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Blood, 1 July 2001, Vol. 98, No. 1, pp. 130-139
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Immunodominant epitopes on glycoprotein IIb-IIIa recognized by
autoreactive T cells in patients with immune
thrombocytopenic purpura
Masataka Kuwana,
Junichi Kaburaki,
Hidero Kitasato,
Miyako Kato,
Shinichi Kawai,
Yutaka Kawakami, and
Yasuo Ikeda
From the Institute for Advanced Medical Research and
the Department of Internal Medicine, Keio University School of
Medicine, Tokyo; the Department of Internal Medicine, Tokyo Electric
Power Company Hospital; the Department of Microbiology, Kitasato
University School of Medicine, Sagamihara; and the Institute of Medical
Science, St Marianna University School of Medicine, Kawasaki, Japan.
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Abstract |
It was recently reported that autoreactive CD4+ T cells
to glycoprotein IIb-IIIa (GPIIb-IIIa) mediate antiplatelet autoantibody production in patients with immune thrombocytopenic purpura (ITP). To
further examine the antigenic specificity of the GPIIb-IIIa-reactive T
cells, 6 recombinant fragments encoding different portions of GPIIb
or GPIIIa were generated and tested for their ability to stimulate
antigen-specific T-cell proliferation and anti-GPIIb-IIIa antibody
production in vitro. T cells from the peripheral blood of 25 patients
with ITP and 10 healthy donors proliferated in response to recombinant
GPIIb-IIIa fragments in various combinations. The amino-terminal
portions of both GPIIb and GPIIIa (IIb18-259 and IIIa22-262) were
frequently recognized (60% and 64%, respectively) compared with other
fragments (4%-28%) in patients with ITP, but this tendency was not
detected in healthy donors. In subsequent analyses in patients
with ITP, T-cell reactivities to IIb18-259 and IIIa22-262 were
consistently detected, whereas those to other fragments were sometimes
lost. In vitro antigenic stimulation of peripheral blood mononuclear
cells with IIb18-259 or IIIa22-262 promoted the synthesis of
anti-GPIIb-IIIa antibodies in patients with ITP, but not in healthy
donors. Of 15 CD4+ T-cell lines specific for
platelet-derived GPIIb-IIIa generated from 5 patients with ITP, 13 lines recognized IIb18-259, IIIa22-262, or both. T-cell lines
reactive to IIb18-259 or IIIa22-262 promoted the production of
anti-GPIIb-IIIa antibodies that were capable of binding to normal
platelet surfaces. These results indicate that the immunodominant
epitopes recognized by pathogenic CD4+ T cells in patients
with ITP are located within the amino-terminal portions of both
GPIIb and GPIIIa.
(Blood. 2001;98:130-139)
© 2001 by The American Society of Hematology.
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Introduction |
Chronic immune thrombocytopenic purpura (ITP) is an
autoimmune disease characterized by increased platelet clearance caused by antiplatelet autoantibodies.1,2 These antibodies bind to circulating platelets, resulting in platelet destruction by the
reticuloendothelial system. The major target of the antiplatelet autoantibodies is platelet membrane glycoprotein IIb-IIIa
(GPIIb-IIIa),3-5 also designated
IIb 3 integrin or CD41/CD61, which is a
calcium-dependent heterodimeric membrane receptor for fibrinogen and
other ligands.6 Although earlier studies reported the
presence of platelet-reactive T cells in patients with
ITP,7-9 we have recently found that GPIIb-IIIa is one of
the major target antigens recognized by platelet-reactive CD4+ T cells.10 GPIIb-IIIa-reactive
CD4+ T cells in patients with ITP have a helper activity
that promotes the production of anti-GPIIb-IIIa antibodies capable of
binding to normal platelets, indicating that these autoreactive T cells are involved in the production of pathogenic antiplatelet
autoantibodies in patients with ITP.10
Our previous study demonstrated that GPIIb-IIIa-reactive T cells
respond to chemically modified GPIIb-IIIa and recombinant GPIIb-IIIa
fragments expressed in bacteria, but not to GPIIb-IIIa in its native
form.10 One possible explanation for this finding is that
autoreactive T cells recognize "cryptic" epitopes on GPIIb-IIIa that are not produced from native GPIIb-IIIa by the normal processing pathway. However, the precise locations of these cryptic epitopes have
not been reported. T-cell epitope mapping on GPIIb-IIIa is potentially
useful for detecting foreign cross-reactive proteins that elicit
autoantibody responses to GPIIb-IIIa11 and for developing therapeutic strategies that suppress harmful T-cell responses in
patients with ITP.12 In this study, a series of
recombinant fragments encompassing different portions of GPIIb and
GPIIIa were constructed, and the responses of T cells from patients
with ITP to these fragments were analyzed.
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Patients, materials, and methods |
Patients and controls
T cells from the peripheral blood of 25 patients with chronic
ITP (3 men, 22 women) were analyzed. Chronic ITP was defined as
thrombocytopenia (platelet count less than 150 × 109/L)
persisting longer than 6 months, normal or increased levels of bone
marrow megakaryocytes without morphologic evidence for dysplasia, and
no secondary immune or nonimmune disease that could account for the
thrombocytopenic state.1,2 The mean age at examination was
50.4 years (range, 21-71 years), and the mean platelet count was
36 × 109/L (range, 17 × 109-68 × 109). Anti-GPIIb-IIIa antibodies
in plasma and in platelet eluates were positive in 12 and 22 patients,
respectively, but 3 patients had negative findings for anti-GPIIb-IIIa
antibodies. Seven patients had newly diagnosed disease, and the
remaining 18 patients underwent various treatment regimens. At the time
of blood examination, 12 patients had been on low-dose corticosteroids
for 1.2 to 22.0 years. Previous medical treatment was with
corticosteroids in 6 patients, splenectomy in 7, danazol in 2, slow
infusion of vincristine in 1, and azathioprine in 1 (Table
1). Peripheral blood samples from 14 patients with ITP were examined on 2 or 3 occasions at intervals
ranging from 3 to 24 months.
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Table 1.
T-cell proliferation and in vitro anti-GPIIb-IIIa
antibody production in response to recombinant GPIIb-IIIa fragments in
25 patients with immune thrombocytopenic purpura
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Control T cells were obtained from 10 healthy donors (4 men, 6 women)
who had no history of ITP and showed a T-cell proliferative response to
trypsin-digested GPIIb-IIIa. The mean age at examination was 36.7 years
(range, 23-56 years). All healthy controls had normal platelet counts
and were negative for anti-GPIIb-IIIa antibodies in plasma and
platelet eluates.
Human leukocyte antigen (HLA)-DRB1, -DQB1, and -DPB1 alleles were
determined for all patients and controls, using polymerase chain
reaction (PCR) followed by analysis of restriction fragment length
polymorphisms.13,14 Written informed consent approved by
the Institutional Review Board guidelines was granted by all study participants.
GPIIb-IIIa preparations for T-cell stimulation
Human GPIIb-IIIa was purified from outdated platelet
concentrates using affinity chromatography and chemically modified by treatment with porcine trypsin (0.1 µg/mL) as described
previously.10 Phosphate-buffered saline (PBS) containing
porcine trypsin in the absence of GPIIb-IIIa was also prepared for use
as a control antigen.
Seven different portions of GPIIb and GPIIIa were expressed as
recombinant glutathione S-transferase (GST) fusion
proteins.15 These included IIb18-259, IIb244-575,
and IIb566-841, which encompass amino acid residues 18-259, 244-575, and 566-841, respectively, of the 871 amino acids of GPIIb. The
GPIIIa fragments used were IIIa22-262, IIIa254-462, IIIa455-723, and
IIIa708-762, which encompass amino acid residues 22-262, 254-462, 455-723, and 708-762, respectively, of the 762 amino acids of GPIIIa. A
series of GPIIb and GPIIIa complementary DNA (cDNA) constructs
prepared by reverse transcription and PCR from the bone marrow cells of
a patient with ITP were subcloned into the 3' end of the
Schistosoma japonicum GST gene in the bacterial expression
plasmid vector, pGEX 6P-1 (Amersham Pharmacia Biotech, Piscataway, NJ).
To eliminate possible PCR errors and to verify the translational
frames, both strands of each DNA construct were sequenced on an ABI
Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Expression of the recombinant GST-fusion proteins was induced with 1 mM
isopropyl--D-thiogalactopyranoside, and bacterial lysates containing
recombinant proteins were prepared by sonication in the presence of 8 M
urea. After stepwise dialysis to remove excess denaturant, soluble
bacterial proteins in the presence of 2 M urea were fractionated by
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and
the recombinant GPIIb-IIIa fragments were directly eluted from the
gels.16 Briefly, both the right and the left edges of the
gel were stained with 0.05% Coomassie blue, and the portion of the gel
corresponding to the recombinant protein was excised and crushed by
mechanical compression in PBS. After the gel components were removed by
quick centrifugation, the eluted proteins were dialyzed against PBS and
filter-sterilized. Specificity of the GST-fusion proteins was confirmed
on immunoblots probed with goat anti-GST polyclonal antibodies
(Amersham Pharmacia Biotech). To evaluate the amounts of contaminating
bacterial proteins in the purified preparations, a sample from each
preparation was incubated with glutathione-Sepharose 4B (Amersham
Pharmacia Biotech) for affinity depletion of GST fusion
proteins.15 Purified preparations before and after
affinity depletion were fractionated on SDS-10% polyacrylamide gels,
stained with 0.05% Coomassie blue or Silver Staining Plus kit (Bio-Rad Laboratories, Hercules, CA), and subsequently analyzed using a Molecular Imager FX (Bio-Rad Laboratories). Protein bands absorbed by
incubation with glutathione-Sepharose beads were regarded as recombinant GST-GPIIb-IIIa fusion proteins.
Detection of anti-GPIIb-IIIa antibodies
The level of immunoglobulin G (IgG) anti-GPIIb-IIIa antibodies
in plasma, platelet eluates, and culture supernatants was measured by
enzyme-linked immunosorbent assay (ELISA) using affinity-purified GPIIb-IIIa as an antigen, as described elsewhere.5,10 All samples were tested in duplicate, and the results were calculated as
the duplicate mean. Cutoff values for plasma and platelet eluates were
considered the mean plus 3 × SD of 20 samples from healthy donors.
