Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG–Fab regions, allowing neighboring IgG–Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1–Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1–Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1–Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.

No abstract

Innate immune memory, also termed “trained immunity” in vertebrates, has been recently described in a large variety of plants and animals. In most cases, trained innate immunity is induced by pathogens or pathogen-associated molecular patterns (PAMPs), and is associated with long-term epigenetic, metabolic, and functional reprogramming. Interestingly, recent findings indicate that food components can mimic PAMPs effects and induce trained immunity. The aim of this study was to investigate whether bovine milk or its components can induce trained immunity in human monocytes. To this aim, monocytes were exposed for 24 h to β-glucan, Toll-like receptor (TLR)-ligands, bovine milk, milk fractions, bovine lactoferrin (bLF), and bovine Immunoglobulin G (bIgG). After washing away the stimulus and a resting period of five days, the cells were re-stimulated with TLR ligands and Tumor necrosis factor (TNF-) and interleukin (IL)-6 production was measured. Training with β-glucan resulted in higher cytokine production after TLR1/2, TLR4, and TLR7/8 stimulation. When monocytes trained with raw milk were re-stimulated with TLR1/2 ligand Pam3CSK4, trained cells produced more IL-6 compared to non-trained cells. Training with bIgG resulted in higher cytokine production after TLR4 and TLR7/8 stimulation. These results show that bovine milk and bIgG can induce trained immunity in human monocytes. This confirms the hypothesis that diet components can influence the long-term responsiveness of the innate immune system.

Class‐switching of B cells to IgA can be induced via both T‐cell‐dependent and T‐cell‐independent mechanisms. IgA is most predominantly produced mucosally and is important for combating infections and allergies. In contrast to mice, humans have two forms of IgA; IgA1 and IgA2 with diverse tissue distribution. In early life, IgA levels might be sub‐optimal especially during the fall season when bacterial and viral infections are more common. Therefore, we investigated using human B cells whether T‐cell‐independent factors ‐promoting cell survival, class switching and immunoglobulin secretion‐ BAFF, APRIL, IL‐10 and retinoic acid can boost IgA production in the context of viral or bacterial infection. To this end total and naive peripheral blood B cells were stimulated with these factors for 6 days in the presence or absence of TLR7/8 agonist R848 (mimicking viral infection) or TLR9 agonist CpG‐ODN (mimicking bacterial infection). We show that BAFF significantly augments IgA2 production in TLR7/8 stimulated mature, but not naïve B cells. In addition, BAFF augments IL‐10 production and viability in TLR7/8 and TLR9 stimulated mature B cells. These data warrant further investigation of its role in immune regulation both in the periphery and mucosal tissues in early life or during disease.

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.

The onset of severe SARS-CoV-2 infection is characterized by the presence of afucosylated IgG1 responses against the viral spike (S) protein, which can trigger exacerbated inflammatory responses. Here, we studied IgG glycosylation after BNT162b2 SARS-CoV-2 mRNA vaccination to explore whether vaccine-induced S protein expression on host cells also generates afucosylated IgG1 responses. SARS-CoV-2 naive individuals initially showed a transient afucosylated anti-S IgG1 response after the first dose, albeit to a lower extent than severely ill COVID-19 patients. In contrast, previously infected, antigen-experienced individuals had low afucosylation levels, which slightly increased after immunization. Afucosylation levels after the first dose correlated with low fucosyltransferase 8 (FUT8) expression levels in a defined plasma cell subset. Remarkably, IgG afucosylation levels after primary vaccination correlated significantly with IgG levels after the second dose. Further studies are needed to assess efficacy, inflammatory potential, and protective capacity of afucosylated IgG responses.One sentence summaryA transient afucosylated IgG response to the BNT162b2 mRNA vaccine was observed in naive but not in antigen-experienced individuals, which predicted antibody titers upon the second dose.

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.