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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 208-215
Complexes of Heparin and Platelet Factor 4 Specifically Stimulate T
Cells From Patients With Heparin-Induced Thrombocytopenia/Thrombosis
By
S. Bacsi,
R. De Palma,
G.P. Visentin,
J. Gorski, and
R.H. Aster
From The Blood Research Institute, The Blood Center of
Southeastern Wisconsin; the Departments of Cell Biology, and
Medicine and Pathology, Medical College of Wisconsin, Milwaukee,
WI; and the Fondazione Salvatore Maugeri, Laboratorio di Medicina
Sperimentale, Pavia, Italy.
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ABSTRACT |
Heparin-induced thrombocytopenia with thrombosis (HITT) is
associated with antibodies specific for complexes consisting of heparin
and platelet factor 4 (PF4). Studies in individual patients with HITT
have demonstrated immunoglobulin (Ig) class switching from IgM to the
IgG or IgA isotypes. This transition is thought to require helper T
cells, but no studies of the cellular or molecular basis of this
process have yet been reported. To characterize T-cell involvement in
HITT, peripheral blood mononuclear cells (PBMC) from two patients with
classical HITT obtained shortly after the acute episode were
restimulated with heparin:PF4 complexes, PF4 alone, heparin alone, and
medium alone in the presence of autologous antigen-presenting cells
(APC). Responding T cells were then examined using the technique of
"spectratyping," in which sequences encoding CDR3 domains of
individual V beta (BV) families are amplified and separated by gel
electrophoresis. After 14 days in culture with antigen (heparin:PF4
complexes), but not after culture with PF4, heparin, or medium alone,
patient cells, but not cells from normal subjects, preferentially
expressed T-cell receptor (TCR)-containing  chains of the BV 5.1 family. Nucleotide sequencing of BV 5.1 TCR CDR3 showed that each
patient had a personal repertoire, but also shared a tetrapeptide motif
(PGTG). These findings provide evidence that the humoral immune
response associated with HITT is driven by helper T cells that
presumably recognize peptides derived from PF4. Identification of a
common -chain CDR3 motif in responding T cells from each of two
patients suggests that a limited number of helper TCRs may be used to
mount an antibody response to heparin:PF4 complexes. TCR spectratyping
appears to offer a new way to examine the molecular basis of pathologic
immune responses and may be useful in further studies of HITT and other immune-mediated hematologic disorders.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEPARIN-INDUCED thrombocytopenia with
thrombosis (HITT) is a major complication of treatment with
unfractionated heparin. Although the molecular basis of this disorder
is not yet fully understood, it is now known that HITT is closely
associated with the production of antibodies specific for complexes
containing heparin and platelet factor 4 (PF4), a heparin-binding
protein normally found in platelet  granules,1-4 and it
is thought that immune complexes consisting of antibody, PF4, and
heparin play a key role in pathogenesis.2,5 More than one
third of patients treated with unfractionated heparin during open heart
surgery produce antibodies reactive with heparin:PF4
complexes,6,7 but for reasons that are not yet clear, only
a minority develop immune thrombocytopenia and/or thrombosis. Although
the two components of the immunogenic complexes (heparin and PF4) are
normal body constituents, antibodies reactive with heparin:PF4
complexes have not yet been described in persons not exposed to heparin exogenously.
In patients who mount a humoral immune response to heparin:PF4
complexes, a primary IgM response is usually followed by secondary formation of IgG and/or IgA antibodies, indicating involvement of
T-helper cells.6,8 A newly developed technique,
spectratyping, enables analysis of T-cell receptor (TCR) utilization
during in vitro stimulation by antigen.9 We used this
approach to analyze the response of T cells from patients with HITT to
heparin:PF4 complexes processed by autologous antigen-presenting cells
(APC) during in vitro culture. In two HITT patients, we observed
expansion of T cells expressing TCR with V beta (BV) chains of the 5.1 family and, by sequencing, identified a shared CDR3 peptide motif.
