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

Abstract

Epstein-Barr virus (EBV) is a B lymphotrophic human herpes virus which is the causative agent of Infectious Mononucleosis (IM) (Henle et al., 1968) and is aetiologically associated with Burkitt's lymphoma (Epstein & Achong, 1979) and nasopharyngeal carcinoma (Epstein, 1978). In most individuals infection occurs subclinically during childhood (Evans et al., 1968) and leads to the production of antibodies to virus-determined antigens which thereafter persist for life (Hewetson et al., 1973). Following either IM or sub-clinical infection EB virus persists in the body, and can be found in saliva (Golden et al., 1973) and in lymphoid tissue (Nilsson et al., 1971). This persistent infection with EBV is probably, at least in part, controlled by EBV-specific memory T cells which have been demonstrated in the peripheral blood of seropositive individuals (Moss et al., 1978). These T cells are activated in cultures of EB virusinfected peripheral blood mononuclear cells to produce cytotoxic cells which then cause regression of proliferating foci of the EB virus-infected B cells within the culture (Rickinson et al., 1979) in an HLA-restricted manner (Rickinson et al., 1980).In the present study we have used the fluorescence activated cell sorter (FACS) to separate peripheral blood cells stained with monoclonal antibodies into subsets with specific activities. These subsets have then been assayed for their capacity to cause regression of autologous EBV-infected B cell targets. Materials and methodsMedium RPMI 1640 medium containing 2mM glutamine, 100 IU penicillin and 100 IU streptomycin was used throughout. 5 mM HEPES buffer and 2% calf serum were added for all cell preparation procedures, and 20% v/v foetal calf serum (FCS) was added for all culture procedures. DonorsNormal healthy adults who were seropositive for antibodies to EB virus were selected as leucocyte donors.Cell preparation Whole blood was diluted with an equal volume of medium and centrifuged on a Ficoll-hypaque gradient. The peripheral blood mononuclear cells (PBMC) were harvested from the interface band and washed twice in medium. E rosettes were formed using AET-treated sheep red cells by the method of Kaplan & Clark (1974) and the E rosette-positive population (E+) was separated from the E rosette-negative population (E-) on a percoll gradient (Callard & Smith, 1981). The E-cells were harvested from the interface band and the E+ cells were recovered from the pellet by lysis of the red cells with 0.83% ammonium chloride. Both cell populations were washed twice in medium before use.(C) The

CD4+T-Cell Effectors Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation

“…Most previous in vitro studies have monitored PBMC culture responses to EBV via a regression assay, in which the readout is microscopic enumeration of transformed-cell foci after 4 weeks in culture or isolation of transformed lymphoblastoid lines (37,40). As a quantitative and rapid assay for T-cell control, we initially assessed cell proliferation in EBV-infected and mock-inoculated PBMC cultures by measuring [ 3 H]dT incorporation 17 to 18 days after inoculation (13,26,33,55).…”

Exposure to Holoendemic Malaria Results in Suppression of Epstein‐Barr Virus–Specific T Cell Immunosurveillance in Kenyan Children

“…In countries such as Kenya, EBV infection generally occurs by 2 years of age [8][9][10][11] and persists for life as a latent infection in memory B cells [12]. The lytic phase of viral replication is thought to occur after plasma cell differentiation [13][14][15]. The virus is periodically shed in the saliva, suggesting that there is ongoing lytic-cycle reactivation [16].…”

Regression of Epstein-Barr Virus-Induced B-Cell Transformation In Vitro Involves Virus-Specific CD8+T Cells as the Principal Effectors and a Novel CD4+T-Cell Reactivity

“…Much of the early literature using this system indicated that regression was associated with the appearance of EBV latent antigen-specific T cells that were operationally HLA class I-restricted in cytotoxicity assays on LCL targets (28,29,38,50) and were therefore assumed to be CD8 ϩ T cells. Indeed, the first experiments separating CD4 ϩ and CD8 ϩ T cells from PBMC cultures strongly supported this assumption (7). A more recent analysis using natural killer (NK) and T-cell subset depletions, however, suggested that CD4 ϩ T cells were the principal effectors of regression (33).…”