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Blood, 15 August 2002, Vol. 100, No. 4, pp. 1388-1398
IMMUNOBIOLOGY
Genetic analysis of autoantibodies in idiopathic thrombocytopenic
purpura reveals evidence of clonal expansion and somatic
mutation
Jessica H. Roark,
James B. Bussel,
Douglas B. Cines, and
Don L. Siegel
From the Department of Pathology and Laboratory
Medicine, University of Pennsylvania School of Medicine, Philadelphia;
and the Division of Pediatric Hematology/Oncology, Joan and Sanford I. Weill Medical College, Cornell University, New York, NY.
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Abstract |
Although idiopathic thrombocytopenic purpura (ITP) is the most
common autoimmune hematologic disorder, little is known about the
associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we
used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig)
gene usage, clonality, and antigen specificity. Phage display libraries
were constructed from splenocytes from 2 patients with chronic ITP, and
competitive cell-surface selection was used to isolate several dozen
unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in
both patients were associated almost exclusively with rearrangements of
a single Ig heavy-chain variable-region gene (VH3-30),
despite an apparent diversity of antigen specificities. Comparative
analysis of platelet-reactive Fab Ig gene rearrangements from each
patient suggested that they evolved from a restricted number of B-cell
clones through somatic mutation with high replacement-to-silent mutation ratios. Although VH3-30-encoded heavy chains were
found with light chains encoded by several different Ig genes,
molecular repairing experiments showed exquisite restriction on the
specific heavy- and light-chain pairings that permitted platelet
reactivity. Together, these data suggest that the development of
platelet-reactive antibodies associated with ITP is driven by an
encounter with diverse platelet antigens through the clonal expansion
of B cells using genetically restricted and highly specific
combinations of heavy- and light-chain gene products. The
extraordinarily high usage of the VH3-30 heavy-chain gene
in these patients has implications for the pathogenesis, diagnosis, and
management of chronic ITP.
(Blood. 2002;100:1388-1398)
© 2002 by The American Society of Hematology.
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Introduction |
Idiopathic thrombocytopenic purpura (ITP) is a
common immunohematologic disorder caused by platelet-reactive
autoantibodies.1 The clearance of antibody-coated
platelets by tissue macrophages is accelerated, and in some cases, the
antibodies also impair platelet production. Childhood-type ITP is
self-limiting in about 80% of cases and may be associated with a
previous viral infection. Adult-onset ITP is a chronic illness in more
than 70% of cases and may occur in association with other disorders,
including systemic lupus erythematosus (SLE), lymphoproliferative
diseases, common variable immunodeficiency (CVID) disease, and human
immunodeficiency virus (HIV) infection. The decision to treat patients
with ITP takes into account the patient's age and disease
severity and the anticipated natural history of the disorder. Therapy
is initially directed toward impeding the clearance of antibody-coated
platelets by using glucocorticoids, splenectomy, anti-blood group D
[(anti-Rh(D)] immunoglobulin (Ig), intravenous  -globulin (IVIG),
and other treatments. Immunosuppressive therapy is nonspecific,
often toxic, and typically reserved for patients with refractory disease.
Numerous studies have been performed to characterize the pathogenic
autoantibodies responsible for platelet destruction and thereby provide
a reliable way to diagnose ITP, understand its pathogenesis, and
predict responsiveness to therapy. IgG antibodies that react with
platelet glycoprotein (GP) IIb/IIIa and GPIb/IX have been identified in
some patient serum samples and platelet eluates2-5;
however, other platelet antigens also appear to be targeted,5-13 and in many cases, the antibody specificity
cannot be determined or even detected.1 Furthermore, there
is no formal proof that any single subset of antibodies for example,
those directed at GPIIb/IIIaare responsible for platelet destruction. Consequently, the clinical utility of measuring serum or
platelet-elutable Ig is unknown and does not have a definitive role in
the diagnosis or treatment of ITP or in distinguishing between the
adult-onset and childhood-onset forms of the disease.14 As
a result, the diagnosis of ITP remains one of exclusion and the
usefulness of available platelet-antibody tests to confirm or exclude
the diagnosis independent of other criteria has not been
established.1
This situation illustrates the difficulty involved in characterizing a
pathologic autoimmune response by analyzing polyclonal serum.
To understand clonality, genetic origin, somatic mutation, and the
molecular basis of pathogenicity, repertoires of IgG antiplatelet autoantibodies, eg, those produced in vitro from the B cells of affected patients, must be studied. Conventional B-cell immortalization approaches for cloning human monoclonal antibodies result in low transformation frequencies and have a propensity for generating IgM-producing clones, thus causing a sampling bias.15,16
Consequently, all but one17 of the reported human
antiplatelet autoantibodies isolated from patients with ITP have been
of the IgM class and no more than 2 or 3 unique antibodies have been
isolated from a given patient.11,12,18-20 As a result, it
has been difficult to assess the genetic diversity among ITP-associated
autoantibodies within an individual patient, among patients, and in
different clinical settings.
To address this problem, we combined antibody phage display, a
molecular approach for cloning human immune repertoires,21 with a novel competitive cell-surface-selection scheme22
to isolate and study repertoires of IgG antiplatelet autoantibodies from 2 unrelated patients with chronic ITP. Using this strategy, we
isolated dozens of IgG platelet-reactive autoantibodies from each
patient, thus permitting a comprehensive analysis of their genetic
origin, extent of somatic mutation, and clonal relatedness. Our results
suggest that the development of platelet-reactive autoantibodies is
driven by an encounter with diverse platelet antigens through the
clonal expansion of B cells using genetically restricted and highly
specific combinations of heavy- and light-chain gene products.
