Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Interferon-gamma, or IFN-γ, a cytokine secreted by the lymphocytes T cells and natural killer cells or NK cells, mediates the immune response against pathogens.

To study in situ IFN-γ production, take a mouse transplanted with fluorescently-labeled T cells engineered to express a receptor recognizing ovalbumin — a model antigen.

Take a suspension of pathogenic bacteria — genetically modified to express ovalbumin — and inject it intravenously into the mouse, causing a systemic infection. The circulating bacteria reach the spleen — a secondary lymphoid organ — which aids pathogen removal by facilitating interaction with immune cells.

Antigen-presenting cells engulf the bacteria and display the antigen. T cells bind to the antigen via its receptor, are activated, and produce IFN-γ.

Macrophage cells, infected with the bacteria, release immune mediators and upregulate activating ligands on their surface. Upon interaction with these signals, NK cells get activated and produce IFN-γ.

Inject an intracellular protein transport inhibitor to restrict IFN-γ secretion, causing its intracellular accumulation.

Harvest the mouse spleen. Fix the tissue and freeze it to aid in sectioning. Using a cryo-microtome, generate thin tissue sections, and place them on glass slides. Add NK cell-specific antibodies, which bind to the surface markers, and IFN-γ-specific antibodies, which stain the intracellular IFN-γ. These antibodies are labeled with different fluorophores.

Place the slide under a fluorescence microscope to detect IFN-γ inside the NK cells and T cells.