The generation of LMP2a-specific cytotoxic T lymphocytes for the treatmentof patients with Epstein-Barr virus-positive Hodgkin disease

Based upon the success of using polyclonal, Epstein-Barr virus (EBV)-specif ic CTL lines for the prophylaxis and treatment of patients with post-transp lant lymphoproliferative disease (PTLPD), there is now considerable incenti ve to develop CTL directed against the subdominant EBV antigens EBNA1, LMP1 and LMP2, which are expressed by the tumor cells of Hodgkin disease and na sopharyngeal carcinoma. To develop a system for generating LMP2a-specific C TL in vitro, we transfected autologous immature dendritic cells (DC), which had been grown in the absence of serum, with LMP2a RNA in the presence of the cationic lipid DOTAP. This transfection method did not adversely affect the DC in terms of immunophenotyping and they expressed high levels of HLA class I and II and critical costimulatory molecules. These LMP2a(+) DC, as compared to DC which had been transfected with irrelevant RNA, were shown to be highly immunostimulatory in autologous mixed lymphocyte reactions and , importantly, could stimulate the generation of CD8(+) and CD4(+) CTL whic h exclusively recognized LMP2a-expressing targets. This specific cytotoxici ty was confirmed using antibody blocking experiments and cytotoxicity assay s of the separated T cell subsets. Using this DC-based system we could also reactivate LMP2a-specific memory in EBV-seropositive donors whose polyclon al CTL response to LCL stimulation did not contain a LMP2a-specific compone nt.