To prepare CAR T cells, Lenti-X cells were transiently transfected with the HIV7 CAR vector, as well as psPAX2 (Addgene plasmid no. 12260) and pMD2.G (Addgene plasmid no. 12259) packaging plasmids. One day later (day 1), primary T cells were activated using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) and cultured in CTL supplemented with IL-2 (50 U/ml). On the next day (day 2), lentiviral supernatant was harvested from Lenti-X cells, filtered using 0.45-μm polyethersulfone (PES) syringe filters (Millipore), and added to activated T cells. Polybrene (Millipore) was added to reach a final concentration of 4.4 μg/ml, and cells were spinoculated at 800g and 32°C for 90 min. Viral supernatant was replaced 8 hours later with fresh CTL supplemented with IL-2 (50 IU/ml). Half-media changes were then performed every 48 hours using CTL supplemented with IL-2 (50 U/ml). Dynabeads were removed on day 6; CD8+EGFRt+-transduced T cells were FACS (fluorescence-activated cell sorting)–purified on a FACSAriaII (BD Biosciences) on day 9.

To prepare K562/CD19 cells, Lenti-X cells were transiently transfected with psPAX2, pMD2.G, and an HIV7 lentiviral vector encoding CD19. To prepare K562/ROR1 cells, Lenti-X cells were transiently transfected with MLV g/p, 10A1, and an mp71 retroviral vector encoding ROR1. To prepare Raji/ffluc cells, Lenti-X cells were transiently transfected with psPAX2, pMD2.G, and an HIV7 lentiviral vector encoding GFP and firefly luciferase. Two days later, viral supernatant was filtered using a 0.45-μm PES syringe filter and added to K562 or Raji cells. Five days later, transduced cells were stained with mAbs specific for CD19 (HIB19, BioLegend) or ROR1 (2A2, Miltenyi Biotec) and FACS-purified on a FACSAria II to greater than 97% purity.

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