EBV-CTLs expressing the CD30CAR retain their ability to kill EBV⁺ tumor cells. (A)The mean (± SD) of ⁵¹Cr release from target cells exposed to EBV-CTLs from 8 donors transduced with an irrelevant CAR (left) and CD30CAR (right) is shown. CD30CAR⁺ CTLs lysed autologous LCLs (□) and the EBV⁻/CD30⁺ HD-derived cell line HDLM-2 (●; P < .05), whereas control CTLs showed significant lysis only of autologous LCLs. Autoreactivity was excluded by the absence of lysis of autologous PHA blasts (*). (B) CD30CAR⁺ CTLs (■) retain their killing activity against autologous LCLs and acquire the ability to kill allogeneic LCLs (* P < .05). As LCLs express CD30, this suggests that the observed lysis of allogeneic LCLs is mediated by the CAR. Bars indicate SD. Panel C shows that killing (at 20:1 E/T ratio) by CD30CAR⁺ CTLs of autologous LCLs (■) is not inhibited by incubation with class I MHC MAb (▩). This suggests that that killing of LCLs can still be mediated by the engagement of the CAR with the CD30 molecule expressed on LCLs. Bars indicate SD. (D) The expression of CD30 antigen on LCLs after depletion or enrichment for the CD30 molecule using immunomagnetic beads (MACS system) in 1 representative donor is shown. The percentage of specific ⁵¹Cr release at 40:1 E/T ratio of CTLs against CD30-depleted and CD30-selected autologous LCLs in 3 donors is shown in the bottom panel. Bars indicate SD.