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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3094-3100
Rapid Determination of Epstein-Barr Virus-Specific CD8+
T-Cell Frequencies by Flow Cytometry
Kiyotaka Kuzushima,
Yo Hoshino,
Ken Fujii,
Naoaki Yokoyama,
Masatoshi Fujita,
Tohru Kiyono,
Hiroshi Kimura,
Tsuneo Morishima,
Yasuo Morishima, and
Tatsuya Tsurumi
From the Laboratory of Viral Oncology, Aichi Cancer Center Research
Institute, Nagoya, Japan; the Department of Hematology and
Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan; and the
Department of Pediatrics, Nagoya University School of Medicine, Nagoya,
Japan.
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  (IFN) 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 IFN-producing T cells.
The class I restriction of IFN 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.
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