Abstract
Humans have circulating antibodies against diverse glycans containing N-glycolylneuraminic acid (Neu5Gc) due to function-loss mutation of the CMAH gene. This xenogenic non-human carbohydrate is abundant in red meat, xenografts and biotherapeutics. Low levels of diet-derived Neu5Gc is also present on normal human endothelial cells, and together with anti-Neu5Gc antibodies could potentially mediate “xenosialitis” chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a drug containing polyclonal IgG glycoproteins commonly used as an immunosuppressant in human transplantation and autoimmune diseases. In type-1 diabetes patients, infusion of Neu5Gc-glycosylated ATG caused increased global anti-Neu5Gc response. Here, for the first time we explore changes in anti-Neu5Gc IgG repertoire following the immunization elicited by ATG, compared with the basal antibodies repertoire that reflect exposure to dietary-Neu5Gc. We used glycan microarrays with multiple Neu5Gc-glycans and controls to elucidate eventual differences in ATG-elicited repertoire, before/after ATG administration and track their kinetics (0, 1, 18 and 24 months). Response of all basal-pre-existing Neu5Gc-specific antibodies rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18–24 months. Induced-antibodies showed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including endogenous glycolipids, that was further validated by affinity-purified anti-Neu5Gc antibodies from patients’ sera. These findings strongly suggest that ATG-induced anti-Neu5Gc IgGs represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies.
Highlights
Humans develop a comprehensive immune response against Neu5Gc, a foreign dietary sialic acid form, with significant potential implications on chronic inflammationmediated diseases such as cancer, atherosclerosis and xeno-transplantation [1,2,3,4]
Antibody secretion is initiated by activated-B cells that differentiate into short- or long-lived plasma cells
To diversify the repertoire of antigen-specific cells and optimize their affinity, activated-B cells transiently repress plasma cell (PC) differentiation followed by class-switch recombination, or that they enter the germinal center (GC) for somatic hypermutation and class-switch recombination to yield high-affinity clonal variants that differentiate into either quiescent memory-B cells or plasma cells [25]
Summary
Humans develop a comprehensive immune response against Neu5Gc, a foreign dietary sialic acid form, with significant potential implications on chronic inflammationmediated diseases such as cancer, atherosclerosis and xeno-transplantation [1,2,3,4]. Neu5Gc and Neu5Ac, that differ by a single oxygen, are the two major sialic acids forms in mammals, which are ubiquitously expressed at the tip of diverse carbohydrate chains (glycans) on glycoproteins and glycolipids [1, 15,16,17]. It is challenging to measure anti-Neu5Gc antibodies, because they recognize multiple Neu5Gc-glycan epitopes, on a large collection of Neu5Gccontaining sugar chains. This variety results from Neu5Gc attachment to diverse underlying sugar-chains, with different linkages, glycan conjugation to protein/ lipid-carriers and their diverse cell-surface density. To estimate overall anti-Neu5Gc response, a simple ELISA Inhibition Assay (EIA [7]) has been developed based on reactivity against multiple mouse Neu5Gc-containing glycoproteins, yet detailed information can only be achieved by measuring responses to distinct Neu5Gcglycans with Neu5Ac-glycans counterparts serving as controls, facilitated by large scale analysis using printed arrays [9, 18, 19]
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