Gestur Vidarsson

IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A

The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so‐called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti‐FVIII antibodies during ITI. FVIII‐specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti‐FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti‐FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low‐titre inhibitor patients, anti‐FVIII antibodies consisted primarily of subclass IgG1 whereas, anti‐FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti‐FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti‐FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Background The majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. Design and Methods In this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of anti-spacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients. Results Antibody profiling revealed that anti-ADAMTS13 immunoglobulin G1 and immunoglobulin G4 predominate in plasma of patients with acquired thrombotic thrombocytopenic purpura. Analysis of anti-spacer domain antibodies revealed that Arg568 and Phe592, in addition to residues Arg660, Tyr661, and Tyr665, also contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost reactivity towards the spacer domain following introduction of multiple alanine substitutions at Arg568, Phe592, Arg660, Tyr661 and Tyr665. Anti-TSP2-8 and anti-CUB1-2 domain-directed antibodies were present in, respectively, 17% and 35% of the patients’ samples analyzed. Conclusions Immunoglobulin G directed towards a single antigenic surface comprising residues Arg568, Phe592, Arg660, Tyr661 and Tyr665 predominates in the plasma of patients with acquired thrombotic thrombocytopenic purpura.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region

Significance Structural variation of antibodies is generally defined in terms of amino acid composition, neglecting posttranslational modifications such as N-linked glycosylation. Little is known about the role of the glycans that are present in about 15% of variable domains. However, recent studies suggest that variable domain glycans exhibit distinct patterns according to (patho)physiological conditions, and can have immunomodulatory effects. Here we highlight a physiological role for variable domain glycans that is predetermined in the germline antibody repertoire: We show that variable domain N-linked glycans are acquired during somatic hypermutation at positions predisposed in the germline and may be positively selected during affinity maturation, representing an additional mechanism of secondary antibody diversification that contributes to the extent of the B-cell antibody repertoire. A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N-linked glycans, a process conditional on the introduction of consensus amino acid motifs (N-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Conserved FcγR- glycan discriminates between fucosylated and afucosylated IgG in humans and mice

HIGHLIGHTSThe N162 glycan is conserved between the orthologous human Fc&ggr;RIIIa and mouse Fc&ggr;RIV.This glycan endows the receptors to discriminate between fucosylated and afucosylated IgG.Human afucosylated IgG/Fc&ggr;RIIIa affinity is further directed by additional galactosylation.Mouse afucosylated IgG/Fc&ggr;RIV affinity remains unaffected by additional galactosylation. ABSTRACT The binding strength between IgG and Fc&ggr;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&ggr;RIIIa up to ˜20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up 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&ggr;RIIIa and hFc&ggr;RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc&ggr;R, mFc&ggr;RIV. The N162‐glycan in mFc&ggr;RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc&ggr;RIIIa. However, unlike hFc&ggr;RIIIa, mFc&ggr;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&ggr;RIII family and its orthologous mouse Fc&ggr;RIV to be functionally conserved.

Glycoengineering HIV-1 Env creates ‘supercharged’ and ‘hybrid’ glycans to increase neutralizing antibody potency, breadth and saturation

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.

Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73-1, ELH2-1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73-1 and TR8C11 bind to the cysteine-spacer domains, while the antibody ELH2-1 recognizes the T2-T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

Mechanisms of inside-out signaling of the high-affinity IgG receptor FcγRI

Cytokine stimulation drives nanoscale reorganization of surface FcγRI to augment antibody-dependent cellular functions. Inside-out Fc receptor activation Important for the activation of integrin adhesion molecules, “inside-out” activation is triggered by cytoplasmic signals that increase the affinity of the integrin for its ligand. Using super-resolution microscopy, Brandsma et al. found that cytokine stimulation similarly augments Fc receptor avidity by increasing the size of FcγRI clusters on the surface of monocytes. Pharmacological inhibitors of actin rearrangement and the phosphatase PP1 reduced receptor clustering. Cytokine-driven FcγRI clustering also correlated with the increased FcγR-dependent cytolytic activity of human neutrophils. Together, these findings suggest that FcR clustering stimulated by cytokines drives the inside-out activation of FcγRI. Fc receptors (FcRs) are an important bridge between the innate and adaptive immune system. Fc gamma receptor I (FcγRI; CD64), the high-affinity receptor for immunoglobulin G (IgG), plays roles in inflammation, autoimmune responses, and immunotherapy. Stimulation of myeloid cells with cytokines, such as tumor necrosis factor–α ( TNFα) and interferon-γ ( IFNγ), increases the binding of FcγRI to immune complexes (ICs), such as antibody-opsonized pathogens or tumor cells, through a process known as “inside-out” signaling. Using super-resolution imaging, we found that stimulation of cells with IL-3 also enhanced the clustering of FcγRI both before and after exposure to ICs. This increased clustering was dependent on an intact actin cytoskeleton. We found that chemical inhibition of the activity of the phosphatase PP1 reduced FcγRI inside-out signaling, although the phosphorylation of FcγRI itself was unaffected. Furthermore, the antibody-dependent cytotoxic activity of human neutrophils toward CD20-expressing tumor cells was increased after stimulation with TNFα and IFNγ. These results suggest that nanoscale reorganization of FcγRI, stimulated by cytokine-induced, inside-out signaling, enhances FcγRI cellular effector functions.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport

&NA; We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fc&ggr; receptors (Fc&ggr;RI–III) and C1q, thereby eliminating their destructive effector functions (constant region G1&Dgr;nab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half‐life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto‐maternal alloimmune disorders. Here, we show that G1&Dgr;nab mutants retain pH‐dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0‐fold and 1.6‐fold for the two antibodies investigated. The transport of the modified G1&Dgr;nab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild‐type, except that no difference was observed with human umbilical vein endothelial cells. G1&Dgr;nab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1&Dgr;nab mutations are away from the FcRn‐binding site, they have long‐distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model: FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA‐4, PD1 and PD‐L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti‐CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti‐PD‐L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti‐PD‐L1 showed similar therapeutic efficacy when compared to each other in either wild‐type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti‐PD‐L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD‐L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti‐PD‐L1 antibody therapy but might play a role in some tumor models.

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.