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.