Detection and quantification of blood-derived CD8 + T lymphocytes secreting tumor necrosis factor α in response to HLA-A2.1-binding melanoma and viral peptide antigens

• Published in: Journal of immunological methods 1996-05, Vol.191 (2), p.131-142
• Main Authors: Herr, Wolfgang, Schneider, Jörg, Lohse, Ansgar W., Meyer zum Büschenfelde, Karl-Hermann, Wölfel, Thomas
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Amino Acid Sequence; Antigens, Neoplasm - chemistry; Antigens, Neoplasm - immunology; Biological and medical sciences; CD8 + T lymphocyte; CD8-Positive T-Lymphocytes - chemistry; CD8-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - metabolism; ELISPOT assay; Enzyme-Linked Immunosorbent Assay - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; HIV; HIV Antigens - chemistry; HIV Antigens - immunology; HLA-A2 Antigen - chemistry; HLA-A2 Antigen - immunology; HLA-A2.1; Humans; Kinetics; Lymphocyte Activation; Lymphocyte Count; Melanoma - immunology; Melanoma antigen; Molecular immunology; Molecular Sequence Data; Peptide Fragments - immunology; Protein Binding - immunology; Techniques; Tumor Cells, Cultured; Tumor necrosis factor α; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(96)00007-5

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 × CEM.T2 cells. Tumor necrosis factor α (TNF-α) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-α 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-α spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-α 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.