Routine detection of Epstein-Barr virus specific T-cells in the peripheralblood by flow cytometry

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peri pheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cyt okine producing) T-cells are then identified by how cytometry. To date, thi s technique has not been reliably used to detect Epstein-Barr virus (EBV)-s pecific T-cells primarily due to the superantigen/mitogenic properties of t he virus which non-specifically activate T-cells. By modifying culture cond itions under which the antigens are presented, we have overcome this limita tion and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by Row cytometry requires an ex tremely strong signal (such as culture in the presence of PMA and ionomycin ). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, bu t antigen presentation will continue uninhibited. Using this method. EBV-sp ecific T-cells may be legitimately detected using flow cytometry. No reduct ion in the numbers of antigen-specific T-cells was observed by the early re moval of target antigen when verified using cytomegalovirus antigen (a viru s with no non-specific T-cell activation properties). In EBV-seropositive i ndividuals, the phenotype of the EBV-specific cytokine producing T-cells wa s evaluated using four-color flow cytometry and found to be CD45(+), CD3(+) , CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimula tion of circulating previously unactivated memory T-cells. No cytokine prod uction was observed in CD4(+) T-cells from EBV-seronegative individuals, co nfirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFN gamma /IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactiv ation. (C) 2001 Elsevier Science B.V. All rights reserved.