Investigating regulatory T cell responses to MAP antigen stimulation using a monocyte-derived macrophage antigen presentation system (P6078)

Abstract

Abstract Johne’s disease is a chronic wasting disease of wild and domestic ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Previously, we proposed that the chronic nature of Johne’s disease may result in development of a regulatory T cell (Treg) population in the host that functions to shift the immune balance away from a pro-inflammatory Th1-like response to an ineffective Th2-like immune response. However, studying the function and specificity of Tregs is difficult due to low abundance of Tregs in the periphery. Furthering this obstacle, Tregs are usually anergic and the proportion of Tregs that develop in response to any one infectious agent is small. To face these challenges, we developed an in vitro method using a combination of TGF-β, interleukin 2, and rapamycin to stimulate expansion of MAP-reactive Tregs from CD4+CD25+ peripheral blood mononuclear cells in contact with MAP-infected monocyte-derived macrophages. Enriched Tregs may subsequently be used in Treg-mediated suppression assays to explore differences in naïve T cell responses to MAP antigen stimulation, including induction of FoxP3 expression. Following PBMC stimulation, qPCR and flow cytometry are used to analyze expression of FoxP3 as well as various relevant cytokines including: IL-1, IFNγ, IL-10, and TGF-β. Results are compared between cattle that are test-negative and test-positive for Johne’s disease.