SEARCH

SEARCH BY CITATION

Keywords:

  • antigen-presenting cells;
  • factor VIII;
  • hemophilia;
  • inhibitor formation;
  • MHC class II

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

Summary.  Only a fraction of patients with hemophilia A develop a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII. Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen-presenting cells (APCs) have expanded significantly. Although the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII-derived peptides on major histocompatibility complex (MHC) class II, and this peptide–MHC class II complex may then be recognized by cognate effector CD4+ T cells, leading to anti-FVIII antibody production. Here we aim to summarize recent knowledge gained about FVIII processing and presentation by APCs, as well as the diversity of the FVIII-specific T-cell repertoire in mice and humans. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of the immune recognition of FVIII and the cellular mechanisms of anti-FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

Professional antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages and B cells endocytose proteins that are degraded along the endocytic pathway and then presented on major histocompatibility complex (MHC) class II molecules to CD4+ T cells. Apart from internalized foreign antigens, many peptides presented on MHC class II are derived from proteins residing in intracellular compartments that are sampled by autophagy. Peptides from proteins degraded in the endosome are loaded on MHC class II in the so-called MHC class II peptide-loading compartment and subsequently transported to the plasma membrane (Fig. 1). In DCs, MHC class II expression at the cell surface increases following their maturation, which also results in upregulation of costimulatory molecules required for activation of naïve CD4+ T cells. The complex mechanisms involved in antigen presentation and their coordinated interplay with pattern-recognition receptors of the innate immune system have been exploited by modern medicine, e.g. by harnessing individuals with pre-existing immunity (neutralizing antibodies) against incoming microbial challenges [1,2]. A growing appreciation of the skewing of immune responses by pattern-recognition receptors of the innate immune system has advanced the field of rational design of vaccines [1,2]. Undesired immune responses have been observed following repeated administration of a number of therapeutic proteins, including anti-tumor necrosis factor-α antibodies such as adalimumab or infliximab, coagulation factors VIII and IX, and erythropoietin [3]. In view of the ubiquitous presentation of self-peptides and non-self-peptides on MHC class II, one can easily appreciate that peptides derived from biotherapeutic proteins are also presented on MHC class II. However, it is more difficult to envision how biotherapeutics, in the absence of immunologic ‘danger signals’, would induce activation of APCs, thereby promoting the upregulation of costimulatory molecules required for activation of naïve CD4+ T cells.

image

Figure 1.  Major histocompatibility complex (MHC) class II presentation pathway. Dendritic cells endocytose antigens that subsequently undergo proteolytic degradation in endosomal compartments. Transport of newly synthesized MHC class II molecules from the endoplasmic reticulum (ER) is facilitated by the invariant chain (not shown), which is processed into a so-called CLIP peptide that occupies the binding groove of MHC class II. In the MHC class II compartment, the CLIP peptide is exchanged for antigen-derived peptides. Following peptide loading, MHC class II molecules are transported to the cell surface, and antigen-derived peptides are presented to CD4+ T cells. TCR, T-cell receptor.

Download figure to PowerPoint

This review will explore the issue of neutralizing anti-drug antibodies in the context of current knowledge of FVIII immunogenicity. Approximately 25% of patients with the severe form of the X-linked bleeding disorder hemophilia A (defined by FVIII procoagulant activity < 1% of normal) develop an immune response resulting in the formation of neutralizing anti-FVIII antibodies. A number of recent reviews have summarized genetic and non-genetic treatment-related risk factors that contribute to inhibitor formation in hemophilia A [4–7]. Guidelines for the treatment of patients with hemophilia A with inhibitors have been provided in a recent paper by Kempton and White [8]. Inhibitor formation, somatic hypermutations and subclass switching of anti-FVIII antibodies are considered to be CD4+ T-cell-dependent processes in both hemophilic mice [9–11] and patients with hemophilia A [12–17]. Activation of FVIII-specific T cells is preceded by the uptake of FVIII by APCs and the subsequent presentation of FVIII-derived peptides on MHC class II molecules on the surface of these APCs [18]. It has been suggested that the HLA alleles DRB1*15 and DQB1*0602 correlate with an increased risk of inhibitor development in hemophilic patients [5,19]. However, the association between MHC class II molecules and FVIII antibody formation is not strong, and this reflects a central and intriguing aspect of anti-FVIII immune responses: a large majority of patients with hemophilia A achieve functional immune tolerance to FVIII, either following their initial infusions or after eradication of an inhibitor response via immune tolerance induction (intensive FVIII therapy) or immunosuppression. The promiscuity of FVIII-derived peptides in terms of binding to different HLA alleles [20], and the huge diversity in HLA types as compared with the number of hemophilic patients included in earlier studies investigating the role of HLA in inhibitor risk, also make the establishment of statistically significant associations with individual HLA types challenging.

This review focuses on the most recent data available on FVIII endocytosis and processing by APCs, as well as the presentation of FVIII-specific peptides to T cells. We discuss new insights into mechanisms of FVIII endocytosis by DCs in humans and mice, and describe recent investigations into what determines the immunogenicity of FVIII. We also summarize efforts made to interfere with the immune recognition of FVIII and with events subsequent to FVIII uptake by APCs that influence anti-FVIII antibody formation. Finally, we discuss investigations into the presentation of FVIII-specific peptides and the diversity and functionality of the subsequent FVIII-specific T-cell repertoire.

