IgG-Fc glycosylation before and after rituximab treatment in immune thrombocytopenia

The interactions of antibodies with myeloid Fc gamma receptors and the complement system are regulated by an Asn297-linked glycan in the Fc portion of IgG. Alterations of serum IgG-Fc glycosylation have been reported in various autoimmune diseases, and correlate with treatment response and disease activity. We hypothesized that IgG-Fc glycosylation is altered in immune thrombocytopenia (ITP) and associates with response to anti-CD20 monoclonal antibody treatment (rituximab). IgG-Fc glycosylation was analyzed by liquid chromatography-mass spectrometry. We found that IgG-Fc glycosylation was identical between refractory ITP patients (HOVON64 trial; N = 108) and healthy controls (N = 120). Two months after rituximab treatment, we observed a shift in Fc glycosylation, with a mean 1.7% reduction in galactosylation for IgG1 and IgG4 and a mean 1.5% increase for bisection in IgG1, IgG2/3 and IgG4 (adjusted p < 1.7 x 10(-3) and p < 2 x 10(-4), respectively). Neither baseline nor longitudinal changes in IgG-Fc glycosylation after rituximab were associated with clinical treatment response. We conclude that IgG-Fc glycosylation in refractory ITP is similar to healthy controls and does not predict treatment responses to rituximab. The observed changes two months after treatment suggest that rituximab may influence total serum IgG-Fc glycosylation. Overall, our study suggests that the pathophysiology of refractory ITP may differ from other autoimmune diseases.Proteomic

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with 60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency—but only in the presence of H435–IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435–IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435–IgG3 to be a candidate for monoclonal antibody therapies

Biological stratification of clinical disease courses in childhood immune thrombocytopenia

Background In childhood immune thrombocytopenia (ITP), an autoimmune bleeding disorder, there is a need for better prediction of individual disease courses and treatment outcomes.Objective To predict the response to intravenous immunoglobulins (IVIg) and ITP disease course using genetic and immune markers.Methods Children aged younger than 7 years with newly diagnosed ITP (N = 147) from the Treatment With or Without IVIG for Kids with ITP study were included, which randomized children to an IVIg or observation group. A total of 46 variables were available: clinical characteristics, targeted genotyping, lymphocyte immune phenotyping, and platelet autoantibodies.Results In the treatment arm, 48/80 children (60%) showed a complete response (platelets >= 100 x 10(9)/L) that lasted for at least 1 month (complete sustained response [CSR]) and 32 exhibited no or a temporary response (absence of a sustained response [ASR]). For a biological risk score, five variables were selected by regularized logistic regression that predicted ASR vs CSR: (1) hemoglobin; (2) platelet count; (3) genetic polymorphisms of Fc-receptor (Fc gamma R) IIc; (4) the presence of immunoglobulin G (IgG) anti-platelet antibodies; and (5) preceding vaccination. The ASR sensitivity was 0.91 (95% confidence interval, 0.80-1.00) and specificity was 0.67 (95% confidence interval, 0.53-0.80). In the 67 patients of the observation arm, this biological score was also associated with recovery during 1 year of follow-up. The addition of the biological score to a predefined clinical score further improved the discrimination of favorable ITP disease courses.Conclusions The prediction of disease courses and IVIg treatment responses in ITP is improved by using both clinical and biological stratification.Clinical epidemiolog

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies

A clinical prediction score for transient versus persistent childhood immune thrombocytopenia

Background Childhood immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. The prognosis (transient, persistent, or chronic ITP) remains difficult to predict. The morbidity is most pronounced in children with persistent and chronic ITP. Clinical characteristics are associated with ITP outcomes, but there are no validated multivariate prediction models.Objective Development and external validatation of the Childhood ITP Recovery Score to predict transient versus persistent ITP in children with newly diagnosed ITP.Methods Patients with a diagnosis platelet count = 100 x 10(9)/L 3 months after diagnosis) versus persistent ITP. Age, sex, mucosal bleeding, preceding infection/vaccination, insidious onset, and diagnosis platelet count were used as predictors.Results In external validation, the score predicted transient versus persistent ITP at 3 months follow-up with an area under the receiver operating characteristic curve of 0.71. In patients predicted to have a high chance of recovery, we observed 85%, 90%, and 95% recovered 3, 6, and 12 months after the diagnosis. For patients predicted to have a low chance of recovery, this was 32%, 46%, and 71%. The score also predicted cessation of bleeding symptoms and the response to intravenous immunoglobulins (IVIg).Conclusion The Childhood ITP Recovery Score predicts prognosis and may be useful to individualize clinical management. In future research, the additional predictive value of biomarkers can be compared to this score. A risk calculator is available ().Clinical epidemiolog

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

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available. Proteomic

Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination

Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce Fc gamma RIIIa-dependent natural killer cell degranulation.Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (Fc gamma R) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to Fc gamma RIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces Fc gamma RIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria.Proteomic