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-γ. 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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