Generation in vitro of HLA‐restricted EB virus‐specific cytotoxic human T cells by autologous lymphoblastoid cell lines: The roles of previous EB virus infection and foetal calf serum
Authors: Misko I. S. , Kane R. G. , Pope J. H. 
Published: February 2007

The cytotoxic response of human lymphocytes to the autologous lymphoblastoid cell line (LCL) was investigated with five Epstein‐Barr virus (EBV)‐seropositive and four seronegative donors and an additional donor who seroconverted during the course of the study. LCL stimulation was carried out usually at a stimulator‐to‐responder cell ratio which varied from 1:200 to 1:20,000. Cytotoxic T cells from 7‐ and 14‐day cultures were assayed on autologous and allogeneic LCL target cells and the K562 line, using the 51Cr release assay. Without exception, the cytotoxic T cells generated in 7‐ and 14‐day LCL‐stimulated cultures from seropositive donors were strongly lytic for the autologous LCL. With some donors, the cytotoxic response increased with increasing autologous LCL stimulation, while with others even the lowest level of stimulation induced a potent response. Overall, the cytotoxic T cells generated in cultures from seropositive donors showed a striking preferential lysis of the autologous LCL and cross‐reactive killing of allogeneic LCL target cells matched for certain HLA‐B antigens. Lysis of the K562 line, an indicator target for natural‐killer‐like activity, was consistently negligible. In comparison to the considerable self‐preferred lysis obtained with T cells from LCL‐stimulated cultures, the level of autologous killing by T cells generated in control unstimulated cultures was relatively low. The cytotoxicity patterns obtained with cytotoxic T cells generated in cultures from seronegative donors were markedly different from those obtained with seropositive donors. The level of autologous killing by T cells harvested from LCL‐stimulated cultures was considerably lower than that obtained with seropositive donors and often did not increase above that seen in control cultures, except at the highest stimulatory dose. Moreover, preferential lysis of the autologous LCL target cells by T cells from LCL‐stimulated cultures was essentially due to the foetal calf serum (FCS)‐induced cytotoxicity and there was no obvious HLA‐antigen‐restricted killing of allogeneic target cells. Lysis of K562 target cells was generally more pronounced at the highest level of stimulation than that observed with seropositive donors. The distinctive cytotoxic T‐cell response patterns observed with the seropositive and seronegative donor cultures were both observed with cultures from an individual donor who was tested before and after infectious mononucleosis. Our cytotoxicity data are interpreted as indicating that with LCL‐stimulated cultures from seropositive donors the cytotoxic T‐cell response to autologous LCL is predominantly directed against EBV‐associated antigens and is HLA‐antigen restricted. In contrast, with seronegative donor cultures, the cytotoxic T‐cell response is primarily due to FCS‐induced cytotoxicity, although a response against other LCL‐associated antigens may be superimposed in cultures stimulated with high levels of autologous LCL.