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Molecular determinants of FVIII immunogenicity in hemophilia A
Wróblewska, A.
Publication date
2013
Link to publication
Citation for published version (APA):
Wróblewska, A. (2013). Molecular determinants of FVIII immunogenicity in hemophilia A.
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2
Dangerous liaisons:
how the immune system deals with
factor VIII
Aleksandra Wroblewska, Birgit M. Reipert, Kathleen P. Pratt and Jan Voorberg
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Abstract
Only a fraction of hemophilia A patients develops a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII (FVIII). 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. While 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 peptides on 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 on FVIII processing and presentation by APCs, as well as the diversity of the FVIII-specific T-cell repertoire in mice and men. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of 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.
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IntroductionProfessional APCs such as dendritic cells (DCs), macrophages and B cells endocytose proteins that are degraded along the endocytic pathway and then presented on 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 (Figure 1). In DCs, MHC class II expression at the cell surface increases following their maturation, which also results in up-regulation of co-stimulatory molecules required for activation of naive 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-TNF-alpha antibodies such as adalimumab or infliximab, coagulation factors VIII and IX and erythropoietin. 3 In view of the ubiquitous
presentation of self- 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 up-regulation of co-stimulatory molecules required for activation of naïve CD4+ T cells.
This review will explore the issue of neutralizing anti-drug antibodies in the context of current knowledge of blood coagulation factor VIII (FVIII) immunogenicity. Approximately 25% of patients with the severe form of the X-linked bleeding disorder hemophilia A (defined by FVIII pro-coagulant activity < 1% 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 treatment of hemophilia A patients with inhibitors have been
provided in a recent paper by Kempton and White. 8 Inhibitor formation as well
as 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 hemophilia A patients. 12-17 Activation of FVIII-specific T cells is preceded
by the uptake of FVIII by antigen-presenting cells (APCs) and the subsequent presentation of FVIII peptides on MHC class II molecules on the surface of these APCs. 18 HLA alleles DRB1*15 and DQB1*0602 have been suggested to correlate
with increased risk for inhibitor development in hemophilia 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
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responses: a large majority of hemophilia A patients achieve functional immune tolerance to FVIII, either following their initial infusions or after eradicating an inhibitor response via immune tolerance induction (intensive FVIII therapy) or immunosuppression. The promiscuity of FVIII peptides in terms of binding to different HLA alleles 20, and the huge diversity in HLA types when compared
to the number of hemophilia patients included in earlier studies investigating the role of HLA in inhibitor risks, 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 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 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
Factor VIII 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 hemophilia A patients who do not have pre-existing immune tolerance to
Figure 1. MHC class II presentation pathway. DCs endocytose antigens
that subsequently undergo proteolytic degradation in endosomal compartments. Transport of newly synthesized MHC class II molecules from the 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.
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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 pro-coagulant function, e.g. by blocking its interaction with thrombin-activated platelet membranes and microparticles derived from these membranes that expose negatively-charged phosphatidylserine 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 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 (N239) and in the light chain (N2118). 18 Blockage with
mannanleadstopartialinhibitionofFVIIIendocytosisbyimmaturehumanDCs.18
Furthermore, strongly reduced proliferation of FVIII-specific human CD4+ T-cell
clone D9E9 was seen after pulsing 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 and co-workers demonstrated that mannan did not block the uptake of FVIII by human or murine DCs, and siRNA-mediated knock-down 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.
Low-density lipoprotein receptor-related protein (LRP), a broadly expressed scavenger molecule, has been implicated in FVIII clearance. 25-29 Lysine 2092
and phenylalanine 2093 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 monoclonal antibody KM33, which targets the FVIII C1 domain, was recently shown to inhibit FVIII endocytosis by human as well as murine DCs (Figure 2A). 24 Moreover, in vivo administration
of KM33 prevented neutralizing antibody production 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 due to its low affinity binding of FVIII. 24 FVIII residues Arg2090, Lys2092 and Phe2093
(Figure 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- and B-cell responses as compared to 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.