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Materials and methods |
Platelet preparations
Platelet-rich plasma (PRP) was prepared by centrifuging
(500g) freshly isolated whole blood collected in sodium
citrate (final concentration, 10.5 mM/L) containing 3 µM/L
prostaglandin E1 (PGE1; Sigma Chemicals, St
Louis, MO) at room temperature for 15 minutes. For some experiments,
platelets were obtained from fresh banked platelet concentrates derived
from CP2D-anticoagulated whole blood (Fenwall; Baxter Healthcare,
Deerfield, IL). PRP from both sources was washed 3 times in
acid-citrate-dextrose (ACD; 145 mM/L sodium chloride, 5 mM/L citric
acid, 9 mM/L sodium citrate, and 17 mM/L dextrose [pH 6.5])
supplemented with 1% wt/vol bovine serum albumin (BSA).
Patients
Fab/phage display libraries were constructed from splenic
mononuclear cells from 2 unrelated adults with chronic ITP (ITP patient
A and ITP patient B) and one control patient with thrombocytopenia but
not ITP. Both patient A (a 56-year-old man) and patient B (a
43-year-old woman) had ITP refractory to prednisone and IVIG for at
least 8 months. After splenectomy, platelet counts rose to the normal
range. Patient A subsequently died of unrelated causes, and patient B
has been in clinical remission for more than 4 years. Splenocytes from
the control patient, a 65-year-old man with nonimmune, multifactorial
thrombocytopenia, were harvested at autopsy after he died of
respiratory failure.
Construction of Fab/phage display libraries
Using previously described methods for cloning IgG1
 and  immune repertoires,23 we prepared total RNA
from about 108 splenocytes, amplified heavy- and
light-chain-rearranged Ig gene segments by reverse
transcriptase-polymerase chain reaction, and cloned the DNA into a
phagemid expression vector (pComb3H; Scripps Institute, La Jolla, CA).
After electroporation into XL1-Blue bacteria (Stratagene, La Jolla) and
coinfection with VCSM13 helper phage (Stratagene), Ig DNA was packaged
into filamentous phage particles that expressed the human Fab molecules
fused to the pIII bacteriophage coat protein.
Panning Fab/phage display libraries
Fab/phage display libraries were enriched for platelet-reactive
Fabs by a modification of a previously described method using competitive cell-surface selection and magnetically activated cell
sorting.22 Platelets were washed free of BSA in
phosphate-buffered saline (PBS) and PGE1, resuspended to a
concentration of 5 × 108/mL, and surface biotinylated by
adding sulfo-N-hydroxysuccinimide biotin (Pierce, Rockford, IL) to 400 µg/mL. After 2 washes with ACD and BSA, 2 × 108
biotinylated platelets were incubated with 20 µL streptavidin-coated paramagnetic microbeads (Miltenyi Biotec, Sunnyvale, CA) for 10 minutes at room temperature in a total volume of 100 µL ACD, BSA, and
PGE1. ACD-BSA buffer (1 mL) containing about 5-fold excess (by surface area) human red blood cells (RBCs; 1 × 107)
was added. The cell admixture was centrifuged and resuspended in 50 µL ACD, BSA, and PGE1 containing about
3 × 1011 colony-forming units of Fab/phage display
library. After a 2-hour incubation at room temperature with
intermittent mixing, the suspension of platelets, RBCs, and phage was
loaded on a MiniMACS column (Mitenyi Biotec, Germany) pre-equilibrated
with ACD and BSA. Column washes (to remove RBCs and irrelevant
Fab-phage), elution of platelet-bound Fab-phage, and amplification of
panned libraries were performed as described
previously.22
Production of soluble antiplatelet Fab Ig
To screen, isolate, and characterize individual monoclonal
platelet-binding Fabs, randomly picked bacterial colonies derived from
phage titering plates were grown to an optical density600 of 0.5, isopropyl- -D-thiogalactopyranoside (1 mM/L) was
added, and cultures were shaken overnight at 30°C. Soluble Fabs were isolated from bacterial pellets by osmotic shock24 and used in flow cytometric experiments and enzyme-linked immunosorbent assays
(ELISAs) without further purification. Where indicated, soluble Fabs
were purified by nickel-chelation chromatography.23 Aliquots of bacterial pellets were used to prepare plasmid DNA (Qiawell
Plus; Qiagen, Valencia, CA) for nucleotide sequencing or antibody chain
shuffling. Heavy- and light-chain DNA was sequenced and analyzed as
described previously.24 Because of the large number of
sequences (> 60), only alignments of the predicted amino acid
sequences for a subset of antibodies are shown here; the rest of the
sequence data are provided on the Blood website; see the
Supplemental Data Set link at the top of the online article.
Characterization of antibody binding by flow cytometry
Platelets were stained by using 5 µL PRP (~ 5 × 106 platelets) and 50 µL Fab. After a 30-minute
incubation, platelets were washed with ACD and BSA, and bound antibody
was detected by using a phycoerythrin (PE)-conjugated
F(ab')2 fragment of goat antihuman F(ab')2-specific Ig (Jackson ImmunoResearch, West Grove,
PA) diluted 1:25 in wash buffer. Samples were analyzed by using a
microfluorometer (FACScan; Becton Dickinson, Mountain View, CA).
