title: Detection of Mycobacterium-specific interferon-gamma-producing human T lymphocytes by flow cytometry.
abstract: Flow cytometry has proven to be a useful tool for the investigation of cytokine synthesis by selected cell subpopulations. While most reports have used mitogen stimulation or long-term cultures with antigen, we describe here a novel method to allow the detection of rare mycobacterial antigen-specific cytokine synthesizing cells within one day. The most important feature of this method is the use of an FITC-conjugated isotype-matched control antibody to identify and exclude cells which fluoresce non-specifically. With this technique, we demonstrate interferon-gamma (IFN-gamma) staining in 785 cells per 1 x 10(5) T cells counted, in mycobacterial antigen-stimulated peripheral blood mononuclear cells from a BCG-vaccinated subject. In comparison, only 14 IFN-gamma-staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1 x 10(5) T cells are stained by an irrelevant control antibody. Specific responses are detectable after 12 h of in vitro culture, and peak at 24 h. In volunteer health care workers, IFN-gamma staining correlated with IFN-gamma production using a published ELISPOT assay (r=0.927). IFN-gamma staining was also higher in PBMC from mantoux skin test-positive volunteers, compared to cells from skin test-negative subjects (p=0.0045). Flow cytometry following short-term culture can thus be used for enumeration of antigen-specific IFN-gamma synthesizing cells.
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
authors.forename: P A
authors.initials: PA
authors.lastname: Tilley
authors.forename: J N
authors.initials: JN
authors.lastname: Menon
mesh_terms: D000328:Adult; D001500:BCG Vaccine / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000096:biosynthesis*; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009161:Mycobacterium / Q000276:immunology*; D009163:Mycobacterium bovis / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D001500:BCG Vaccine; D007371:Interferon-gamma