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Modulating the effect of von Willebrand factor on uptake of FVIII by APCs
FVIII circulates in plasma as a large glycoprotein complexed with its multimeric chaperone molecule, von Willebrand Factor (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 using mouse models of hemophilia A have indicated that pre-incubation of recombinant FVIII with VWF leads to a reduction in titers of inhibitory anti-FVIII antibodies following FVIII infusions. 23,34-36 However,
results of these studies should be interpreted carefully since human VWF is also immunogenic in mice. 34,37 Immune responses to infused human VWF may result
in lower anti-FVIII antibody titers simply due to 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 (Figure 2A). 24,39 These experimental results have relevance to epidemiological
studies that have suggested a reduced prevalence of inhibitors in hemophilia A patients 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. Due to the high on-rate of the FVIII-VWF complex, one would expect that infused FVIII associates rapidly with endogenous VWF. 42 Randomized, prospective
clinical studies, such as the RES.I.ST (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) and SIPPET (Survey of Inhibitors in Plasma-Product Exposed Toddlers) studies, are addressing potential benefit of VWF containing concentrates for hemophilia A treatment. 43,44
On the immunogenicity of bio-engineered FVIII derivatives
PEGylation is a common method 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-DCs, followed by significantly reduced proliferation and production of IFNg 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 as compared to its recombinant non-PEGylated counterpart. On the other hand, a recent report by van Helden et al. indicated that PEGylation of FVIII could also result in a FVIII protein that expresses higher immunogenicity in hemophilic mouse models. 48 Also, incorporation of
PEGylated lipids into complexes of FVIII and phosphatidylinositol resulted in increased immunogenicity following intravenousinfusioninamurinehemophilia Amodel.49GeneticfusionsofFVIIItotheFcfragmentofIgG1havebeenutilized
toprolongthehalf-lifeofFVIII. 50,51Prolongation of therapeutic protein-Fc fusion
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and FcRn in the early endosome; recycling of endosomes re-exposes IgG1-Fc to physiological 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 compared to endocytosis of unmodified FVIII, which may be followed by FVIII peptide presentation on the 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 hemophilia A mice with pre-existing inhibitors. 53 Initial data on the immunogenicity of FVIII-Fc proteins
in murine hemophilia A models has recently been reported in two meeting abstracts. 54,55 These initial reports suggested a slightly reduced immunogenicity
of the FVIII-Fc fusions, as compared to a B-domain deleted FVIII, when they were administered at therapeutic dosages (50 – 100 IU/kg). Infusion of supra-physiological dosages (250 IU/kg), however, resulted in significantly higher inhibitor titres when compared to B-domain deleted FVIII. 54,55 Additional studies
are needed to shed light on the immunogenicity of FVIII-Fc fusions and other bio-engineered FVIII derivatives, including the delivery methods and dosages.
Activation of the immune system by FVIII: looking for the “danger signals”.
Activation of naive CD4+ T cells requires presentation of antigenic peptides by
mature DCs in the context of MHC class II molecules and co-stimulatory signals. Although FVIII is endocytosed efficiently, it is still not clear what sometimes prompts the immune system to raise a high titre antibody response against it, especially since it is administered intravenously, which is a normally tolerogenic route, in the absence of any adjuvant. Pfistershammer and colleagues 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
A1 A2
A3
C1
C2 Arg2090 Lys2092Phe2093
KM33 VWF FVIII FVIII with C1 domain mutation APC A B
Figure 2. Modulation of FVIII endocytosis by antigen-presenting cells. A. Interference of FVIII uptake by
antigen-presenting cells. C1 domain-directed monoclonal anti-FVIII antibody KM33 as well as the FVIII chaperone molecule, VWF, abolish uptake of FVIII by 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 antigen-presenting cells. 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 residues Arg2090, Lys2092 and Phe2093 highlighted in red. Models were based on FVIII crystal structure (PDB code 3cdz) and prepared using PyMol imaging software.
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change following incubation of monocyte-derived DCs with FVIII or FVIII-VWF complexes. 56
FVIII exerts its pro-coagulant effect at a critical control point in the coagulation cascade, and coagulation itself has been shown to be a hallmark of sepsis as well as 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 pro-inflammatory 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 are the primary site in the lymphatic compartment that intersects inflammation and coagulation. 64 PAR1 signalling and cross-talk between PAR1 and sphingosine
1-phosphate (S1P) activates DCs and promotes systemic inflammation. 64
Pro-coagulant activity of FVIII leads to thrombin generation, which can potentially act as a “danger signal” and thus contribute to FVIII immunogenicity. Skupsky and colleagues 65 demonstrated that co-injections of FVIII and OVA caused
mice to mount an immune response to the latter, which when injected alone is poorly immunogenic. Furthermore, co-administration of OVA and thrombin was sufficient to provoke an immune response to OVA. Anticoagulant warfarin as well as the direct thrombin inhibitor hirudin also significantly reduced B- 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 primary immune response induction 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 pro-coagulant activity were tested for their ability to raise an immune response in FVIII- and FVIII/VWF-deficient mice. 67 FVIII R372A/R1689A was inactive due to substitutions at thrombin
and factor X proteolytic activation sites, whereas the other variant tested, FVIII V634M, although cleaved by thrombin, displayed less than 1% of the pro-coagulant activity of FVIII wild-type. Various injection protocols demonstrated that FVIII wild-type and V634M were equally immunogenic, independently from FVIII dosages and mouse models used. FVIII R372A/R1689A was slightly less immunogenic than the other two variants tested, however this could be potentially explained by its limited release from VWF. These recent experimental results suggest that the pro-coagulant function of FVIII may not be the major determinant of its immunogenicity.
