title: Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry.
abstract: 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 gamma (IFNgamma) 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 IFNgamma-producing T cells. The class I restriction of IFNgamma 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.
authors.affiliation: Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kuzushima
authors.forename: Y
authors.initials: Y
authors.lastname: Hoshino
authors.forename: K
authors.initials: K
authors.lastname: Fujii
authors.forename: N
authors.initials: N
authors.lastname: Yokoyama
authors.forename: M
authors.initials: M
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Kiyono
authors.forename: H
authors.initials: H
authors.lastname: Kimura
authors.forename: T
authors.initials: T
authors.lastname: Morishima
authors.forename: Y
authors.initials: Y
authors.lastname: Morishima
authors.forename: T
authors.initials: T
authors.lastname: Tsurumi
mesh_terms: D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology*; D002469:Cell Separation / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000956:Antigens, Viral; D007371:Interferon-gamma