EBV-CTLs redirected with CD30CAR retain their polyclonal EBV specificity. (A)The frequencies of tetramers recognizing lytic (BZLF1-RAK) or latent (EBNA3C-RPP and LMP2-CLG) EBV-associated antigens in control and transgenic EBV-CTLs generated from 3 different donors are shown. The bottom panels show that the same frequency of EBV-specific tetramers is maintained after transduction with CD30CAR. (B) The CD30CAR is also detectable on tetramer⁺ CTLs. (C) CFSE-labeled control (left panels) and CD30CAR⁺ CTLs (right panels) nonstimulated (top panels), stimulated with EBV⁺ (middle panels), or stimulated with CD30⁺ cells (bottom panels) are shown. After LCL stimulation (middle panels), both control and CD30CAR⁺ EBV-tetramer⁺ CTLs proliferate, as shown by decrease of CFSE⁺ cells. After stimulation with CD30⁺ cells (bottom panels), only CAR⁺ CTLs proliferate. (D) The frequency (mean ±SD) of T cells responding to EBV-specific peptides in control and CD30CAR⁺ EBV-CTLs from a representative donor, assessed by IFN-γ Elispot assay, is shown. Tetramer and Elispot analyses are representative of a total of 5 donors.