Glycan microarray reveal induced IgGs repertoire shift against a dietary carbohydrate in response to rabbit anti-human thymocyte therapy

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.

Quantitative and qualitative changes in anti‐Neu5Gc antibody response following rabbit anti‐thymocyte IgG induction in kidney allograft recipients

“…The detailed analysis of the arrays performed on eight patients in this study showed a clear increase in the anti‐Neu5Gc IgG repertoire and reactivity at 6 months post‐ATG, in contrast to the controls. Despite the major immunosuppression, these data fit with those obtained in non‐immunosuppressed patients . These results thus sustain the possibility that ATG can extend the pre‐existing anti‐Neu5Gc antibodies’ repertoire to new specificities, leaving xenosialitis conceivable.…”

“…Animal‐derived molecules or engineered tissues are highly immunogenic. ATG, a widely used polyclonal rabbit IgG preparation that expresses α‐Gal and Neu5Gc‐glycans according to a mass spectrometry analysis, induces a vigorous response against these two xeno‐antigens in non‐immunosuppressed patients . However, as for allo‐antibodies, the immune response against these foreign glycans was dramatically delayed and decreased by modern immunosuppressive cocktails, as recently described .…”

“…The diversity of Neu5Gc epitopes in glycans is enormous in contrast to α‐Gal, generating a collection of epitopes with a terminal Neu5Gc deciphered using sialoglycan arrays . In addition, whereas pre‐existing anti‐Neu5Gc antibodies (ie, diet induced) are present in all humans, ATG‐elicited anti‐Neu5Gc IgGs recognize new specificities and with higher affinities that could potentially induce a local inflammation, which had been coined “xenosialitis” . This inflammation may theoretically result from the presence, even at a low level, of the Neu5Gc antigen itself on endothelial cells .…”

“…Non‐immunosuppressed patients with severe burns treated by engineered pig skin dressings develop a long‐lasting increase in their anti‐Neu5Gc antibody levels . Similarly, an intravenous (IV) injection of rabbit anti‐human thymocyte IgGs, which display α‐Gal and Neu5Gc carbohydrate antigens detectable by mass spectrometry analysis, also induce a vigorous immune response against α‐Gal and Neu5Gc antigens in non‐immunosuppressed patients causing serum sickness in almost all cases . In contrast, kidney recipients receiving ATG as an induction treatment in association with a cocktail of modern immunosuppressive agents develop a late anti‐Neu5Gc response, and this response is stronger in patients who develop an early serum sickness disease (SSD) .…”

Elicited and pre‐existing anti‐Neu5Gc antibodies differentially affect human endothelial cells transcriptome

“…High titers of elicited anti‐Neu5Gc Abs are also generated following exposure to animal‐derived products or proteins, such as after polyclonal rabbit anti‐human T‐cell globulin (ATG) infusion . Such elicited anti‐Neu5Gc Abs also display higher affinity and an extended repertoire of responses against new Neu5Gc epitopes than diet‐derived anti‐Neu5Gc Abs, and some are not present before immunization, as demonstrated by sialoglycan microarrays . Thus, in addition to the expected deleterious interaction with the immunizing animal‐derived material, such elicited anti‐Neu5Gc Abs can also result in the formation of in situ immune complexes and chronic inflammation in the micro‐vicinity of the recipients' own Neu5Gc‐positive endothelial cells (ECs) .…”