The Cobbohydrates Laboratory (i.e. the laboratory of Brian A. Cobb, PhD) was founded on July 1, 2005 at Case Western Reserve University School of Medicine in the Department of Pathology. Dr. Cobb was recruited from Harvard Medical School by John B. Lowe, MD, the (at the time) newly appointed Chair of Pathology.
Much of the initial work in the lab focused upon the underlying mechanism of T cell activation by microbial polysaccharides. The foundation for this was the discovery (while Dr. Cobb was still a postdoctoral fellow in Dennis Kasper’s laboratory) that the capsular polysaccharide PSA from Bacteroides fragilis activates CD4 T cells via processing and presentation by the class II major histocompatibility complex (MHCII) in professional antigen presenting cells (APCs) (1). This was the first time a non-protein antigen was shown to be presented by the canonical MHCII glycoprotein. We further discovered that the oxidative action of nitric oxide downstream of iNOS induction was a critical step in PSA processing to low molecular weight fragments (2,3), analogous to protein processing by proteases in the lysosome. Mechanistic studies revealed that the interactions between MHCII and processed PSA were of high affinity (2), dependent upon PSA conformation (4), and competitive with peptides (1,2,3,4). Through these studies, we also found that the conserved complex N-linked glycans on MHCII were necessary for robust PSA binding and presentation, and therefore T cell activation (5).
Beyond the presentation pathway in APCs, we have also focused a great deal of effort in understanding the biological impact of PSA-mediate T cell activation and the mechanism by which those T cells function. We discovered that T cells recognize MHCII-presented PSA in a clonal fashion (6), and that the responding T cell population are highly suppressive in inflammatory models such as asthma (7) and the murine model for multiple sclerosis called EAE (experimental autoimmune encephalomyelitis)(8,9). More recently, we found that the type of T cells expanded by PSA recognition function to suppress the immune system (and therefore inflammatory disease) through cooperation with traditional FoxP3+ regulatory T cells (Tregs)(9,10). Responder T cells secrete (among other cytokines like IFNγ), IL-4 and IL-2. These two cytokines signal to Tregs in an unanticipated combinatorial fashion via STAT5 to elicit Treg proliferation and synergistic IL-10 expression and release (9). However, after 16 years of active research on PSA and the associated mechanisms and pathways, we have essentially ended the project and are now focused on two independent areas of investigation.
On the heels of our work with MHCII glycosylation and its impact on PSA presentation, we began a project in 2013 focused on IgG. IgG carries a single conserved site of N-linked glycosylation within the Fc domain, and many laboratories over the years have shown that the composition of that glycan changes as a function of disease (particularly inflammation) and those changes impact the biological function of the antibody (11). For example, when IgG is sialylated (i.e. carries glycans with α2,6-linked terminal sialic acids), the antibody tends to have a suppressive activity, whereas the lack of sialic acid and galactose trends towards pro-inflammatory activities. In 2016, we discovered that the sialyltransferase responsible for IgG sialylation (ST6Gal1) is dispensable in B cells (12). This remarkable observation means that although B cells are uniquely able to express IgG, they are not responsible for modulating the activity of IgG through sialylation. This discovery continues to fuel our research into understanding how and where IgG sialylation is manipulated and regulated.
In the course of our studies to understand IgG sialylation, we serendipitously discovered that changes in liver (hepatocyte) sialylation leads to a change in liver-resident macrophage polarization and the T cell compartment in model mice (8,13). These published findings have led to a new arm of the Cobbohydrates laboratory in which we are exploring how glycans in the tissue microenvironment impacts the “effector class” of the resident immune cells – particularly the macrophages.
A major gap in the “bench to bedside” paradigm is the ability to harness the glycome for the development of novel therapeutics. Although decades of research in glycobiology have established glycomic changes associated with disease, almost nothing is known about how those changes arise or the functions they play in disease initiation or progression. Based on our published findings, we are working on a model for glycomic compositional regulation of soluble secreted glycoproteins that provides a clear path for the development of the first generation of glycan-modulating therapies for a wide range of diseases. The model is based on the notion that the glycans of glycoproteins can be remodeled after release from the originating cell, and if correct, our findings will redefine the glycobiology dogma in which glycomic changes are dependent upon the slow process of protein turnover and de novo synthesis to one that is highly dynamic, rapid, and specific to the immunologic environment.
The current project centers on the molecular action, regulation and necessary microenvironment for ST6Gal1 to add α2,6-linked sialic acids onto glycans with available terminal galactose residues. Our proposal also focuses upon the B cell-secreted glycoprotein/antibody IgG. This is a critical pathway to understand because ST6Gal1 is the sole enzyme that determines whether anti-inflammatory α2,6-sialyl-IgG or pro-inflammatory asialyl-IgG is produced at any given time, thereby making it a key immunomodulatory factor. Even if this model for glycoprotein glycan remodeling is limited to sialylation, such a pathway could influence immune pathways such as leukocyte trafficking, the distinction between self and non-self by siglecs, synthesis of the ABO blood groups, transplantation, IgG functionality and many others, and thereby raise the possibility that dynamic regulation of the glycome could be therapeutically designed for the treatment of diseases ranging from inflammatory disorders and autoimmunity to cancer.
Tissue-resident macrophages play important roles in maintain tissue homeostasis. They very broadly fall into two categories: classically-activated and pro-inflammatory macrophages (M1) and alternatively-activated and anti- inflammatory macrophages (M2). Within immune privileged tissues, such as the lung and liver, macrophages, such as Kupffer cells, are typically described as M2. Our published findings suggest that the M2 phenotype in the liver depends at least in part upon the glycome of the surrounding parenchyma, particularly the hepatocytes. We have found that α2,6-linked sialic acids upon hepatocyte surface glycans promotes normal M2 polarization, but that loss of this sialylation drives M1 polarization and subsequent aberrant T cell activation which leads to increased inflammatory disease susceptibility in a mouse model whereby ST6Gal1 was selectively ablated in hepatocytes.
The current project seeks to determine the mechanism by which α2,6-sialylated glycans in the liver drives changes in resident macrophage phenotype and T cell activation. The proposed studies are broken into three aims, with the first two focused upon the influence of ST6Gal1-synthesized α2,6-sialylated glycans on macrophage function and signaling, and the third focused upon the mechanism underlying increased T cell activation and disease in the absence of sialylation. We believe that these studies will ultimately introduce a novel immune checkpoint receptor responsive to glycans that promotes M2 polarization and leads to immune homeostasis.
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