Expression of anti-CD7 CAR in human peripheral blood T cells results in fratricide, which is prevented by CD7 downregulation. (A) The percentage of viable T cells recovered 24 hours after electroporation with or without anti-CD7 CAR mRNA (n = 7). Viable cells were counted by flow cytometry. (B) The percentage of viable T cells recovered 24 hours after CAR transduction with a retroviral vector as compared with cells from the same donors transduced with GFP alone (Mock) (n = 10). (C) The percentage of viable CAR- or mock-transduced T cells recovered during the week after transduction. Shown are follow-up results for 5 of the 10 experiments shown in panel B. (D) The percentage of CD107a in T cells after electroporation with or without anti-CD7 CAR mRNA. Mean (± SD) of triplicate measurements are shown. (E) Schematic representation of anti-CD7 PEBL constructs. (F) Representative flow cytometric histograms illustrate CD7 expression in T lymphocytes after retroviral transduction of 3 anti-CD7 PEBLs or mock-transduced GFP alone (Mock). T cells were stained with anti-CD7–PE (M-T701; BD Biosciences). (G) The percentage of CD7 expression in T cells retrovirally transduced with the anti-CD7 PEBL-1 or mock-transduced (n = 5). (H) Flow cytometric dot plots illustrate downregulation of CD7 expression in T cells by PEBL transduction together with expression of anti-CD7–41BB-CD3ζ CAR 12 hours after electroporation with or without CAR mRNA. Cells were stained with biotin-conjugated goat anti-mouse F(ab′)2 antibody and streptavidin-APC (Jackson ImmunoResearch Laboratories). (I) The percentage of viable T cells transduced with anti-CD7 PEBL recovered 24 hours after electroporation of anti-CD7 CAR mRNA as compared with cells electroporated with the anti-CD7 CAR mRNA, but transduced with a vector without anti-CD7 PEBL (n = 6). The number of viable cells was measured by flow cytometry. **P < .01; ***P < .001.