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-Ba rr virus (EBV)-specific CD8(+) T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotox ic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumula tion of interferon gamma (IFN gamma) is detected by multiparameter flow cyt ometric analysis. EBV-specific CD8(+) T-cell frequencies ranged between 0.6 3% and 1.29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specif ic T-cell lines, it was shown that flow cytometric analysis is more sensiti ve than limiting dilution analysis for CTL precursors and enzyme-linked imm unospot assay detecting IFN gamma-producing T cells. The class I restrictio n of IFN gamma production was confirmed using an anti-class I monoclonal an tibody (MoAb). Information on other cytokine production of EBV-specific CTL s could be obtained using combinations of anti-cytokine MoAbs. The sensitiv e and rapid nature of the flow cytometric assay for EBV-specific CD8(+) T-c ell frequency has significant advantages for evaluation of EBV-specific CD8 (+) T-cell responses in PBMCs of patients with EBV-related diseases. a 1999 by The American Society of Hematology.