These findings indicate that complexes of heparin:PF4 can specifically drive a T-cell response in peripheral blood mononuclear cells (PBMC)
from patients with HITT in an in vitro culture and suggest possible
commonality of this response in different patients. Spectratyping, combined with the determination of nucleotide sequences in TCR complementarity-determining regions (CDR) regions of responding T
cells, appears to offer a useful means to examine the molecular basis
of the cellular immune response in HITT and other immune hematologic disorders.
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MATERIALS AND METHODS |
Patients.
Patient 1 was a 50-year-old woman who presented with a right femoral
artery thrombosis. Thrombolytic therapy was unsuccessful and an ilial
femoral bypass graft was implanted with partial restoration of venous
return. She was treated with unfractionated porcine heparin, but the
graft thrombosed after 1 week, which led to gangrene in the lower leg
that required an above-knee amputation. About 1 week later, while still
on heparin, she developed a deep vein thrombosis in the left leg and a
decrease in platelet count to 30,000/µL. A serotonin release
assay10 was "borderline" positive, but her serum gave
a strongly positive reaction against complexes of heparin: PF4 in an
enzyme-linked immunosorbent assay (ELISA),2 and heparin was
discontinued. She later experienced a pulmonary embolus, after which an
inferior vena caval filter was put in place. She was discharged 1 month
after surgery and is currently doing well.
Patient 2 was a 69-year-old woman who presented with acute onset of
pain in the left foot. Physical examination showed ischemia of the left
lower leg and contrast studies demonstrated atherosclerotic changes in
the aorta. It was concluded that she had had an arterial embolus,
possibly from an atherosclerotic plaque, and thrombolytic therapy and
heparin were instituted. Over a period of 1 week, the patient's
platelet level decreased progressively from normal to 30,000/µL, at
which time she developed thrombosis of the left lower leg. Strongly
positive reactions for heparin-induced antibodies were obtained by
serotonin release10 and heparin:PF4 ELISA2 tests. Heparin was discontinued, after which the platelet count returned to normal in 4 days. However, a left below-knee amputation was
necessary because of gangrenous changes. About 10 days later, decreased
blood flow and cyanosis developed in the right lower leg without
associated thrombocytopenia. She was treated with local infusion of
urokinase and plasma exchange for 3 successive days, after which
circulation was restored. She was discharged after 1 month and is
currently doing well.
Informed consent, approved by the Human Research Review committee of
the Blood Research Institute, was obtained for procurement of all blood samples.
Culture conditions.
A quantity of 3 × 106 PBMC was cultured in RPMI medium at
106 cells/mL supplemented with 1 mmol/L
L-glutamine and 10% fetal bovine serum (FBS) in six-well
flat bottom plates at 37°C in a humidified 5% CO2
incubator. Cultures were maintained for a total of 14 days. An
equal number of irradiated (3,000 rad) autologous PBMC as a source of
APC was added on day 7. Recombinant human IL-2 (Genzyme,
Cambridge, MA) 50 IU/mL was added every 3 days to all cultures. The
cultured PBMC were challenged upon initial plating and every 3 days
thereafter with unfractionated heparin (0.25 U/mL final), purified
human PF4 (5 µg/mL final), heparin:PF4 complexes (0.25 IU/mL added to
PF4 5.0 µg/mL), or media alone.
RNA and cDNA preparation.
Fourteen-day and noncultured PBMC were harvested or thawed for RNA
preparation using the guanidium hydrochloride-containing Trizol Reagent
(Life Technologies, GIBCO-BRL, Gaithersburg, MD). First-strand cDNA synthesis was performed using oligo (dT) as a primer
for reverse transcription (RT) of 1 µg total RNA using MMLV-RT (Moloney murine leukemia virus-reverse
transcriptase; Life Technologies) as described
previously.11
Polymerase chain reaction (PCR) amplification.
