Immune Complexes Enhance Uptake of Blood Coagulation Factor VIII by Antigen Presenting Cells (original) (raw)
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Journal of Thrombosis and Haemostasis
Background: Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. Objectives: Use dendritic cell (DC)-protein processing/presentation assays with massspectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, * RSFSQNSRHPSTRQKQFNATTIPEN 764 TTLQ 768
Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain
Journal of Allergy and Clinical Immunology, 2012
Background: The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4 1 T cells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor-related protein-1 (LRP1) have been implicated in FVIII uptake. Objective: This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. Methods: FVIII uptake was performed with human monocytederived and murine bone marrow-derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA-induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm in vitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. Results: Upon treatment of DCs with mannan or LRP ligand a2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA-mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 in vivo. Conclusion: Together, our observations emphasize the physiological significance of KM33-targeted residues within the C1 domain in the uptake of FVIII by DCs in vitro and in vivo. (J Allergy Clin Immunol 2012;129:501-9.)
Factor VIII: Perspectives on Immunogenicity and Tolerogenic Strategies for Hemophilia A Patients
International Journal of Molecular and Cellular Medicine (IJMCM), 2020
A major complication in treating hemophilia A is the development of neutralizing antibodies (inhibitors) against therapeutic administered factor VIII (FVIII), which occurs in approximately 20-30% of patients with severe disease. These inhibitors render FVIII replacement therapy ineffective and increase the morbidity and mortality risk. The currently accepted method to eradicate inhibitors is immune tolerance induction (ITI), and frequent intensive administration of FVIII until inhibitor titers drop. Current ITI protocols are extremely costly and not effective in all patients. During the last decade, many types of research have been accomplished to clarify the mechanisms that mediate immune tolerance induction. Novel experimental therapies including monoclonal antibodies, viral vector-mediated gene therapy, regulatory T cell induction using immunosuppressive drugs, and nanoparticle-based immune modulation show promising results in hemophilia A clinical trials. This review focuses on treatment options towards the anti-FVIII immune responses and current novel therapies in clinical trials.
Blood, 2012
Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and re-tained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/ K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4 ؉ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A. (Blood. 2012;119(22):5294-5300)
Journal of Thrombosis and Haemostasis, 2017
Essentials • Anti-factor (F) VIII antibody formation is a major complication in the treatment of hemophilia A. • We investigated uptake of FVIII and FVIII immune complex by bone marrow derived dendritic cells. • Immune complex formation increased uptake of FVIII 3-4 fold in a Fcc receptor dependent manner. • FVIII immune complex binding to Fcc receptors may modulate immune tolerance induction. Summary. Background: A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives: Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods: BMDCs were pulsed with FVIII/ FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results: BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcc receptor (FccR) antibody 2.4G2, indicating functional involvement of FccR. No internalization of FVIII-IC was observed in BMDCs lacking FccRI, FccRIIb, FccRIII and FccRIV. Genetic ablation of FccRIIb, FccRIII or FccRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FccRI showed lower FVIII-IC uptake levels when compared with other single FccR null BMDCs. Expression of the inhibitory FccRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions: FccR are critical in the internalization of FVIII-IC by BMDCs and multiple FccR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FccR.
British Journal of Haematology, 2008
IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A Haemophilia A is an X-linked bleeding disorder that is caused by a deficiency or dysfunction of human blood clotting factor VIII (FVIII). In about 25% of severely affected haemophiliacs, treatment is complicated by the development of inhibitory antibodies against infused FVIII (Lollar, 2004). Inhibitor development renders patients unresponsive to FVIII replacement therapy. Acute bleeds must therefore be treated with FVIII bypassing agents that include recombinant activated factor VII (FVIIa) and activated prothrombin complex concentrates (Lollar, 2004; Reipert et al, 2007). As prophylaxis is the preferred treatment to prevent emergency bleeds and haemophilic arthropathy, immune tolerance induction (ITI) for eradication of FVIII inhibitors is the strategy of choice for haemophilia A patients with inhibitors (Leissinger, 2006). ITI treatment comprises frequent administration of high or intermediate doses of FVIII, which generally results in a gradual decline in inhibitor titre (Brackmann & Gormsen, 1977; Lollar, 2004; Reipert et al, 2007). Retrospective data from two international registries indicate that the success rate for ITI varies between 70-80% (DiMichele & Kroner, 2002; Mariani et al, 2003; Lillicrap, 2006). Outcome of ITI is influenced by the inhibitor titre at the onset of treatment, the historical peak titre and the peak titre during treatment (DiMichele & Kroner, 2002; Mariani et al, 2003; Lillicrap, 2006). Factor VIII inhibitors consist of a polyclonal population of antibodies that is targeted to multiple antigenic sites present within the A2, A3 and C2 domains of FVIII (Lollar, 2004). Subclass analysis by immunoblot and enzyme-linked immunosorbent assay (ELISA) revealed that FVIII inhibitors comprise subtypes IgG1, IgG2 and IgG4 (Fulcher et al, 1987; Gilles et al, 1993). Generation of high-affinity inhibitory antibodies of subclass IgG requires help from antigen-specific
Immunology Letters, 2007
A number of diseases are treated by passive administration of human proteins. Human coagulation factor VIII (FVIII) is one such protein which is adminsitered to hemophilia A patients in order to manage and treat hemorrhagic incidences. This mode of therapy suffers from the side effect of generating anti-FVIII antibodies (inhibitors) which neutralizes the function of the infused FVIII. At a time when efficient viral screening procedures are at place, development of inhibitors poses the greatest threat to such a therapy. Various predisposing factors, both patient and product-related, are responsible for the development of inhibitory antibodies. A proper understanding of these "risk-factors" would eventually help to better design therapeutic regimen to tackle hemophilia A.