Endocytosis and processing of FVIII by APCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

FVIII is internalized by APCs such as DCs and macrophages, where it is processed efficiently into peptides, some of which may then be presented on MHC class II molecules at the cell surface [20]. If a given MHC class II peptide is then recognized by a T-cell receptor on an effector T cell, as can happen in patients with hemophilia A who do not have pre-existing immune tolerance to FVIII, this can lead to subsequent activation of FVIII-specific T and B cells and the production of antibodies. Inhibitory anti-FVIII antibodies interfere with its procoagulant function, e.g. by blocking its interaction with thrombin-activated platelet membranes and microparticles derived from these membranes that expose negatively charged phosphatidylserine (PS) on their surface [21]. The complexity of the B-cell responses to FVIII has been described in two recent reviews [7,22]. Here, we focus on critical determinants on FVIII that are involved in its uptake by APCs. Endocytosis of FVIII by APCs requires bivalent cations, as it can be inhibited by EDTA [18]. The macrophage mannose receptor has been suggested to be an important interaction partner for FVIII. Mannose-ending glycans are linked to residues in the heavy chain (Asn239) and in the light chain (Asn2118) [18]. Blockage with mannan leads to partial inhibition of FVIII endocytosis by immature human DCs [18]. Furthermore, strongly reduced proliferation of the FVIII-specific human CD4+ T-cell clone D9E9 was seen after pulsing of DCs with FVIII in the presence of mannan [18]. In a more recent report, the same authors concluded that mannan did not block the uptake of FVIII by murine bone marrow-derived DCs [23]. In an independent study, Herczenik et al. demonstrated that mannan did not block the uptake of FVIII by human or murine DCs, and small interfering RNA-mediated knockdown of mannose receptor expression on DCs did not affect FVIII endocytosis [24]. These recent studies imply that additional receptors can contribute to endocytosis of FVIII by DCs.

LDL receptor-related protein (LRP), a broadly expressed scavenger molecule, has been implicated in FVIII clearance [25–29]. Lys2092 and Phe2093 in FVIII have been shown to play a role in its binding to LRP [30]. Moreover, it has been suggested that disruption of FVIII–LRP binding could potentially lead to a prolonged half-life of FVIII in vivo [25]. However, LRP has not been implicated in the uptake of FVIII by DCs [18,24,31]. FVIII binds with high affinity to negatively charged phospholipid membranes, but lactadherin, which competes with FVIII for binding to these membranes, does not block FVIII endocytosis by DCs [24]. Therefore, PS-rich membranes do not seem to play a significant role in FVIII uptake. The mAb KM33, which targets the FVIII C1 domain, was recently shown to inhibit FVIII endocytosis by both human and murine DCs [24] (Fig. 2A). Moreover, in vivo administration of KM33 prevented the production of neutralizing antibody against FVIII [24]. The in vitro and in vivo inhibitory effect of KM33 suggests that this antibody targets an epitope on the FVIII surface that is essential for its uptake by APCs. KM33 also interferes with binding of FVIII to LRP, although, as mentioned above, this receptor does not seem to be involved in FVIII endocytosis, most probably because of its low-affinity binding of FVIII [24]. The FVIII residues Arg2090, Lys2092 and Phe2093 (Fig. 2B) have been implicated in KM33 binding to FVIII [32,33]. Moreover, infusions of FVIII variant proteins with alanine substitutions at these three positions in FVIII-deficient mice led to reduced T-cell and B-cell responses as compared with wild-type FVIII [33]. Taken together, these findings suggest that an as yet unidentified cellular component that interacts with an exposed loop in the FVIII C1 domain promotes endocytosis of FVIII by APCs.

image

Figure 2.  Modulation of FVIII endocytosis by antigen-presenting cells. (A) Interference of factor VIII uptake by antigen-presenting cells (APCs). The C1 domain-directed monoclonal anti-FVIII antibody KM33 and the FVIII chaperone molecule, von Willebrand factor (VWF), abolish uptake of FVIII by dendritic cells (DCs). Mutations in the C1 domain were shown to diminish FVIII endocytosis by both DCs and macrophages. (B) Three-dimensional structure of FVIII, highlighting sites important for its recognition by APCs. The left panel provides an overview of the domain organization of the crystallized FVIII protein. The right panel shows a close-up view of the C1 and C2 domains, with Arg2090, Lys2092 and Phe2093 highlighted in red. Models were based on the FVIII crystal structure (Protein Data Bank code: 3cdz) and prepared with pymol imaging software.

Download figure to PowerPoint

Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

FVIII circulates in plasma as a large glycoprotein complexed with its multimeric chaperone molecule, VWF. VWF protects FVIII from premature activation as well as degradation or inactivation by circulating proteases. It also regulates FVIII catabolism, and transports it to sites of injury. A number of studies performed with mouse models of hemophilia A have indicated that preincubation of recombinant FVIII with VWF leads to a reduction in titers of inhibitory anti-FVIII antibodies following FVIII infusions [23,34–36]. However, the results of these studies should be interpreted carefully, as human VWF is also immunogenic in mice [34,37]. Immune responses to infused human VWF may result in lower anti-FVIII antibody titers simply because of antigenic competition [38]. In vitro studies performed with human APCs clearly showed that VWF protects FVIII from endocytosis by these cells, and leads to decreased FVIII-specific T-cell proliferation [24,39] (Fig. 2A). These experimental results have relevance to epidemiologic studies that have suggested a reduced prevalence of inhibitors in patients with hemophilia A treated with VWF-containing FVIII concentrates [40]. However, this clinical observation was not reproduced in a larger cohort study, which concluded that the risk of inhibitor development is similar for plasma-derived and recombinant FVIII [41]. Moreover, patients with hemophilia A have normal levels of circulating VWF. Owing to the high on-rate of the FVIII–VWF complex, one would expect infused FVIII to associate rapidly with endogenous VWF [42]. Randomized, prospective clinical studies, such as the Randomization study of First Time Immune Tolerance Induction in Patients with Severe Type A Hemophilia with Inhibitor at High Risk of Failure: Comparison of Induction of Immune Tolerance With Factor VIII Concentrates With or Without von Willebrand Factor (RES.I.ST) and the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) studies, are addressing the potential benefit of VWF-containing concentrates for hemophilia A treatment [43,44].