We anticipate that bystander bacterial or viral challenges can provide co-stimulatory signals to provoke immune responses against FVIII. High-intensity FVIII treatment due to excessive bleeding episodes has also been linked to inhibitor development. 68 Intensive treatment may allow FVIII to compete more
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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 subsequent T-cell response
Recently, a plethora of new data have emerged regarding the presentation of FVIII peptides by APCs to T cells, both in 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 and colleagues 20 explored the repertoire of FVIII 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 which many promiscuous epitopes appeared, whereas others 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 using mouse models of hemophilia A, e.g. immunodominant T-cell epitopes in FVIII were identified using E16-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, while 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
due to 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 FVIII-specific T-cell repertoire between mice and men.
Steinitz and colleagues 74 recently utilized these humanized hemophilic mice to
analyze FVIII 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, which might have been due to induction of peripheral tolerance
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in these animals. However, when FVIII was co-injected with LPS, all of the mice responded and developed inhibitory antibodies. In the future, this new hemophilia 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.
As an alternative to mass spectrometry or humanized mice models, Moise and colleagues 78 have used bioinformatic tools to predict HLA-DR epitopes in the
C2 domain of FVIII. Furthermore, they validated these predictions using HLA binding assays and by immunization studies using 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 mild hemophilia A patients with and without clinically significant inhibitors have been identified by 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 residues 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 due to a single amino acid mismatch with endogenously expressed FVIII. Based on these observations we hypothesize that inhibitor formation in mild hemophilia A is induced following presentation of a FVIII peptide containing the wild-type (infused) sequence that overlaps 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 mild hemophilia A patient 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 severity hemophilia A. 83 Currently known FVIII-derived
peptides that either contain potential T-cell epitopes (due to their presentation by human or humanized mouse model APCs), or that contain T-cell epitopes (confirmed on the basis of human or humanized mouse model T-cell responses) are summarized in Table 1.
Apotentiallyclinicallyrelevantsituationanalogoustoinhibitordevelopmentin mildhemophilia A exists, in which infused FVIII may provoke immune responses due to single amino acid differences with the patient’s hemophilic FVIII. Human genome sequencing projects, as well as sequencing studies of the gene encoding FVIII 84, are identifying an increasing number of non-synonymous single nucleotide
polymorphisms in FVIII. It is conceivable that when some hemophilia A patients, 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
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FVIII domains Residues Sequence Presented by APCs Described T-cell epitope References
Heavy chain 5-13 YYLGAVELS 20
A1 (1-372) 83-103 TVVITLKNMASHPVSLHAVGV 20, 74 128-136 VFPGGSHTY 20 227-247 AWPKMHTVNGYVNRSLPGLIG 74 246-254 IGCHRKSVY 20 293-301 FLTAQTLLM 20 368-376 FIQIRSVAK 20 A2 (373-740) 455-481 GEVGDTLLIIFKNQASRPYNIYPHGIT 20, 74 521-541 PTKSDPRCLTRYYSSFVNME 14, 74 729-737 YLLSKNNAI 20 B (741-1648) 782-790 IQNVSSSDL 20 991-999 FKVSISLLK 20 1101-1109 FLPESARWI 20 1129-1137 LVSLGPEKS 20 1209-1217 VVLPQIHTV 20 1227-1235 LFLLSTRQN 20 1382-1405 QANRSPLPIAKVSSFPSIRPIYLT 74 1492-1500 VELLPKVHI 20 1548-1554 FLRVATESS 20 1598-1606 ILSLNACES 20
Light chain 1731-1750 KKVVFQEFTDGSFTQPLYRG 71
A3 (1649-2020) 1766-1786 EVEDNIMVTFRNQASRPYSFY 20, 74 1783-1791 YSFYSSLIS 20 1801-1820 EPRKNFVKPNETKTYFWKVQ 20, 71 1883-1891 FDETKSWYF 20 1996-2004 WRVECLIGE 20 2001-2026 LIGEHLHAGMSTLFLVYSNKCQTPLG 20, 74 C1 (2021-2173) 2053-2061 LHYSGSINA 20 2081-2089 IHGIKTQGA 20 2098-2112 ISQFIIMYSLDGKKW 13, 20 2141-2161 NPPIIARYIRLHPTHYSIRST 20, 71, 74 C2 (2174-2332) 2191-2220 TASSYFTNMFATWSPSKARLHLQGRSNAWR 14, 20, 72, 78, 81 2254-2269 SMYVKEFLISSSQDGH 20, 78, 81
for T-cell stimulation to occur. The 32 core FVIII peptides recently shown to bind human MHC class II 20 did not include peptides with amino acids residues (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 hemophilia A patients as a result of exposure to these mismatched FVIII sequences.
Table 1. FVIII-derived peptides presented on MHC class II molecules. Peptides have been divided into two categories: (1) reported to be presented by antigen-presenting cells and (2) 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 papers by van Haren et al. 20, Moise et al. 78 and Steinitz
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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 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 derived peptides might also be presented by multiple MHC class II alleles. 20,85,86 Current research efforts
are being directed towards better understanding the immunologic mechanisms leading to inhibitor development versus 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 likely be required in order to significantly reduce the immunogenicity of FVIII. Successful outcome of this approach is critically dependent on the diversity and hierarchy of CD4+
T-cell responses in hemophilia A patients. 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.
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