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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-05-1369.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular and Vascular Biology,
University of Leuven, Belgium; Département
d'Ingénierie et d'Etudes des protéines, CEA-Saclay,
Gif-sur-Yvette, France; and Ludwig Institute for Cancer
Research, Brussels, Belgium.
Mild/moderate hemophilia A patients carrying certain mutations in
the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor
occurrence. To analyze the mechanisms responsible for inhibitor
development in such patients, we characterized FVIII-specific CD4+ T-cell clones derived from a mild hemophilia A patient
carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All
T-cell clones recognized synthetic peptides encompassing Arg2150. The
peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of
inhibitor formation. Peptide I2144-T2161 also bound to other DR
molecules such as DRB1*0101 and DRB1*0701, indicating that the
peptide binds to major histocompatibility complex (MHC) class II
molecules expressed in more than 60% of the population. None of the
T-cell clones recognized recombinant FVIII carrying the substitution
Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the
mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as
the native peptide. These observations demonstrate that T cells of this
patient with mutation Arg2150His distinguish between self- and
wild-type FVIII and provide a plausible mechanism for the frequent
occurrence of an inhibitor in patients carrying this substitution. A
similar phenomenon may occur with other mutations associated to an
increased incidence of inhibitor formation.
(Blood. 2003;101:1351-1358) Hemophilia A is an X-linked hereditary bleeding
disorder due to a lack or insufficient amount of functional factor VIII
(FVIII). FVIII is a 330 kDa glycoprotein produced by the liver as a
single polypeptide chain of 2332 amino acids that subsequently
undergoes proteolytic processing. The circulating molecule consists of
2 chains. The heavy chain consists of the A1 and A2 domains and variable lengths of the B domain; the light chain consists of the A3,
C1, and C2 domains. Patients with hemophilia A are treated by
administration of recombinant or plasma-derived FVIII concentrates. This substitution therapy induces the production of FVIII inhibitor antibodies in about 20% of patients with severe hemophilia A (FVIII less than 1%).1
The reason why only some patients with severe hemophilia A produce
inhibitors is poorly understood. Product-related factors play a
role2 but do not account for all cases. Patient-related factors have been identified. Some of the FVIII gene defects such as
large deletions, inversions, or nonsense mutations are accompanied by a
higher risk of inhibitor formation,3,4 but the association is only weak. Clearly, additional predisposing factors are yet to be identified.
The capacity to mount an immune response to soluble proteins depends on
efficient presentation of protein-derived peptides to specific T cells.
This takes place in the context of major histocompatibility
complex (MHC) class II presentation. Early attempts to identify
an association between some human leukocyte antigen (HLA)
haplotypes and the capacity to elicit inhibitory antibodies were not
conclusive. A weak association has, however, been discovered between
the HLA haplotype DRB1*1501, DQB1*0602, and DQA1*0102 and the presence
of inhibitory antibodies.5-7
Several lines of evidence indicate that the immune response to FVIII is
T-cell dependent. First, hypermutations are consistently observed in
the genes coding for the variable part of anti-FVIII antibodies
obtained either by immortalization of peripheral blood lymphocytes of
patients with inhibitor8 or by phage display technology.9 This indicates that B cells producing
anti-FVIII antibodies undergo affinity maturation processes, which
require specific T-cell help.10 Second, the observation
that a large proportion of anti-FVIII antibodies belong to the IgG4
subclass11 also points to a role of T cells in the
development of the humoral response to FVIII, because isotypic
switching is T-cell dependent.
Finally, more direct evidence that the humoral response to FVIII is
T-cell dependent was provided by the observation that in patients with
an established humoral response to FVIII, HIV infection leads to
disappearance of FVIII inhibitor when T-cell counts
decline.12
FVIII-specific T cells have been identified in the peripheral blood of
hemophilia A patients with inhibitor using T-cell proliferation assays
with native FVIII.13 The epitopes recognized by such T
cells were studied using synthetic peptides covering the entire FVIII
molecule.14 Epitopes were mapped in all domains of the FVIII molecule. Surprisingly, T cells proliferating in response to
FVIII peptides were also identified in hemophilia A patients without
inhibitor and in healthy individuals. Only minor qualitative or
quantitative differences were identified between FVIII-specific T cells
from healthy individuals and from hemophilia A patients with or without
inhibitor.14 Thus, the cellular regulatory mechanisms behind the development of inhibitors to FVIII in some hemophilia A
patients remain to be elucidated.