On the immunogenicity of bio-engineered FVIII derivatives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

PEGylation is a common method used to reduce the immunogenicity and antigenicity of protein therapeutics [45]. Site-specific PEGylation through engineered free cysteines in the heavy and light chains of FVIII has prolonged its half-life in animal models of hemophilia A [46]. This PEGylated FVIII variant showed diminished endocytosis by human monocyte-derived DCs, followed by significantly reduced proliferation and production of interferon-γ by FVIII-specific T cells [47]. Moreover, studies performed in hemophilic mice, rats and rabbits showed a lower incidence of anti-FVIII antibodies in animals infused with PEGylated FVIII than in those infused with its recombinant non-PEGylated counterpart. On the other hand, a recent report by van Helden et al. [48] indicated that PEGylation of FVIII could also result in an FVIII protein that expresses higher immunogenicity in hemophilic mouse models. Also, incorporation of PEGylated lipids into complexes of FVIII and phosphatidylinositol resulted in increased immunogenicity following intravenous infusion in a murine hemophilia A model [49]. Genetic fusions of FVIII to the Fc fragment of IgG1 have been utilized to prolong the half-life of FVIII [50,51]. Prolongation of therapeutic protein–Fc fusion half-lives requires effective complex formation between the Fc portion of IgG1 and FcRn in the early endosome; recycling of endosomes re-exposes IgG1-Fc to physiologic pH at the cell surface, resulting in its dissociation from FcRn. FcRn is also expressed on APCs [52]. Therefore, internalization of FVIII–Fc fusion proteins by APCs most likely proceeds via a different mechanism than endocytosis of unmodified FVIII, which may be followed by FVIII-derived peptide presentation on MHC class II and subsequent T-cell stimulation. In this respect, it is interesting to note that retroviral transduction of B-cell blasts with Ig fusions harboring the A2 and C2 domains of FVIII can restore tolerance in mice with hemophilia A with pre-existing inhibitors [53]. Initial data on the immunogenicity of FVIII–Fc fusion proteins in murine hemophilia A models has recently been reported in two meeting abstracts [54,55]. These initial reports suggested slightly reduced immunogenicity of the FVIII–Fc fusion proteins as compared with a B-domain-deleted FVIII when they were administered at therapeutic dosages (50–100 IU kg−1). Infusion of supraphysiologic dosages (250 IU kg−1), however, resulted in significantly higher inhibitor titers than infusion of B-domain-deleted FVIII [54,55]. Additional studies are needed to shed light on the immunogenicity of FVIII–Fc fusion proteins and other bio-engineered FVIII derivatives, including the delivery methods and dosages.

Activation of the immune system by FVIII: looking for the ‘danger signals’

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

Activation of naive CD4+ T cells requires presentation of antigenic peptides by mature DCs in the context of MHC class II molecules and costimulatory signals. Although FVIII is endocytosed efficiently, it is still not clear what sometimes prompts the immune system to raise a high-titer antibody response against it, especially as it is administered intravenously, which is normally a tolerogenic route, in the absence of any adjuvant. Pfistershammer et al. [56] demonstrated that neither FVIII alone, in its native circulating or thrombin-activated from, nor FVIII in complex with VWF present danger signals to the immune system. Cytokine expression profiles, cellular maturation markers and the ability of FVIII to stimulate autologous or allogenic T cells did not change following incubation of monocyte-derived DCs with FVIII or FVIII–VWF complexes [56].

FVIII exerts its procoagulant effect at a critical control point in the coagulation cascade, and coagulation itself has been shown to be a hallmark of both sepsis and viral hemorrhagic fevers [57,58]. The administration of anticoagulants, such as recombinant activated protein C, significantly reduces mortality in patients with severe sepsis [59,60]. Coagulation induces a proinflammatory response: thrombin [61] and other coagulation factors [62,63] can directly activate endothelial cells, platelets and white blood cells via protease-activated receptors (PARs). Moreover, DCs constitute the primary site in the lymphatic compartment that links inflammation and coagulation [64]. PAR1 signaling and crosstalk between PAR1 and sphingosine 1-phosphate activates DCs and promotes systemic inflammation [64]. The procoagulant activity of FVIII leads to thrombin generation, which can potentially act as a ‘danger signal’ and thus contribute to FVIII immunogenicity. Skupsky et al. [65] demonstrated that coinjections of FVIII and ovalbumin (OVA) caused mice to mount an immune response to the latter, which, when injected alone, is poorly immunogenic. Furthermore, coadministration of OVA and thrombin was sufficient to provoke an immune response to OVA. The anticoagulant warfarin and the direct thrombin inhibitor hirudin also significantly reduced B-cell and T-cell responses to FVIII. Moreover, heat inactivation of FVIII, which reduces the number of B-cell epitopes but preserves the linear T-cell epitopes, also resulted in reduced immunogenicity of FVIII following infusions into mice. It is interesting to speculate that heat-treated FVIII may also be recognized and therefore endocytosed less efficiently by DCs, the most potent APCs in the induction of the primary immune response, and/or by memory B cells, which mediate recall responses to FVIII after initial antibody development. However, heat treatment (pasteurization) may also increase the immunogenicity of FVIII, as was found when patients were treated with one lot of a heat-pasteurized product [66].

In a recent study, FVIII mutants defective in procoagulant activity were tested for their ability to raise an immune response in FVIII-deficient and FVIII/VWF-deficient mice [67]. FVIII R372A/R1689A was inactive, owing to substitutions at the thrombin and FX proteolytic activation sites, whereas the other variant tested, FVIII V634M, although cleaved by thrombin, showed < 1% of the procoagulant activity of wild-type FVIII. The use of various injection protocols demonstrated that wild-type FVIII and FVIII V634M were equally immunogenic, independently of the FVIII dosages and mouse models used. FVIII R372A/R1689A was slightly less immunogenic than the other two variants tested; however, this could potentially be explained by its limited release from VWF. These recent experimental results suggest that the procoagulant function of FVIII may not be the major determinant of its immunogenicity.

We would expect bystander bacterial or viral challenges to provide costimulatory signals to provoke immune responses against FVIII. High-intensity FVIII treatment because of excessive bleeding episodes has also been linked to inhibitor development [68]. Intensive treatment may allow FVIII to compete more efficiently with other antigens for uptake by APCs, resulting in more efficient presentation of FVIII-derived peptides to CD4+ T cells. The reported association between intensity of treatment and inhibitor development has influenced clinical risk assessment and decision-making regarding initial FVIII exposures. Many clinicians now consider the potential benefits of beginning early prophylaxis and of delaying FVIII replacement therapy, when possible, in the face of clinical conditions that could potentially lead to activation of the immune system [69].

Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

Recently, a plethora of new data have emerged regarding the presentation of FVIII-derived peptides by APCs to T cells, in both humans and mice, using a broad spectrum of tools, including mass spectrometry, humanized mouse models, and bioinformatics. Activation of T-helper cells is dependent on the proper presentation of antigen-derived peptides on MHC class II molecules expressed on APCs. van Haren et al. [20] explored the repertoire of FVIII-derived peptides presented on MHC class II molecules on human monocyte-derived DCs from four HLA-typed healthy donors, using mass spectrometry. Thirty-two core peptides were identified; among these were many promiscuous epitopes, although some were presented in a donor-specific manner. Interestingly, a number of peptides identified in this study were previously reported as immunodominant T-cell epitopes [13,70,71].

Many studies of the FVIII-specific T-cell repertoire have been carried out with mouse models of hemophilia A; for example, immunodominant T-cell epitopes in FVIII were identified with rhe use of E16-knockout (KO) mice [72]. Regardless of the route of FVIII administration (subcutaneous or intravenous), T cells recognized peptides corresponding to residues 2191–2220 in the FVIII C2 domain, whereas none of the C1 domain peptides induced T-cell proliferation. This same FVIII region interacts with VWF and activated phospholipid membranes. More recently, the development of a humanized hemophilic E17 HLA-DRB1*1501 mouse model has been described [73,74]. Humanized mice have been utilized to study the regulation of HLA class II-restricted immune responses to various antigens, and they are highly suitable for in vivo research into the mechanistic basis of human diseases associated with activation of CD4+ T cells [75]. HLA-DRB1*1501 was selected because of a strong connection between this haplotype and many immunologic diseases [76], as well as a previously noted link between inhibitor incidence and DRB1*1501 in patients with severe hemophilia A [5,19,77]. Despite some obvious limitations, such new models can be used to analyze the differences in the FVIII-specific T-cell repertoire between mice and humans.

Steinitz et al. [74] recently utilized these humanized hemophilic mice to analyze FVIII-derived peptides presented by HLA-DRB1*1501 (Table 1). Eight different immunodominant regions were identified, and subsequent in vitro binding assays also demonstrated that most of these epitopes were promiscuous. Interestingly, although the application route did not alter the FVIII-specific T-cell repertoire, it did influence the incidence of a neutralizing antibody response to FVIII. A subset of DRB1*1501 mice did not respond to FVIII infusions following intravenous administration, possibly because of induction of peripheral tolerance in these animals. However, when FVIII was coinjected with lipolysaccharide, all of the mice responded and developed inhibitory antibodies. In the future, this new hemophilic mouse model, complemented with models expressing alternative MHC class II molecules, could serve to unravel the complex mechanisms that induce immune responses or tolerance to infused FVIII.

Table 1.   Factor VIII-derived peptides presented on major histocompatibility complex class II molecules
FVIII domainsResiduesSequencePresented by APCsDescribed T-cell epitopeReferences
  1. Peptides have been divided into two categories: (i) reported to be presented by APCs and (ii) described as T-cell epitopes. Light and dark bars indicate peptides that meet the criteria for one or two of these categories, respectively. Detailed information regarding HLA binding of individual peptides can be found in van Haren et al. [20], Moise et al. [78], and Steinitz et al. [74].

Heavy chain
 A1 (1–372)5–13YYLGAVELS   20
83–103TVVITLKNMASHPVSLHAVGV   20,74
128–136VFPGGSHTY   20
227–247AWPKMHTVNGYVNRSLPGLIG   74
246–254IGCHRKSVY   20
293–301FLTAQTLLM   20
368–376FIQIRSVAK   20
 A2 (373–740)455–481GEVGDTLLIIFKNQASRPYNIYPHGIT   20,74
521–541PTKSDPRCLTRYYSSFVNME   14,74
729–737YLLSKNNAI   20
 B (741–1648)782–790IQNVSSSDL   20
991–999FKVSISLLK   20
1101–1109FLPESARWI   20
1129–1137LVSLGPEKS   20
1209–1217VVLPQIHTV   20
1227–1235LFLLSTRQN   20
1382–1405QANRSPLPIAKVSSFPSIRPIYLT   74
1492–1500VELLPKVHI   20
1548–1554FLRVATESS   20
1598–1606ILSLNACES   20
Light chain
 A3 (1649–2020)1731–1750KKVVFQEFTDGSFTQPLYRG   71
1766–1786EVEDNIMVTFRNQASRPYSFY   20,74
1783–1791YSFYSSLIS   20
1801–1820EPRKNFVKPNETKTYFWKVQ   20,71
1883–1891FDETKSWYF   20
1996–2004WRVECLIGE   20
2001–2026LIGEHLHAGMSTLFLVYSNKCQTPLG   20,74
 C1 (2021–2173)2053–2061LHYSGSINA   20
2081–2089IHGIKTQGA   20
2098–2112ISQFIIMYSLDGKKW   13,20
2141–2161NPPIIARYIRLHPTHYSIRST   20,71,74
 C2 (2174–2332)2191–2220TASSYFTNMFATWSPSKARLHLQGRSNAWR   14,20,72,78,81
2254–2269SMYVKEFLISSSQDGH   20,78,81

As an alternative to mass spectrometry or humanized mouse models, Moise et al. [78] have used bioinformatics tools to predict HLA-DR epitopes in the C2 domain of FVIII. Furthermore, they validated these predictions by using HLA binding assays and by immunization studies with various hemophilic mouse models. Reding and co-workers pioneered the analysis of CD4+ T-cell responses in patients with hemophilia A [71,79]. By using overlapping synthetic peptides, they identified potential CD4+ T-cell epitopes in the A3 and C2 domains of FVIII. More recently, T-cell epitopes in the A2, C1 and C2 domains of patients with mild hemophilia A with and without clinically significant inhibitors have been identified by the isolation and characterization of T-cell clones and polyclonal lines that recognize peptides containing the wild-type FVIII sequence at the site of the hemophilic missense mutation (at FVIII residue 593, 2150, or 2201) [14,15,70,80,81].

