Fc glycosylation of alloantibodies in platelet refractoriness
Authors:   , van Osch Thijs Luc Junior 
Published: January 2024

Platelet transfusions are essential for the prevention and treatment of hemorrhagic complications in patients with platelet dysfunction or thrombocytopenia. However, patients requiring frequent platelet transfusions are at risk for alloimmunization, which can lead to immune platelet refractoriness. These alloantibodies are formed after alloantigen exposure during pregnancy, incompatible transfusions or transplantations, and most frequently against Class I Human Leukocyte Antigens (HLA), but occasionally also towards Human Platelet Antigens (HPA) and ABO blood groups. These alloantibodies will bind to the corresponding antigens expressed on donor platelets, leading to rapid clearance via multiple immunological processes, such as FcγR-mediated phagocytosis and complement-mediated cytotoxicity. However, even if alloantibodies are formed, not all patients become necessarily refractory to platelet transfusions. Hence, more research is required into antibody characteristics, which can explain these inter-donor variation in antibody responses.

In this thesis we evaluated the role of Fc glycosylation of anti-platelet antibodies in platelet refractoriness. IgG contains a highly conserved N-linked glycan in its Fc region, which is essential for the antibody's function, structure, and stability. The glycan consists of highly variable extensions of a bi-antennary core structure and the configuration of these sugar residues can strongly affect the antibody's effector functions and therefore the clinical course of the immune response.

In Chapter 4, we characterized the glycosylation profile of anti-HLA antibodies in haemato-oncological patients receiving platelet transfusions. We found IgG-Fc glycosylation of anti-HLA antibodies to be highly variable between patients, especially with respect to galactosylation and sialylation, which were significantly increased for the majority of the patients. Furthermore the level of galactosylation and sialylation of anti-HLA antibodies negatively correlated with the transfusion outcome. Aside galactosylation and sialylation, afucosylation of HLA-specific antibodies was also observed for two individuals.

Previously, it was shown that both elevated antibody galactosylation and sialylation increases the potency of IgG antibodies to activate the complement system. At the time, it was known that monomeric IgG requires hexamerization, via noncovalent Fc:Fc interactions, to form an high avidity interaction platform for the first classical complement component, C1q, to bind. In Chapter 2 we showed that Fc galactosylation promotes IgG1 hexamerization and thereby complement activation. We observed that increased Fc galactosylation elevates C1q-binding, and downstream complement activity for wildtype IgG1, but not for antibodies with increased hexamerization potential.

In addition to Fc glycosylation IgG hexamerization and thereby complement activation, is also dependent on numerous other factors, including the antigen’s size, expression level, spatial distribution and mobility but also the antibody’s titer, epitope position, binding angle, hinge length and flexibility. In Chapter 3 we showed that individual anti-HLA or anti-HPA-1a monoclonal antibodies are incapable of activating the classical complement pathway on the surface of platelets. In contrast, when specific combinations of anti-HLA mAbs with different specificities are used simultaneously, complement activation was strongly enhanced even in low antibody concentrations. The combination of mAbs increases the amount of opsonization, facilitating the formation of hetero-hexamers, which is further enhanced by Fc galactosylation and sialylation, leading to efficient complement activation.
Although the majority of human IgG responses are dominated by fucosylated IgG, for some antigen-specific antibody responses much lower fucosylation levels are detected. Afucosylation has been shown to increase the affinity for FcγRIIIa/b up to 40-fold, which can turn into even larger functional effects for associated effector functions, including antibody dependent cellular cytotoxicity and – phagocytosis, and ultimately disease severity. In Chapter 5, we investigated the effect of Fc fucosylation, galactosylation and sialylation of anti-HLA antibodies on phagocytosis of opsonized platelets by monocyte-derived macrophages. Interestingly, we did not observe any differences in phagocytosis between differently glycosylated anti-HLA monoclonal antibodies, as our in vitro model appeared to be primarily FcγRI-mediated.