Rapid determination of Epstein-Barr virus-specific CD8⁺ T-cell frequencies by flow cytometry

We have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8⁺ T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon γ (IFNγ) is detected by multiparameter flow cytometric analysis. EBV-specific CD8⁺ T-cell frequencies ranged between 0.63% and 1.29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV- specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme- linked immunospot assay detecting IFNγ-producing T cells. The class I restriction of IFNγ production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV- specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8⁺ T-cell frequency has significant advantages for evaluation of EBV- specific CD8⁺ T-cell responses in PBMCs of patients with EBV-related diseases.