In contrast to the situation of patients with severe hemophilia A, the
development of inhibitor is rare in mild/moderate hemophilia A patients
(FVIII: 1% to 40%).15 Nevertheless, patients carrying certain mutations present an incidence of inhibitor formation comparable to that of patients with severe hemophilia A. Such mutations
have been located in the amino-terminal region of the A2 domain, the
carboxy-terminal part of the C1 domain, and the amino-terminal part of
the C2 domain.16 The development of inhibitors in
mild/moderate hemophilia A patients is a major complication, because
the immune response is frequently directed toward both exogenous normal
FVIII and to the patient's own mutated FVIII. Under such
circumstances, the bleeding phenotype of the patient becomes similar to
that of a patient with severe hemophilia A.16
However, recent analysis of polyclonal and monoclonal anti-FVIII
antibodies derived from patients with mild/moderate hemophilia A and
inhibitor demonstrated that certain antibodies recognized exclusively
epitope(s) present on wild-type FVIII but not on mutated counterparts
carrying substitution Arg593Cys or Arg2150His.17-20
We have taken advantage of such a situation to investigate the
mechanisms responsible for the development of the immune response to
FVIII. Thus, we have analyzed at the clonal level the T-cell response
to FVIII in a mild hemophilia A patient who had developed a strong
immune response to wild-type FVIII while remaining tolerant to his own
FVIII. FVIII-specific T-cell clones have been derived from peripheral
blood CD4+ T cells. The characterization of the epitope
specificity and MHC class II restriction of the T-cell clones
demonstrated that the C1 domain of normal FVIII contains T-cell
epitopes that are absent in FVIII of mild/moderate hemophilia A
patients carrying the mutation Arg2150His in the C1 domain.
Reagents and cell lines
Human plasma-derived FVIII (pdFVIII) was obtained from the Belgian Red
Cross (Brussels, Belgium) and recombinant FVIII (rFVIII) from Wyeth
(Madison, NJ) and Baxter (Deerfield, IL). Human rFVIII light and heavy
chains were obtained by courtesy of Dr Mirella Ezban (Novo Nordisk,
Copenhagen, Denmark). Recombinant FVIII fragments were expressed in
Escherichia coli as fusion proteins with
glutathione-S-transferase and purified on a glutathione column, as
described.8 The chimeric fragments corresponding to the
heavy chain contained amino acid residues 1 to 330, 318 to 398, and 379 to 758, whereas the light chain fragments corresponded to residues 1637 to 1700, 1692 to 2043, 1981 to 2222 (C1 domain), and 2125 to 2332 (carboxy-terminal part of the C1 domain and C2 domain). FVIII peptides
were synthesized on solid phase using F-moc for transient NH2-terminal
protection and were characterized using mass spectrometry. All peptides
were more than 80% pure, as indicated by analytical high-performance liquid chromatography (HPLC). Lyophilized peptides were
dissolved in dimethyl sulfoxide (DMSO) before dilution in
culture medium. Recombinant FVIII wild-type and Arg2150His
B-domain-deleted FVIII were produced as previously
described.21
The FVIII-specific human lymphoblastoid cell lines BO2C118
and LE2E921 were grown in Dulbecco modified Eagle media
(MEM)/Nutrient Mix F12 (Life Technologies, Paisley, United
Kingdom) supplemented with 10% fetal calf serum (FCS), 1.5 g/L
glucose, 4 mM L-glutamine, 1% Caryoser (Bio Sepra,
Cergy-Saint-Christophe, France), and 80 mg/L gentamycin
(Life Technologies).
