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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-04-1240.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1460-1468
IMMUNOBIOLOGY
Tetramer-assisted identification and characterization of epitopes
recognized by HLA A*2402-restricted Epstein-Barr virus-specific
CD8+ T cells
Kiyotaka Kuzushima,
Naomi Hayashi,
Ayumi Kudoh,
Yoshiki Akatsuka,
Kunio Tsujimura,
Yasuo Morishima, and
Tatsuya Tsurumi
From the Divisions of Immunology and Virology, Aichi
Cancer Center Research Institute, and the Department of Hematology and
Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan.
We determined cytotoxic T lymphocyte (CTL) epitopes through
screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8+ T cells as
responders. In addition, to confirm that the epitopes were generated
after endogenous processing and presentation of the EBV proteins, a
novel T-cell receptor (TCR) down-regulation assay was introduced, in
which a fluorescent tetrameric major histocompatibility complex
(MHC)/peptide complex was employed for detecting TCR
down-regulation after stimulation with the epitope presented on
antigen-presenting cells. Through such screening, 3 HLA
A*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and
DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24-positive fibroblast
cells infected with recombinant vaccinia viruses expressing LMP2.
Furthermore, ELISPOT assays with an epitope-specific CTL clone
demonstrated that the presentation was partially restored by
pretreatment of the fibroblast cells with interferon- . The epitope
was presented on transporters associated with antigen processing
(TAP)-negative T2 cells transfected with plasmids encoding HLA A*2402
and the minimal epitope, indicating that the presentation is TAP
independent. In conclusion, the 3 epitopes thus defined could be useful
for studying EBV-specific CD8+ T-cell responses among
populations positive for HLA A*2402.
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