title: A sensitive ELISPOT assay for detection of CD8+ T lymphocytes specific for HLA class I-binding peptide epitopes derived from influenza proteins in the blood of healthy donors and melanoma patients.
abstract: An enzyme-linked immunospot (ELISPOT) assay was adapted to detect peptide-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs). In HLA-A1-, HLA-A2-, and/or HLA-A3-positive individuals, we determined the release of IFN-gamma on a single cell level in response to three different peptide epitopes derived from the influenza matrix protein and nuclear protein containing the HLA-A2.1- and HLA-A1- or HLA-A3-binding motif, respectively. Comparison of the ELISPOT assay with the standard chromium release assay revealed a close correlation between the number of peptide-specific IFN-gamma-releasing T cells in PBMCs and the level of specific cytotoxicity after 14 days of in vitro expansion. The ELISPOT assay detected T cells with specificity for the HLA-A2. 1-binding epitope derived from the matrix protein in 76% of HLA-A2-positive healthy individuals (n = 25); the median frequency was 41 in 10(6) PBMCs. We also detected peptide-specific T cells in 10 of 12 HLA-A2-positive patients with metastatic melanoma with a median frequency of 20.5 in 10(6) PBMCs. In 10 of 24 HLA-A3-positive individuals and in 2 of 14 HLA-A1-positive individuals, peptide-specific T cells for a HLA-A3- and a HLA-A1-binding epitope derived from the nucleoprotein, respectively, were present. In conclusion, the ELISPOT assay may be suitable to monitor a peptide-specific T-cell response in vaccination protocols using peptides derived from tumor or viral antigens.
authors.affiliation: Department of Hematology/Oncology, Medizinische Klinik V, University of Heidelberg, 69115 Heidelberg, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Scheibenbogen
authors.forename: K H
authors.initials: KH
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Mayer
authors.forename: S
authors.initials: S
authors.lastname: Stevanovic
authors.forename: U
authors.initials: U
authors.lastname: Moebius
authors.forename: W
authors.initials: W
authors.lastname: Herr
authors.forename: H G
authors.initials: HG
authors.lastname: Rammensee
authors.forename: U
authors.initials: U
authors.lastname: Keilholz
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D015788:HLA-A1 Antigen / Q000378:metabolism; D015791:HLA-A3 Antigen / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D008545:Melanoma / Q000097:blood / Q000276:immunology*; D009698:Nucleoproteins / Q000097:blood / Q000276:immunology; D009975:Orthomyxoviridae / Q000276:immunology* / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D018984:Epitopes, T-Lymphocyte; D015788:HLA-A1 Antigen; D015791:HLA-A3 Antigen; D015395:Histocompatibility Antigens Class I; D009698:Nucleoproteins; D010455:Peptides; D014764:Viral Proteins; D007371:Interferon-gamma