Quantitative assessment by flow cytometry of T-lymphocytes producing antigen-specific γ-interferon in Brucella immune cattle

Peripheral blood mononuclear cells (PBMC) isolated from cattle infected with Brucella secreted γ-interferon (IFN-γ) after antigen-specific stimulation with Brucellergene, which is a mixture of cytoplasmic proteins of rough Brucella melitensis B115. Following the depletion of the monocyte-macrophages from the PBMC, the enriched lymphocyte populations stimulated with Brucellergene did not produce IFN-γ. Two-colour immunofluorescence staining of intracellular IFN-γ and bovine cell surface molecules identified the cells producing IFN-γ among the PBMC stimulated with Brucellergene. Moreover, this method could be used to estimate the number of T-cells specifically producing IFN-γ. For a given animal, there is a significant correlation (p < 0.05) between the production of IFN measured by an ELISA of the supernatant of whole blood stimulated with Brucellergene and the number of T-cells producing IFN-γ after in vitro stimulation with Brucellergene. The development of the immunofluorescence staining technique provides a new tool for analysing and for measuring the T-cell immune response in cattle.