Interestingly, the results of these studies indicate that the wild-type sequence as present in the infused FVIII contains a neo-epitope that arises because of a single amino acid mismatch with endogenously expressed FVIII. On the basis of these observations, we hypothesize that inhibitor formation in mild hemophilia A is induced following presentation of an FVIII-derived peptide containing the wild-type (infused) sequence that overlaps with the missense site in a patient with an HLA type that can bind to this peptide. In other words, the neo-epitope occurs at the single region in FVIII to which a patient with mild hemophilia A would not have developed immune tolerance through clonal deletion or anergic pathways in utero [82]. The number of potential T-cell epitopes increases significantly in severe hemophilia A, as these patients do not express or circulate a full-length, functional FVIII protein. Accordingly, the inhibitor incidence is higher in severe than in mild or moderate hemophilia A [83]. Currently known FVIII-derived peptides that either contain potential T-cell epitopes (because of their presentation by human or humanized mouse model APCs) or contain T-cell epitopes (confirmed on the basis of human or humanized mouse model T-cell responses) are summarized in Table 1.

A potentially clinically relevant situation analogous to inhibitor development in mild hemophilia A exists, in which infused FVIII may provoke immune responses because of single amino acid differences from the patient’s hemophilic FVIII. Human genome sequencing projects and sequencing studies of the gene encoding FVIII [84] are identifying an increasing number of non-synonymous single-nucleotide polymorphisms (ns-SNPs) in FVIII. It is conceivable that when some patients with hemophilia A, including those who produce, and hence are tolerant to, a dysfunctional hemophilic FVIII, are infused with therapeutic FVIII, the wild-type sequence at the mismatch site could provoke a T-cell response leading to inhibitor formation. Potential T-cell epitopes must bind effectively to MHC class II receptors in order for T-cell stimulation to occur. The 32 core FVIII-derived peptides recently shown to bind human MHC class II [20] did not include peptides with amino acids (R484H, R776G, D1241E, and M2238V) encoded by four recently identified ns-SNPs in the F8 gene [20]. Peptide–MHC binding assays, however, indicate that these regions (if presented as naturally processed peptides in vivo) could bind effectively to several class II receptors [85]. Future studies should reveal whether CD4+ T-cell responses arise in some patients with hemophilia A as a result of exposure to these mismatched FVIII sequences.

Summary and future directions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References

