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 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.
Kuzushima
K
K
Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan.
Hoshino
Y
Y
Fujii
K
K
Yokoyama
N
N
Fujita
M
M
Kiyono
T
T
Kimura
H
H
Morishima
T
T
Morishima
Y
Y
Tsurumi
T
T
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
D000956:Antigens, Viral; D007371:Interferon-gamma