). An extracellular murine TCRB-C epitope allows detection of transduced cells with anti-TCRB (H57-597, eBioscience, San Diego, CA). TCR cassettes were inserted into pRRL.PPT.MP.GFPpre (37 (link)). Lentiviral stocks were created by co-transection of HEK293 and used to transduce anti-CD3/anti-CD28 (Dynal, Grand Island, NY)-stimulated CD8 PBMC, which were expanded using anti-CD3 (38 (link)). For bulk CDR3 sequencing, 10⁶ polyclonal HSV-2-reactive CD8 T-cells or 3 × 10³ tetramer-sorted cells were submitted to Adaptive Biotechnologies (Seattle, WA) for TCRB clonoSEQ^™ (39 ).