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

Peripheral blood mononuclear cells (PBMC) isolated from cattle infected wit h Brucella secreted gamma-interferon (IFN-gamma) after antigen-specific sti mulation with Brucellergene, which is a mixture of cytoplasmic proteins of rough Brucella melitensis B115. Following the depletion of the monocyte-mac rophages from the PBMC, the enriched lymphocyte populations stimulated with Brucellergene did not produce IFN-gamma. Two-colour immunofluorescence sta ining of intracellular IFN-gamma and bovine cell surface molecules identifi ed the cells producing IFN-gamma among the PBMC stimulated with Brucellerge ne. Moreover, this method could be used to estimate the number of T-cells s pecifically producing IFN-gamma. 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 num ber of T-cells producing IFN-gamma after in vitro stimulation with Brucelle rgene. The development of the immunofluorescence staining technique provide s a new tool for analysing and for measuring the T-cell immune response in cattle. (C) 1998 Elsevier Science B.V. All rights reserved.