The mouse monoclonal antibody 2B6 and L24322 to HLA-DR
were obtained from the American Type Culture Collection (Manassas, VA).
The monoclonal antibodies to DP (B7/2123) and to DQ
(SPVL324) were kindly provided by Y. van de Wal (Leiden
University Medical Center, Leiden, The Netherlands).
Immortalization of human PBMCs
Expansion of FVIII-specific oligoclonal T-cell lines PBMCs were purified by Lymphoprep density gradient centrifugation (Nycomed Pharma, Oslo, Norway). To generate autologous dendritic cells (DCs), PBMCs were depleted from T lymphocytes by rosetting with sheep erythrocytes treated with 2-aminoethylisothiouronium. Negatively selected cells were incubated for 2 hours at 37°C in culture flasks at a density of 2 × 106/mL in RPMI 1640 medium supplemented with 0.24 mM L-asparagine, 0.55 mM L-arginine, 1.5 mM L-glutamine (AAG), and 1% autologous plasma previously heated at 56°C for 30 minutes. Adherent cells were cultured in the presence of IL-4 (100 U/mL) and GM-CSF (100 ng/mL) in RPMI-1% autologous plasma. Half of the medium was replaced on day 2 and 4 with fresh medium plus interleukins.On day 5, autologous DCs were incubated at 37°C (5% CO2)
for 18 hours in RPMI-1% autologous plasma supplemented with IL-4 (100 U/mL), GM-CSF (100 ng/mL), and TNF- Detection of FVIII-specific T cells by IFN-  (IFN-) in the supernatant was measured by
enzyme-linked immunosorbent assay (ELISA) using reagents from
Medgenix Diagnostics Biosource (Fleurus, Belgium) following the
manufacturer's recommendations.
Isolation of CD4+ T-cell clones The cell lines that recognized antigen-presenting cells loaded with FVIII were cloned by limiting dilution using as stimulating cells FVIII-specific autologous lymphoblastoid cells (LE2E9 cells, 5000 cells per well) loaded with FVIII by incubation for 18 hours with 7 µg/mL FVIII. Allogeneic lymphoblastoid cells (5000 cells per well) were added as feeder cells. Cultures were supplemented with 50 U/mL IL-2. Established clonal CD4+ T-cell lines were grown in complete IMDM supplemented with IL-2 (50 U/mL) and 0.5 µg/mL phytohemagglutinin (PHA) (HA 16; Murex Diagnostics).Characterization of T-cell clones The antigenic peptides recognized by the T-cell clones were identified in a similar assay using autologous lymphoblastoid cell lines pulsed as above with 20 µg/mL FVIII or FVIII fragments or various concentrations of FVIII synthetic peptides.HLA-DR peptide-binding assay Purification of HLA-DR molecules and peptide-binding assays were performed as previously described.25,26 Briefly, HLA-DR molecules purified from EBV homozygous cell lines by affinity chromatography were incubated with different concentrations of competitor peptide and an appropriate biotinylated peptide. Bound peptide was evaluated in a fluorescence assay.25,26 Data were expressed as the peptide concentration that prevented binding of 50% of the labeled peptide (IC50). Maximal binding was determined by incubating the biotinylated peptide with the purified MHC class II molecule in the absence of competitor. Averages were deduced from at least 3 independent experiments. Unlabeled forms of the biotinylated peptides were used as reference peptides to assess the validity of each experiment. Their IC50 variation did not exceed a factor of 3. These reference peptides were also used to take into account the disparity of the binding sensitivity between the different HLA II molecules. For DRB1*0101 a very good binder displays an IC50 around 1 nM although for DRB1*0301 a very good IC50 is around 450 nM. We therefore expressed the data as the ratio between the IC50 of the tested peptides and that of the reference to facilitate the comparison of binding activity from one HLA II to another one. A ratio lower than 10 identifies peptides with a high affinity for a given MHC class II molecule.
