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, Tatsuya Tsurumi

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89 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)3094-3100
Number of pages7
Issue number9
Publication statusPublished - Nov 1 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology


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