Forward- and side-scatter gates for platelet populations were
determined by using murine antihuman GPIIIa (SSA6; Dr J. Bennett,
University of Pennsylvania) counterstained with PE-conjugated goat
antimouse reagent (Southern Biotechnology, Birmingham, AL). Platelets
from 3 unrelated donors with type I Glanzmann thrombasthenia were
provided by Dr M. Poncz (University of Pennsylvania). A stable K562
cell line expressing GPIa/IIa was provided by Dr M. Zutter (Washington
University, St Louis, MO).
Blocking experiments were conducted to compare the repertoires of
recombinant platelet-reactive autoantibodies from ITP patients A and B
with those in the serum of other patients with chronic ITP. Platelet
aliquots were preincubated with each of 19 different ITP serum samples
or a pool of normal serum, then mixed with antibodies from ITP patient
A or B expressed as phage displayed Fabs. Blocking of recombinant
patient autoantibodies by ITP serum was then detected with biotinylated
anti-M13 antibody and PE-streptavidin.24 Binding of
recombinant autoantibodies in the presence of normal serum was defined
as 100%, and inhibition in the presence of ITP serum was normalized to
that value. Administration of IVIG to ITP patients A and B just before
splenectomy precluded use of their serum in competition assays.
Characterization of antibody binding by ELISA and
immunofluorescence
Antibodies to platelet GPIIb/IIIa, GPIb/IX, or GPIa/IIa were
measured by using a PakAuto kit (GTI, Brookfield, WI); those to
cardiolipin were assessed with a QuantaLite kit (Inova, San Diego, CA).
Binding to cytoplasmic or nuclear determinants was assessed by
immunofluorescence with HEp-2 cells (ANA Kit, Antibodies Incorporated,
Davis, CA).
Immunoprecipitation of platelet-Fab immune complexes
Immunoprecipitation of biotinylated platelet membrane proteins
was performed as described previously25 except that Protein L (Pierce) was used instead of Protein A to capture immune complexes. Precipitated material was electrophoresed on 4% to 12% polyacrylamide gels under nonreducing and reducing conditions and electrophoretically blotted on nitrocellulose membranes. Precipitated, biotinylated platelet membrane proteins were detected with biotinylated horseradish peroxidase-avidin complexes (ABC Staining Kit, Pierce).
Light-chain-library shuffling
To randomly pair the H44 heavy chain with a library of light
chains, 10 µg plasmid DNA from clone H44L4 (a GPIIb/IIIa-specific Fab
isolated from ITP patient A) was digested for 6 hours at 37°C with
SacI and XbaI (Roche Molecular Biochemicals,
Indianapolis, IN) to remove the endogenous light-chain L4, and the
heavy-chain-containing vector fragment was gel purified. A preparation
of and light-chain segments from the original, unpanned ITP
patient A library was obtained by digesting an equivalent amount of
plasmid DNA purified from the bacterial pellet obtained during library
preparation with SacI/XbaI and gel purifying the
excised light chains. Vector containing heavy-chain H44 was then
ligated to the library of light chains and electroporated into XL1-Blue
bacteria. Transformants were plated on carbenicillin-containing
Luria-Bertani plates from which antibody clones were randomly selected,
produced as soluble Fab preparations, and assayed for platelet binding
by flow cytometry. Plasmid minipreparations were performed on the
bacterial pellets derived from the expression experiments, and
nucleotide sequencing was done to verify the presence of heavy-chain
H44 and to determine the sequence of the light chain to which it
randomly paired.
Exchanging heavy and light chains among platelet-binding
clones
Light-chain gene segments from clones H36/L76, H44/L4, and
H47/L64 were freed from their respective plasmid DNAs by
SacI/XbaI digestion, and the restriction products
from the 3 clones (ie, 3 heavy-chain-containing plasmids and 3 free
light chains) were combined. Religation regenerated the 3 original Fabs
and created 6 novel heavy-chain-light-chain pairs. After bacterial
transformation, several dozen bacterial clones were randomly selected
to produce Fabs for platelet-binding assays and to isolate plasmid DNA
to determine heavy- and light-chain composition.
Fab binding to modified staphylococcal protein A
Binding of Fabs to the superantigen domain of staphylococcal
protein A (SpA) was measured by ELISA using SpA that had been chemically modified with iodine monochloride to destroy its native Fc-binding domain (designated mod-SpA).26 Mod-SpA (1 µg
in 50 µL) was coated on the wells of a 96-well microplate and
incubated overnight at 4°C. After a rinse with distilled water, wells
were blocked for 1 hour at 37°C with PBS and 1% BSA, and Fab samples were added (50 µL/well). After a 2-hour incubation at 37°C, the wells were washed 3 times with PBS, and a mixture of alkaline phosphatase-conjugated goat antihuman (1:10 000) and (1:5000) light-chain reagents was added (Sigma Chemical). Wells were incubated at 37°C for an additional hour, washed again with PBS, and developed with P-nitrophenyl phosphate.
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Results |
Isolation of monoclonal human platelet autoantibodies
The purpose of this study was to characterize on a genetic level
the repertoires of platelet autoantibodies in chronic ITP. To isolate
the repertoires, Fab/phage display technology was used to avoid the
technical limitations inherent in experimental approaches that rely on
B-cell immortalization to produce human monoclonal antibodies. IgG and libraries were constructed from splenic lymphocytes from 2 patients with chronic ITP and a control patient with multifactorial
thrombocytopenia not due to ITP. The libraries (each comprising
> 2 × 108 independent transformants) were panned
against intact platelets (as opposed to isolated platelet membrane GPs)
to present the libraries with all possible autoantigenic determinants
and to do so in a physiologically relevant manner that would preserve native antigen structure and optimize capture. By employing a magnetically activated competitive cell-surface panning strategy in
which selection of platelet binders was done in the presence of an
irrelevant cell type (RBCs), we prevented the capture of panreactive or
nonspecific Fab-phage.
