Construction of mCAR-hCD19

The sequences for mCAR-hCD19 contained the antigen receptor of human CD19 or HER2 scFv; the murine CD3ζ, CD28, and/or 4-1BB; and the myc tag on the N terminus, as described before (43), and were synthesized by SyngenTech. This chimeric antigen construct was then cloned into a murine stem cell virus–green fluorescent protein (MSCV-GFP) (Clontech) murine retroviral vector (MSCV-myc-CAR-2A). Then, the Platinum-E (Plat-E) Retroviral Packaging Cell Line, Ecotropic cells (Cell Biolabs, RV-101) were transfected with mCAR-hCD19 plasmid and pCL-Eco retrovirus packaging plasmid to obtain retrovirus containing mCAR-hCD19. OT-I CD8⁺ T cells were activated with anti-CD3/CD28 beads (Gibco, 11453D), IL-2 (PerproTech, 212-12), and 55 μM β-mercaptoethanol (Gibco, 21985-023) for 24 hours. Then, OT-I CD8⁺ T cells were infected with the above virus in the presence of RetroNectin (Takara Bio) for 8 hours. Twenty-four hours later, the GFP-positive cells were sorted by flow cytometry using the BD Biosciences FACSAria II to obtain cells expressing high levels of hCD19 or hHER2.