PCR amplification was performed according to Maslanka et
al.9 Briefly, cDNA was amplified for 30 cycles under
nonsaturating PCR conditions with a panel of TCR BV family-specific
primers, and a -chain constant primer in duplex or simplex (for BV
6.1 and 6.2) reactions using 24 TCR BV family-specific primers and pairings as previously described.9 The common constant
primer was labeled at the 5' end with 5'-6 carboxyfluorescein (6-FAM). In addition to the TCR BV family-specific and constant primers, each
reaction contained a -actin-specific primer pair producing a
6-FAM-labeled 230-bp product as an internal PCR control for verification of cDNA integrity and the fidelity of the PCR reactions. The -actin primer sequences were 5'-CCCTGTACGCCTCTGGCCGTACCAC-3' (6-FAM labeled) and 5'-CACGGCATCGTCACCAACTGGG-3', and were added to a
final concentration of 0.025 µmol/L.
TCR spectratyping.
TCR spectratyping was performed as described by Maslanka et
al.9 Briefly, an equivalent volume of PCR-labeled product
was mixed with formamide dye-loading buffer, heated at 94°C for 2 minutes, and applied to a pre-run 5% acrylamide-urea sequencing gel.
Gels were run for 110 minutes at 40 W. After resolution on the gel, the
labeled PCR product was analyzed with a Molecular Dynamics FluorImager
575 (Sunnyvale, CA). TCR spectratyping of a healthy PBMC repertoire
typically results in a banding pattern composed of between seven and
eight bands at three-nucleotide base intervals, reflecting the correct
"in-frame" nature of functionally rearranged -chain TCR gene
products. The limited number of PCR cycles used (30) leads
to the generation of PCR products with a distribution representative of
the starting material, ie, a Gaussian distribution.9 Each
band in a spectratype, while uniform in size, is heterogeneous with
respect to the nucleotide sequences it contains, as would be expected,
given the high degree of diversity required of TCR CDR3 regions.
PCR product cloning and DNA sequencing.
The TCR -chain PCR products corresponding to specific spectratype
bands were cloned using a T/A Cloning Kit (Invitrogen, San Diego, CA)
for PCR fragment cloning. Entire PCR reactions were purified using a
QIAquick PCR Purification Kit (Qiagen, Chatsworth CA) before
cloning. This approach produced a spectrum of clones representative of all band species and their relative frequencies within a particular spectratype PCR reaction. DNA sequence
analysis of clones was performed using a Taq Dye Deoxy-Terminator
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). We
define the unique TCR BV CDR3 nucleotide sequences determined in
this manner to be a "TCR BV clonotype" or simply
"clonotype."
Electroblot hybridization.
Oligonucleotide clonotype-specific probes to TCR CDR3 of interest were
designed based on the DNA sequence of predominant BV 5.1 species
derived from patients 1 and 2. The sequences for the probes used are
underlined in Fig 4. Each clonotype-specific probe, as well as a BV
constant region primer used in each TCR spectratyping PCR reaction, was
radiolabeled using 32P ATP (Dupont NEN, Boston, MA),
polynucleotide kinase and endlabeling buffer according to the
manufacturer's recommendation (New England Biolabs, Beverly, MA). The
BV constant region primer sequence was 5'-CTGTGTTTGAGCCATCAGAAGC-3'.
Spectratypes were electroblotted from acrylamide gels onto positively
charged nylon membranes (Boehringer Mannheim, Indianapolis, IN) and
UV-crosslinked using the auto crosslink setting on a Stratalinker UV
1800 (Stratagene, La Jolla, CA). Electroblots were subsequently
hybridized overnight with agitation at 42°C with
32P-labeled clonotype-specific probe in a solution
containing: 10× Denhardt's solution, 5× SSC (1× SSC = NaCl
0.15 mol/L, Na3 citrate · 2H2O 0.0015 mol/L; pH 7.0), 7% sodium dodecyl sulfate (SDS), 20 mmol/L Na2HPO4, and 100 µg/mL denatured
herring sperm DNA. Posthybridization, the electroblots were washed
twice for 10 minutes at 42°C in a solution containing 3× SSC, 5%
SDS, and 70 mmol/L phosphate buffer pH 7.0, followed by two additional
washes at 42°C in a solution containing 1× SSC and 1% SDS. After a
brief rinse in 2× SSC, the electroblots were subjected to
autoradiography and analyzed using a Molecular Dynamics PhosphoImager
(Sunnyvale, CA).
After clonotype-specific probe hybridization and autoradiography, the
radiolabeled probe was stripped from the electroblots with an alkaline
stripping solution (0.2 N NaOH, 0.1% SDS) at 37°C for 20 minutes before rehybridizing as described earlier with a second
clonotype-specific probe or the BV constant region-specific probe.
Complete stripping of each previous probe was verified by autoradiography.
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RESULTS |
TCR BV spectratypes of noncultured PBMC from the two patients were
similar to those of T cells from normal controls.
Depending on the extent of the T-cell response, it might be possible to
distinguish T cells of interest by a direct comparison of PBMC from a
patient experiencing HITT with those from a normal subject. To test
this, the TCR -chain spectratypes of PBMC from patients 1 and 2 and
from normal subjects were analyzed. In most cases, spectratypes
consisted of seven or eight bands for each BV family, differing in size
by 3 bp and having an intensity distribution that was approximately
Gaussian (Fig 1A, B, and C). Only an
occasional deviation from this pattern was observed in the spectratypes
of noncultured PBMCs, as seen for the BV 14 family of patient 2, where
a high-intensity band appears at the high end of the size distribution
(Fig 1C, Lane 14/19).
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| Fig 1.
T-cell receptor spectratype analysis (multiplex PCR) of
26 BV families using noncultured PBMC from a normal subject (A) and
from patients 1 and 2 (B and C). Primers specific for -actin were
included in each reaction mixture as a check on fidelity. Bands in each
group differ from each other by 3 bp in length because only T cells
with productive (in phase) VDJ rearrangements are found in the
peripheral blood.
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After stimulation of patient PBMC with heparin:PF4 complexes, there
was selection for T cells expressing transcripts of the TCR BV 5.1 subfamily.
As shown in Fig 2A and B, bands that
corresponded to members of the BV 5.1 family were prominent in
spectratypes derived from PBMC of the two patients cultured for 14 days in the presence of heparin:PF4 complexes. When there is a
considerable expansion of one size class of CDR3 and the amount of cDNA
used is high, it is possible to saturate the signal from the expanded
band artifactually increase the signal from other components. To check
this possibility, templates were diluted as much as 100-fold before
spectratype analysis. As shown in Fig 2, the BV 5.1 band is easily
observed at dilutions of 1:100 (Fig 2A) and 1:10 (Fig 2B). No such
pattern was observed in PBMC from either patient cultured with PF4
alone, with heparin alone, or with buffer (data not shown). Spectratype analysis of serially diluted DNA templates from normal subjects whose
PBMC were cultured under the same conditions failed to show selection
for any BV families. In the example shown (Fig 2C), which was developed
at a dilution of 1:10, a few scattered, faint bands are seen, but there
was no focusing of a BV 5.1 band. In patient 1, narrowing of the TCR
spectratype to one or two prominent bands was also observed in BV
families 13, 8, 17, 18, 6.1, and 6.2 (Fig 2A). Quantitation of the
various PCR products using ImageQuant software (Molecular Dynamics)
after normalization against the internal cDNA control (-actin)
showed that the dominant PCR products were those of the BV 5.1 family.
Similarly, in patient 2, using a 1:10 dilution of cDNA template, bands
of the BV 5.1 family were found to dominate weaker bands from the BV
12, 3, 11, 18, 6.1, and 6.2 families (Fig 2B). These findings indicate
that patient T cells that survived in culture for 14 days in the
presence of heparin:PF4 complexes preferentially utilized the BV 5.1 gene for expression of the component of their TCR. In contrast, no particular pattern of receptor utilization was seen in normal PBMC
cultured with heparin:PF4 or in patient cells cultured with PF4 alone
or heparin alone.