Individual Fab clones were randomly selected from platelet-selected
libraries and assessed for platelet binding by flow cytometry. For the
2 patients with ITP, 78 of 294 clones were positive, of which 39 were
determined to be unique antibodies on the basis of the heavy- and
light-chain DNA sequence. In contrast, only 1 of 77 additional clones
randomly selected from the unpanned ITP libraries and none of 59 clones
isolated from the control libraries (16 from the original unpanned and
43 from the platelet-selected libraries) showed platelet reactivity.
We then asked whether the panned ITP Fab libraries would bind to a
cohort of antigens recognized by polyclonal antibodies in serum from
patients with ITP. The capacity of 19 ITP serum samples to block the
binding of phage displayed Fabs was assessed by flow cytometry using
fluorescently labeled anti-M13 (phage) antibody relative to normal
control serum samples. Fab-phage from ITP patient A was inhibited 25% ± 15% (range, 0%-41%) on average; that from ITP patient B was
inhibited 41% ± 17% (range, 14%-74%). Analogous studies with serum
from these patients were precluded by administration of IVIG
immediately before sample collection.
Sequence analysis of platelet autoantibodies
The heavy- and light-chain nucleotide sequences from the 39 unique
platelet autoantibodies were aligned with the V Base Directory of Human
V Gene Segments27 to examine their genetic origins and
possible genetic interrelatedness. As shown in Figure
1 (red boxes), all heavy chains from ITP
patient A (6 of 6) and all but 4 heavy chains from ITP patient B (29 of
33) used VH3-30. Usage of light-chain variable-region genes
was less restricted but comprised a limited set of VL
genes, including the V genes A19/A3, A27, and
L6.
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| Figure 1.
Matrix illustrating the genetic composition of platelet
autoantibodies.
The horizontal axis represents the unique heavy chains (H01 through
H98) and the vertical axis represents the unique and light
chains (L01 through L124) used by antibodies cloned and sequenced from
the patients with ITP and the control patient. The letter at the
intersection of a heavy-chain-light-chain pair indicates the
composition of a platelet-reactive (red box) or platelet-unreactive
(clear box) antibody isolated from ITP patient A or B or control
patient C. For positive clones, H and L designations are indicated. The
order of heavy chains (left to right) and light chains (top to bottom)
was determined by multiple alignments based on amino acid similarity
and then grouped by putative Ig variable-region germline gene and
germline gene family. Note the marked predominant use of the
VH3-30 germline gene to encode platelet-binding antibodies
in both patient repertoires (red-framed area).
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Selective usage of a particular heavy- or light-chain gene in a cohort
of antibodies may occur because of in vivo or in vitro preselection
factors (eg, greater gene usage by the pre-existing pool of B cells or
cloning artifacts) or if an encounter with antigen drives clonal
expansion and somatic mutation of a restricted population of B cells
that use that particular gene. To address the first possibility, we
assessed the diversity of the unpanned ITP patient A and patient B
libraries. Analysis of the heavy and light chains of a random cohort of
43 of the 76 non-platelet-binding clones from the original libraries
found no duplicate sequences and marked heterogeneity in V gene
representation before selection for platelet binding (Figure 1, A and B
clear boxes). Specifically, 20 different VH genes and 20 different VL genes were represented and their distribution
was similar to that typically found for IgG-secreting lymphocytes in
the repertoire of adults.28 The absence of platelet
reactivity of recombinant antibodies from the control library was not
due to inefficient library construction or lack of VH3-30
heavy-chain representation (Figure 1, C boxes), since 26 different
VH genes and 25 different VL genes were used, including 3 antibodies encoded by VH3-30. Therefore, the
highly restricted, near-total use of VH3-30 by the 39 platelet-binding ITP patient autoantibodies did not reflect a skewed
representation of genes within the original pool of splenic
lymphocytes, nor was it the result of a cloning artifact introduced
during construction of the Fab/phage display libraries.
We next addressed the possibility that the increased usage of a given V
gene results from clonal expansion of restricted B-cell populations. To
do this, we exploited the fact that rearranged Ig genes have extensive
diversity; ie, there is only a remote probability that 2 B cells will
not only randomly select an identical combination of VH, D,
and JH (for heavy chain) or VL and
JL (for light chain) gene segments but will also splice the
genes together to create identical junctional regions. Alignments of
the heavy- and light-chain variable-region amino acid sequences of a
cohort of 39 platelet autoantibodies from ITP patients A and B were
performed. Examination of the complete set of heavy-chain
sequences (see the Blood website's Supplemental Data Set)
revealed evidence of clonal expansion for a subset of B cells using
VH3-30 in both patients (Figure
2A). The members of each clone appear to
have resulted from recombination of VH3-30, D1-26, and
JH4b gene segments, and within each clone, they showed
identical junctional regions. The fact that the CDR3 regions of clone A
and clone B were quite distinct indicates that neither resulted from an
interlibrary contaminant.
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| Figure 2.
Alignment of clonally related platelet autoantibody
heavy-chain amino acid sequences and their putative ontogenic trees.
The H and L nomenclature is the same as in Figure 1. (A) Groups of
related sequences comprising expanded heavy-chain clones in each
patient library (clone A and clone B) are enclosed in boxes. The
coalignment with the rest of the 16 unique heavy chains is available on
the Blood website; see the Supplemental Data Set link at the
top of the online article. For clone A, the putative intermediate
heavy-chain sequences are also shown (1, 2, and 3 asterisks). The
number of nucleotide differences from germline VH is
tabulated to the right of each sequence. Because D segments showed poor
homology with known D genes, mutations were not scored in these
regions. Replacement mutations are indicated by letters, identities as
".", and insertions as  , and + to maintain spacing due
to variability in CDR3 length. Sequences derived from the 5' V-region
primers used for library construction22 are marked as >.
CDR-region designations are according to the system of Kabat et
al29; numbering and hypervariable loop designations are
according to the system of Chothia et al.30 (B) Analysis
of nucleotide data in each patient revealed a distinct set of
somatically mutated heavy chains sharing common
VHDJH rearrangements of VH3-30,
D1-26, and JH4b gene segments. Circles represent isolated
and sequenced clones (Figures 1 and 2A); diamonds (for ITP patient A
only) represent putative intermediates. For each member of a patient's
clone, the number of nucleotide mutations from its germline
VH gene is shown in parentheses, and the resulting number
of replacement (R) or silent mutations (S) is shown in brackets. For
each patient clone ontogenic tree, the distance in the horizontal
direction represents the extent of mutation from the proposed
germline origin within the constraints of the
diagram.
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By examining nucleotide alignments with germline genes (data not
shown), we constructed ontogeny trees for the 2 putative clones to
illustrate how the patterns of somatic mutation in the respective heavy
chains may have evolved in vivo (Figure 2B). For the ITP patient A
clone in particular, a parsimonious mutation scheme (ie, postulating
the minimum number of mutations) was used to derive putative
intermediate heavy chains (Figure 2B, 1, 2, and 3 asterisks). The
members of this clone appear to have undergone a marked degree of
somatic mutation (from 4 to 21 nucleotide changes in the VH
segment alone) that resulted in high replacement-to-silent (R:S)
ratios, both hallmarks of an immune response characterized by
antigen-driven selection.31,32 For the ITP patient
B clone, there were fewer mutations overall, but almost every mutation resulted in an amino acid replacement and clonal expansion was apparent. Therefore, the marked usage of VH3-30 in
these cohorts of platelet-binding antibodies resulted at least partly
from a restricted number of autoreactive B cells undergoing clonal
expansion. The use of VH3-30 may also be important in
conferring platelet binding because it encodes H44, a clonally
unrelated heavy chain, and at least one IgM platelet autoantibody
generated by conventional tissue-culture techniques.33
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| Figure 3.
Determination of platelet autoantibody specificity by
ELISA and flow cytometry.
Shown are results for ITP patient A antibody H44L4, which was judged to
be specific for platelet GPIIb/IIIa because of its binding to
immobilized GPIIb/IIIa (but not GPIb/IX or GPIa/IIa; panel A), its
binding to wild-type platelets but not GPIIb/IIIa-deficient platelets
from 3 patients with Glanzmann thrombasthenia (one of 3 examples is
shown in the flow cytogram; panel B), and its immunoprecipitation of
GPIIb/IIIa molecules. Antibody H68L120, an anti-blood group B antibody
isolated from the same original ITP patient A library,38
was used as a negative control as indicated.
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H44 and the remaining heavy chains (H4, H10, H29, and H83) each had its
own unique VHDJH recombination, and except for
H29, somatic mutation occurred in their VH segments as
well. Light chains also underwent somatic mutation (their aligned
sequences are available in the online Blood supplement).
Some light chains in the cohort may be clonally related (eg, L43,
L44, and L45), but because the VLJL junction is
not as diverse as the junctional regions for the heavy chain, clonal
relatedness among light chains is more difficult to prove. Only a few
light chains were present in platelet-reactive Fabs, none of which
appeared to be clonally related.
Identification of recombinant platelet-autoantibody
specificity
Autoantibodies from patients with ITP often recognize complexes
composed of platelet glycoproteins GPIIb/IIIa or
GPIb/IX,2,4,5,7,17,33-37 although
autoantibodies against other identified and unidentified antigens have
been described.5-13 Panning on intact platelets ensures
that all relevant antigens were present during the selection process
and that their native conformation was preserved.
Each of the 39 unique platelet-reactive antibodies showed specificity
for this cell type. None bound to Chinese hamster ovary cells, K562
cells, erythrocytes, or leukocytes on flow cytometric analysis (not
shown). In addition, none showed surface, cytoplasmic, or nuclear
binding to HEp-2 cells on immunofluorescence analysis and none bound to
cardiolipin. However, only the antigen specificity of H44L4 could be
determined with relative unambiguity. In an ELISA, H44L4 reacted with
purified, immobilized GPIIb/IIIa but not with GPIb/IX or GPIa/IIa
(Figure 3A). H44L4 did not recognize platelets from 3 unrelated donors with type I Glanzmann thrombasthenia (Figure 3B), whereas all other platelet-reactive Fabs bound comparably to wild-type platelets and Glanzmann platelets. Furthermore,
H44L4-immunoprecipitated polypeptides migrated in accordance with the
behavior of GPIIb/IIIa under reducing and nonreducing
conditions (Figure 4).
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| Figure 4.
Determination of platelet autoantibody specificity by
immunoprecipitation.
Biotinylated platelets solubilized after incubation with recombinant
Fabs and antigen-Fab complexes were captured on Protein L dextran
beads. Immunoprecipitated material was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis under nonreducing (left) or
reducing (right) conditions, transferred to nitrocellulose, and
detected with enzyme-labeled avidin-biotin complexes. Shown in this
figure are results with ITP patient A-derived antibodies H44L4 and
H46L16. Note that the presence of polypeptide bands with a relative
molecular weight of about 150 kd (unreduced) and about 50 kd and 25 kd
(reduced) represent platelet-bound autologous IgG that was biotinylated
during the platelet-labeling procedure and coprecipitated by
Protein L.
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None of the other Fabs immunoprecipitated polypeptides in a manner
consistent with the behavior of GPIIb/IIIa, a finding in agreement with
the results of the ELISA and flow cytometry analysis using Glanzmann
platelets; nor did any of them react with a stable K562 cell line
expressing GPIa/IIa. However, autoantibodies H46L16, H47L64,
and H48L24, 3 Fabs with clonally related heavy chains, immunoprecipitated polypeptides with molecular weights consistent with
those for GPIb/IX. On ELISA, this set of Fabs did not bind significantly above background levels to purified immobilized GPIb/IX,
but blocking of the relevant epitope by the mouse monoclonal capturing
antibody could not be excluded. Until further analyses are performed,
assignment of GPIb/IX as the specificity for clone A will remain
tentative. Neither 2 antibodies from clone B (H37L50 and H42L38) nor
the 2 non-VH3-30-encoded antibodies (H4L106 and H83L34)
specifically immunoprecipitated labeled protein, perhaps because their
target polypeptides were not biotinylated sufficiently or lost
conformation during solubilization or because their targets are not proteins.
Contribution of the heavy and light chains of H44L4 to
GPIIb/IIIa specificity
For certain antibodies, antigen specificity is determined
primarily by one or the other component chain.39-42
Identification of the platelet GPIIb/IIIa complex as the antigenic
target of Fab H44L4 allowed us to examine the contribution of its
constituent heavy and light chains to antigen recognition. If the
VH3-30 heavy chain of H44L4 is solely responsible for
GPIIb/IIIa binding, then the specific light chain that is used might be
of little relevance, as long as it is permissive. Alternatively, the
fine specificity of the VH3-30 heavy chain might be
modified or actually determined by the paired light
chain.24 The amenability of phage display-derived antibodies to molecular manipulation allowed us to examine this issue
in some detail.
We first paired the H44 heavy chain with a panel of light chains and
surveyed the resultant combinatorial Fabs for their capacity to bind
platelets. To do this, we created a new library in which heavy-chain
H44 was recombined with the entire light-chain repertoire from the
original ITP patient A library. Only one of 101 Fabs expressing the H44
heavy chain paired with random light chains reacted with platelets.
Like the original H44L4 antibody, this recombinant Fab recognized
GPIIb/IIIa on ELISA (data not shown). Sequence analysis confirmed that
H44 was used to encode this Fab. The presence of H44 in 20 randomly
selected nonreactive Fabs was also confirmed. Thus, mere usage of the
H44 heavy chain alone was insufficient to confer GPIIb/IIIa reactivity
on a Fab molecule. This finding suggests that specific
VH-VL pairing is required to impart this
binding specificity.
To examine this idea further, we sequenced the light-chain gene
segments of the platelet-reactive Fab and those encoding the reference
set of 20 H44-expressing Fabs that lacked platelet reactivity. Interestingly, the single positive Fab (H44L125) employed an O12/O2 variable light-chain gene and J4 J-segment gene, as did the original
H44L4 Fab (Figure 5). Indeed,
light-chains L4 and L125 appear to have derived from the same B-cell
clone, because they shared an especially distinctive VJ junction in
which 3 nucleotides had been lost, resulting in deletion of the
germline-encoded proline usually found at amino acid position 95 (Figure 5B). Because this residue lies in the CDR3 region of the light
chain, deletion of 95P may confer or at least contribute to GPIIb/IIIa
specificity. This idea is supported further by the observation that
none of the 3 sampled non-platelet-reactive Fabs that use an O12/O2
light chain (Figure 5B; clones H44L126, H44L127, and H44L128) had a deletion at position 95.
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| Figure 5.
Platelet binding of randomized light chains paired with
platelet autoantibody heavy-chain H44.
The heavy chain of GPIIb/IIIa-specific H44L4 was paired again
with a library of more than 106 light chains derived from
the original, unselected ITP patient A library, and 101 resorted clones
were screened for platelet binding by flow cytometry. (A) Matrix
illustrating the genetic composition of the single retrieved positive
resorted clone (designated H44L125). For comparison, 20 (of the 100)
randomly chosen negative clones (designated H44L126 through H44L145)
and the original H44L4 antibody are tabulated. Numbers in shaded boxes
represent mean fluorescent intensities. Note that the single positive
platelet-binding clone comprises a light chain derived from the same Ig
light-chain gene as the original L4 light chain (012/02), yet
no other 012/02-encoded light chain (eg, L125-L128) conferred binding
when paired with H44. (B) Sequence analysis of cohort of 012/02-encoded
light chains retrieved in resorting experiment shows that light-chain
L125, which reconstitutes platelet binding, may be clonally related to
the original L4 light chain because of a distinctive VJ junction
characterized by loss of an entire amino acid residue at position 95 (boldface region).
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These results indicate that only a limited set of light chains impart
or are permissive for GPIIb/IIIa specificity when paired with a given
VH3-30 gene product. This finding led us to investigate the
limitations in heavy- and light-chain pairings required to generate
platelet-reactive Fabs. As a corollary, we asked whether the specific
light chain actually determines antigen specificity, in view of the
finding that VH3-30 heavy-chain gene usage is so prevalent
among platelet-reactive antibodies.
To address these issues, we exploited the distinctive flow
cytometric (Figure 6A) and
immunoprecipitation patterns of H44L4 (a GPIIb/IIIa-specific Fab that
uses V-O12/O2), H47L64 (a putative GPIb/IX-specific Fab and clone A
member that uses V-A27), and H36L76 (a clone B member that uses
V-L6), each of which uses VH3-30-encoded heavy chains.
By mixing their plasmid DNAs, restriction digesting each light chain
away from its originally associated heavy chain, and religating the
resultant admixture of heavy- and light-chain gene segments, we
generated all 9 possible combinations of the 3 heavy chains and 3 light
chains. Forty-three randomly selected clones, which included several
examples of each combination, were assessed for platelet binding. Only
the heavy- and light-chain combinations that reconstituted the 3 original Fabs bound to platelets (Figure 6B), and their flow cytometric
patterns were indistinguishable from those of the parental molecules
(data not shown). Thus, although VH3-30 is used frequently
by autoantibodies that bind to platelets, it is not only the specific
light chain but also the particular heavy- and light-chain pairing that
imparts platelet reactivity and specificity.
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| Figure 6.
Exchange of light chains among platelet autoantibody
clones.
Heavy and light chains for 3 platelet-binding clones (H44L4, H47L64,
and H36L76) were interchanged to generate 9 possible combinations
(6 novel and 3 reconstituted originals). (A) Flow cytograms comparing
the fluorescent intensities of the 3 index antibodies. (B) Matrix
showing that only reconstituted original heavy-chain-light-chain pairs
conferred platelet binding. Numbers in boxes represent mean fluorescent
intensities.
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Binding of platelet autoantibodies to the superantigen domain
of SpA
The mechanism by which extracorporeal absorption of plasma from
ITP patients with affinity columns containing SpA is sometimes efficacious is unknown, given that the amount of IgG removed is only
about 2% of that removed during plasmapheresis,1,43 a treatment that is rarely effective in chronic ITP.44,45 A
B-cell superantigen site on SpA has been described that is independent of its well-characterized Fc binding site and that interacts with variable regions of antibodies encoded by certain members of the VH3 family, notably VH3-30.46,47
Modification of SpA by iodination completely destroys Fc-binding
activity, whereas Fab-binding activity is retained.26 We
asked whether this modified SpA would bind our platelet autoantibodies
by virtue of their genetic restriction. We found that as we selected
platelet-binding autoantibodies from polyclonal, polyspecific Fab
libraries through sequential rounds of panning, there was concurrent
selection for binding activity to the superantigen domain
of SpA (Figure 7).
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| Figure 7.
Binding of platelet-selected Fabs to mod SpA.
Polyclonal Fab preparations derived from the original unselected ITP
patient A and patient B Fab/phage display libraries (panel A and B,
respectively) and from the libraries after each round of platelet
panning were assayed for platelet binding by flow cytometry (circles,
right set of axes) and for binding to mod-SpA by ELISA (squares, left
set of axes).
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Discussion |
Platelet autoantibodies are encoded by a restricted set of
VH genes
Use of the VH3-30 heavy-chain gene was found to be
highly represented among platelet-reactive Fabs from both patients with ITP compared with its prevalence in the general library and despite differences in antigen specificity
(P < 10 13 by Fisher exact test;
Figures 1 and 4). Interestingly, this same heavy-chain gene was found
to encode an IgM anti-GPIIb autoantibody derived by hybridoma
technology from another patient with ITP,20,33 as well as
several platelet-reactive, IgG phage display-derived antibodies from
ITP patients selected because of their ability to bind to
IVIG.48,49 This marked genetic restriction to the VH3-30 heavy-chain gene for antiplatelet autoantibodies may
provide an explanation for the association of ITP with seemingly
unrelated disorders, such as autoimmune hemolytic anemia, SLE, chronic
lymphocytic leukemia, CVID, and HIV infection, in which
VH3-30 and related gene products are expanded or involved
in disease pathogenesis.50-56
Why use of the VH3-30 heavy chain is overrepresented among
antibodies that show platelet reactivity is a pivotal question. One
possibility is that antibodies encoded by most other VH
gene products are less able to bind to platelets. Such a restriction on
antigen recognition could explain why we did not identify any VH3-23 heavy-chain gene product among platelet-reactive
Fabs, even though it is the most frequently used VH gene in
the repertoire.57-61 However, several antibodies in our
cohort of platelet binders were encoded by VH genes other
than VH3-30, including VH1-02, VH1-46, VH3-21, and VH4-59 (Figure
1 and Blood online supplement). Remarkably, this identical
group of VH genes was found by Boucher et al62
to encode all but one antibody in a large number of human anti-Rh(D)
RBC alloantibodies. As noted by these investigators, products of these
germline genes are among the most cationic in the human VH
repertoire. The resulting constitutive net positive charge may allow
the antibodies to effectively permeate the highly negative RBC  potential, thus permitting contact with antigen.63 Because
platelets have an even greater density of cell-surface negative charges
as a result of their thick glycocalyx rich in acidic
mucopolysaccharides,64,65 platelet-surface charge may play
a similar role in biasing the use of cationic germline VH segments. Therefore, use of cationic VH genes may
facilitate access to the membrane surface, but specificity for a
particular antigen may be determined by heavy-chain CDR3 and light chain.
We investigated this role for light chain by pairing the
VH3-30-encoded H44 heavy-chain product found in our
platelet GPIIb/IIIa-specific Fab with all members of the entire
light-chain repertoire from the same library (~108 light
chains). Only one other platelet-reactive, GPIIb/IIIa-specific Fab was
retrieved (Figure 5). Remarkably, the light chain in this Fab was not
only very similar in sequence to the light chain found in the original
antibody, but it appeared, on the basis of CDR3 analysis, to have
derived from the same B-cell clone in vivo. Furthermore, light chains
from platelet-reactive Fabs that use VH3-30 heavy-chain
genes were not interchangeable. Indeed, when the genes from a set of
platelet-reactive Fabs with differing specificity were permitted to
recombine randomly, only the original combinations of heavy and light
chains led to detectable platelet binding (Figure 6). These
observations suggest that platelet antigen specificity cannot result
from simple pairing of an array of permissive heavy- and light-chain
gene products but requires precise interactions between particular
heavy chains and their light-chain companions.
Role of autoantigen and clonal expansion in chronic ITP
The study of human autoimmune disease is greatly facilitated by
focusing on disorders such as ITP, in which it is clear that the
associated autoantibodies are unequivocally involved in pathogenesis. However, the role played by self-antigens in the evolution of autoreactive antibodies and the clonality of the autoimmune response are not well understood. On the basis of light-chain restriction, previous reports suggested that platelet autoantibodies in chronic ITP
are clonally restricted.66-70 Consistent with these
findings, several features of the platelet-reactive autoantibodies
described in the current study indicate that they arose as part of an
antigen-driven clonal expansion rather than being the result of
polyclonal B-cell activation triggered by nonspecific stimuli. First,
most antibodies isolated from each patient shared a single heavy-chain
VHDJH rearrangement indicating their derivation
from a single B cell (Figure 2B). Second, somatic mutation with high
R:S ratios was evident in heavy- and light-chain variable regions
(Figure 2 and Blood online supplement). Third, each of the
platelet-reactive Fabs was derived from an IgG library, indicating that
isotype switching had occurred, another hallmark of a
T-cell-dependent, antigen-driven immune response. Finally, the
requirement for precise heavy- and light-chain pairing to generate
antigen specificity (Figures 5 and 6) also typifies antigen-driven
immune responses.40,41,71,72
It may be these characteristics that distinguish pathogenic
antiplatelet autoantibodies from "benign" ones cloned from samples from unaffected donors. Such naturally occurring platelet-binding antibodies, in contradistinction to those observed in the current study, are nearly always IgM, are often polyreactive, have little or no
somatic mutation of their variable regions, or show a combination of
these characteristics.73-75 These differences are
analogous to those used to distinguish pathogenic from benign
autoantibodies in murine models of autoimmunity.31,33
Whether the B cells that produce benign antiplatelet autoantibodies are
the clones that go on to lose self-tolerance, switch isotypes,
somatically mutate their variable-region genes, and secrete pathogenic
autoantibodies is not clear. In fact, it may be this clonally unrelated
pool of natural, nonpathologic antiplatelet autoantibodies that
normally functions to keep production of pathologic autoantibodies in
check through a mechanism of competitive tolerance, as has been
proposed for murine rheumatoid factors.76
Clinical and therapeutic implications of VH gene
restriction
Current treatments for chronic ITP are characterized by relatively
nonspecific immune intervention. If restriction of platelet autoantibodies to the VH3-30 heavy-chain gene is confirmed
by studies of additional immune repertoires, exploitation of this restriction may facilitate the design of more targeted forms of immunotherapy. For example, it is known that SpA has a B-cell superantigen sitedistinct from its well-characterized Fc-binding domainthat is specific for the gene products of certain
VH3-encoded Igs, notably
VH3-30.46,47 Consistent with this activity, we found that panning of ITP patient A and B phage display libraries on
platelets resulted in concomitant enrichment for both platelet and
mod-SpA binders (Figure 7). In studies in mice, targeted
deletion of VH3-30 homologs by apoptotic cell death
occurred on in vivo administration of recombinant mod-SpA
superantigen,77,78 suggesting that infusion of small
amounts of mod-SpA might likewise down-regulate production of platelet
autoantibodies in ITP. In this regard, and in light of recent studies
demonstrating shedding of up to 200 µg SpA from SpA-silica columns
during extracorporeal immunoabsorption procedures,79 the
long-term remissions may be a consequence of infused SpA and not the
removal of antibody by the columns per se. Future studies
testing the therapeutic effectiveness of this or other
VH3-30-targeted reagents, such as anti-idiotypic antibodies derived from mice80,81 or humans,49
may provide novel approaches for regulating immune-repertoire
composition. Furthermore, development of reagents for rapid
identification of the genetic origins of platelet autoantibodies may
help predict responsiveness to such novel molecular therapies in
individual patients.
| |
Acknowledgments |
We thank Tylis Chang for his contribution to the early phase of
this study and Stephen Kacir for excellent technical assistance.
| |
Footnotes |
Submitted June 18, 2001; accepted April 10, 2002.
Supported by grant R01-HL61844 from the National Institutes of Health.
The online version of the article contains a data supplement.
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: Don L. Siegel, Blood Bank/Transfusion
Medicine Section, Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Room 510, Stellar-Chance
Building, 422 Curie Blvd, Philadelphia, PA 19104; e-mail:
siegeld{at}mail.med.upenn.edu.
| |
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Abstract
Introduction