LMP2-specific CTL lines derived from lymphoma patients contained functional LMP2-specific T-cell populations. This figure shows the LMP2-specific activity in a CTL line generated from a patient with relapsed lymphoma. The patient is representative of the 10 patients whose CTL lines recognized LMP2. To demonstrate cytolytic specificity of patient CTL lines in vitro, percentage specific ⁵¹Cr release was determined 6 hours after coincubation with HLA-matched fibroblasts transduced with Ad5LMP2 (■), or Ad5GFP (□), autologous LCLs (▴), and allogeneic LCLs (▵). The LMP2-specific CTL line from a representative patient shown in panel B showed killing of autologous LCLs and HLA-matched fibroblasts only if they were transduced with Ad5LMP2 (30% at an E/T ratio of 20:1). Killing was not due to adenovirus-directed CTLs, since fibroblasts infected with recombinant adenovirus encoding GFP were not recognized. There was no killing of HLA-mismatched LCLs, (A) or nontransduced fibroblasts (data not shown). Panel B shows the frequency of LMP2-specific T cells in the CTL line generated from the same patient (HLA A2;29/B13;27). CTLs were costained with PE-conjugated multimers CD8-FITC and CD3-PerCP. Multimers were as follows: LMP2-HLA-A*0201-LLW, HLA-A*0201-FLYALALLL, HLA A29-ILL, and EBNA 3C-HLA-B27-RRI. When compared with the EBV-LCL–activated CTL line, the LMP2-activated CTL line showed an increase in the frequency of T cells reactive with all 3 LMP2 tetramers, but decreased frequency in the EBNA3C tetramer reactivity (B). To assess the breadth and function of the LMP2-specific response, the CTLs were coincubated with overlapping LMP2 peptides and IFN-γ release in response to specific 15-mer and 8-mer peptides was measured in an ELISPOT assay. (C) This patient's polyclonal CTL line was then sorted for CD4⁺ and CD8⁺ T cells. Subsequently, recognition of LMP2 by these separated CD4⁺ and CD8⁺ T cells was determined in an IFN-γ ELISPOT assay using OKT3 blasts pulsed with either LMP2pepmix or CMVpepmix as the APCs (D). Error bars are SD.