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It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.

The binding strength between IgG and Fc gamma R is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human Fc gamma RIIIa up to similar to 20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to similar to 40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFc gamma RIIIa and hFc gamma RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc gamma R, mFc gamma RIV. The N162-glycan in mFc gamma RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc gamma RIIIa. However, unlike hFc gamma RIIIa, mFc gamma RIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human Fc gamma RIII family and its orthologous mouse Fc gamma RIV to be functionally conserved

Ingileif Jonsdottir, Bjorn Nilsson, Kari Stefansson and colleagues perform a genome-wide association study for immunoglobulin levels in Icelandic and Swedish cohorts. They find 38 new variants associated with IgA, IgG, IgM or composite immunoglobulin traits and identify candidate genes underlying the regulation of immunoglobulin levels. Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10−55, β = −0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10−8, β = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

The precise mechanisms underlying anti-inflammatory effects of intravenous immunoglobulin (IVIg) therapies remain elusive. The sialylated IgG fraction within IVIg has been shown to be therapeutically more active in mouse models. Functionally, it has been suggested that IgG undergoes conformational changes upon Fc-sialylation which sterically impede binding to conventional FcγRs, but simultaneously allow binding to human DC-SIGN (SIGN-R1 in mice) and also CD23. These latter C-type lectins have been proposed responsible for the immunomodulatory effects in mouse models. However, there is conflicting evidence supporting direct interactions between sialylated human IgG and CD23/DC-SIGN. While cells expressing human CD23 and DC-SIGN in their native configuration bound their natural ligands IgE and ICAM-3, respectively, no IgG binding was observed, regardless of Fc-glycan sialylation in any context (with or without bisection and/or fucosylation) or presence of sialylated Fab-glycans. This was tested by both by FACS and a novel cellular Surface Plasmon Resonance imaging (cSPRi) approach allowing for monitoring low-affinity but high-avidity interactions. In summary, we find no evidence for human CD23 or DC-SIGN being bona fide receptors to human IgG, regardless of IgG Fc- or Fab-glycosylation status. However, these results do not exclude the possibility that either IgG glycosylation or C-type lectins affect IVIg therapies.

The composition of the conserved N297 glycan in immunoglobulin G (IgG) has been shown to affect antibody effector functions via C1q of the complement system and Fc gamma receptors (FcγR) on immune cells. Changes in the general levels of IgG-glycoforms, such as lowered total IgG galactosylation observed in many autoimmune diseases have been associated with elevated disease severity. Agalactosyslated IgG has therefore been regarded and classified by many as pro-inflammatory. However, and somewhat counterintuitively, agalactosylation has been shown by several groups to decrease affinity for FcγRIII and decrease C1q binding and downstream activation, which seems at odds with this proposed pro-inflammatory nature. In this review, we discuss these circumstances where altered IgG galactosylation/glycosylation is found. We propose a novel model based on these observations and current biochemical evidence, where the levels of IgG galactosylation found in the total bulk IgG affect the threshold required to achieve immune activation by autoantibodies through either C1q or FcγR. Although this model needs experimental verification, it is supported by several clinical observations and reconciles apparent discrepancies in the literature, and suggests a general mechanism in IgG-mediated autoimmune diseases.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to Fc gamma R-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse Fc gamma Rs is available. The orthologous mouse and human Fc gamma Rs share roughly 60-70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse Fc gamma R. Human IgGs bound to mouse Fc gamma R with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3 > IgG1 > IgG4 > IgG2 and Fc gamma RI >> Fc gamma RIV > Fc gamma RIII > Fc gamma RIIb. This suggests human IgG subclasses to have similar relative FcR-mediated biological activities in mice

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene–matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1–4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.

Objectives Recent reports describe interactions between the two most prominent RA-related autoantibodies, RFs and ACPAs. The main aim of the present study was to investigate whether RFs preferentially interact with ACPA-IgG over non-ACPA IgG. Additionally, interactions of RFs with IgG with altered galactose content in the Fc domain were examined, since ACPA-IgGs have been shown to have decreased Fc galactose content in RF+ patients. Methods (Auto)antibody interactions were studied in a surface plasmon resonance imaging assay and with ELISA. Target antibodies were isolated from RA patient plasma (polyclonal ACPA- and non-ACPA-IgG) or recombinantly produced to obtain monoclonal IgG with well-defined Fc galactose content. Interacting autoantibodies were studied using autoantibody positive patient sera and two recombinantly produced IgM-RFs. Results The sera from 41 RF+ RA patients showed similar RF binding to ACPA- and non-ACPA-IgG and no differences in binding to IgG with normal, high or low levels of Fc galactosylation. Two monoclonal IgM-RFs, one interacting with the CH2-CH3 interface and one binding close to the C-terminal end of the CH3 domain showed no influence of the Fc glycan on IgG binding by IgM-RF. Conclusion Although interactions between RF and ACPA may play a role in inflammatory processes in RA, RFs do not preferentially interact with ACPA-IgG over non-ACPA-IgG nor with agalatosylated IgG over IgG with normal or high galactosylation.

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design., Author summary Here we engineered various changes in the sizes and shapes of sugars that decorate HIV surface spike proteins and tested the effects of these changes on virus susceptibility to neutralizing antibodies. In so doing, we were able to define the optimal Env-sugars recognized by prototype bnAbs that recognize various canonical epitope clusters on Env spike proteins. Some bnAbs preferred spike proteins decorated with large, complex glycans. Others preferred smaller glycans that improved their access to underlying protein targets. For similar reasons, germline-reverted versions of bnAbs were also generally more effective when the glycans were small. In some cases, bnAbs acquired an ability to bind to sugars as they matured. A comparison of viruses generated in cell lines and primary cells revealed large differences in bnAb sensitivity, raising questions about clinical relevance of cell line-produced virus for checking vaccine responses and, moreover, the use of these cell lines for manufacturing vaccines. Overall, just as car engines may be modified to be supercharged or hybrid for increased power or efficiency, the sugars of HIV coat proteins may also need to be engineered as 'supercharged' and 'hybrid' or otherwise modified in rational vaccine designs to optimize bnAb recognition.

Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life-threatening disease where fetal platelets are destroyed by maternal antiplatelet IgG alloantibodies. The clinical outcome varies from asymptomatic, to petechiae or intracranial haemorrhage, but no marker has shown reliable correlation with severity, making screening for FNAIT impractical and highly inefficient. We recently found IgG Fc-glycosylation towards platelet and red blood cell antigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core-fucosylation increases the affinity of the pathogenic antibodies to Fc gamma RIIIa and Fc gamma RIIIb, and hence platelet destruction. Here we analysed the N-linked glycans of human platelet antigen (HPA)-1a specific IgG1 with mass spectrometry in large series of FNAIT cases (n = 166) including longitudinal samples (n = 26). Besides a significant decrease in Fc-fucosylation after the first pregnancy (P = 0.0124), Fc-glycosylation levels remained stable during and after pregnancy and in subsequent pregnancies. Multiple logistic regression analysis identified anti-HPA-1a - fucosylation (P = 0.006) combined with galactosylation (P = 0.021) and antibody level (P = 0.038) correlated with bleeding severity, making these parameters a feasible marker in screening for severe cases of FNAIT.