Generation of FVIII-specific T-cell clones Monocyte-derived DCs were loaded with pdFVIII and incubated with TNF- . Thirty microcultures were initiated with
6 × 103 autologous DCs loaded with FVIII and
105 CD4+ T cells and were incubated for 1 week
with IL-6 and IL-12. T cells were restimulated after 1, 2, and 3 weeks
with 6 × 103 DCs loaded with FVIII in the presence of
IL-2 and IL-7. After a resting period of 2 weeks, the responding cells
of each microculture were tested for IL-4 and IFN- production
following stimulation with the autologous FVIII-specific lymphoblastoid
cell line LE2E9 loaded with FVIII. No cell line producing IL-4
following stimulation with FVIII was detected. As shown in Figure
1, 8 microcultures (B2, B3, B10, C3, C4,
D4, D6, and D9) produced significantly higher amounts of IFN- when
incubated with lymphoblastoid cells pulsed with rFVIII than with the
control cells. Because each microculture was started with
105 cells per well and 8 of 30 cultures contained IFN-
following stimulation with FVIII, the frequency of FVIII-specific
precursors is at least 1 per 375 000 CD4+ T cells in
peripheral blood.
FVIII-specific oligoclonal T-cell lines were cloned by limiting
dilution and repeated stimulation with the autologous lymphoblastoid cell line LE2E9 expressing FVIII-specific surface immunoglobulin and
which had been pulsed with FVIII for 18 hours before incubation with T
cells. Three of the 8 oligoclonal cell lines yielded clones producing
IFN- As shown in Figure 2, when the 3 T-cell
clones were incubated with LE2E9 loaded with pdFVIII, high amounts of
IFN-
Epitope mapping of T-cell clones To determine the location of T-cell epitopes in the FVIII molecule, T cells were incubated with recombinant fragments corresponding to various FVIII domains produced in E coli. The isolated light chain and recombinant protein encompassing the native C1 domain induced secretion of IFN- by each of the 3 T-cell
clones. No IFN- secretion was induced using other recombinant
fragments corresponding to the A1, A2, or A3 domains, respectively
(data not shown).
The epitope specificity of the T-cell clones was determined using synthetic peptides. Because the patient's humoral response was limited to epitope(s) present on wild-type FVIII but not on his own FVIII carrying substitution Arg2150His,19 2 synthetic peptides, I2144-T2161 and I2139-T2154, encompassing residue Arg2150 were selected for these experiments. The latter peptide did not stimulate any of the T-cell clones (data not shown). By contrast, the peptide containing residues I2144-T2161 stimulated the 3 T-cell clones. The T-cell epitopes were further defined using truncated synthetic peptides. Deletion of 4 amino-terminal residues (peptide Y2148-T2161) allowed the stimulation of B3:6 and B10:4 but not of D9:E9 (data not shown). A synthetic peptide devoid of the 3 carboxy-terminal residues (I2144-I2158) stimulated B10:4 but only poorly B3:6 and D9:E9 (data not shown), which indicated that the specificities of the 3 T-cell clones are different. MHC class II restriction When the T-cell clones were cultured with the lymphoblastoid cell line LE2E9 and peptide I2144-T2161 in presence of a monoclonal antibody to MHC class II DR molecules, IFN- production was completely abrogated, indicating that MHC class II restriction is mediated by DR
molecules. No inhibition was observed with monoclonal antibodies to MHC
class II DP and DQ molecules (data not shown).
Further identification of MHC class II molecules responsible for presentation of this peptide was obtained using lymphoblastoid cell lines carrying DR molecules corresponding to those of patient LE (ie, allotypes DRB1*1502/DRB5*0102 and DRB1*0401/DRB4*0101). As shown in Table 1, both B3:6 and D9:E9
recognized peptide I2144-T2161 presented by cell lines expressing the
haplotype DRB1*1502 and DRB5*0102. B10:4 was activated by a cell line
expressing the haplotype DRB1*0401 and DRB4*0101. Although in the
epitope mapping experiment, B10:4 recognized the same peptides as B3:6, this difference in MHC class II restriction demonstrates that these 2 T-cell clones are different.
Because of the homology between the haplotype DRB1*1502/DRB5*0102 and DRB1*1501/DRB5*0101, we investigated whether B3:6 and D9:E9 recognized peptide I2144-T2161 presented by DRB1*1501/DRB5*0101. As shown in Table 1, the cell lines BO2C11 expressing the latter allele presented the FVIII peptide as efficiently as cell lines expressing DRB1*1502/DRB5*0102. Binding of peptide I2144-T2164 to purified MHC class II DR molecules Peptide I2144-T2164 is therefore presented by 2 common MHC class II haplotypes: DRB1*1501/DRB5*0101 and DRB1*0401/DRB4*0101. Previous observations suggested that certain peptides bind to a large array of DR molecules.27,28 We therefore investigated in a competitive binding assay the in vitro association of peptide I2144-T2161 to a series of recombinant MHC class II molecules. Alleles expressed at high frequency in the white population were selected for these experiments. Binding was considered positive when the concentration of the competitor peptide required to inhibit 50% of binding of the labeled peptide (IC50) was lower than 1000 nM, a threshold previously used by other groups.25,29,30As shown in Table 2, peptide I2144-T2164
bound to the most commonly expressed DR molecules, except DRB1*03 and
DRB3*01. Noteworthy, the binding affinity for these alleles was high.
Indeed, the reference peptides used in these assays were among the best
binder peptides toward these HLA-DR molecules, and the concentration of
peptide I2144-T2164 required to inhibit 50% of binding of the labeled peptide was either equal to or lower than that required to reach the
same inhibition level with the unlabeled reference peptide. These
results indicate that peptide I2144-T2164 can bind to at least one DR
allele in at least 60% of the population in Europe and the US.
Comparison of FVIII-specific and non-FVIII-specific lymphoblastoid cell lines as antigen-presenting cells Antigen presentation by specific B cells is deemed to be important for the initiation of an immune response to many proteins.31 We therefore studied T-cell activation by lymphoblastoid cell lines as a function of antibody specificity.T-cell activation in presence of LE56, an autologous lymphoblastoid
cell line that does not recognize FVIII, was therefore compared with
that in presence of the FVIII-specific cell line LE2E9. Synthetic
peptide I2144-T2161 was presented to the T-cell clones B10:4 and D9:E9
as efficiently by LE2E9 and LE56 (Figure 3A,C), which indicates that the latter
correctly expressed the relevant MHC class II molecules. However,
compared with LE2E9, LE56 was poorly efficient to present FVIII to the
T-cell clones B10:4 and D9:E9. Indeed, 10- to 100-fold more FVIII was
required to induce comparable levels of IFN-
T-cell activation by FVIII or synthetic peptide carrying substitution Arg2150His Patient LE developed a strong humoral response to FVIII but not to his mutated FVIII. We therefore asked whether the patient's T cells also exhibited a specificity for wild-type but not for autologous FVIII.Activation of the T-cell clones by FVIII peptide I2144-T2161 presented
by LE2E9 was therefore compared with that induced by an analoguous
peptide carrying the Arg2150His substitution. No production of IFN-
Activation of the T-cell clone D9:E9 was further evaluated using normal and Arg2150His rFVIII as source of antigenic peptides. Because non-FVIII-specific lymphoblastoid cell lines were poorly efficient to present the wild-type FVIII molecule to T cells (Figure 3), FVIII-specific lymphoblastoid cell lines were the antigen-presenting cells (APCs) of choice for these experiments. However, the FVIII-specific cell line LE2E9 could not be used because it does not bind Arg2150His FVIII. The BO2C11 cell line was therefore used as APCs because it produces an antibody recognizing the C2 domain, which allows binding of Arg2150His FVIII as well as of native FVIII.8,21 In addition, BO2C11 expresses MHC class II molecules DRB1*1501/DRB5*01 compatible with presentation of peptide I2144-T2161 to D9:E9 (Table 1). D9:E9 activation in presence of peptide Arg2150His I2144-T2161
presented by BO2C11 was lower than activation with the native peptide
(Figure 5A), in agreement with the
reduced activation of the T-cell clone by the mutated peptide presented
by the autologous cell line, LE2E9. No activation of D9:E9 was observed
with Arg2150His FVIII at concentrations up to 20 µg/mL (ie, 100-fold
higher than the FVIII concentration in plasma), whereas with wild-type
FVIII a significant production of IFN-
Binding of peptide Arg2150His I2144-T2164 to purified MHC class II DR molecules To determine how mutation Arg2150His impairs the presentation of peptide I2144-T2161 to T lymphocytes, we compared the binding of native and mutated peptides to immunopurified DR molecules immobilized on ELISA plates. As shown in Table 3, substitution Arg2150His did not reduce the binding to DRB1*0101, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB5*0101, while binding to DRB1*1501 or DRB4*01 was only moderately reduced. This suggests that substitution Arg2150His does not prevent peptide anchoring into MHC class II molecules but rather impairs recognition of the peptide/DR molecule complex by the T-cell receptors.
The objective of this work was to determine the cellular mechanisms responsible for the increased incidence of immune response to FVIII in mild/moderate hemophilia A patients carrying the mutation Arg2150His in the FVIII C1 domain. We therefore characterized FVIII-specific T-cell clones derived from a hemophilia A patient carrying such a mutation and who developed an immune response to normal FVIII but not to his own FVIII. The 3 T-cell clones isolated from this patient recognized epitopes in the FVIII peptide, I2144-T2161, encompassing residue Arg2150. None of these T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His found in the patient FVIII. The patient, from whom the T-cell clones were derived, developed a strong humoral response to normal FVIII but not to his own mutated FVIII molecule. This suggested that amino acid residue(s) involved in FVIII antigenicity is present in the C1 domain. Mutation Arg2150His prevents inhibition of FVIII activity by the patient's polyclonal antibodies and by the human monoclonal antibody LE2E9, which indicates that this mutation alters antigenic determinant(s) recognized by inhibitor antibodies.17,19,20,21 This phenomenon may contribute to the higher incidence of inhibitor formation in mild/moderate hemophilia A patients with mutations in the carboxy-terminal part of the C1 domain. The observation that the T-cell clones derived from the patient did not recognize FVIII carrying the substitution Arg2150His indicates that normal and patient LE FVIII differ with regard to T-cell epitope(s) expression. This also suggested that the T cells recognized an epitope encompassing residue 2150. In agreement with this hypothesis, epitope mapping with recombinant FVIII fragments and synthetic peptides indicated that the 3 T-cell clones recognized epitopes within residues I2144-T2161. This peptide encompasses Arg2150, which is mutated in the patient FVIII gene. In agreement with the observation that the 3 T-cell clones do not recognize FVIII carrying the substitution Arg2150His, the peptide I2144-T2161 carrying that substitution was not recognized by the T-cell clones. Analysis of wild-type and Arg2150His peptide I2144-T2161 binding to immunopurified MHC class II molecule indicated that the mutation only moderately reduces the binding of the antigenic peptide to MHC class II molecules. Accordingly, it is likely that the Arg2150His mutation impairs recognition of the antigenic peptide I2144-T2161 by the T-cell receptors of 3 FVIII-specific T-cell clones rather than preventing association of the peptide with MHC class II molecules. The observation that patient LE B and T cells recognize epitopes within the same region of the FVIII molecule was surprising because B and T cells often recognize epitopes in distinct regions of antigenic proteins,32,33 although certain T cells can provide help to B lymphocytes recognizing antigenic determinants overlapping the T-cell epitopes.34 It is plausible that the limited difference between normal FVIII and patient LE FVIII (the substitution Arg2150His) has directed the selection of overlapping B- and T-cell epitopes. Peptide I2144-T2161 is an interesting antigenic peptide because it binds to a large array of DR molecules. This peptide was presented by DR molecules of the patient's haplotype DRB1*1502/DRB5*0102 or DRB1*04/DRB4*01. The 2 T-cell clones recognizing peptide I2144-T2161 in the context of DRB1*1502/DRB5*0102 also recognized the FVIII peptide presented by DRB1*1501/DRB5*01, in agreement with the high homology existing between these haplotypes. Both T-cell clones were also activated when the native FVIII protein was presented by a lymphoblastoid cell line BO2C11 expressing these DR molecules and a high-affinity anti-FVIII antibody. Study of peptide I2144-T2161 binding to immunopurified DR molecules demonstrated that this peptide can bind to the most commonly expressed DR molecules but not to DR3 (DRB1*03/DRB3*01). Peptides binding to a large number of DR molecules were previously identified for other proteins and called "universal" T-cell antigenic peptide.27,28,35 Few of these peptides bind to DRB1*03, similarly