T cells were obtained from peripheral blood apheresis samples from consenting healthy donor adults. CD4+ and CD8+ T cell populations were isolated from the samples via immunoaffinity-based enrichment, activated in a ratio of 1:1, transduced with a lentiviral vector encoding the various CD19-specific CAR constructs, and then expanded. All CAR constructs used the CD19-directed fmc63 scFv on an IgG4 hinge, CD28 transmembrane domain, CD3ζ stimulatory domain, and 4-1BB costimulatory domain. Cells were then cryopreserved, stored at temperatures below −130°C, and thawed before use. To account for any variability in transduction efficiency, numbers of transduced cells were normalized across all experiments.

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