title: Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing.
abstract: Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli beta-galactosidase in mouse or human target cells, and an Elispot to detect release of beta-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-gamma secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.
authors.affiliation: David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, New York, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Snyder
authors.forename: William J
authors.initials: WJ
authors.lastname: Bowers
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Livingstone
authors.forename: F Eun-Hyung
authors.initials: FE
authors.lastname: Lee
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Federoff
authors.forename: Tim R
authors.initials: TR
authors.lastname: Mosmann
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018139:Simplexvirus / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D007371:Interferon-gamma; D001616:beta-Galactosidase