Design, expression, and signaling of the anti-CD7 CAR. (A) Schema of the anti-CD7–41BB-CD3ζ construct. (B) Flow cytometric analysis of Jurkat cells transduced with either GFP alone (Mock) or GFP plus anti-CD7 CAR. Dot plots illustrate GFP fluorescence, and CAR expression after staining with biotin-conjugated goat anti-mouse F(ab′)2 antibody and streptavidin-APC (Jackson ImmunoResearch Laboratories). (C) Western blot analysis of CAR expression in Jurkat cells. Cell lysates of mock- and CAR-transduced Jurkat cells were separated on a 10% polyacrylamide gel under reducing or nonreducing conditions. The blotted membrane was probed with mouse anti-human CD3ζ antibody (8D3; BD Biosciences) and goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (R&D Systems). Antibody binding was revealed with Clarity Western ECL Substrate (Bio-Rad). (D) Anti-CD7 CAR induces expression of activation markers on ligation. Bars show the mean (± SD) of CD25 and CD69 MFI in CAR- and mock-transduced Jurkat cells after 24 hours with or without CD7⁺ MOLT-4 cells. P values by Student t test are shown for significant differences (*P = .016; ***P < .001). (E) Representative flow cytometric histograms of the experiments shown in panel D.