This review has discussed recent clinical and basic science investigations that, together, illustrate the heterogeneity of the CD4+ T-cell repertoire in anti-FVIII immune responses. Promiscuously presented FVIII-derived peptides have so far been identified in the FVIII A2, A3 and C1 domains. In vitro peptide–HLA class II binding studies, however, suggest that additional FVIII-derived peptides might also be presented by multiple MHC class II alleles [20,85,86]. Current research efforts are being directed towards obtaining a better understanding the immunologic mechanisms leading to inhibitor development vs. immune tolerance to FVIII, and on utilizing this information to improve patient outcomes. For example, it may be possible to design less immunogenic FVIII proteins targeted to patients with higher-risk HLA and hemophilia genotypes by modifying anchor residues crucial for MHC class II binding, in order to abrogate the presentation of immunodominant peptides. However, alteration of multiple residues would probably be required to significantly reduce the immunogenicity of FVIII. A successful outcome of this approach is critically dependent on the diversity and hierarchy of CD4+ T-cell responses in patients with hemophilia A. We expect that continuing research into the basis of FVIII immunogenicity, and into novel approaches to promote immune tolerance to infused FVIII, will translate eventually into new therapies to improve patient outcomes.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Endocytosis and processing of FVIII by APCs
  5. Modulating effect of von Willebrand factor (VWF) on the uptake of FVIII by APCs
  6. On the immunogenicity of bio-engineered FVIII derivatives
  7. Activation of the immune system by FVIII: looking for the ‘danger signals’
  8. Presentation of FVIII-specific peptides by APCs and the subsequent T-cell response
  9. Summary and future directions
  10. Disclosure of Conflict of Interests
  11. References
  • 1
    Ahmed R, Pulendran B. Learning vaccinology from viral infections. J Exp Med 2011; 208: 23479.
  • 2
    Kasturi SP, Skountzou I, Albrecht RA, Koutsonanos D, Hua T, Nakaya HI, Ravindran R, Stewart S, Alam M, Kwissa M, Villinger F, Murthy N, Steel J, Jacob J, Hogan RJ, Garcia-Sastre A, Compans R, Pulendran B. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 2011; 470: 5437.
  • 3
    Jahn EM, Schneider CK. How to systematically evaluate immunogenicity of therapeutic proteins – regulatory considerations. N Biotechnol 2009; 25: 2806.
  • 4
    Astermark J, Altisent C, Batorova A, Diniz MJ, Gringeri A, Holme PA, Karafoulidou A, Lopez-Fernandez MF, Reipert BM, Rocino A, Schiavoni M, von Depka M, Windyga J, Fijnvandraat K. Non-genetic risk factors and the development of inhibitors in haemophilia: a comprehensive review and consensus report. Haemophilia 2010; 16: 74766.
  • 5
    Pavlova A, Delev D, Lacroix-Desmazes S, Schwaab R, Mende M, Fimmers R, Astermark J, Oldenburg J. Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A. J Thromb Haemost 2009; 7: 200615.
  • 6
    Gouw SC, van den Berg HM. The multifactorial etiology of inhibitor development in hemophilia: genetics and environment. Semin Thromb Hemost 2009; 35: 72334.
  • 7
    Pratt KP. Inhibitory antibodies in hemophilia A. Curr Opin Hematol 2012; 19: 399405.
  • 8
    Kempton CL, White GC II. How we treat a hemophilia A patient with a factor VIII inhibitor. Blood 2009; 113: 1117.
  • 9
    Qian J, Collins M, Sharpe AH, Hoyer LW. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000; 95: 13249.
  • 10
    Reipert BM, Sasgary M, Ahmad RU, Auer W, Turecek PL, Schwarz HP. Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance. Thromb Haemost 2001; 86: 134552.
  • 11
    Wu H, Reding M, Qian J, Okita DK, Parker E, Lollar P, Hoyer LW, Conti-Fine BM. Mechanism of the immune response to human factor VIII in murine hemophilia A. Thromb Haemost 2001; 85: 12533.
  • 12
    Ragni MV, Bontempo FA, Lewis JH. Disappearance of inhibitor to factor VIII in HIV-infected hemophiliacs with progression to AIDS or severe ARC. Transfusion 1989; 29: 4479.
  • 13
    Jones TD, Phillips WJ, Smith BJ, Bamford CA, Nayee PD, Baglin TP, Gaston JS, Baker MP. Identification and removal of a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII. J Thromb Haemost 2005; 3: 9911000.
  • 14
    James EA, Kwok WW, Ettinger RA, Thompson AR, Pratt KP. T-cell responses over time in a mild hemophilia A inhibitor subject: epitope identification and transient immunogenicity of the corresponding self-peptide. J Thromb Haemost 2007; 5: 2399407.
  • 15
    James EA, van Haren SD, Ettinger RA, Fijnvandraat K, Liberman JA, Kwok WW, Voorberg J, Pratt KP. T-cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site. J Thromb Haemost 2011; 9: 68999.
  • 16
    van den Brink EN, Bril WS, Turenhout EA, Zuurveld M, Bovenschen N, Peters M, Yee TT, Mertens K, Lewis DA, Ortel TL, Lollar P, Scandella D, Voorberg J. Two classes of germline genes both derived from the V(H)1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII. Blood 2002; 99: 282834.
  • 17
    Jacquemin MG, Desqueper BG, Benhida A, Vander EL, Hoylaerts MF, Bakkus M, Thielemans K, Arnout J, Peerlinck K, Gilles JG, Vermylen J, Saint-Remy JM. Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood 1998; 92: 496506.
  • 18
    Dasgupta S, Navarrete AM, Bayry J, Delignat S, Wootla B, Andre S, Christophe O, Nascimbeni M, Jacquemin M, Martinez-Pomares L, Geijtenbeek TB, Moris A, Saint-Remy JM, Kazatchkine MD, Kaveri SV, Lacroix-Desmazes S. A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes. Proc Natl Acad Sci USA 2007; 104: 896570.
  • 19
    Oldenburg J, Picard JK, Schwaab R, Brackmann HH, Tuddenham EG, Simpson E. HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII. Thromb Haemost 1997; 77: 23842.
  • 20
    van Haren SD, Herczenik E, ten Brinke A, Mertens K, Voorberg J, Meijer AB. HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII. Mol Cell Proteomics 2011; 10: M110.002246.
  • 21
    Lollar P. Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX. J Thromb Haemost 2004; 2: 108295.
  • 22
    van Helden PM, van Haren SD, Fijnvandraat K, van den Berg HM, Voorberg J. Factor VIII-specific B cell responses in haemophilia A patients with inhibitors. Haemophilia 2010; 16: 3543.
  • 23
    Delignat S, Repessé Y, Navarrete AM, Meslier Y, Gupta N, Christophe OD, Kaveri SV, Lacroix-Desmazes S. Immunoprotective effect of von Willebrand factor towards therapeutic factor VIII in experimental haemophilia A. Haemophilia 2012; 18: 24854.
  • 24
    Herczenik E, van Haren SD, Wroblewska A, Kaijen P, van den Biggelaar M, Meijer AB, Martinez-Pomares L, ten Brinke A, Voorberg J. Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain. J Allergy Clin Immunol 2012; 129: 5019.e5.
  • 25
    Bovenschen N, van Dijk KW, Havekes LM, Mertens K, van Vlijmen BJ. Clearance of coagulation factor VIII in very low-density lipoprotein receptor knockout mice. Br J Haematol 2004; 126: 7225.
  • 26
    Bovenschen N, Mertens K, Hu L, Havekes LM, van Vlijmen BJ. LDL receptor cooperates with LDL receptor-related protein in regulating plasma levels of coagulation factor VIII in vivo. Blood 2005; 106: 90612.
  • 27
    Saenko EL, Yakhyaev AV, Mikhailenko I, Strickland DK, Sarafanov AG. Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism. J Biol Chem 1999; 274: 3768592.
  • 28
    Sarafanov AG, Ananyeva NM, Shima M, Saenko EL. Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein. J Biol Chem 2001; 276: 119709.
  • 29
    Sarafanov AG, Makogonenko EM, Pechik IV, Radtke KP, Khrenov AV, Ananyeva NM, Strickland DK, Saenko EL. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein. Biochemistry 2006; 45: 182940.
  • 30
    Meems H, van den Biggelaar M, Rondaij M, van der Zwaan C, Mertens K, Meijer AB. C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII. Int J Biochem Cell Biol 2011; 43: 111421.
  • 31
    Dasgupta S, Navarrete AM, Andre S, Wootla B, Delignat S, Repesse Y, Bayry J, Nicoletti A, Saenko EL, d’Oiron R, Jacquemin M, Saint-Remy JM, Kaveri SV, Lacroix-Desmazes S. Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation. Haematologica 2008; 93: 839.
  • 32
    Meems H, Meijer AB, Cullinan DB, Mertens K, Gilbert GE. Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity. Blood 2009; 114: 393846.
  • 33
    Wroblewska A, van Haren SD, Herczenik E, Kaijen P, Ruminska A, Jin S-Y, Zheng XL, van den Biggelaar M, ten Brinke A, Meijer AB, Voorberg J. Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice. Blood 2012; 119: 5294300.
  • 34
    Behrmann M, Pasi J, Saint-Remy JM, Kotitschke R, Kloft M. von Willebrand factor modulates factor VIII immunogenicity: comparative study of different factor VIII concentrates in a haemophilia A mouse model. Thromb Haemost 2002; 88: 2219.
  • 35
    Delignat S, Dasgupta S, Andre S, Navarrete AM, Kaveri SV, Bayry J, Andre MH, Chtourou S, Tellier Z, Lacroix-Desmazes S. Comparison of the immunogenicity of different therapeutic preparations of human factor VIII in the murine model of hemophilia A. Haematologica 2007; 92: 14236.
  • 36
    Kallas A, Kuuse S, Maimets T, Pooga M. von Willebrand factor and transforming growth factor-beta modulate immune response against coagulation factor VIII in FVIII-deficient mice. Thromb Res 2007; 120: 91119.
  • 37
    Qadura M, Waters B, Burnett E, Chegeni R, Bradshaw S, Hough C, Othman M, Lillicrap D. Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice. Blood 2009; 114: 87180.
  • 38
    Reipert BM, Schoppmann A, Schwarz HP. A caution on the use of murine hemophilia models for comparative immunogenicity studies of FVIII products with different protein compositions. Thromb Haemost 2003; 89: 111012.
  • 39
    Dasgupta S, Repesse Y, Bayry J, Navarrete AM, Wootla B, Delignat S, Irinopoulou T, Kamate C, Saint-Remy JM, Jacquemin M, Lenting PJ, Borel-Derlon A, Kaveri SV, Lacroix-Desmazes S. VWF protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors. Blood 2007; 109: 61012.
  • 40
    Goudemand J, Rothschild C, Demiguel V, Vinciguerrat C, Lambert T, Chambost H, Borel-Derlon A, Claeyssens S, Laurian Y, Calvez T. Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A. Blood 2006; 107: 4651.
  • 41
    Gouw SC, van der Bom JG, Auerswald G, Ettinghausen CE, Tedgard U, van den Berg HM. Recombinant versus plasma-derived factor VIII products and the development of inhibitors in previously untreated patients with severe hemophilia A: the CANAL cohort study. Blood 2007; 109: 46937.
  • 42
    Vlot AJ, Koppelman SJ, van den Berg MH, Bouma BN, Sixma JJ. The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor. Blood 1995; 85: 31507.
  • 43
    Mannucci PM, Gringeri A, Peyvandi F, Santagostino E. Factor VIII products and inhibitor development: the SIPPET study (survey of inhibitors in plasma-product exposed toddlers). Haemophilia 2007; 13: 658.
  • 44
    Gringeri A. VWF/FVIII concentrates in high-risk immunotolerance: the RESIST study. Haemophilia 2007; 5: 737.
  • 45
    Gaberc-Porekar V, Zore I, Podobnik B, Menart V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel 2008; 11: 24250.
  • 46
    Mei B, Pan C, Jiang H, Tjandra H, Strauss J, Chen Y, Liu T, Zhang X, Severs J, Newgren J, Chen J, Gu JM, Subramanyam B, Fournel MA, Pierce GF, Murphy JE. Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment. Blood 2010; 116: 2709.
  • 47
    Paz P, Xie J, Fuelle L, Shiroma D, Wu J, Liu P, Koellnberger M, Jacquemin M, Lavendhomme R, Laux V, Murphy J, Aswad F. PEGylated FVIII exhibits reduced immunogenicity in hemophilia A mice and in vitro in human cells. Haemophilia 2012; 18: PO-WE-125. Available at: http://www.journalth.com/about.php.
  • 48
    van Helden PM, Unterthurner S, Hermann C, Schuster M, Ahmad RU, Schiviz AN, Weiller M, Antoine G, Turecek PL, Muchitsch EM, Schwarz HP, Reipert BM. Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model. Blood 2011; 118: 3698707.
  • 49
    Peng A, Kosloski MP, Nakamura G, Ding H, Balu-Iyer SV. PEGylation of a factor VIII–phosphatidylinositol complex: pharmacokinetics and immunogenicity in hemophilia A mice. AAPS J 2012; 14: 3542.
  • 50
    Powell JS, Josephson NC, Quon D, Ragni MV, Cheng G, Li E, Jiang H, Li L, Dumont JA, Goyal J, Zhang X, Sommer J, McCue J, Barbetti M, Luk A, Pierce GF. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients. Blood 2012; 119: 30317.
  • 51
    Dumont JA, Liu T, Low SC, Zhang X, Kamphaus G, Sakorafas P, Fraley C, Drager D, Reidy T, McCue J, Franck HW, Merricks EP, Nichols TC, Bitonti AJ, Pierce GF, Jiang H. Prolonged activity of a recombinant factor VIII–Fc fusion protein in hemophilia A mice and dogs. Blood 2012; 119: 302430.
  • 52
    Akilesh S, Christianson GJ, Roopenian DC, Shaw AS. Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism. J Immunol 2007; 179: 45808.
  • 53
    Lei TC, Scott DW. Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins. Blood 2005; 105: 486570.
