title: Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens.
abstract: We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.
authors.affiliation: Medizinische Klinik, Johannes Gutenberg-Universität Mainz, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Herr
authors.forename: J
authors.initials: J
authors.lastname: Schneider
authors.forename: A W
authors.initials: AW
authors.lastname: Lohse
authors.forename: K H
authors.initials: KH
authors.lastname: Meyer zum Büschenfelde
authors.forename: T
authors.initials: T
authors.lastname: Wölfel
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015488:HIV Antigens / Q000737:chemistry / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D008545:Melanoma / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D015488:HIV Antigens; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha