Preclinical CAR T Cell Therapy

Six- to eight-week-old male or female NSG mice were obtained from the Jackson Laboratory or bred in-house. Mice were engrafted via tail vein with 5×10⁵ CD19⁺ Raji/ffluc cells, and 7 days later, injected intravenously with PBS or a defined product of purified CD8⁺ and CD4⁺ CD19-specific CAR T cells mixed together in a 1:1 ratio. Bioluminescence imaging was performed as described (49 (link)). Mice were either followed for survival or sacrificed on day 20 for analysis of T cell frequencies and phenotypes by flow cytometry. Peripheral blood was extracted, red blood cells were lysed using ACK Lysing Buffer (ThermoFisher), and remaining cells were stained with fluorochrome-labeled mAbs. Bone marrow was isolated from hind-limbs by mechanical disruption followed by red blood cell lysis, and staining with fluorochrome-labeled mAbs. Mice handlers were blinded to group allocation. The Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee approved all experimental procedures.