Transduction of PBLs with a lentiviral vector containing a CD19 CAR. (A) A diagram of the CD19 CAR, consisting of the variable region light chain (V_L) and heavy chain (V_H) of the single chain variable fragment (scFv), CD8 hinge domain, CD28 costimulatory molecule, and TCR-ζ signaling domain attached to a truncated CD34 molecule (CD34t) with a T2A self-cleaving peptide inserted into the pLVX-E1a-N1 lentiviral vector. (B) Flow cytometry analysis of CD3⁺ cells transduced with the CD19 CAR-containing lentiviral vector either after no treatment, IL-7 culture, or CD3 activation. These results are representative of 10 experiments using cells from 10 different donors. (C) CD19 CAR T cells transduced either after IL-7 treatment or CD3 activation were cocultured with CD19⁺ Raji cells in a 1:1 ratio. CD19 CAR T cells cultured with T2 or media alone were used as negative controls. IFN-γ and TNF-α release in supernatants were measured by ELISA. Graphs depict 3 experiments using 3 different donors with each data point representing the triplicate mean and error bars indicating standard deviation. ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon gamma; TCR, T cell receptor; TNF-α, tumor necrosis factor α; WPRE, woodchuck hepatitis virus posttranscriptional response element.