Analysis of the antigen-specific IFN-γ producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 106 cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-γ production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-γ in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3–4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-γ producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-γ and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-γ following stimulation with PPD, live or killed BCG. animals analysed, the relative percentage of circulating IFN-γ producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-γ secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-γ only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-γ only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-γ production showing the key role of this cell population for the specific IFN-γ production.