Generation and Expansion of CAR T Cells

CAR T cells were produced using our research-grade protocol(25 (link)) for indicated experiments. Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from the Michael Amini Transfusion Medicine Center at COH (IRB: 15283). CD8 and/or CD8 T naïve cells were isolated from PBMCs with Human Naïve CD8+ or Naive T Cell Isolation Kits, respectively (Stemcell Technologies, Vancouver, Canada). T cells were cultured in vivo-15 (Lonza, Basel, Switzerland) supplemented with 10% human serum (Valley biomedical, Winchester, Virginia, USA) and 100U/mL human IL-2, and activated with Human T-Activator CD3/CD28 beads (Life Technologies, Carlsbad, California, USA) for 24 hours before transduction with lentivirus at MOI 1. Cultures were maintained at 0.5–1×10⁶ cells/mL in medium containing 100 U/mL IL-2. After 7 days, CD3/CD28 beads were removed by Dynamag-2. GFP-positive single CAR T cells or EGFR-positive dual CAR T cells were enriched by FACS, activated and expanded with CD3/CD28 bead stimulation for another 7 days. CD3/CD28 beads were removed by Dynamag-2 before use. T cells from each donor were used to produce un-transduced T-cell controls (mock) in parallel to CAR T cells.