Reactivation of epstein‐barr virus‐specific cytotoxic t cells by in vitro stimulation with the autologous lymphoblastoid cell line

Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co‐cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB‐virus‐transformed B‐cell line. The fresh cells present in co‐cultures from EB virus antibody‐negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co‐cultures from seropositive donors. Larger‐scale co‐cultures, set up at a ratio of 80–100 fresh UM cells to one autologous virus‐transformed B cell, were harvested after 8 to 12 days and the T‐cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co‐cultures and were strongly active against the autologous virus‐transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome‐negative target cell lines chosen as sensitive indicators of non‐specific cytotoxicity. Recognition of allogeneic EB‐virus‐transformed cells was restricted to those whose HLA‐A and/or B and/or C antigen expression matched that of the effector cells themselves; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB‐virus‐specific HLA‐restricted memory T cells, present in the blood of previously‐infected individuals, can be reactivated in vitro using the established autologous virus‐transformed cell line as a stimulus. The reactivated cytotoxic cells appear to recognize a virus‐induced lymphocyte‐detected membrane antigen, LYD‐MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious mononucleosis.