to peptide I2144-T2161. The latter peptide can thus be antigenic in patients carrying DRB1*0101, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, or DRB1*1501, which are each expressed in about 2% to 20% of the white population. Accordingly, this peptide could be presented to T lymphocytes of at least 60% of that population. The isolation of FVIII-specific T-cell clones from the blood of a moderate hemophilia A patient with inhibitor is in agreement with previous publications in which T cells proliferating specifically upon incubation with FVIII were observed in peripheral blood of hemophilia A patients with inhibitor.13,14 The epitopes recognized by such T cells were located by incubating T cells with peptides overlapping the whole FVIII molecule.14 In severe hemophilia A patients, FVIII-specific T cells recognizing a large array of peptides covering the entire FVIII molecule were detected. However, stimulation with FVIII peptides also identified FVIII-specific CD4+ T cells in the blood of hemophilia A patients without inhibitor and in healthy individuals. There was only a limited quantitative difference between healthy individuals and hemophilia A patients with or without inhibitor in proliferation of FVIII-specific T cells.14 Different hypotheses can be formulated to account for the limited difference reported between individuals with and without inhibitor with regard to FVIII-specific T cells. First, it is plausible that stimulation with peptides does not provide a full evaluation of the size of the T-cell population recognizing each individual peptide, which could lead to an underestimation of the number of FVIII-specific T cells in patients with inhibitor. In addition, some FVIII peptides used to stimulate T cells may correspond to cryptic epitopes. Such epitopes elicit a T-cell response when the immune system is challenged with short peptides encompassing these residues but not with the native protein containing the corresponding peptidic sequences.36,37 To stimulate T cells in a manner representative of what may happen following treatment of patients with FVIII, we isolated T-cell clones from patient LE by repeated stimulation with intact FVIII molecules, thereby avoiding potential bias in capture, processing, and/or cleavage of the protein. The restricted epitope specificity of the clones generated in the present study strikingly differs from that observed when peptides are used to stimulate CD4+ T cells in blood of hemophilia A patients or of healthy individuals. The 3 T-cell clones derived from patient LE blood recognized the same region within the FVIII C1 domain, suggesting that such cells represent a prominent T-cell population that is associated to the development of an inhibitory immune response to FVIII. This observation suggests that 2 populations of FVIII-specific T cells may coexist: one harmless, found in healthy individuals and in hemophilia A patients irrespective of inhibitor status, and the second pathogenic, which may be present only in patients with inhibitor antibodies. FVIII-specific T cells were cloned using the secretion of interleukin
as screening assay. In the initial screening of patient LE oligoclonal
T-cell lines, both IFN- The FVIII amounts administered for substitution therapy in hemophilia A patients are relatively low, with FVIII concentrations peaking between 0.1 to 0.2 µg/mL plasma. This raises the question of how the immune system efficiently presents such small amounts of antigen to T lymphocytes. Studies carried out in mice have demonstrated that, for most proteins, the initiation of a T-cell response is dependent on B lymphocytes.38 The ability of antigen-specific B cells to present FVIII to T cells was therefore evaluated in vitro using FVIII-specific B-cell lines as APCs. About 10-fold less FVIII was required to activate T cells with lymphoblastoid cell lines expressing surface FVIII-specific monoclonal antibody than with a nonspecific cell line. This is in agreement with the ability of B lymphocytes to concentrate antigen through binding on surface immunoglobulins.39 Epidemiologic studies have identified a higher incidence of