Generation of CD19-targeting 4-1BB CAR T cells

A 32A9-based second generation 4-1BB CAR fragment was synthesized (GenScript, Nanjing, Jiangsu). To detect the expression of CAR, an EGFP coding sequence was added upstream of the 32A9-CAR fragment separated by an F2A sequence. The whole sequence was subcloned into the pLVX lentiviral vector. The same version of FMC63 4-1BB CAR targeting CD19 was used as a negative control [38 (link)]. To produce viral supernatant, HEK293T cells were cotransfected with CAR lentiviral vector, packaging plasmid and envelope plasmid using Lipofectamine 2000 according to the manufacturer’s protocol. The supernatant was collected and concentrated 72 h later. PBMCs were stimulated for 24 h with anti-CD3/anti-CD28 antibody-coated beads (Invitrogen, Carlsbad, CA) at a 2:1 bead-to-T cell ratio in growth medium supplemented with 50 U/ml IL-2 (GenScript, Nanjing, Jiangsu). Activated T cells were then transduced with the lentivirus expressing CARs (MOI = 10). Cells were counted and fed fresh growth medium every other day. The transduction efficiency was detected by GFP fluorescence with flow cytometry.