Discussion
Targeting IgE with therapeutic anti-IgE antibodies such as omalizumab is a validated strategy to treat IgE-mediated diseases. Naturally occurring anti-IgE IgG antibodies have been identified in patients with various diseases, including asthma and atopic dermatitis, but also healthy individuals. Our recent work demonstrated that a single immunization with IgE-allergen complexes elicits an anti-IgE IgG response without adding adjuvants. The resulting anti-IgE IgGs bind glycosylated IgE significantly better than deglycosylated IgE. Here we show that booster immunizations can even enhance the anti-IgE IgG response suggesting that repeated exposure to IgE-allergen IC might lead to a beneficial anti-IgE IgG response. Glycosylation of IgE plays a vital role in this process. The use of glycosylated IgE leads to an increased anti-IgE IgG and even an increased anti-Fel d 1 IgG response compared to deglycosylated IgE. The adjuvant effect of IgE for antigens has been previously demonstrated, but here we show that glycans are critical for this effect. The reduced immunogenicity of deglycosylated IgE might be counterintuitive. Removal of the glycans makes the IgE less soluble and could result in the formation of aggregates. It is known that aggregates can be important for antigenicity. Hence, it is reasonable to assume that glycan-recognizing receptors are involved in the process since the interaction between IgE and CD23 is glycan independent and CD23KO mice also show an anti-IgE response. The fact that IgE is the most glycosylated immunoglobulin further strengthens this assumption. About 12% of its molecular weight is carbohydrate. Moreover, glycosylation of proteins influences their serum kinetic. A possible candidate could be the lectin galectin-9, which is known to bind IgE. Even though it has an anti-allergic effect by preventing IgE-antigen complex formation, it is not clear whether it can bind IgE-antigen complexes or not. However, it is known to induce DC maturation and promote T cell proliferation and Th1 cytokine production. This would explain the higher anti-IgE IgG response in mice immunized with glycosylated IgE-ICs. Other receptors in mice that can bind IgE are FcγRIIIA and FcγRIV. Specifically for FcγRIV, one study has shown that this receptor binds IgE when IgE is present as a complex but not monomeric IgE. Moreover, the engagement of FcγRIV led to the antigen presentation to T cells However, whether glycosylation of IgE plays a role in this process is unclear. Interestingly, two amino acids (K117 and E132) essential for FcεRI binding to IgE are conserved in FcγRIV. Since IgE glycosylation is required for binding to FcεRI, it may also be necessary for binding to FcγRIV.
Further differences can be observed in the ability of anti-IgE and anti-IgE(PNG) IgG antibodies to clear IgE from serum and prevent binding to effector cells. The results show that anti-IgE IgG antibodies are significantly better at controlling the binding of IgE to FcεRI of basophils than anti-IgE(PNG) antibodies. This indicates that IgE glycosylation affects the quantity of anti-IgE antibodies generated and their quality. An explanation for this could be the difference in affinity since it is higher for the anti-IgE IgG antibodies than anti-IgE(PNG) antibodies. It could be that glycans are essential for interacting with anti-IgE IgG to IgE. Thus, in the case of missing glycans, the anti-IgE IgG binding site could be altered, resulting in decreased affinity. Another explanation could be that deglycosylation leads to a conformational change. This change would result in different conformational epitopes between glycosylated and deglycosylated IgE.
Passive immunization with anti-IgE IgG and anti-IgE(PNG) IgG allowed us to see if the difference in serum clearance is also reflected in the ability to protect mice from anaphylaxis upon allergen challenges. Our results show that both anti-IgE IgG antibodies protect BALB/c WT mice compared with controls. However, protection was always better in the anti-IgE IgG group than in the anti-IgE(PNG) group. These results are consistent with the clearance experiment results, where significantly more IgE was found on the basophils of the anti-IgE(PNG) IgG group than in those of the anti-IgE-IgG group. To better understand the protection mechanism, allergen challenges were also performed in FcγRIIBKO and CD23KO mice. Previous studies have shown that co-aggregation of FcγRIIb with FcεRI inhibits IgE-mediated degranulation of effector cells. Moreover, it has been observed that allergen-specific desensitization (SIT) induced allergen-specific IgGs, which act via FcγRIIb. However, since the FcγRIIbKO mice were as protected as the WT mice, we speculate that the protection is mainly mediated by the neutralization of IgE via CD23. In both B cells and monocyte-derived DCs, CD23 was shown to bind and internalize IgE-ICs better than IgE alone and could therefore play a role in the clearance of the complexes. Indeed, CD23KO mice were not as well protected as wild-type Balb/c mice, suggesting that CD23 is important for the clearance of IgE-IgG complexes. Indeed, our serum kinetic results in CD23KO confirmed that most complexes are eliminated via CD23. Not enough elimination of complexes leads to the accumulation of IgE-IgG complexes and then to their dissociation, during which the released IgE can bind preferentially to FcεRI because of its higher affinity for FcεRI than for the IgG antibodies (see Graphical Abstract). This results in the sensitization of effector cells, which can degranulate upon encountering the allergen. Additionally, while IgE-ICs do not trigger empty FcεRI-mediated degranulation, IgE pre-sensitized basophils/mast cells can be degranulated by IgE-ICs. A lack of clearance could thus favor degranulation of IgE pre-sensitized effector cells.
In conclusion, we have demonstrated the importance of IgE glycosylation for the anti-IgE-IgG response and a possible pathway to eliminate IgG-IgE complexes via CD23 in a mouse model. Further research is needed to investigate the effects of IgE glycosylation on anti-IgE IgG antibodies and serum IgE levels in humans. In particular, the extent to which different IgE glycosylation patterns affect anti-IgE IgG antibodies should also be investigated. More insights into the role of IgE glycosylation for anti-IgE IgG antibodies could help to find even more efficient strategies against type I hypersensitivity diseases.
References and Notes: