Molecular determinants of FVIII immunogenicity in hemophilia A - Chapter 1: General introduction

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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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Hemophilia A

Hemophilia A is hereditary X-linked disorder caused by dysfunction or absence

of blood coagulation factor VIII. The bleeding syndrome has been recognized

as early as in the fifth century, but only since 1937 was linked to a deficiency

of a plasma component, named in 1962 “factor VIII”.

_{Depending on the}

severity of the disease, it can be divided into three categories: mild (5-25% of

normal

plasma

levels

of FVIII),

moderate (1-5%)

and severe

(<1%)

hemophilia A.

In its severe form it can lead to spontaneous joint and muscle bleeds, which

consequently can cause deformation of joints requiring surgical intervention.

Trauma can cause life-threatening internal bleedings and hemorrhages. Treating

hemophilia became possible in 1964, when Judith Graham Pool, working at

Stanford University, described how to obtain cryoprecipitate from human

plasma.

_{Since then, so-called replacement therapy comprising regular injections}

of FVIII of either recombinant or plasma-derived origin became commonly

used in hemophilia care. However, such treatment is hampered by high costs

of the FVIII products – it is estimated that more than 75% of the hemophilia

community

worldwide

receive

either

inadequate

or

no

treatment

whatsoever.

In response to frequent FVIII infusions, a subset of patients develops anti-FVIII

antibodies. In severe hemophilia A, inhibitors develop after a median of 10

to 15 days of treatment with FVIII.

_{After 50 exposure days, the cumulative}

incidence of inhibitors reaches a plateau, after which the occurrence of

inhibitors is exceptional.

_{Anti-FVIII antibodies can rapidly inhibit FVIII function,}

rendering FVIII infusion therapy ineffective. Inhibitory antibodies interfere with

binding of FVIII to other coagulation factors such as factor IIa, IXa and X or to

phospholipids.

_{Non-inhibitory antibodies can also compromise hemophilia}

treatment by influencing FVIII stability and/or its pharmacokinetics by interfering

with binding to von Willebrand factor (VWF).

_{The majority of inhibitory}

antibodies directed towards FVIII are of subclass IgG1 and IgG4.

Factor VIII: structure and function

FVIII is synthesized as a polypeptide chain comprising a signal peptide of 19

amino acids and a mature protein of 2332 residues. FVIII consists of three A, two

C domains and one unique B domain that are arranged in the following order:

A1-a1-A2-a2-B-a3-A3-C1-C2 (Figure 1).

_{The a1 (residues 337-372), a2 (residues}

711-740) and a3 (residues 1649-1689) regions bordering the A domains are

enriched for aspartic and glutamic acid residues.

_{In plasma, FVIII circulates as a}

hetero-dimer consisting of a 90-220 kDa heavy chain that is non-covalently linked

to a 80 kDa light chain (Figure 1).

_{Cleavage at Arg1648 by as yet unidentified}

protease releases the light chain (a3-A3-C1-C2); additional processing at various

positions (including Arg740 and Arg1312) within the B domain yields a heavy

chain (A1-a1-A2-a2-B) that is heterogeneous in size. In the circulation, FVIII is

tightly associated with its carrier protein – von Willebrand factor (VWF), which

protects it from proteolytic degradation and premature clearance.

_Sulfation

of Tyr1680 within the acidic a3 region is required for high affinity binding of

FVIII to VWF.

_{Also residues in the C1 and C2 domain have been implicated}

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in binding of FVIII to VWF.

_{FVIII circulates as an inactive precursor that}

can only act as a cofactor for the FIXa-dependent conversion of FX following its

activation by thrombin or FXa.

_{Cleavage by thrombin at Arg1689 releases the}

acidic a3 region which promotes rapid disassembly of the FVIII-VWF complex

_;

cleavage at Arg372 and Arg740 results in a hetero-trimeric molecule comprising

the A3-C1-C2, A1-a1 and A2-a2 domains.

_{The resulting hetero-trimer can}

efficiently catalyze the conversion of factor X to Xa by factor IXa on phospholipid

surfaces.

_{Dissociation of the A2 domain from hetero-trimeric FVIII results in}

a rapid decline of FVIII cofactor activity.

_{Also cleavage of FVIII by activated}

protein C at positions Arg336 and Arg562 abolishes cofactor activity of activated

FVIII.

_{Residues 1811-1818 in the A3 domain and residues 558-565 in the}

A2 domain have been shown to contribute to the binding of factor IXa.

Limited information is available with respect to the binding site for factor X on

activated FVIII; binding sites for factor X in the C2 domain and the a1 region have

been proposed.

_{It is now well-established that both the C1 and C2 domain}

contribute to binding of FVIII to negatively charged phospholipids.

_{Analysis of}

the three-dimensional structure of the C2 domain and site-directed mutagenesis

have firmly implicated a role for Met2199, Phe2200, Leu2251 and Leu2252 in

the binding of FVIII to negatively charged phospholipids.

_{More recent}

studies have identified an exposed surface loop in the C1 domain harbouring

Arg2090, Gln2091, Lys2092 and Phe2093 that modulates binding of FVIII to

surfaces containing a low percentage of negatively charged,

phosphatidylserine-containing phospholipids.

_{Figure 1 provides an overview of interactive sites}

on FVIII for its major binding partners.

FVIII precursor form

FVIII circulating in plasma

Heavy chain Light chain

Me2+ A1 A2 A3 C1 C2

Figure 1. Structure of blood coagulation factor VIII (FVIII). Upper left panel shows

schematic domain organization of FVIII; lower left panel – schematic representation of circulating FVIII, where the heavy (A1-A2-B) and the light (A3-C1-C2) chain are non-covalently linked via a metal ion-dependent interaction between the A1 and A3 domain. In activated FVIII, the A1 and A3 domains remain non-covalently bound, while the A2 domain is weakly associated with the A1/A3-C1-C2 dimer. Right panel provides a crystal structure of B domain-deleted FVIII (pdb code 3cdz). Interactive sites for phospholipids, VWF, FIXa and FX are indicated in the model.

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Epitope mapping studies have revealed that inhibitory antibodies that develop

in patients with hemophilia A bind to the A2 and C2 domain of FVIII.

_More

detailed binding studies have shown that residues 484-508 provide a major binding

site for anti-A2 domain antibodies.

_{The mode of action of anti-A2 inhibitors is not}

yet entirely clear; an early study by Lollar and co-workers suggested that anti-A2

antibodies act as non-competitive inhibitors of intrinsic Factor X activation complex

by blocking the conversion of FXase/FX complex to the transition state.

_Follow-up

studies using isolated A2 domain suggested that anti-A2 antibodies directly inhibit

the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this

subunit to cofactor activity.

_{Anti-C2 domain antibodies have been shown to bind to}

exposed residues overlapping with the phospholipid binding site.

_{Co-crystalization}

of a human monoclonal anti-C2 domain antibody, BO2C11, revealed that Arg2215,

Arg2220, Met2199, Phe2200, Val2223, Leu2251 and Leu2252 comprise contact

residues for this inhibitor.

_{Inhibitory antibodies directed towards residues}

1811-1818 in the A3 domain have also been detected in patients with hemophilia A.

Antibodies binding to these sites have been shown to limit the binding of FIXa to

FVIII.

_{Moreover, in a small number of patients antibodies directed towards the}

acidic a1-region have been identified.

Replacement therapy and inhibitor development in hemophilia A

The development of inhibitors occurs in approximately 5% of mild or moderate

hemophilia A patients, and in 25% of severe hemophilia A patients.

The low prevalence of inhibitor development in patients with mild and moderate

hemophilia A is most likely caused by the presence of endogenous levels of

circulating FVIII which render this group of patients tolerant to subsequent

replacement therapy.

_{The lack of endogenous levels of circulating FVIII most}

likely underlies the increased frequency of FVIII inhibitors in patients with

severe hemophilia A. A large number of studies have addressed the correlation

between FVIII genotype and inhibitor development.

_{Patients carrying intron}

22 inversions, nonsense mutations or large deletions are more prone to develop

inhibitors than those with small deletions and missense mutations.

_Nonsense

mutations that affect the light chain of FVIII are more frequently associated with

inhibitor development than those present in the FVIII heavy chain.

_Formation

of inhibitors in mild hemophilia associates with a limited number of high-risk

FVIII mutations within the A2, C1 or C2 domain, namely Arg593Cys, Arg2150His

or Trp2229Cys.

_{Why only a fraction of patients generate antibodies against}

FVIII is still poorly understood. Both treatment-related and genetic risk factors

have been shown to contribute to inhibitor development in hemophilia A.

Intensity of FVIII treatment is an acknowledged risk factor, as FVIII administered

to treat bleeding episodes or to support surgery delivers high concentrations

of FVIII that could promote antibody development.

_{Apart from FVIII gene}

mutations

_{, polymorphisms within the IL-10}

_{and TNFA gene}

_{have been}

associated with inhibitor development. Interestingly, a C/T polymorphism in the

promoter region of the CTLA-4 gene

_{was overrepresented in hemophilia A}

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formation. Several studies have explored whether inhibitor development is linked

to type of FVIII product. There are conflicting findings about the immunogenicity

of recombinant versus plasma derived FVIII.

_{Results from a large}

multi-center cohort study suggest that the risk of inhibitor formation is similar for

recombinant and plasma derived FVIII products.

_{Also the von Willebrand}

factor content of therapeutic FVIII products seems not to be associated with the

risk of inhibitor development.

FVIII and the immune system

It is now well-established that the formation of high-affinity IgG molecules

requires FVIII-specific CD4

_{T-cell help.}

_{Initial evidence for involvement of T}

cells in the development of inhibitors in hemophilia A patients came from studies

involving HIV-positive individuals, that due to diminished number of CD4

_{T cells}

showed also decline in anti-FVIII antibody responses.

_{The first step leading}

to activation of specific T- and B-cell responses is recognition of antigen by

specialized antigen-presenting cells (APCs). Subsequently, antigen is processed

into small peptides which are loaded on MHC class II. Presentation of

derived peptides on the surface of APCs triggers the activation of

antigen-specific CD4

_{T cells (Figure 2). In the past years, several studies have addressed}

how FVIII is processed by APCs.

_{Although a role for several receptors has}

been suggested (for a review see Chapter 2)

_{, the exact mechanism of FVIII}

endocytosis by APCs remains unclear. APCs, equipped with receptors recognizing

foreign, pathogen-derived molecules (so-called pathogen-recognition receptors,

PRRs), are able to sense “danger”, which prompts maturation of these cells.

Upon maturation, they upregulate a number of co-stimulatory molecules such

as CD40, CTLA-4, CD80 and CD86 and due to their simultaneous ability to release

cytokines APCs can activate and modulate antigen-specific T cell responses

(Figure 2). The importance of co-stimulatory mechanism for FVIII-specific T-cell

activation has been illustrated by several studies on the blockade of CD40/CD40L

interactions in vivo.

_{CD40/CD40L ligation provides a key event to induce}

humoral responses against antigens

_{; furthermore, blockage of CD40/CD40L}

interaction leads to long-lasting tolerance in mice.

_{However, even though}

the disruption of CD40/CD40L interaction by pre-administration of a monoclonal

antibody targeting CD40L resulted in deficient immune responses against FVIII

in vivo, it failed to induce long-lasting tolerance.

_{Clinical trials suggested that}

administration of a humanized anti-CD40 ligand antibody (hu5c8) can block

anamnestic responses to factor VIII; however it remained unclear whether that

effect would persist and result in long-lasting tolerance.

_{Moreover, due to}

tromboembolic complications in patients treated with hu5c8, this approach for

treatment has been discontinued.

During TCR activation in a particular cytokine milieu, naive CD4

_{T cells may}

differentiate into one of several lineages of T helper (Th) cells including Th1, Th2,

Th17 and regulatory T cells (Tregs), as defined by their pattern of cytokine production

and function. Generally, Th1 cells promote cellular immunity; Th2 cells mediate

humoral immunity; Th17 cells play an important role in clearing pathogens during

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host defense reactions and in inducing tissue inflammation in autoimmune disease

94

_{; Tregs are capable of inducing tolerance by suppressing T- and B-cell responses.}

T-cell reponses against FVIII are of a polyclonal origin and directed against multiple

epitopes present in different domains of FVIII.

_Th1

_{, Th2}

_{as well as Th17}

_cells

have been shown to contribute to FVIII-specific immune responses in hemophilia

subjects. Eventually, FVIII-specific T cells are able to activate FVIII-specific B cells

and subsequently induce affinity maturation and class-switching of immunoglobulin

genes. As a consequence, FVIII-specific long-living plasma cells and memory B cells

are generated, which are able to rapidly respond to re-exposure to FVIII.

FVIII-specific B cell responses are also, similar to T-cell responses, of a polyclonal origin

and directed against various epitopes. Determination of classes and subclasses of

anti-FVIII antibodies revealed dominant contribution of IgGs and their subclasses

– IgG1, IgG2 and IgG4.

_{Generated antibodies can inhibit FVIII function by}

interfering with its interaction with other coagulation factors (mainly A2 and A3-C1

antibodies) and/or phospholipids (C1/C2-directed antibodies), as described earlier.

It has been reported, that a subset of anti-factor VIII IgG hydrolyzes FVIII. These

so-called catalytic antibodies were found in over 50% of inhibitor-positive patients with

severe hemophilia A, but not in inhibitor-negative individuals.

_{FVIII-hydrolyzing IgG}

from each patient tested exhibit multiple cleavage sites on FVIII and the specificity

of cleavage varied from one patient to another.

_{Circulating antibodies that do not}

inhibit FVIII function can influence FVIII half-life, either by interfering with binding

of FVIII to VWF or by formation of immune complexes that can be efficiently cleared

via Fc receptors.

Figure 2. Simplified overview of the development of humoral immune responses. Antigen is endocytosed by

antigen-presenting cell (such as dendritic cell), processed and presented on MHC class II. For efficient activation, co-stimulatory molecules present on APCs and T cell receptors need to interact, while cytokines released by APCs determine the future direction of ongoing T-cell responses. Once primed, T cells can activate B cells in an antigen-specific manner, which leads to formation of long-living plasma cells producing high-affinity antibodies.

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Immune tolerance induction (ITI) therapy – eradication of inhibitors

Development of inhibitors is a serious complication in hemophilia care. The

magnitude of antibody responses is quantified by a functional assay and

expressed in Bethesda units (BU). Patients with low titer of inhibitors (< 5 BU/

ml) can be treated with higher and/or more frequent doses of FVIII, which leads

to saturation of pre-existing inhibitors and still provides enough FVIII to restore

hemostasis and normal coagulation. For patients with higher inhibitor titers

(>5 BU/ml), simple infusion therapy becomes ineffective, therefore bypassing

agents such as activated factor VII or activated prothrombin-complex

concentrates need to be used.

_{Simultaneously, eradication of inhibitors and}

immune tolerance induction (ITI) therapy is introduced. ITI comprises regular

injections of FVIII for a period varying from several weeks up to two years and

since its first description in 1977

_{remains the only strategy that proved to both}

eradicate FVIII inhibitors as well as lead to induction of FVIII-specific immune

tolerance. The most commonly used protocols, known as the Bonn

_{, the Van}

Creveld

_{and the Malmö}

_{protocols, although considerably different}

_,

result in comparable success rates (up to 87%). A recent study showed that

high-dose ITI leads to faster recovery and tolerance induction, accompanied by fewer

bleeding episodes as compared to low-dose ITI.

_{Van Helden and co-workers}

showed a correlation between distribution of IgG-subclasses of anti-FVIII

antibodies and outcome of ITI therapy. A predominance of IgG4 antibodies was

observed in patients who needed prolonged ITI treatment.

_{Also complications,}

such as venous catheter infection, can prolong the course of ITI or even lead to its

failure.

_{The immunological mechanisms underlying success of ITI therapy}

remain unclear. In a naïve, non-primed environment, chronic exposure to high

doses of antigen would activate regulatory T cells able to suppress

antigen-specific (in this case FVIII-antigen-specific) effector T cells, resulting in tolerance

induction.

_{Consequently, no T cell help would be provided to FVIII-specific}

B cells that could not differentiate into long-living antibody producing plasma

cells and, as a result, would be depleted. This scenario is however unlikely to

happen in patients with pre-existing antibodies that have enriched populations

of FVIII-specific memory T and B cells. In this case, memory B cells serve as highly

efficient antigen-presenting cells, able to effectively re-stimulate FVIII-specific

memory T cells. Moreover, upon encounter with antigen, memory B cells rapidly

differentiate into specific antibody producing plasma cells, enriching already

pre-existing pool of such cells. However, the amount of antigen seems to be

crucial factor for an optimal stimulation of memory B cells.

