Infection and Immunity

Antibody affinity maturation and isotype switching in B cells controls the adaptive humoral immune system. Recently, we and others discovered the existence of a third layer of regulation, which involves N-linked glycosylation of the antibody Fc tail. Multiple different glycosylation patterns are possible, generating a glycan code that determines the affinity for Fc-receptors (FcγR). As a consequence, antibody molecules of the same amino acid sequence can exhibit distinct effector functions. This is most apparent for IgG-fucosylation, with ~94% of plasma IgG having this sugar. Removal of this fucose generates afucosylated IgG which improves binding to FcγRIII 40x and elevates its potency to activate immune cells. This fact is already being exploited for antibody-mediated therapy of cancer. Although such immune responses were not known to exist (IgG in plasma is almost exclusively fucosylated), we found that especially immune-responses towards foreign blood-cells can result in unique afucosylated IgG-responses, resulting in worse clinical outcome. Such responses have recently also been reported against HIV in elite controllers and Dengue. We hypothesize that alloimmune-responses mimic evolutionary conserved responses against pathogen-infected cells (not viral particles but host cells infected by enveloped viruses like HIV), through co-recognition of self, possibly on the B cell level (e.g. BCR and SIRP-CD47). These responses are then triggered upon alloimmune reactions such as transfusion and in pregnancy. Our pilot experiments with antibodies against enveloped viral antigens (HIV, CMV, HBV) developed during natural infection support this. Furthermore, our results suggest that Ig-glycosylation is remembered for decades. How this is acquired is unknown. The main aim of this proposal is to gain a comprehensive understanding on what steers IgG-glycosylation, with emphasis of fucosylation, and the basic mechanisms on how it is regulated.

To achieve this, the following key objectives will be tackled:

What are the requirements to achieve hypofucosylated immune response? Here we will systematically test the abovementioned main hypotheses what the requirements are for achieving low fucosylation. In particular we will explore whether the trigger is a foreign antigen present as a surface of otherwise autologous cells (provided by pathogens or allogeneic mismatch), both in experimental B cell-culture systems, in mice and in human immune responses.
Identify regulatory mechanisms in B cell development steering IgG-glycosylation and thereby IgG-function. Here we will interrogate our antigen-specific human B cells using our established single-cell culture methods for IgG-glycosylation patterns. By RNAseq we will analyze which genes (including glycosyl-transferases and glycosidases, but also unbiased) are responsible for the differential glycosylation profiles. This will give us leads and provide the basis for alternative hypothesis on how this information is stored in B cells.

The results of this study will provide a roadmap of genes and factors dictating Ig-glycosylation. By also determining whether individual B cell clones commit to a specific Ig-glyco-pattern and stably pass this on to their progeny or whether Ig-glycosylation requires external signals, we reveal the working-mechanism of this immunological memory. This will allow for targeted approaches directed at changing the metabolic pathways involved to alter immunoglobulin glycan phenotypes and thereby disease activities. These results may also provide fundamental knowledge on why some patients make extraordinarily potent anti-RBC and platelet IgG through altered glycosylation and some not, information that might be utilized for diagnosis to identify those at risk

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Mads Larsen