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

Abstract

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 5'Cr release assay. Without exception, the cytotoxic T cells generated in 7-and 14-day LCLstimulated cultures from seropositive donors were strongly lytic for the autologous L C L 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. Lysir of the K562 line, an indicator target for natural-killerlike activity, was consistently negligible. In comparison to the considerable self-preferred lysis obtained with T cells from LCLstimulated 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 LCLstimulated 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 LCLstirnulated cultures from seropositive donors the cytotoxic T-cell response to autologous LCL is predominantly directed against EBV-asrociated 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 L C L

Characterisation of Epstein-Barr virus-specific memory T cells from the peripheral blood of seropositive individuals

“…Our findings may reflect the fact that these cytotoxic T cells are constantly activated in vivo by the low numbers of EBV-carrying B lymphocytes known to be present in the circulation of all seropositive individuals (Nilsson et al, 1971) and that T-T interactions are important only in the generation of primary responses. Since FCS has been shown to selectively induce the generation of cytotoxic T cells with a preferential lysis for autologous cell lines (Misko et al, 1982) we carried out one further separation experiment using the Leu 2a antibody in which all the cell cultures were set up in medium containing 20% AB serum from a seronegative donor in place of FCS. In this experiment all the regression activity again occurred in the cultures receiving Leu 2a+ cells, and thus an active role for FCS in the generation of cytotoxic T cells in our experiments was excluded.…”

Cell-mediated immunity against epstein-barr virus infected B lymphocytes

“…Target panels showed great variation in the sensitivity of the individual lines, which correlated to their HL A make-up in that lytic effects were restricted to those which expressed HLA-A and HLA B antigens matching with the effectors [31,32]. The HLA-restricted cytotoxicity was substantiated by the inhibitory effect of monoclonal antibodies specific for common determinants on HLA A, B, and C molecules [63].…”

“…(1) After infection of the total lymphocyte population of seropositive donors, the cultures which showed the regression of BEB v growth contained T cells with lytic potential [34,44]. (2) Cocultivation of T cells with autologous LCLEB v activated them for cytotoxic potential [31,43,57]. In the majority of experiments these effects have been revealed in short-term 51Cr release assays.…”

“…In the second system, used more often, T cells were cocultivated with autologous LCL [31,43]. This system is complex and unless performed under special conditions it is difficult to show the EBV-specific component in the lytic assay because lymphoblastoid lines are known to act as polyclonal activators [4,17].…”

Cross-reactivity of self-HLA-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes for allo-HLA determinants.

“…1 legend and unpublished results). It was necessary to check this particular point since there are circumstances in which fetal calf serum-directed cytotoxicity can contaminate otherwise EB virus-specific cytotoxic T cell preparations (36,37).…”