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| Fig 2.
Polyacrylamide gel shows TCR spectratype analysis of T
cells from patient 1 (A), patient 2 (B), and a normal subject (C) after
14 days of culture in the presence of heparin:PF4. The spectratypes
shown were generated using template cDNA diluted 1:100 (patient 1) and
1:10 (patient 2 and normal subject) to illustrate the dominant PCR
products.
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Sequence analysis of the dominant BV 5.1 TCR spectratype bands showed
a CDR3 motif shared by both patients, which suggests antigen-driven
skewing of the T-cell repertoire.
The fact that both patients utilized the BV5.1 family made it
interesting to determine whether similar TCR CDR3 sequences were
involved in the response in culture. This was accomplished by
determining the sequences of the CDR3 for the BV5.1 PCR product. Each
patient's undiluted cDNA template derived from PBMC cultured in the
presence of antigen (heparin:PF4) was amplified with primers specific
for the BV 5.1 TCR family and was cloned and sequenced as described in
Materials and Methods. Deduced amino acid sequences of the identified
BV 5.1 CDR3 loops and adjacent regions are shown in Fig
3. The dominant BV 5.1 PCR product from
patient 1 encoded a CDR3 region 10 amino acids in length (using the
nomenclature of Rock et al12), utilized BJ 2.3, and was
represented at a frequency of 86% (38 of 44 sequenced clones) within
the PCR product of this length. Minor clonotypes, encoding CDR3 regions
of the same size, were identified at frequencies of 9% (four of 44 sequenced clones) and 5% (two of 44 sequenced clones). A second BV 5.1 PCR product encoding a CDR3 region nine amino acids in length was found
to consist of two clonotypes, each at a frequency of 50% (four of
eight sequenced clones), utilizing the BJ regions 2.7 or 1.6. Thus,
three high-frequency ( 50%) clonotypes were found in patient 1. The
dominant one was associated with the most prominent PCR product and had
the CDR3 loop sequence SLEDREVDTQ joined to BJ 2.3. The remaining two
had the CDR3 loop sequences SPGTGTGEQ and SLGGYNSPL joined to BJ 2.7 and 1.6, respectively.
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| Fig 3.
Predicted amino acid sequences of BV 5.1 CDR 3 domains of
T cells from patients 1 and 2 following 14 days of culture with antigen
(heparin:PF4). For each unique sequence identified (clonotype), a
portion of the BV and BJ regions and the complete CDR3 loop region is
shown. "Clonotype frequency" is the percentage of clones
sequenced that encoded the amino acid sequences shown. A common CDR3
loop region motif (P G T G) shared by responding T cells of
the 2 patients is highlighted in bold.
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The prominent BV 5.1 PCR product from patient 2 encoded a CDR3 region
10 amino acids in length, was present at a frequency of 70% (7 of 10 clones sequenced), and had the CDR3 sequence SPGTGVTDTQ, utilizing BJ
2.3. A second TCR clonotype encoding a CDR3 of the same length was
present at a frequency of 20% (2 of 10 sequenced clones) and had the
CDR3 sequence SSWGDPLGEQ, utilizing BJ 2.7. No other BV 5.1 PCR
products were identified by cloning, despite the detection of a few
other minor bands in spectratypes prepared from undiluted cDNA templates.
The shared BV 5.1 motif found in patients 1 and 2 consisted of four
identical amino acid residues (PGTG) on the amino proximal side of the
dominant TCR clonotype CDR3 region having the amino acid sequences
SPGTGTGEQ in the case of patient 1 (Probe 1b, Fig
4) and SPGTGVTDTQ in the case of
patient 2 (Probe 2, Fig 4). Examination of the DNA sequence for each of
the CDR3 regions showed that nucleotide sequences encoding the first
and second amino acids (PG) in each CDR3 were generated by random introduction of nucleotides during V(D)J recombination of the TCR chain, while the next two amino acids (TG) were derived from a D region
(D1.1) contribution during V(D)J recombination. This finding suggests
that there was selection for T cells bearing a common TCR motif during
14 days of culture with antigen (heparin:PF4 complexes). The common
serine residue preceding the PGTG motif is not considered to be a
component of the motif because it originates from a genomic DNA
sequence of the BV 5.1 gene, rather than from a random recombination
event.
