TCR Engineering for Adoptive Therapy

Full-length TCR α and β chain genes were cloned by 5’-RACE PCR method and sequenced as previously described [28 (link)]. TCR β chain gene was genetically fused to α chain gene via T2A site and cloned into a murine stem cell virus-derived retroviral vector. Retroviral vector production and transduction were performed as previously described [26 (link)]. Transduction efficiency was determined by staining with A*02/NY-ESO-1_157-165 (SLLMWITQC) tetramer (iTAg MHC tetramer, MBL), anti-human TCR Vβ8 mAb (clone JR2, BioLegend) for 19305DP-TCR, and anti-human TCR Vβ3 mAb (clone CH92, Beckman Coulter) for CD8SP (CD8SP1) or control HLA-DR*01:01 (DR*01)-restricted NY-ESO-1-specific TCR [29 (link)]. In some experiments, CD4⁺ or CD8⁺ T cells were depleted from PBMC by anti-biotin magnetic beads (Dynabeads, Life Technologies) following staining with biotin-conjugated anti-CD4 (clone OKT4, BioLegend) or anti-CD8 (clone HIT8a, BioLegend) mAb prior to activation with PHA. For in vivo experiments, T cells were harvested 20–22 h after the second infection.