Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-γ-producing CD8⁺ cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4⁺ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients. Received: 4 October 2000