  • 54
    Liu T, Hoehn T, Patarroyo-White S, Pierce G, Jiang H. Evaluation of antibody responses to rFVIIIFc compared to Xyntha and Advate in hemophilia A mice. Haemophilia. 2012; 18: FP-WE-04.2-5. Available at: http://www.journalth.com/about.php.
  • 55
    Krishnamoorthy S, Valee S, Liu T, Drager D, Light D, Pierce G, Jiang H. Cell-mediated immune response to recombinant factor VIII–Fc in hemophilia A mice. Haemophilia 2012; 18: PO-WE-113, Available at: http://www.journalth.com/about.php.
  • 56
    Pfistershammer K, Stockl J, Siekmann J, Turecek PL, Schwarz HP, Reipert BM. Recombinant factor VIII and factor VIII–von Willebrand factor complex do not present danger signals for human dendritic cells. Thromb Haemost 2006; 96: 30916.
  • 57
    Esmon CT. Interactions between the innate immune and blood coagulation systems. Trends Immunol 2004; 25: 53642.
  • 58
    Ruf W. Emerging roles of tissue factor in viral hemorrhagic fever. Trends Immunol 2004; 25: 4614.
  • 59
    Bernard GR, Vincent J-L, Laterre P-F, LaRosa SP, Dhainaut J-F, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, Fisher CJ. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699709.
  • 60
    Opal SM. The nexus between systemic inflammation and disordered coagulation in sepsis. J Endotoxin Res 2004; 10: 1259.
  • 61
    Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 87987.
  • 62
    van der Poll T, Levi M, Hack CE, ten Cate H, van Deventer SJ, Eerenberg AJ, de Groot ER, Jansen J, Gallati H, Büller HR. Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees. J Exp Med 1994; 179: 12539.
  • 63
    Riewald M, Kravchenko VV, Petrovan RJ, O’Brien PJ, Brass LF, Ulevitch RJ, Ruf W. Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1. Blood 2001; 97: 310916.
  • 64
    Niessen F, Schaffner F, Furlan-Freguia C, Pawlinski R, Bhattacharjee G, Chun J, Derian CK, Andrade-Gordon P, Rosen H, Ruf W. Dendritic cell PAR1–S1P3 signalling couples coagulation and inflammation. Nature 2008; 452: 6548.
  • 65
    Skupsky J, Zhang AH, Su Y, Scott DW. A role for thrombin in the initiation of the immune response to therapeutic factor VIII. Blood 2009; 114: 47418.
  • 66
    Sawamoto Y, Prescott R, Zhong D, Saenko EL, Mauser-Bunschoten E, Peerlinck K, van den Berg M, Scandella D. Dominant C2 domain epitope specificity of inhibitor antibodies elicited by a heat pasteurized product, factor VIII CPS-P, in previously treated hemophilia A patients without inhibitors. Thromb Haemost 1998; 79: 628.
  • 67
    Meeks SL, Cox CL, Healey JF, Parker ET, Doshi BS, Gangadharan B, Barrow RT, Lollar P. A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function. Blood 2012; 120: 251220.
  • 68
    Gouw SC, van den Berg HM, le Cessie S, van der Bom JG. Treatment characteristics and the risk of inhibitor development: a multicenter cohort study among previously untreated patients with severe hemophilia A. J Thromb Haemost 2007; 5: 138390.
  • 69
    Mancuso ME, Graca L, Auerswald G, Santagostino E. Haemophilia care in children – benefits of early prophylaxis for inhibitor prevention. Haemophilia 2009; 1: 814.
  • 70
    Jacquemin M, Vantomme V, Buhot C, Lavend’homme R, Burny W, Demotte N, Chaux P, Peerlinck K, Vermylen J, Maillere B, van der Bruggen P, Saint-Remy JM. CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A. Blood 2003; 101: 13518.
  • 71
    Reding MT, Okita DK, Diethelm-Okita BM, Anderson TA, Conti-Fine BM. Epitope repertoire of human CD4(+) T cells on the A3 domain of coagulation factor VIII. J Thromb Haemost 2004; 2: 138594.
  • 72
    Pratt KP, Qian J, Ellaban E, Okita DK, Diethelm-Okita BM, Conti-Fine B, Scott DW. Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site. Thromb Haemost 2004; 92: 5228.
  • 73
    Reipert BM, Steinitz KN, van Helden PM, Unterthurner S, Schuster M, Ahmad RU, Ilas J, Schwarz HP. Opportunities and limitations of mouse models humanized for HLA class II antigens. J Thromb Haemost 2009; 7(Suppl. 1): 927.
  • 74
    Steinitz KN, van Helden PM, Binder B, Wraith DC, Unterthurner S, Hermann C, Schuster M, Ahmad RU, Weiller M, Lubich C, de la Rosa M, Schwarz HP, Reipert BM. CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice. Blood 2012; 119: 407382.
  • 75
    Gregersen JW, Holmes S, Fugger L. Humanized animal models for autoimmune diseases. Tissue Antigens 2004; 63: 38394.
  • 76
    Mangalam AK, Rajagopalan G, Taneja V, David CS. HLA class II transgenic mice mimic human inflammatory diseases. Adv Immunol 2008; 97: 65147.
  • 77
    Hay CR, Ollier W, Pepper L, Cumming A, Keeney S, Goodeve AC, Colvin BT, Hill FG, Preston FE, Peake IR. HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party. Thromb Haemost 1997; 77: 2347.
  • 78
    Moise L, Song C, Martin WD, Tassone R, De Groot AS, Scott DW. Effect of HLA DR epitope de-immunization of factor VIII in vitro and in vivo. Clin Immunol 2012; 142: 32031.
  • 79
    Reding MT, Okita DK, Diethelm-Okita BM, Anderson TA, Conti-Fine BM. Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIII. J Thromb Haemost 2003; 1: 177784.
  • 80
    Ettinger RA, James EA, Kwok WW, Thompson AR, Pratt KP. Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti-factor VIII immune responses. Blood 2009; 114: 14238.
  • 81
    Ettinger RA, James EA, Kwok WW, Thompson AR, Pratt KP. HLA-DR-restricted T-cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P. Haemophilia 2010; 16: 4455.
  • 82
    Hay CR. Factor VIII inhibitors in mild and moderate-severity haemophilia A. Haemophilia 1998; 4: 55863.
  • 83
    Gouw SC, van den Berg HM, Oldenburg J, Astermark J, de Groot PG, Margaglione M, Thompson AR, van Heerde W, Boekhorst J, Miller CH, le Cessie S, van der Bom JG. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis. Blood 2012; 119: 292234.
  • 84
    Viel KR, Ameri A, Abshire TC, Iyer RV, Watts RG, Lutcher C, Channell C, Cole SA, Fernstrom KM, Nakaya S, Kasper CK, Thompson AR, Almasy L, Howard TE. Inhibitors of factor VIII in black patients with hemophilia. N Engl J Med 2009; 360: 161827.
  • 85
    Gunasekera D, Ettinger RA, Hughes RJ, Epstein MS, Barrett JC, Thompson AA, Withycombe J, Pratt KP. Potential immunogenicity of amino acid sequences encoded by Ns-SNPs in factor VIII. ASH Annual Meeting Abstracts 2011; 118: 3329.
  • 86
    Liu MER, James E, Lewis K, Pratt K. Identification of potential T-cell epitopes in factor VIII using peptide microarrays. Haemophilia. 2012; 18: PO-WE-132, Available at http://www.journalth.com/about.php.