Catalytic activity of antibodies against factor VIII in patients with hemophilia A
Nature medicine, 1999
Hemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient factor VIII (FVIII) molecules, which, in its severe form, is a life-threatening and crippling hemorrhagic disease. Infusion of homologous FVIII to patients with severe hemophilia A results, in 25% of patients, in the emergence of alloantibodies against FVIII (inhibitors)( ref. 1) that inhibit FVIII procoagulant activity by steric hindrance of the interaction of FVIII either with stabilizing molecules, with molecules essential for its activity or with activating molecules. Here, we report on the proteolysis of FVIII by alloantibodies of two patients with severe hemophilia A, demonstrating a previously unknown mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. The kinetic parameters of FVIII hydrolysis indicate a functional role for the catalytic immune response in the inactivation of FVIII in vivo. The characterization of alloantibodies against FVIII as site...
Haemophilia, 2007
In patients with haemophilia A, factor VIII (FVIII) therapy leads to the development of anti-FVIII alloantibodies that inhibit FVIII pro-coagulant activity, in up to 25% of the cases. At a time when efficient viral screening procedures are at place, development of inhibitors poses the greatest threat to haemophilia A patients. Various risk factors, both patient and product-related, are responsible for the development of inhibitory antibodies. The role of FVIII-specific CD4+ T lymphocytes in the initiation of the humoral immune response to exogenous FVIII has been well. In view of their capacity to stimulate naïve T cells, dendritic cells (DCs) play a central role in the initiation of the primary immune response. Thus, in the context of a primary alloimmunization against FVIII, i.e. when FVIII-specific B lymphocytes are not there to take up FVIII from the circulation and to serve as antigen presenting cells (APCs), DCs are the only cell type that internalize FVIII, leading to activation of FVIII-specific CD4+ T lymphocytes. von Willebrand factor (VWF) present in plasmaderived FVIII therapeutic concentrates, is known to act as a chaperone molecule for procoagulant FVIII. In addition to its role in reducing the ÔantigenicityÕ of FVIII, the role of VWF in the reduction of the ÔimmunogenicityÕ of therapeutic FVIII in patients with haemophilia A has also been suggested. We have recently demonstrated that VWF protects FVIII from being endocytosed by human DCs and subsequently being presented to FVIII-specific T cells. We propose that VWF may reduce the immunogenicity of FVIII by preventing, upstream from the activation of immune effectors, the entry of FVIII in professional antigen presenting cells.
Proteolytic antibodies activate factor IX in patients with acquired hemophilia
Blood, 2011
Acquired hemophilia is a rare bleeding disorder characterized by the spontaneous occurrence of inhibitory antibodies against endogenous factor VIII (FVIII). IgG from some patients with acquired hemophilia hydrolyze FVIII. Because of the complex etiology of the disease, no clinical parameter, including the presence of FVIII-hydrolyzing IgG, has been associated with patient's survival or death. Here, we demonstrate the presence of anti-FIX antibodies in acquired hemophilia pa-tients. IgG from some patients were found to hydrolyze FIX. In most cases, IgGmediated FIX-hydrolysis resulted in FIX activation. IgG-mediated hydrolysis of FIX thus led to the significant generation of activated FIX in 25 of 65 patients. Based on the estimated kinetic parameters, patients' IgG activated up to 0.3nM FIX in 24 hours, an amount that restored thrombin generation in vitro provided the presence of more than or equal to 3% residual FVIII activity in plasma. This work identi-fies proteolytic IgG as novel molecules able to activate FIX under pathologic conditions. IgG-mediated FIX activation is a prevalent phenomenon among acquired hemophilia patients. The presence of FIXactivating IgG may partly compensate for the antibody-mediated inhibition of endogenous FVIII in restoring thrombin generation. This clinical trial was registered at www. clinicaltrials.gov as #NCT00213473. (Blood.