Reactivation of viral-specific T cells is not impaired in the presence of EBV-CTLs expressing the CD30CAR. Autologous EBV-CTLs engineered to express the CD30CAR were added to cultures of PBMCs stimulated to reactivate CMV- or adenovirus-specific CTLs. (A) The percentage of pp65-tetramer⁺ T cells generated in a representative donor by day 9 of culture in the presence of nontransduced EBV-CTLs (top plot), EBV-CTLs transduced with an irrelevant CAR (middle plot), or the CD30 CAR⁺ (bottom plot) are shown. (B) Cells from the coculture were stained with the goat anti–human IgG (H + L) Ab to demonstrate the continued presence of the CD30CAR⁺ CTLs throughout the culture. As expected, no CAR⁺ CTLs were detectable in cocultures where NT CTLs were added. In contrast, 21% and 25% CAR⁺ CTLs were detectable at the end of the cocultures where irrelevant CAR or CD30CAR⁺ CTLs were added, respectively. (C) The IFN-γ–specific Elispot assay of coculture from 2 representative donors is shown. Mean frequency (± SD) of IFN-γ–producing T cells in response to the CMV-specific peptides NLV and TRP is shown. (D) The percentage of adeno-tetramer⁺ T cells (shown is the analysis with the 2 available tetramers) is shown in the only donor whose viral-specific response was reduced when CD30CAR⁺ EBV-CTLs were added to the culture (see also Table 3).