GPIIb-IIIa-reactive T-cell lines
GPIIb-IIIa-reactive T-cell lines were generated from patients
with ITP using a previously described method17 with some
modifications. Briefly, peripheral blood mononuclear cells (PBMCs) were
isolated from heparinized venous blood using Lymphoprep (Nycomed Pharma AS, Oslo, Norway) density gradient centrifugation and were cultured in
RPMI 1640 containing 8% pooled human AB serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin in the presence of
trypsin-digested, platelet-derived GPIIb-IIIa (5 µg/mL) in a
humidified atmosphere of 5% CO2 at 37°C. On day 3, 20 U/mL interleukin-2 (IL-2) (Life Technologies, Grand Island, NY) was
added to the cultures. Cells were restimulated with trypsin-digested
GPIIb-IIIa and IL-2 (50 U/mL) and irradiated (30 Gy) autologous PBMCs
in fresh medium on day 10. Seven days after the second stimulation, T-cell blasts were cloned by limiting dilution at 1 to 100 cells/well using round-bottomed, 96-well plates in the presence of
trypsin-digested GPIIb-IIIa, IL-2, and irradiated PBMCs. Wells that
contained growing cells were selected, and these cells were expanded by
repeated stimulation with trypsin-digested GPIIb-IIIa, IL-2, and
autologous Epstein-Barr virus-transformed lymphoblastoid B
cells irradiated at 100 Gy. The specificity of each T-cell line
was assessed by antigen-induced T-cell proliferation assays, and T-cell
lines that proliferated in response to trypsin-digested GPIIb-IIIa, but
not to mock-treated PBS containing trypsin alone, were selected as
GPIIb-IIIa-specific T-cell lines. Cell surface expression of CD4 and
CD8 was examined by flow cytometry using fluorescein
isothiocyanate-conjugated monoclonal antibodies (mAbs) against CD4 and
CD8 (Becton Dickinson, San Jose, CA).
T-cell proliferation assay
The antigenic specificity of T cells was determined by
antigen-induced T-cell proliferation.17,18 PBMCs were
cultured in the presence or absence of antigen for 7 days unless
otherwise indicated. GPIIb-IIIa-reactive T-cell lines were cultured
with irradiated autologous lymphoblastoid B cells in the presence or absence of antigen for 3 days. After a final 16-hour incubation with
0.5 µCi/well of 3H-thymidine, the cells were harvested,
and 3H-thymidine incorporation was determined in a TopCount
microplate scintillation counter (Packard, Meriden, CT). Antigens were
used at a concentration of 5 µg/mL and included native GPIIb-IIIa, trypsin-digested GPIIb-IIIa, mock-treated PBS, GST, tetanus toxoid (List Biological Laboratories, Campbell, CA), and individual
recombinant GPIIb-IIIa fragments. All cultures were prepared in
triplicate, and all values represent the mean of triplicate
determinations. For bulk PBMC cultures,  cpm was calculated by
subtracting the cpm value obtained in cultures incubated without
antigen from those obtained in cultures with antigen. Antigen-specific
T-cell proliferation in response to each recombinant GPIIb-IIIa
fragment was expressed as a stimulation index, which was calculated as the cpm incorporated into cultures with recombinant GPIIb-IIIa fragment divided by the cpm incorporated into cultures with GST. A
positive response was defined as having a stimulation index greater
than 3. Standard deviations were less than 20% of the mean or less
than 100 cpm, unless indicated otherwise. To examine the inhibitory
effects of anti-HLA class II mAb on antigen-specific T-cell
proliferation, mAbs were added at the start of the
cultures.10 Anti-HLA-DR (L243; IgG2a), anti-HLA-DQ (1a3;
IgG2a), anti-HLA-DP (B7/21; IgG3), and isotype-matched control mAbs
(Leinco Technologies, Ballwin, MO) were dialyzed against PBS and used
at a final concentration of 1 µg/mL.
In vitro assay for anti-GPIIb-IIIa antibody production
An in vitro assay to analyze the antigen-induced
anti-GPIIb-IIIa antibody synthesis in PBMC cultures or in cultures of
GPIIb-IIIa-reactive T-cell lines and autologous peripheral blood B
cells was carried out as described.10,19 Briefly, PBMCs
(5 × 105/well) or GPIIb-IIIa-reactive T cells
(2 × 105) plus autologous peripheral blood B cells
(105) were cultured in complete medium with or without
antigen in the presence of pokeweed mitogen (1 µg/mL) for 10 days.
Individual recombinant GPIIb-IIIa fragments and GST were used at 5 µg/mL as antigens. In some experiments, anti-interferon  (IFN-) (25718.11; IgG2a), anti-IL-4 (34019.111; IgG2b), or
anti-IL-6 (6708.111; IgG1) mAb (Genzyme Techne, Cambridge, MA) were
added at the initiation of the culture. IgG anti-GPIIb-IIIa antibody
levels in undiluted culture supernatants were measured by ELISA using
purified GPIIb-IIIa as an antigen source, as described above. All
cultures were prepared in duplicate, and anti-GPIIb-IIIa antibody
results represent the mean of duplicate values. Significant
anti-GPIIb-IIIa antibody production in response to antigenic
stimulation with recombinant GPIIb-IIIa fragments was defined as having
both a value greater than 2 for the anti-GPIIb-IIIa antibody levels in
cultures with a recombinant GPIIb-IIIa fragment divided by the
anti-GPIIb-IIIa antibody levels in cultures with GST and an increase
in OD450 (optical density) greater than 0.1 associated with
antigenic stimulation. In some experiments, anti-GPIIb-IIIa antibodies
produced in culture supernatants were absorbed by incubation with
platelets, erythrocytes, or PBMCs obtained from 2 independent healthy
donors, as described elsewhere.10 Standard deviations were
less than 20% of the mean or less than 0.01 (OD450),
unless indicated otherwise.
