CD123-specific CARs can be expressed in healthy donor human T cells. (A) Schematic diagram of the CAR containing a modified immunoglobulin G4 hinge, a modified transmembrane and intracellular signaling domain of CD28, and the CD3ζ signaling domain. The T2A ribosomal skip sequence and the EGFRt transduction marker are also indicated. (B) Representative phenotype of mock- and lenti-transduced T cells derived from a single healthy donor. After immunomagnetic selection and 1 cycle of expansion, CAR-modified T cells were stained with biotinylated anti-Fc or biotinylated anti-cetuximab followed by phycoerythrin-conjugated streptavidin and anti-TCRα/β, anti-CD4, or anti-CD8 and analyzed by flow cytometry. Quadrant placement is based on staining with isotype controls, and the percentage of cells falling in each quadrant is indicated. (C) Expression of indicated cell surface markers from 3 different healthy donor T-cell lines following immunomagnetic selection and 1 cycle of expansion. Data represent mean values ± standard error of the mean (SEM).