Effector functions of differentially glycosylated anti-Bet v 1 human IgG subclasses

Allergy to birch pollen is characterized by the development of IgE and IgG antibodies (Abs) against the major allergen Bet v 1. Successful allergen-specific immunotherapy (SIT; hyposensibilization) to combat birch pollen allergy is often associated with a large increase in Bet v 1-reactive serum IgG Abs, mainly IgG1 and IgG4. The common idea about the function of these anti-Bet v 1 IgG Abs is that they capture the allergen and inhibit its binding to the pro-inflammatory IgE bound to mast cells. However, it has recently been shown that the Fc N-glycosylation pattern of IgG Abs determines their pro- or anti-inflammatory effector functions: agalactosylated (non-galactosylated and non-sialylated; G0) IgG Abs are pro-inflammatory and galactosylated plus sialylated IgG Abs are anti-inflammatory.In this context, we have shown that sialylated IgG Abs are induced after successful tolerance induction with protein antigens in mice and after SIT in humans, suggesting that sialylated allergen-specific IgG Abs are involved in this SIT-dependent tolerance induction. Further results indicated that sialylated, but not G0, anti-ovalbumin (OVA) murine monoclonal IgG1 Abs, can reduce allergic airway inflammation in an OVA-dependent mouse model. Furthermore, these studies indicated that immune complexes containing antigen-specific sialylated IgG Abs are able to inhibit dendritic cell maturation and therefore priming of pro-inflammatory T cells.In this study, we want to clone and produce differentially glycosylated monoclonal humanized and real human anti-Bet v 1 IgG1 and IgG4 Abs to investigate their inhibitory potential in human immune cell culture assays and, in parallel with differentially glycosylated murine anti-Bet v 1 IgG1 Abs, in a Bet v 1-dependent mouse allergy model. In this mouse model, we also want to characterize the receptor complexes of the differentially glycosylated IgG Abs.Additionally, we want to analyze the Fc glycosylation patterns of human anti-Bet v 1 serum IgG Abs before, during and after subcutaneous SIT.Overall, we want to identify the optimal Fc glycosylated allergen-specific human IgG subclass to inhibit allergic reactions and characterize its functional role. This will be important for the validation of successful SIT, the possible development of Abs for passive immunization and the optimization of individualized SIT protocols.

IgE antibodies (Abs) can mediate allergic reactions, including systemic anaphylaxis, by activating the high affinity Fcepsilon receptor (R) I on mast cells and basophils, leading to release of inflammatory mediators. In contrast, allergen-specific IgG Abs, which are also induced by allergen-specific immunotherapies (AIT), can inhibit IgE-mediated anaphylaxis caused by low levels of allergen through allergen-masking and crosslinking of the Fcepsilon RI with the classical IgG inhibitory receptor Fcgamma RIIb. However, when allergen levels are high, IgG Abs for example to medical drugs also have the potential to mediate anaphylaxis by activating classical activating Fcgamma Rs on different immune cell types. The effector functions of IgG Abs depend on their subclass and the type of Fc Nglycosylation. Agalactosylated (non-galactosylated) IgG Abs are associated with inflammation, whereas galactosylation and terminal sialylation of IgG Abs can suppress inflammation. However, the effects of IgG subclass and Fc glycosylation pattern in allergy remain unclear. The results of this project showed that the IgG subclass and the IgG Fc glycosylation pattern had only a slight effect on extent of inhibition of an IgE-mediated llergic reaction via Fcgamma RIIb. However, we identified that the severity of IgG-mediated systemic anaphylaxis, which required challenge with a higher antigen dose, was IgG subclass- and Fc glycosylation-dependent. Sialylation of IgG Abs reduced their potential to induce an IgG-mediated anaphylaxis. This inhibitory effect was dependent on the C-type lectin receptor SignR1.In conclusion, the IgG Ab subclass distribution and their Fc glycosylation pattern might predict the potential of IgG-mediated allergic reactions against medical drugs or also during AIT, when allergen doses are high. We further analyzed how conventional AIT with birch pollen extract and the adjuvant aluminium hydroxide (alum) affects the IgG subclass and Fc glycosylation pattern of anti-Bet v 1 (Betula verrucosa 1; the major birch pollen allergen) Abs in birch pollen allergic patients. In untreated patients, Bet v 1-specific IgG4 titers were constantly low, while IgE but also IgG1 titers increased during the pollen season. In contrast, during AIT, levels of Bet v 1-specific IgG1 increased in the first 12 months but decreased afterwards, while Bet v 1-specific IgG4 titers persistently increased. However, the Fc glycosylation profile of Bet v 1-specific serum IgG Abs from untreated and AIT-treated patients remained stable and were highly galactosylated and sialylated. Consistent with an inverse relationship between IgG sialylation and inflammatory potential, we found that in vitro de-sialylation of native Bet v 1-specific IgG Abs from the serum of AIT-treated patients strongly increased their ability to activate neutrophils in vitro. These observations suggest that conventional AIT with alum induces sialylated IgG(4) Abs that probably have low potential to induce IgG-mediated allergic reactions, when the allergen dose would be high. However, studies remain required to assess how Fc glycosylation modulates the effector functions of human IgG1 and IgG4 and how new AIT protocols with distinct adjuvants, will influence the human IgG subclass distribution and Fc glycosylation pattern and consequently, the risk of IgG-mediated allergic reactions, when the allergen dose would be high. Taken together, our data suggest that although IgG subclass and glycosylation pattern have relatively little effect on IgG Ab blocking of IgE-mediated anaphylaxis, increased sialylation of IgG(4) Abs should decrease the risk of IgG-induced anaphylaxis in the presence of high allergen doses.