title: Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells.
abstract: 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-gamma. 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.
authors.affiliation: Division of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan. kkuzushi@aichi-cc.jp
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Kuzushima
authors.forename: Naomi
authors.initials: N
authors.lastname: Hayashi
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kudoh
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Akatsuka
authors.forename: Kunio
authors.initials: K
authors.lastname: Tsujimura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Morishima
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tsurumi
mesh_terms: D000465:Algorithms; D017951:Antigen Presentation; D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology*; D059871:HLA-A24 Antigen; D004854:Herpesvirus 4, Human / Q000276:immunology*; D017874:Immediate-Early Proteins / Q000276:immunology; D007371:Interferon-gamma / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D015534:Trans-Activators / Q000276:immunology; D014162:Transfection; D014763:Viral Matrix Proteins / Q000276:immunology; D014764:Viral Proteins
chemical_list: D000956:Antigens, Viral; C068382:BRLF1 protein, Human herpesvirus 4; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D000939:Epitopes; D015234:HLA-A Antigens; C438506:HLA-A*24:02 antigen; D059871:HLA-A24 Antigen; D017874:Immediate-Early Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C090236:SM protein, Human herpesvirus 4; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D007371:Interferon-gamma