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Detection of Mycobacterium‐specific interferon‐gamma‐producing human T lymphocytes by flow cytometry
Author(s) -
Tilley Peter A. G.,
Me Juthika N.
Publication year - 2000
Publication title -
apmis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.909
H-Index - 88
eISSN - 1600-0463
pISSN - 0903-4641
DOI - 10.1034/j.1600-0463.2000.d01-6.x
Subject(s) - elispot , flow cytometry , antigen , peripheral blood mononuclear cell , interferon gamma , biology , immunology , antibody , staining , microbiology and biotechnology , cytokine , t cell , in vitro , cd8 , immune system , biochemistry , genetics
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‐γ (IFN‐γ) staining in 785 cells per 1times10 5 T cells counted, in mycobacterial antigen‐stimulated peripheral blood mononuclear cells from a BCG‐vaccinated subject. In comparison, only 14 IFN‐γ‐staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1times10 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‐γ staining correlated with IFN‐γ production using a published ELISPOT assay (r=0.927). IFN‐γ 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‐γ synthesizing cells.