Ivan Peyron

Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46–3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30–3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development

Antibody-mediated catalysis: induction and therapeutic relevance.

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases.

Hunting down factor VIII in the immunopeptidome.

Major histocompatibility complex class II (MHCII)-restricted peptide presentation is crucial for the selection and subsequent proliferation of antigen specific CD4+ T cells. While selection of antigen-specific CD4+ T cells is beneficial in the context of vaccination, emergence of antigen CD4+ T cells following administration of therapeutic proteins like factor VIII (FVIII) is not desirable. The mechanism of uptake, processing and presentation of FVIII by antigen-presenting cells (APCs) has been the subject of intense study over the past 10 years. Multiple receptors have been implicated in the uptake of FVIII by APCs. A crucial determinant directing its entry in APCs resides in the C1 domain of FVIII. Until recently, our knowledge on the repertoire of FVIII derived presented on MHCII was limited. Peptide sequences on FVIII recognized by CD4+ T cells have been identified using MHCII tetramers as well as by directly monitoring peptide-induced proliferation of CD4+ T cells. More recently, the repertoire of naturally presented peptides derived from FVIII has been identified by pulsing of immature dendritic cells with FVIII. In a complementary approach HLA-DRB1(∗)15 transgenic mice were used to identify HLA-DRB1(∗)15 restricted CD4+ T cells reactive towards human FVIII. In this review we summarize our current knowledge on FVIII derived peptides that are presented on MHCII and discuss the relevance of these findings for the etiology of inhibitor development in patients with hemophilia A.

Haemarthrosis and arthropathy do not favour the development of factor VIII inhibitors in severe haemophilia A mice

presence of normal factor X levels (88.1%) in our patient made acquired factor X deficiency a highly unlikely cause of his bleeding episodes. In addition, our patient did not have any prior history of vasculopathy or any alternative process that could contribute to his haemorrhagic symptoms. As such, vascular disruption due to light-chain deposition remains the most plausible mechanism behind his recurrent, visceral hematomas. It is also important to note that, despite the spontaneous haemorrhagic episodes the patient experienced, invasive procedures such as kidney and bone marrow biopsies were safely performed. In conclusion, when a patient with haemophilia experiences an unexplained increase in the frequency and severity of bleeding, clinicians should rule additional pathologies and use all the diagnostic tools at their disposal.

Heme binds to factor VIII and inhibits its interaction with activated factor IX

Summary.  Background:  Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions.

To serve and protect: The modulatory role of von Willebrand factor on factor VIII immunogenicity

Hemophilia A is a bleeding disorder characterized by the absence or dysfunction of blood coagulation factor VIII (FVIII). Patients are treated with regular infusions of FVIII concentrate. In response to treatment, approximately 30% of patients with severe hemophilia A develop inhibitory antibodies targeting FVIII. Both patient and treatment related risk factors for inhibitor development have been described. Multiple studies comparing the immunogenicity of recombinant and plasma-derived FVIII have yielded conflicting results. The randomized controlled SIPPET (Survey of Inhibitors in Plasma-Product Exposed Toddlers) trial demonstrated an increased risk of inhibitor development of recombinant FVIII when compared to von Willebrand factor (VWF)-containing plasma-derived FVIII. Presently, it is unclear which mechanism underlies the reduced immunogenicity of plasma-derived FVIII. In this review we address the potential role of VWF on FVIII immunogenicity and we discuss how VWF affects the immune recognition, processing and presentation of FVIII. We also briefly discuss the potential impact of glycan-composition on FVIII immunogenicity. It is well established that VWF shields the uptake of FVIII by antigen presenting cells. We have recently shown that VWF binds to the surface of dendritic cells. Here, we present a novel model in which surface bound FVIII-VWF complexes regulate the internalization of FVIII. Binding of FVIII to VWF is critically dependent on sulfation of Tyr1699 (HVGS numbering) in the light chain of FVIII. Incomplete sulfation of Tyr1699 has been suggested to occur in several recombinant FVIII products resulting in a loss of VWF binding. We hypothesize that this results in alternative pathways of FVIII internalization by antigen presenting cells which are not regulated by VWF. This hypothetical mechanism may explain the reduced immunogenicity of VWF containing plasma-derived FVIII concentrates as found in the SIPPET study.

Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells

The development of anti-factor VIII antibodies is a major complication of the treatment of patients with hemophilia A. Generation of high affinity anti-factor VIII antibodies is dependent on help provided by CD4+ T cells that recognize factor VIII-derived peptides presented on class II major histocompatibility complex on the surface of antigen-presenting cells. In order to identify the immune-dominant epitopes that can be presented to CD4+ T cells, we previously developed a mass spectrometry-based method to identify factor VIII-derived peptides that are presented on human leukocyte antigen (HLA)-DR. In the present work, we compared the repertoire of FVIII-derived peptide presented on HLA-DR and HLA-DQ. Monocyte-derived dendritic cells from nine HLA-typed healthy donors were pulsed with recombinant factor VIII. HLA-DR and HLA-DQ molecules were purified using monoclonal antibodies. Our data show that HLA-DQ and HLA-DR present a similar repertoire of factor VIII-derived peptides. However, the number of peptides associated with HLA-DQ was lower than that with HLA-DR. We also identified a peptide, within the acidic a3 domains of factor VIII, which is presented with higher frequency on HLA-DQ. Interestingly, this peptide was found to have a higher predicted affinity for HLA-DQ than for HLA-DR. Taken together, our data suggest that HLA-DQ participates in the presentation of factor VIII peptides, thereby contributing to the development of inhibitory antibodies in a proportion of patients with severe hemophilia A.

Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion

Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.

IVIg treatment reduces catalytic antibody titers of renal transplanted patients.

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.

Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.