Generation of AAV-CAR Tregs
(A) Schematic representation of transgene construct for AAV-CAR T cell. The AAV-specific scFv is fused to a human IgG1 CH2-CH3 hinge, followed by intracellular CD28 (human) and CD137 (4-1BB) (human) co-stimulatory domains, and CD3ζ (human), under the control of the EF1-alpha promoter. Truncated CD19 (external domain only) were added following the E2A self-cleavage peptide sequences. (B) Schematic representation of the AAV-CAR Treg construct. Murine FoxP3 cDNA, and truncated CD19 (external domain only) were added following the E2A self-cleavage peptide sequences. (C) Schematic diagram of T cell isolation and lentiviral transduction. Pan T cells are isolated from human blood or mouse spleen and activated for 2 days followed by lentiviral-CAR transduction; mixed population of transduced and non-transduced T cells are used in all experiments.