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Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an in silico analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura., (Copyright © 2018 Ferrata Storti Foundation.)

Essentials Initial immune cell interactions leading to factor (F) VIII immunity are not well characterized. We assessed cellular interactions and expression profiles in hemophilia A mice. MARCO+, followed by SIGLEC1+ and SIGNR1+ macrophages co-localize most with human FVIII. The splenic transcriptome highlights potential therapeutic targets to prevent inhibitors., Summary: Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 μg (2000-8000 IU kg -1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 μg (240 IU kg -1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO + cells compared with SIGLEC1 + and SIGNR1 + in the splenic marginal zone. FVIII exhibited little co-localization with CD11c + dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO + ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation., (© 2017 International Society on Thrombosis and Haemostasis.)

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/β-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM., (© 2018 by The American Society of Hematology.)

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., (Copyright© 2018 Ferrata Storti Foundation.)

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., (Copyright © 2017. Published by Elsevier Ltd.)

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 Fcγ receptor dependent manner. FVIII immune complex binding to Fcγ 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-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR 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 FcγR., (© 2016 International Society on Thrombosis and Haemostasis.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II., (Copyright© Ferrata Storti Foundation.)