Cytokine production assay
For the determination of cytokine profiles in individual
GPIIb-IIIa-reactive T-cell lines, T cells were cultured with
phytohemagglutinin (1 µg/mL) and an anti-CD3 mAb (OKT3; 30 ng/mL) for
48 hours, and the supernatants were collected and stored at  80°C
until analysis. The levels of human IFN-, IL-4, and IL-6 in the
culture supernatants were measured in duplicate using ELISA kits
(BioSource International, Camarillo, CA) according to the
manufacturer's instructions.
Statistical analysis
All comparisons between the 2 groups were tested for statistical
significance using the Fisher 2-tailed exact test or the Student
t test. Correlation coefficient (r) was determined
using a single regression model.
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Results |
Expression and purification of recombinant GPIIb and
GPIIIa fragments
All recombinant fusion proteins except IIIa455-723 were
successfully purified and used as antigens for T-cell stimulation. We
failed to purify IIIa455-723, which contains cysteine-rich, tandemly
repeated domains of GPIIIa, because only a trace amount of recombinant
protein was produced by bacteria transfected with cDNA encoding this
region. IIb18-259, IIb244-575, IIb566-841, IIIa22-262,
IIIa254-462, and IIIa708-762 were expressed in large quantities, but
the yields by affinity purification were low because of the formation
of insoluble aggregates. Therefore, these recombinant fragments and GST
were purified directly from bacterial lysates fractionated on
SDS-polyacrylamide gels. As shown in Figure
1, each purified preparation represented
major protein band consistent with the predicted molecular weight mass,
with or without additional bands. These extra protein bands were
completely absorbed by incubation with glutathione-Sepharose beads
and, therefore, were degradation products of recombinant GPIIb-IIIa
fragments. Densitometric analysis on silver-stained gels revealed that
more than 92% of the proteins in our preparations were recombinant
GPIIb-IIIa fragments.
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| Figure 1.
Analysis of recombinant human GPIIb-IIIa fragments by
SDS-polyacrylamide gel electrophoresis.
Purified GST and recombinant human GPIIb-IIIa fusion proteins were
fractionated on a 10% polyacrylamide-SDS gel and stained with
Coomassie blue.
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T-cell proliferative responses to GPIIb-IIIa fragments
The proliferative responses of peripheral blood T cells with the 6 recombinant GPIIb-IIIa fragments were examined, and representative results from 3 patients with ITP are shown in Figure
2. ITP2 responded to IIb18-259,
IIb566-841, and IIIa708-762; ITP18 responded to IIIa22-262; and
ITP21 responded to IIb18-259. Table 1 summarizes the T-cell
proliferative responses to recombinant GPIIb-IIIa fragments in 25 patients with ITP. Each patient showed a significant response to at
least one fragment, though 3 patients (ITP12, ITP18, and ITP22) were
negative for anti-GPIIb-IIIa antibodies at the time of examination. T
cells from the patients with ITP recognized the GPIIb-IIIa fragments in
various combinations, and 12 patients responded to 2 or more fragments.
Amino-terminal regions of both IIb and IIIa (IIb18-259 and
IIIa22-262) were frequently recognized (60% and 64%, respectively),
compared with the other fragments (IIb566-841, IIb244-575,
IIIa254-462, and IIIa708-762), which were recognized by 28%, 12%,
4%, and 12% of the patients, respectively. T-cell proliferative
responses to IIb18-259 or IIIa22-262 were detected in all but one
patient. These findings indicate that several distinct T-cell epitopes
are present on GPIIb-IIIa, but the immunodominant T-cell epitopes are
located within IIb18-259 and IIIa22-262.
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| Figure 2.
Peripheral blood T-cell proliferative responses to recombinant
GPIIb-IIIa fragments.
PBMCs from patients with ITP were stimulated with GST or individual
GPIIb-IIIa fragments for 7 days, and 3H-thymidine
incorporation was measured by liquid-scintillation counting.
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All healthy donors who responded to trypsin-digested GPIIb-IIIa also
showed T-cell proliferative responses to 2 or more recombinant GPIIb-IIIa fragments in various combinations (Table
2). In contrast to findings in patients
with ITP, all 6 recombinant fragments were recognized in similar
frequencies. It was of note that T-cell responses to IIb18-259 and
IIIa22-262 were detected in 3 healthy donors each, whereas
IIb566-841 was recognized in 6 healthy controls. To examine the
kinetics of T-cell proliferative responses to IIb18-259 and
IIIa22-262 in healthy donors versus patients with ITP, T-cell responses
to these fragments were measured on day 5 and day 7 in responders,
including 4 patients with ITP and 3 healthy donors (Figure
3). T-cell responses to IIb18-259 and
IIIa22-262 were detected on day 5 and day 7 in patients with ITP,
whereas T cells from healthy donors did not show a T-cell response on
day 5 but responded on day 7.
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Table 2.
T-cell proliferation and in vitro anti-GPIIb-IIIa
antibody production in response to recombinant GPIIb-IIIa fragments
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| Figure 3.
Peripheral blood T-cell proliferative responses to
IIb18-259 and IIIa22-262 on day 5 and day 7.
PBMCs from 4 patients with ITP (closed circles) and 3 healthy donors
(open circles) were stimulated with GST or GPIIb-IIIa fragments, and
3H-thymidine incorporation was measured on day 5 and day 7 by liquid-scintillation counting. T-cell proliferative responses
induced by IIb18-259 and IIIa22-262 are expressed by
stimulation index.
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HLA and clinical associations with T-cell reactivities to
GPIIb-IIIa fragments
An association between the T-cell reactivities to individual
GPIIb-IIIa fragments and the HLA-DRB1, -DQB1, and -DPB1 alleles was
examined. No statistically significant associations were found in
patients with ITP. However, when patients with ITP and healthy donors
were combined, T-cell reactivity to IIb18-259 was detected in 9 of
10 of them with DRB1*0901 and in 9 of 25 without DRB1*0901 (P = .007).
Thirteen patients with ITP had T-cell responses to one fragment, and
the remaining 12 patients had responses to 2 or more fragments. When
demographic and laboratory findings were compared between these 2 patient groups, no differences were found in sex, age at the time of
diagnosis, platelet count at the time of blood examination, levels of
anti-GPIIb-IIIa antibodies in plasma and platelet eluates, or current
and previous treatment regimens (data not shown). However, the time
between diagnosis and blood examination for patients who responded to
one fragment was significantly longer than that for patients who
responded to 2 or more fragments (135.1 ± 91.7 vs 50.3 ± 60.2
months; P = .01). Figure 4
illustrates that a significant negative correlation between the number
of immunoreactive fragments and the time between diagnosis and blood
examination was detected (r = 0.52;
P = .008).
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| Figure 4.
Negative correlation between the number of
immunoreactive GPIIb-IIIa fragments and the time between diagnosis and
blood examination.
The number of immunoreactive GPIIb-IIIa fragments was negatively
correlated with the time between diagnosis and blood examination in 25 patients with ITP (r = 0.52; P = .008).
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Serial analysis of T-cell reactivities to GPIIb-IIIa fragments in
patients with ITP
T-cell proliferative responses to GPIIb-IIIa fragments were
serially examined in 14 patients with ITP. Immunoreactive fragments detected at the initial and later examinations were concordant in 11 patients. None of the patients developed T-cell responses to additional
fragments during the follow-up, but 3 patients demonstrated a loss of
fragment reactivity. ITP4 responded to 4 fragments IIb18-259, IIb566-841, IIIa22-262, and IIIa708-762at the first examination but responded to IIb18-259 and IIIa22-262 at follow-up examinations 10 and 18 months later. ITP2 lost T-cell reactivity to IIIa708-762, and
ITP7 lost reactivity to IIIa254-462 at the follow-up examinations. It
was noted that T-cell reactivities to immunodominant fragments were
consistently detected in all 14 patients examined. In contrast, T-cell
reactivities lost during follow-up were to nondominant fragments.
In vitro anti-GPIIb-IIIa antibody production in response to
GPIIb-IIIa fragments
Anti-GPIIb-IIIa antibody production was measured in PBMC cultures
with recombinant GPIIb-IIIa fragments, and representative results are shown in Figure 5. PBMCs from
ITP4 produced anti-GPIIb-IIIa antibodies in response to recombinant
fragments IIb18-259, IIb566-841, and IIIa22-262. Samples from
this patient showed T-cell proliferative responses to these 3 fragments
in addition to IIIa708-762, which did not promote in vitro
anti-GPIIb-IIIa antibody production. In vitro anti-GPIIb-IIIa
antibody production was detected in ITP19 and ITP20 in response to
IIIa22-262 and IIb18-259, respectively. For these patients, the
fragments inducing T-cell proliferation were consistent with those
promoting in vitro anti-GPIIb-IIIa antibody production. Levels of
anti-GPIIb-IIIa antibodies in PBMC culture supernatants stimulated
with GST or individual GPIIb-IIIa fragments for 15 patients with ITP
and 7 healthy donors are summarized in Tables 1 and 2, respectively. In
patients with ITP, in vitro anti-GPIIb-IIIa antibody production was
induced by antigenic stimulation with at least one of the GPIIb-IIIa
fragments. In contrast, none of the PBMCs from healthy donors produced
anti-GPIIb-IIIa antibodies, though they showed T-cell proliferation to
2 or more GPIIb-IIIa fragments. To examine whether anti-GPIIb-IIIa
antibodies produced in in vitro cultures react with platelets, 10 randomly selected culture supernatants containing
anti-GPIIb-IIIa antibodies were incubated with platelets,
erythrocytes, or PBMCs obtained from healthy donors. Anti-GPIIb-IIIa
antibody reactivity was suppressed by incubation with platelets, but
not by incubation with erythrocytes or PBMCs (data not shown).
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| Figure 5.
In vitro anti-GPIIb-IIIa antibody production in PBMC cultures in
response to recombinant GPIIb-IIIa fragments.
PBMCs from patients with ITP were cultured with GST or individual
recombinant GPIIb-IIIa fragments for 10 days, and anti-GPIIb-IIIa
antibody levels were measured by ELISA.
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When the results of in vitro anti-GPIIb-IIIa antibody production were
compared with those of T-cell proliferation assays, it was found that
the immunodominant fragments IIb18-259 and IIIa22-262 promoted
anti-GPIIb-IIIa antibody production exclusively in responders to these
fragments in the proliferation assay. In contrast, anti-GPIIb-IIIa
antibody production was detected in none of the PBMC cultures with
IIb244-575, whereas samples from 2 patients showed a T-cell
proliferative response to IIb244-575 (ITP9 and ITP15). Similarly,
IIb566-841 failed to induce anti-GPIIb-IIIa antibody production in
2 of 4 responders to IIb566-841 in the proliferation assay (ITP24
and ITP25). Samples from none of the patients with ITP, including ITP4,
induced anti-GPIIb-IIIa antibody production in response to
IIIa708-762, though ITP4 showed a T-cell proliferative response to this fragment.
Characterization of GPIIb-IIIa-reactive T-cell lines
Fifteen T-cell lines specific for platelet-derived GPIIb-IIIa were
established from 5 patients with ITP. All these lines had a
CD4+CD8 phenotype and responded to
trypsin-digested GPIIb-IIIa, but not to the mock-treated PBS control.
Antigen-induced proliferative responses in representative
GPIIb-IIIa-reactive T-cell lines are shown in Figure
6. Lines WY3 and FS4 responded to
IIIa22-262 and IIb18-259, respectively, in addition to
trypsin-digested GPIIb-IIIa. Eight T-cell lines reacted with one
GPIIb-IIIa fragment (2 with IIb18-259, 1 with IIb566-841, and 5 with IIIa22-262), and 6 lines reacted with 2 or 3 fragments (5 with
IIb18-259 and IIIa22-262, and 1 with IIb18-259, IIb566-841,
and IIIa22-262). One GPIIb-IIIa-reactive T-cell line did not respond
to any of the 6 fragments. In total, 13 of 15 T-cell lines that were
reactive with platelet-derived GPIIb-IIIa recognized IIb18-259,
IIIa22-262, or both, indicating again that T cells reactive with these
2 amino-terminal fragments are the predominant GPIIb-IIIa-reactive T
cells in the peripheral blood of patients with ITP.
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| Figure 6.
Proliferative responses to a series of GPIIb-IIIa
preparations in GPIIb-IIIa-reactive CD4+ T-cell lines.
GPIIb-IIIa-reactive T-cell lines were cultured with autologous
antigen-presenting cells in the presence or absence of various
antigens, including tetanus toxoid, mock-treated PBS containing trypsin
alone, native GPIIb-IIIa, trypsin-digested GPIIb-IIIa, GST, or
individual recombinant GPIIb-IIIa fragments for 7 days, and
3H-thymidine incorporation was measured by
liquid-scintillation counting. Only standard deviation > 20% of the
mean and 100 cpm is shown as an extended line above a bar.
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HLA class II restriction, T-cell helper activity, and cytokine profiles
were further analyzed in the 8 GPIIb-IIIa-reactive T-cell lines that
responded to a single GPIIb-IIIa fragment (Table 3). HLA class II restriction was
determined according to the inhibitory effect of an anti-HLA class II
mAb on antigen-induced T-cell proliferation. As shown in Figure
7, IIIa22-262-induced proliferation was
inhibited by an anti-HLA-DR mAb in line WY3 and by both anti-HLA-DR
and anti-HLA-DP mAb in line WY9. All GPIIb-IIIa-reactive T-cell lines
were restricted by HLA-DR, but line WY9 was restricted by both HLA-DR
and HLA-DP.
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Table 3.
Immunoreactive GPIIb-IIIa fragment, human leukocyte
antigen class II restriction, T-cell helper activity inducing
anti-GPIIb-IIIa antibody production, and cytokine profiles in
GPIIb-IIIa-reactive CD4+ T-cell lines generated from
patients with immune thrombocytopenic purpura
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| Figure 7.
Effects of anti-HLA class II mAb on antigen-induced
proliferation of GPIIb-IIIa-reactive CD4+ T-cell lines.
GPIIb-IIIa-reactive T-cell lines were cultured with autologous
antigen-presenting cells and IIIa22-262 for 3 days in the presence or
absence of anti-HLA-DR, anti-HLA-DQ, anti-HLA-DP, or isotype-control
mAbs, and 3H-thymidine incorporation was measured by
liquid-scintillation counting. Anti-HLA class II mAb and
isotype-control mAbs were added at the initiation of cultures.
Only standard deviation > 20% of the mean and 100 cpm is shown as an
extended line above a bar.
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Representative results of the helper activity promoting
anti-GPIIb-IIIa antibody production in GPIIb-IIIa-reactive T-cell lines are shown in Figure 8A. The
IIb18-259-specific T-cell line SiM4 and the IIIa22-262-specific
T-cell line WY9 promoted anti-GPIIb-IIIa antibody production from
autologous B cells in the presence of IIb18-259 and IIIa22-262,
respectively. Anti-GPIIb-IIIa antibodies synthesized in these cultures
were specifically absorbed by preincubation with normal platelets
(Table 3), indicating that the anti-GPIIb-IIIa antibodies produced in
vitro had the pathogenic activity of binding to platelet surfaces. In
contrast, the IIb566-841-specific T-cell line SuM7 had only a
minimal effect on anti-GPIIb-IIIa antibody production on antigenic
stimulation with IIb566-841. When cytokine expression profiles for
IFN-, IL-4, and IL-6 were examined in 6 GPIIb-IIIa-reactive
CD4+ T-cell lines, all lines produced IFN- with no or
minimal IL-4 expression, indicating a Th1- or a Th0-like phenotype
(Table 3). The capacity of these cell lines to produce individual
cytokines was then compared with their T-cell helper activity to
promote anti-GPIIb-IIIa antibody production. SuM7, which produced only a trace amount of IL-6, lacked significant helper activity, whereas the
remaining 5 lines, which were capable of producing a larger amount of
IL-6 (more than 100 pg/mL), promoted anti-GPIIb-IIIa antibody
production. These findings suggest that T-cell-derived IL-6 plays an
important role in activating B cells. To confirm this, we examined
whether neutralization of IL-6 by an anti-IL-6 mAb inhibited the
T-cell helper activity that promotes anti-GPIIb-IIIa antibody
production. As shown in Figure 8B, the anti-IL-6 mAb inhibited
anti-GPIIb-IIIa antibody production in a dose-dependent manner, but
anti-IFN- and anti-IL-4 mAb had no effect.
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| Figure 8.
T-cell helper activity promoting anti-GPIIb-IIIa
antibody production from B cells in GPIIb-IIIa-reactive
CD4+ T-cell lines.
(A) GPIIb-IIIa-reactive T-cell lines were cultured with autologous
peripheral blood B cells in the presence or absence of GST or
individual recombinant GPIIb-IIIa fragments for 10 days. (B)
GPIIb-IIIa-reactive T-cell lines were cultured with autologous
peripheral blood B cells and the antigenic GPIIb-IIIa fragment in the
presence or absence of anti-IL-6 (1, 5, 25, and 50 µg/mL),
anti-IFN- (25 µg/mL), anti-IL-4 (25 µg/mL), or isotype-control
(25 µg/mL) mAbs for 10 days. The level of anti-GPIIb-IIIa antibodies
in culture supernatants was measured by ELISA.
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Discussion |
In this study, we examined T-cell responses to a series of
recombinant GPIIb-IIIa fragments in 25 patients with ITP. Our results indicate that the immunodominant epitopes recognized by
GPIIb-IIIa-reactive T cells in patients with ITP are located within
the amino-terminal regions of both GPIIb and GPIIIa, though several
T-cell epitopes are located in other portions of the molecule as well.
These findings were drawn from 2 independent assay systems, one
measuring antigen-induced T-cell proliferation and the other
determining the T-cell helper activity promoting autoantibody
production in response to antigenic stimulation. Furthermore, the
results obtained from bulk peripheral blood T cells were principally
concordant with those obtained from CD4+ T-cell lines
specific for platelet-derived GPIIb-IIIa. Our results further suggest
that autoreactive T cells to the immunodominant epitopes within the
amino-terminal portions of GPIIb and GPIIIa are present in the
peripheral blood of patients with ITP for a long period and are
involved in the production of anti-GPIIb-IIIa antibodies.
Anti-GPIIb-IIIa antibodies synthesized in our in vitro cultures
appeared to be pathogenic because they could bind to normal platelet
surfaces. In this regard, it was interesting to examine whether
anti-GPIIb-IIIa antibodies produced in vitro-induced thrombocytopenia
in mice, as has been shown in HIV-1-associated ITP.20,21
It was less likely that an additional dominant T-cell epitope was
present within the cysteine-rich domain of GPIIIa because 14 of 15 T-cell lines specific for platelet-derived GPIIb-IIIa recognized at
least 1 of the 6 recombinant fragments used in this study.
The current study further confirmed the presence of autoreactive T
cells to GPIIb-IIIa in healthy donors without having detectable anti-GPIIb-IIIa antibodies.10,22 The kinetics of T-cell
responses to immunodominant GPIIb-IIIa fragments in patients with ITP
were accelerated compared with that of healthy donors, as observed in a
T-cell response to trypsin-digested GPIIb-IIIa.10
Anti-GPIIb-IIIa antibody synthesis was observed in PBMC cultures of
patients with ITP, but not in cultures of healthy donors, in response
to recombinant GPIIb-IIIa fragments. This is analogous to autoantibody
responses to topoisomerase I in scleroderma23 and to
2-glycoprotein I in antiphospholid syndrome.24 Using an
in vitro culture system consisting of topoisomerase I-specific T-cell
clones and peripheral blood B cells from patients with scleroderma and
healthy donors in different combinations, we have recently shown that
topoisomerase I-specific T-cell clones established from healthy donors
are capable of driving patients' B cells to produce
anti-topoisomerase I antibodies.19 Thus, the failure of
PBMCs from healthy donors to produce anti-GPIIb-IIIa antibodies in in
vitro cultures is probably owing to the absence of circulating B cells
capable of producing anti-GPIIb-IIIa antibodies.
B-cell epitopes on GPIIb-IIIa recognized by autoantibodies from
patients with ITP have been extensively analyzed by examining competitive binding between human antibodies and murine mAbs as well as
B-cell reactivities to enzyme-cleaved GPIIb-IIIa fragments or synthetic
peptides.25-32 Previously reported antigenic epitopes include a 33-kd chymotryptic core fragment of GPIIIa, encoding cysteine-rich domains,25 a carboxyl-terminal cytoplasmic
portion of GPIIIa,26 a 65-kd chymotryptic
carboxyl-terminal fragment of IIb,27 and amino acid
residues 231-238 of IIb,28 which are included in
IIb18-259. However, several recent studies showed that
anti-GPIIb-IIIa antibodies, especially platelet-associated antibodies,
recognize cation-dependent conformational epitopes expressed
exclusively on the GPIIb-IIIa complex.29-32 McMillan et
al33 just recently reported that anti-GPIIb-IIIa
antibodies bind primarily to the Ca++-binding site on
GPIIb. These findings demonstrate that several distinct B-cell
epitopes, including continuous and conformational determinants, are
present on GPIIb-IIIa. Taken together, the recognition of multiple
epitopes on GPIIb-IIIa by autoreactive T and B cells indicates that the
entire GPIIb-IIIa molecule is a target for the autoimmune response in
patients with ITP.
Here, we found that anti-GPIIb-IIIa antibody production was induced in
vitro by antigenic stimulation with IIb18-259 or IIIa22-262 exclusively in patients who showed T-cell proliferative responses to
these fragments. In contrast, nondominant GPIIb-IIIa fragments failed
to induce the production of anti-GPIIb-IIIa antibodies in some samples
that had responded to these fragments in the proliferation assay. This
discrepancy might be explained by a low frequency of autoreactive T
cells to nondominant fragments in peripheral blood, which would have
insufficient power to activate B cells in in vitro cultures. However,
analysis using GPIIb-IIIa-reactive T-cell lines showed that the helper
activity that induces anti-GPIIb-IIIa antibody production was variable
among T-cell lines and was correlated with the amount of IL-6 produced
on stimulation. This finding is consistent with our recent report that
the T-cell helper activity that promotes autoantibody production in
topoisomerase I-specific CD4+ T-cell clones generated by
scleroderma patients is largely dependent on their cytokine profiles,
and especially on their ability to produce IL-6.19 In this
study, the T-cell line SuM7, which was reactive with the nondominant
fragment IIb566-841, expressed a trace amount of IL-6 and failed to
induce significant anti-GPIIb-IIIa antibody production, whereas T-cell
lines that responded to the immunodominant fragments expressed a higher
level of IL-6 and had greater helper activity. Therefore, it is also
possible that autoreactive T cells to immunodominant epitopes have
stronger helper activity than do those to nondominant epitopes, though the number of T-cell lines analyzed in this study was too small to draw
a conclusion.
Findings from the current study and our previous report10
suggest that the epitopes recognized by GPIIb-IIIa-reactive T cells
are cryptic but the factors that induce the expression of cryptic
determinants and activate GPIIb-IIIa-reactive CD4+ T cells
in patients with ITP are unknown. Lehmann et al34 proposed that, in autoimmunity, de novo presentation of a previously cryptic self-determinant is induced by up-regulated antigen presentation capacity and shifts in peptide hierarchy. In this process,
antigen-presenting cells in a local milieu play a central role through
an activated antigen-processing pathway and increased expression of
adhesion and costimulatory molecules. In addition, shifts in peptide
hierarchy resulting in the expression of cryptic self-peptides are
shown to be induced by events that affect the normal processing of
self-proteins, such as an unusual cleavage or a complex formation with
other proteins.35-37 These modifications are presumed to
subsequently mask or unmask cleavage sites for proteinases and
reductases in endosomes, resulting in the expression of previously
cryptic self-peptides. In this regard, it is plausible that the changes
of structural conformation of GPIIb and GPIIIa in recombinant
GPIIb-IIIa fusion proteins by fusion with GST or lack of glycosylation
contribute to the expression of the cryptic self-peptides in the
current study. Our results here indicate that immunodominant T-cell
epitopes are located within the amino-terminal extracellular domains of both GPIIb and GPIIIa, which are known to be exposed on the surface of the molecule. In particular, IIb18-259 and IIIa22-262 contain the
Ca++-binding site and Arg-Gly-Asp binding site,
respectively.38 Therefore, it is possible that the
amino-terminal regions of GPIIb and GPIIIa are preferentially
attacked by chemicals or foreign proteins that can change the molecular
context and lead to the efficient presentation of previously cryptic
peptides in these regions.
It is interesting to note that the number of immunoreactive GPIIb-IIIa
fragments was negatively correlated with the time between diagnosis and
blood examination. Serial analysis of GPIIb-IIIa fragment-induced
T-cell proliferation revealed that 3 of 14 patients lost T-cell
reactivity to the previously recognized fragments during follow-up.
These findings suggest that patients with ITP have T cells that are
responsive to a variety of epitopes on GPIIb-IIIa early in the course
of the disease and that their T-cell repertoires to the immunodominant
epitopes are selectively expanded, whereas those to nonimmunodominant
epitopes fade during the course of the disease. It has been shown in
animal models that an autoimmune response is initiated to one epitope
and subsequently spreads to other sites on the same molecules, a
concept called epitope spreading.39,40 However, epitope
spreading is rarely observed in patients, probably because it has
already occurred before the clinical onset of the disease. On the other
hand, a reduction in isotype expression and epitope reactivity of
autoantibody responses during follow-up was observed in patients with
mixed connective tissue disease41 and
scleroderma,42 independent of treatment. It was also noted
that peripheral blood T cells from healthy donors reacted with 2 or
more GPIIb-IIIa fragments, but IIb18-259 and IIIa22-262 were not
dominant. Taken together, it is possible that T-cell responses to
GPIIb-IIIa are skewed to immunodominant epitopes in the amino-terminal
portions of both GPIIb and GPIIIa during the pathogenic process of ITP.
In summary, our results indicate that GPIIb-IIIa-reactive
CD4+ T cells in patients with ITP recognize several
distinct epitopes on GPIIb-IIIa, but the amino-terminal extracellular
domains of both GPIIb and GPIIIa are the major targets for
autoreactive T cells that mediate antiplatelet autoantibody production.
Further investigation to identify the T-cell epitope peptides within
the immunodominant regions may provide a clue to the pathogenesis of
chronic ITP.
| |
Acknowledgments |
We thank Yuka Okazaki and Kyoko Kimura for their expert
technical assistance and Dr Kenichi Furihata for helpful suggestions.
| |
Footnotes |
Submitted December 4, 2000; accepted March 7, 2001.
Supported by the Keio University Medical Science Fund (M.K.), by a
grant from the Japanese Ministry of Health and Welfare (Y.I.), and by
grants from the Ministry of Education, Science, Sports and Culture of
Japan (S.K. and Y.I.).
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: Masataka Kuwana, Institute for Advanced Medical
Research, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan; e-mail: kuwanam{at}sc.itc.keio.ac.jp.
| |
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Abstract
Introduction