Effective antibody (Ab) neutralization of infectious pathogens is critically dependent on B cell differentiation in specialized lymphoid niches called germinal centers (GC). A GC is a multi-cellular immunologic nest exquisitely designed to control the fate and function of long-lived, high affinity Ab-producing B cells. In GCs, activated GC B cells, through the assistance of follicular T helper and dendritic cells, actively mutate their Ab receptors and differentiate in B cells capable of producing high affinity Abs. Given that GC B cell receptors and their counter-receptor ligands have been well-described, there is surprisingly little information on how these cell surface molecules are structurally regulated via post-translational glycosylations. Interestingly, a hallmark feature on the GC B cell surface is the stage-specific expression of a glycan moiety T antigen (TAg), known functionally as a regulator of effector T cell fate. Whether TAg or other heretofore unknown glycan determinants, we believe that GC B cells also express discrete glycan moieties that help regulate their fate in GCs and prime their ability to differentiate and transition into high affinity Ab producers. In this exploratory research project, we will identify signature glycomic features, including membrane glycoprotein scaffolds capable of transmitting survival signals, on native human GC B cells. Using freshly-isolated human GC B cells, including pre- and post-GC B cell subsets, we obtained exciting preliminary data showing that GC B cells indeed exhibited a glycomic genotype and phenotype distinguishable from pre- and post-GC B cell subsets. GC B cells were not only selectively marked by TAg, but also displayed a conspicuous high level of poly-N-acetyllactosamines (polylac). Moreover, we found that immunoregulatory galectin (Gal)-3, which is known to avidly bind polylacs, was highly-expressed in GCs. Functional assessments on putative glycomic gene regulators of TAg and polylac expression indicated that ?2,3 sialyltransferase, ST3Gal-1, functions as a dual negative regulator of TAg and O-glycan polylac synthesis. Analysis of polylac-enriched GC B cell glycoprotein also revealed polylac expression principally on a known Gal-3 ligand and pro-survival receptor, CD45.
SPECIFIC AIMS of this grant are: 1.) To analyze glycomic features of human GC B cells and 2.) To investigate the function of O-glycosylation in GC-dependent immune responses. Our overarching hypothesis is that GC B cell-specific O-glycan TAg/polylacs serve as regulators of GC B cell fate in a Gal-3- dependent manner. We will employ innovative experimental approaches to study native human pre-GC, GC and post-GC B cells and leverage our collective expertise in glyco-immunology to define the glycomic signature and its impact on GC B cells. Our results will provide new insights on glycosylation programs regulating human B cell differentiation and related development of a humoral immune response. Our findings will also implicate glycomic regulators on GC B cells that could alter autoimmunity and/or B-lymphomagenesis.
Development of effective antibody (Ab) immune protection against pathogens is regulated in specialized tissue microenvironments called germinal centers (GC). GCs are dynamic multi-cellular immune domains containing activated B-lymphocytes (GC B cells), which develop into B cells capable of producing potent, long-lasting Abs. Unfortunately, knowledge of how GC B cell surface carbohydrates prime their ability to become effective Ab-producing cells is still poorly understood. In this grant, we will study B cell surface carbohydrates and how they influence GC B cell survival and downstream Ab responses. Our results will offer a new perspective on how to generate effective Ab responses during infection or vaccination or how to regulate autoimmune disease or B cell lymphomas.
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