_{Studies using}

FVIII-deficient mice showed that concentrations of FVIII below the physiologic plasma

concentration of 0.1 μg/ml (1 U/mL) restimulate FVIII-specific memory B cells

and induce their differentiation into antibody-secreting cells (ASCs).

_However,

high concentrations (above 0.1 μg/ml), inhibit memory B cell restimulation and

prevent the formation of ASCs.

_{The inhibition of FVIII-specific memory B-cell}

responses seems to be irreversible and not mediated by FVIII-specific T cells. Such

depletion of memory B-cell can be an early event in the inhibitors eradication

in patients undergoing ITI therapy, who receive high doses of FVIII. Gilles and

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colleagues

_{followed two patients with FVIII inhibitors during their course of}

ITI and suggested that induction of anti-idiotypic antibodies, neutralizing the

existing anti-FVIII antibodies, might be one of the reasons underlying success of

ITI.

Animal models of hemophilia A

In

1995

Bi

and

co-workers

_introduced

_two

_mouse

_models

_for

_{hemophilia A,}

which over the years proved to be very useful for broadening our knowledge

on induction and modulation of immune responses against FVIII. Targeted

disruption of exon 16 (E16-KO mice) or exon 17 (E17-KO) resulted in the absence

of functional FVIII in the circulation (<1% normal FVIII activity). Both strains

displayed a bleeding tendency

_{, which could be corrected by infusions with}

human FVIII.

_{Similar to hemophilia A patients, FVIII-deficient mice develop}

an immune response following repetitive intravenous injections of therapeutic

doses of FVIII.

_{Generated antibodies are directed both to the light as}

well

as

to

the

heavy

chain

of

FVIII

_,

_persist

_in

_the

_circulation

_for

_a

_long

_time

_and

their development is strictly dependent on CD4

_{T helper cells.}

_Cytokine

profiles of factor VIII-specific T cells indicate that the regulation of the

anti-FVIII antibody response in hemophilic mice involves both Th1- and Th2-type

cells.

_{Over the years, studies involving FVIII-deficient mice enabled us to}

gain more knowledge regarding modulation of immune response to FVIII and

tolerance induction. Furthermore, it allowed for evaluation of a number of

novel therapeutic approaches to prevent or eradicate inhibitor development in

hemophilia A. Administration of complexes of FVIII and phosphatidylserine (PS)

liposomes resulted in reduced antibody formation against FVIII in hemophilia A

mice.

_{PS associated with apoptotic cells is known to induce anti-inflammatory}

responses in APCs

_{, moreover, PS liposomes reduce the maturation,}

pro-inflammatory cytokine production and T cell priming of APCs.

_{Formation of}

complex between FVIII and phospholipid molecules is mediated by residues

located in the C1 and C2 domains of FVIII.

_{Therefore, a possible alternative}

explanation of the observed inhibitory effect is that PS occupies residues of FVIII

that are crucial for its endocytosis by APCs. CTLA4-IgG, blocking the co-stimulatory

interaction between B7 and CD28, has been shown to transiently inhibit

anti-FVIII antibody formation in hemophilia A mice.

_{An independent study revealed}

that simultaneous blockage of CD40-CD40L and B7-CD28 pathways abolishes

development of inhibitors and promotes long-term immune tolerance specific

for FVIII.

_{CD3 antibody has been studied as a tolerance-inducing agent for}

several autoimmune and inflammatory diseases.

_{It modulates the}

CD3–T-cell receptor (TCR) complex and leads to anergy or apoptosis of effector T CD3–T-cells,

or to the expansion of regulatory CD4

_CD25

_{T cells.}

_{Consequently, anti-CD3}

treatment proved to successfully prevent inhibitor formation in hemophilia A

mice with both BALB/c and C57BL/6 background.

_{Rituximab, a therapeutic}

anti-CD20 antibody, has been used for several years to treat patients with

inhibitors, although with various success rates.

