CD7 downregulation by PEBL does not alter T-cell phenotype, proliferation, and functionality. (A) The percentage of CD4 and CD8 cells 7 to 14 days after retroviral transduction with either anti-CD7 PEBL or GFP alone (Mock). Each symbol corresponds to a different T-cell donor. (B) The growth rate of PEBL- and mock-transduced T cells (from 3 donors) maintained with 200 IU/mL IL-2 for 14 days. Symbols represent the mean (± SD) of triplicate measurements. (C) PEBL- and mock-transduced T cells were electroporated with either anti-CD19–41BB-CD3ζ CAR mRNA or no mRNA. Flow cytometric dot plots illustrate GFP and CAR expression 12 hours after electroporation. CAR was detected with biotin-conjugated goat anti-mouse F(ab′)2 antibody and streptavidin-APC (Jackson ImmunoResearch Laboratories). (D) Cytotoxicity of PEBL- or mock-transduced T cells, electroporated with or without anti-CD19 CAR mRNA, against CD19⁺ ALL cells (OP-1). Bars show the mean (± SD) of 4-hour cytotoxicity at a 1:1 E:T ratio. (E) CD107a expression in T cells from experiments identical to those described in panel D. (F) IFN-γ production in PEBL- or mock-transduced T cells, electroporated with or without anti-CD19 CAR mRNA, and cocultured with OP-1 for 6 hours at an E:T ratio of 1:1. Bars represent the mean (± SD) of triplicate experiments. ***P < .001; ****P < .0001.