Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against Human Leukocyte Antigens (HLA) and to a lesser extent to Human Platelet Antigens (HPA). These antibodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc Receptor (FcγR)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness (PR). Strikingly, not all patients with anti-HLA or -HPA antibodies develop PR upon unmatched platelet transfusions. Previously, we found IgG Fc glycosylation of anti-HLA antibodies to be highly variable between patients with PR, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and -HPA-1a monoclonal antibodies (mAbs) with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA mAbs with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a mAbs interact synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and to a lesser extent sialylation significantly increased the complement activating properties of anti- HLA and anti-HPA-1a mAbs. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens and the type of Fc glycosylation, can provide an optimal stoichiometry for C1q binding and subsequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement mediated clearance of donor platelets as observed in PR.

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Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.

Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti–major histocompatibility complex (MHC) class I antibody 34-1-2S (anti–H-2Kd) causes TRALI in BALB/c mice (MHC class I haplotype H-2Kd), whereas SF1.1.10 (anti–H-2Kd) does not. In C57BL/6 mice (MHC class I haplotype H-2Kb), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti–H-2Kb) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALI-causing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2Kd. Regarding binding affinity to H-2Kb, only AF6-88.5.5.3 potently bound to H-2Kb, whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to FcγRs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- and Fc-binding affinities for antigen and FcγRs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction.

Background The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%–15% of patients with chronic platelet support. Interestingly, anti‐HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti‐HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR‐mediated phagocytosis and/or complement activation, which both are affected by the IgG‐Fc glycosylation. Objectives To characterize the Fc glycosylation profile of anti‐HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome. Patients/Methods We screened and captured anti‐HLA class I antibodies (anti‐HLA A2, anti‐HLA A24, and anti‐HLA B7) developed after platelet transfusions in hemato‐oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography‐mass spectrometry, we analyzed the glycosylation profiles of total and anti‐HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments. Results The glycosylation profile of anti‐HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients. Conclusions These differences in composition of anti‐HLA Fc‐glycosylation profiles could potentially explain the variation in clinical severity between patients.