Retroviral vector design and expression of 19z1 and cytokine transgenes in human primary T cells. (A) Schematic diagram of bicistronic oncoretroviral vectors used for 19z1 and cytokine overexpression. 19z1 indicates human CD19-specific chimeric antigen receptor; black box, CD8 leader sequence; V_H, variable heavy chain; gray box, (Gly₃Ser)₄ linker; V_L, variable light chain; CD8, CD8 hinge and transmembrane domains; ζ-chain, TCR ζ-chain cytoplasmic domain; P2A, porcine teschovirus-1–derived 2A peptide; ΔLNGFR, doubly mutated human low-affinity nerve growth factor receptor; LTR, long terminal repeat; ψ, packaging signal; and pA, polyadenylation signal. (B) Cell surface expression of 19z1, CD3, CD4, and CD8 by transduced T cells 6 days after AAPC stimulation. Numbers at the bottom of the upper right quadrant represent the 19z1 MFI of the CD3⁺19z1⁺ subset. CD4/CD8 dot plots have been gated on the CD3⁺19z1⁺ subset. Dot plots are representative of 8 to 10 experiments using 6 different donors. (C) Secretion of IL-2, IL-7, IL-15, or IL-21 by transduced T cells. Equivalent numbers of 19z1⁺ T cells were washed and cultured in the presence or absence of an antireceptor blocking antibody. Culture supernatants were assayed for cytokine content by ELISA 12 hours later. Data shown are the average of 2 donors. ND indicates below limit of detection. (D) Overall accumulation of transduced T cells in response to tumor antigen exposure. T cells were transduced and exposed weekly to AAPCs. In parallel, control 19z1-ΔLNGFR T cells were cultured with titrating amounts of exogenously added IL-2, IL-7, IL-15, or IL-21. The number of viable 19z1⁺ T cells was assessed at the indicated time points. Arrows denote AAPC restimulation. The bottom right graph represents an overlay of the accumulation of cytokine-transduced T cells. Data are average (± SEM) of 3 donors.