The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens.
Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8+ T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8+ T cells. We conclude that the combined use of the TNF-alpha ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.
Herr
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Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität Mainz, Germany.
Linn
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Leister
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Wandel
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Meyer zum Büschenfelde
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Wölfel
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D000328:Adult; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003587:Cytomegalovirus / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018655:Lymphocyte Count / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D008875:Middle Aged; D010455:Peptides / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000276:immunology
D015789:HLA-A2 Antigen; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus