Growth kinetics, immunophenotype, and functionality of EBV-CTL lines are retained after transduction with CD30CAR. EBV-CTL lines were expanded from PBMCs obtained from 8 healthy EBV-seropositive donors by weekly stimulation with irradiated autologous LCLs and biweekly feeding with rhIL-2. EBV-CTLs were transduced with the CD30CAR or an irrelevant CAR (control CTLs) after the third stimulation. (A) CD30CAR expression was evaluated by flow cytometry using a goat anti–human IgG (H + L) Ab (—) or the isotype control (- - -). (B) The growth of EBV-CTLs was transduced with the CD30CAR (■) or an irrelevant CAR (□). The arrow indicates time of retroviral transduction. (C) The expression of CAR on transgenic CTLs over time is shown. The number of stimulations after transduction is indicated. Bars represent average (± SD) for the 8 donors. (D) The immunophenotype of the EBV-CTLs was transduced with the CD30CAR (■) compared with EBV-CTLs transduced with an irrelevant CAR (□). ▩ show the phenotype of the CD30CAR⁺ CTLs stimulated with CD30⁺ tumor cells. Means (± SD) are shown for the 8 donors. (E) CD30CAR can be detected on both CD8⁺-transduced (left plot) and CD4⁺-transduced (right plot) EBV-CTLs.