Aleksandar Madjovski

Objective: To assess the role of N-linked glycosylation in the development of CNS autoimmunity. Background: The susceptibility to develop autoimmunity within the Central Nervous System is increased in the absence of N-linked glycosylation. Design/Methods: Surface expression of N-glycans was determined by flow cytometry after staining with PHA-L in naïve SJL mice and mice with PLP139-151 induced experimental autoimmune encephalomyelitis. N-glycan expression was further assessed on dendritic cells after in vitro stimulation with LPS and during the development of induced regulatory T cells using FoxP3-GFP transgenic mice. Suppession of N-glycosylation was achieved by incubation with the alpha 1,2-mannosidase inhibitor kifunensine. iTregs were further assessed by flow cytometry and using T cell suppression assays. Results: N-glycans were detected on Th1, Th2, Th17 cells and Tregs, B cells, and CD11c+CD4+ and CD11c+CD4- dendritic cells in naïve mice. N-glycan expression was increased on Th1, Th2, Th17 cells and Tregs, B cells and dendritic cells in peripheral lymphoid organs during the development of experimental autoimmune encephalomyelitis (EAE). Dendritic cells increased N-glycan expression after TLR-4 mediated stimulation and reduction of N-glycosylation by kifunensine inhibited dendritic cell activation. CD4+ T lymphocytes increased N-glycan expression after TCR-stimulation and inhibition of N-glycosylation suppressed their differentiation into induced regulatory T cells. Inhibition of N-glycosylation in Tregs led to reduced expression of CTLA-4, CCR6, and PD-1 and inhibited their ability to suppress effector T cell proliferation. These data indicate that N-glycosylation is critical for differentiation and function of inducible regulatory T cells. This mechanism may contribute to the reduced susceptibility to develop CNS autoimmunity in the absence of N-glycans.