title: Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens.
abstract: CD8(+) cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-gamma ELISPOT assay to the quantitative analysis of EBV-specific CD8(+) cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8(+) cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/10(6) CD8(+) cells). LMP2 peptide-responsive CD8(+) cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/10(6) PBMC). BMLF1 peptide-responsive CD8(+) cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/10(6) PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8(+) cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8(+) cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8(+) cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8(+) responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines. (Blood. 2000;95:241-248)
authors.affiliation: Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: V M
authors.initials: VM
authors.lastname: Lemas
authors.forename: I W
authors.initials: IW
authors.lastname: Flinn
authors.forename: C
authors.initials: C
authors.lastname: Krone
authors.forename: R F
authors.initials: RF
authors.lastname: Ambinder
mesh_terms: D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000494:pharmacology; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; C403195:glycyl-leucyl-cysteinyl-threonyl-leucyl-valyl-alanyl-methionyl-leucine