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Detection of Mycobacterium-specific interferon-gamma-producing human T lymphocytes by flow cytometry
1Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
Insights
This study introduces a rapid flow cytometry method to detect rare mycobacterial antigen-specific cytokine-producing cells. This technique accurately identifies interferon-gamma (IFN-γ) synthesis in T cells within 24 hours.
Area of Science:
- Immunology
- Cellular Biology
- Medical Diagnostics
Background:
- Flow cytometry is valuable for studying cytokine synthesis in specific cell populations.
- Existing methods often require mitogen stimulation or prolonged antigen cultures.
- A need exists for a faster method to detect antigen-specific cytokine production.
Purpose of the Study:
- To develop and validate a novel, rapid flow cytometry method for detecting rare mycobacterial antigen-specific cytokine-producing cells.
- To enable the enumeration of these cells within a single day.
- To improve the efficiency of immunological assays for mycobacterial infections.
Main Methods:
- Utilized flow cytometry with FITC-conjugated isotype-matched control antibodies to identify and exclude non-specific fluorescence.
- Cultured peripheral blood mononuclear cells (PBMCs) from BCG-vaccinated subjects with mycobacterial antigens for 12-24 hours.
- Quantified interferon-gamma (IFN-γ) producing cells and correlated results with ELISPOT assays and Mantoux skin tests.
Main Results:
- Demonstrated detection of 785 IFN-γ-producing cells per 10^5 T cells in stimulated PBMCs, compared to 14 in unstimulated cultures.
- Observed specific responses detectable after 12 hours and peaking at 24 hours.
- Found a strong correlation (r=0.927) between flow cytometry IFN-γ staining and ELISPOT assay results.
- Showed significantly higher IFN-γ staining in PBMCs from Mantoux skin test-positive subjects (p=0.0045).
Conclusions:
- Short-term culture flow cytometry is an effective method for enumerating antigen-specific IFN-γ synthesizing cells.
- This technique offers a rapid and reliable alternative for assessing cellular immune responses to mycobacterial antigens.
- The method holds potential for diagnosing and monitoring mycobacterial infections and evaluating vaccine efficacy.
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.