inhibitor development in severe hemophilia A patients with inversion of intron 22 and expressing the HLA haplotype DRB1*1501, DQB1*0602, and DQA1*0102, with only the association with DQA1*0102 reaching statistical significance. The association with DRB1*1501 did not reach statistical significance, possibly because this allele is common in the white population. The fact that a T-cell epitope containing peptide such as peptide I2144-T2161 is able to bind to a large array of DR molecules may be one of the reasons why only weak associations are observed between inhibitor development and MHC class II molecules. Although one T-cell clone recognized peptide I2144-T2161 in the context of the haplotype DRB1*04/DRB4*01, the frequency of this haplotype was not increased in patients with inhibitor included in the above studies. The difference between DRB1*1501 and DRB1*04/DRB4*01 with regard to the incidence of inhibitor formation may thus be due to the number of different FVIII peptides that can bind to the respective alleles. In this regard, it is of interest that an FVIII peptide from the A3 domain has been eluted from DRB1*1501 immunopurified from a lymphoblastoid cell line cultured in vitro.40 No FVIII peptide was eluted from similar cell lines expressing DR3, DR4, or DR7.40 The success in establishing T-cell clones from patient LE suggests that characterization of T cells in mild/moderate hemophilia A patients with inhibitor to normal but not self-FVIII may be a promising approach to characterize FVIII-specific T cells. It is possible that a restricted epitope specificity of FVIII-specific T cells is characteristic of hemophilia A patients who express a mutant form of FVIII that bears a single amino acid substitution (or perhaps a small deletion) and whose FVIII mutation affects a T-cell epitope. When exposed to wild-type FVIII, the immune system of these patients might recognize only that portion of FVIII carrying the mutation, because that is the only part of FVIII to which their T cells have not been tolerized. Thus, generation and characterization of T-cell clones from mild/moderate hemophilia A patients with inhibitors, where the T-cell response may be more focused, may enable identification of the T-cell epitopes in the FVIII molecule. Once such epitopes are identified, it might then be productive to look for responses to these T-cell epitopes in severe hemophilia A patients, where the response is likely to be more complex because of a lack of tolerance to the whole FVIII molecule. In conclusion, this work allowed the isolation and characterization of the first human FVIII specific T-cell clones. All T-cell clones recognized epitopes located in the C1 domain of normal FVIII but not in FVIII with the substitution Arg2150His. In addition to the previously reported difference between B-cell epitope(s) on normal and Arg2150His FVIII, this phenomenon is likely to have predisposed the patient from whom the T-cell clones were derived to develop an immune response to normal FVIII administered as substitution therapy. Given the binding of the antigenic peptide encompassing Arg2150 to a large array of HLA-DR molecules, such a phenomenon could also be at play in many mild/moderate hemophilia A patients carrying a mutation of Arg2150 and explain why these patients have an increased propensity to develop an FVIII inhibitor.
Submitted May 10, 2002; accepted September 6, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-05-1369.
Supported in part by grant G.0292.98 of the Flemisch Research Foundation (F.W.O.). J.V. is the holder of the Dr J. Choay chair in Haemostasis Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marc Jacquemin, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium; e-mail: marc.jacquemin{at}med.kuleuven.ac.be.
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Abstract
Introduction
) or the
non-FVIII-specific cell line LE56 (
). The indicated concentrations
of peptide I2144-T2161 (A,C) or of FVIII (B,D) were added to the
cultures. After 16 hours of incubation, IFN-
) or Arg2150His (
) I2144-T2161 peptide
at the indicated concentrations. After 16 hours of incubation, IFN-