Lentiviral TCR Transduction of T Cells

Buffy coat was obtained from local Shepard blood center. PBMCs were harvested by centrifugation on a Lymphoprep Ficoll gradient, diluted to 1 × 10⁷ cells/ml, aliquoted, and frozen. Lentiviral vectors expressing TCRs were prepared by 4 a plasmid co-transfection as previously described (20 (link)–22 (link)). Total T cells were isolated from PBMC by negative isolation kit (STEMCELL Technologies, Vancouver, BC, Canada) and then transduced by lentiviral vectors at MOI of 20–40 as described (15 (link)). Between 12–15 days after transduction, tetramer (NIH Tetramer Core Facility) staining was conducted to measure the percentage of TCR+ T cells. The TCR-Ts were then aliquoted and frozen.