The role of glycosylation in regulatory T-cell biology

Abstract

Regulatory T-cells (T-reg) play a pivotal role in maintaining immune homeostasis and tolerance. There has been a significant amount of research devoted to understanding their physiological mechanisms of action and the causes of their dysfunction in disease settings as well as to developing strategies for their therapeutic application. Glycosylation is ubiquitous in all mammalian cells and surface glycan expression is involved in the regulation of many biological processes. While it is currently well accepted that surface glycan expression influences T-cell development, trafficking and function, little is known about the relevance of glycosylation to T-reg biology. The main goal of this thesis was to characterize the surface glycosylation characteristics of T-reg and to determine how the surface glycan expression profile of T-reg correlates with their suppressive functions. Initially, the surface glycomes and relevant protein marker expression patterns of T-reg and conventional (non-regulatory) T-cells were compared in mouse lymphoid organs and human white blood cells by multi-colour flow cytometry using a panel of carbohydrate-binding proteins (lectins) and fluorescently-labelled antibodies. Next, to evaluate the suppressive potencies of different T-reg sub-populations, T-cells and antigen presenting cells were isolated by fluorescence-activated cell sorting and were co-cultured to perform a range of functional assays. The T-reg sub-populations evaluated by purification and co-culture included those with differential levels of surface glycosylation in vivo as well as those in which surface glycosylation was manipulated by treatment with the enzymes neuraminidase and PNGase F. The experimental results first revealed that T-reg surface glycosylation differs significantly from that of conventional T-cells in the resting state and is further modified by activation stimuli. Following this, correlations were identified between higher surface expression levels of certain glycan epitopes and higher expression of proteins known to be involved in mediating T-reg suppressive functions. Next, it was shown that T-reg with high-level binding of the lectins GSL-I and PHA-L have greater suppressive potency than those with lower lectin binding levels. Finally, enzymatic manipulation of mouse T-reg surface glycosylation resulting in decreased surface expression of sialic acids or N-glycans was found to significantly reduce their capacity to suppress conventional T-cell activation through contact-dependent mechanisms. The novel findings of the thesis demonstrate an important role for glycosylation in determining T-reg phenotype and function. They also indicate the potential to apply knowledge of surface glycan expression profiles to better define and purify T-reg subsets with enhanced immunotherapeutic potency.