_{In hemophilia A mice,}

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titers in FVIII-challenged animals.

_{However, hyporesponsiveness to FVIII was}

sustained over time only when an anti-CD20 antibody of subclass IgG1 was used,

which, in contrast to the other isotype tested – IgG2a, did not deplete marginal

zone B cells.

_{Moreover, treatment with anti-CD20 IgG1, but not IgG2a, was}

accompanied by substantial increase of splenic regulatory T cells, implicating a

possible tolerogenic role for the remaining B cell population.

_{A recent study}

by Sack and colleagues, where anti-CD20 treatment was combined with

AAV-based gene therapy, showed that B cell depletion can render FVIII-deficient mice

hyporesponsive to FVIII, but results were dependent on the genetic background

of the strain used.

_{Transduction with retroviral constructs encoding FVIII}

A2- or C2-domain in frame with an IgG heavy chain backbone of B cell blasts

has been shown to induce immune tolerance to FVIII in hemophilic mice.

Mice treated with transduced B cells showed reduced inhibitor titers, which

were persistently low after additional challenges with FVIII. Furthermore, the

lower antibody titers correlated with an increased frequency of FVIII-specific

regulatory T cells.

_{Factor VIII deficient mice have also been successfully}

used to study the presence and persistence of memory B cells, as in detailed

described in the previous paragraph.

Bril and colleagues

_{generated transgenic mice expressing human FVIII with the}

Arg593 to Cys mutation, which is associated with mild hemophilia phenotype.

Unlike E16-KO or E17-KO mice, these animals did not develop inhibitor titers upon

repetitive intravenous injections of human FVIII, however induction of an immune

response took place after subcutaneous FVIII administration in the presence of a

strong adjuvant.

_{A similar model was described by van Helden and colleagues who}

constructed a transgenic mouse expressing full length human FVIII.

_{In accordance}

with findings by Bril and co-workers no inhibitor formation was observed in this model

following the intravenous administration of human FVIII. Interestingly, infusion of

PEGylated FVIII evoked an immune response in transgenic mice expressing human

FVIII. These results suggest that transgenic mouse models that express human

FVIII are useful models for assessing the potential immunogenicity of genetically or

chemically modified FVIII variants. More recently, the development of a humanized

hemophilic E17 HLA-DRB1*1501 mouse model has been described.

_Humanized

mice have been utilized to study the regulation of HLA class II-restricted immune

responses to various antigens and they proved to be highly suitable for in vivo

research into the mechanistic basis of human diseases associated with activation of

CD4

_{T cells.}

_{HLA-DRB1*1501 was selected due to a strong connection between}

this haplotype and many immunologic diseases

_{, as well as a previously noted}

link

between

inhibitor

incidence

and

DRB1*1501

in

patients

with

severe

hemophilia

A.

_{Despite some obvious limitations, such new models can be used to analyze the}

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Scope of the thesis

Understanding the recognition of FVIII by antigen-presenting cells and subsequent

activation of specific T and B cells is essential for development of new strategies

for treatment and/or prevention of inhibitor formation to FVIII in hemophilia

A patients. In this thesis we explored the mechanism of FVIII endocytosis by

both human and mouse dendritic cells (Chapter 3 and Chapter 6). A search for

structural determinants important for immune recognition of FVIII revealed that

C1 domain-targeting monoclonal antibody KM33 prevents the uptake of FVIII

by APCs and delays the formation of anti-FVIII antibodies in hemophilia A mice.

Chapter 4 follows up on this finding and shows that modification of C1 domain

residues crucial for KM33 binding diminishes FVIII uptake by dendritic cells.

Upon in vivo administration in FVIII-deficient mice these C1 domain variants

developed significantly lower anti-FVIII antibody titers and reduced CD4

_{T cell}

responses. In Chapter 5 we address the role of immune complex formation in

FVIII endocytosis by APCs and its influence on subsequent FVIII-specific T cell

responses. Chapter 6 elaborates in more detail on the possible mechanism

of FVIII endocytosis by human dendritic cells and the potential role of the C2

domain in this process. Together our findings provide more insight into immune

recognition of FVIII that can be utilized to develop novel strategies for treatment

or prevention of inhibitor formation in hemophilia A.

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