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| Fig 4.
DNA and predicted amino acid sequences of TCR BV 5.1 clonotypes selected for hybridization experiments. For each clonotype,
a portion of the BV and BJ regions and the complete CDR3 loop region is
shown. The CDR3 length and BJ utilization are indicated for each
clonotype. CDR3-specific oligonucleotide probe names are shown on the
left and the sequence for each is underlined. A shared CDR3 loop region
motif (P G T G) present in both HITT patients is highlighted
in bold.
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The dominant clonotypes are specific for heparin:PF4 complexes.
Although the clonotype sequences were determined in cultures containing
heparin:PF4, it was important to test for their specificity. To do so,
probes identifying each clonotype were used to test the results of
cultures in which T cells were stimulated with heparin, PF4, media
alone, or heparin:PF4 complexes. BV 5.1 TCR spectratypes of PBMC from
patients 1 and 2, uncultured and cultured under various conditions, are
shown in Figs 5A and
6A. With
noncultured cells, a Gaussian distribution was obtained as expected,
whereas patterns obtained from cells cultured with media alone, heparin alone, PF4 alone, or heparin:PF4 complexes consisted of several prominent bands of various sizes with no apparent relationship one to
another. The sporadic appearance of other bands in Figs 5A and 6A is a
consequence of specific or nonspecific stimulation of T cells subsets.
In the case of nonspecific stimulation, T-cell subsets may survive or
expand in response to some component of the culture medium other than
specific antigen. This type of "chatter" in the spectratype
signal is typically observed when nondiluted 14-day-cultured DNA
templates are tested (Figs 5A and 6A). Figures 5A (plasmid 1a, plasmid
1b lanes) and 6A (plasmid 2 lane) show bands obtained by direct
amplification of DNA in plasmids carrying the BV 5.1 CDR3 sequences
identified by cloning (see Fig 4). The clonotype content of bands
derived from T cells cultured under different conditions (Figs 5A and
6A) was then examined by exposing the gels to radiolabeled probes
complementary to the dominant BV 5.1 CDR3 nucleotide sequences. As
shown in Fig 5B and C, probes complementary to the dominant clonotypes
identified in patient 1 (Fig 4, sequences 1a and 1b) hybridized only
with bands obtained after culture with heparin:PF4 complexes and with
the appropriate plasmids. Similarly, the probe specific for the
dominant clonotype identified in patient 2 (Fig 4, sequence 2)
hybridized only with a single band obtained after culture with antigen
(heparin:PF4) and with the appropriate plasmid.
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| Fig 5.
Analysis of clonotype specificity of cultured PBMC from
patient 1. (A) Fluorescent spectratype. The nature of the sample
analyzed is identified above each lane. The first 4 lanes are from
14-day cultures containing no addition (media), media alone, heparin
alone (heparin), PF4 alone (PF4), or heparin:PF4 complexes (Hep/PF4).
The next lane is from uncultured PBMC. The next 2 lanes are from the
plasmid DNA clones used to generate the clonotype sequences. The lane
marked "PCR blank" is a water control. The actin control
amplification is shown as a separate subpanel. (B) Hybridization of DNA
from the gel shown in A with a probe for clonotype 1b (radioautogram).
The gel was electroblotted onto a nylon membrane and treated as
described in Materials and Methods. (C) Rehybridization of the
electroblot with a probe specific for clonotype 1a. The membrane was
stripped of the previous probe and rehybridized as described. (D)
Rehybridization of the membrane with a TCR constant region probe
after stripping of the previous probe.
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| Fig 6.
Analysis of clonotype specificity of cultured PBMC from
patient 2. The lanes are labeled as described in Fig 5, except that
only 1 plasmid control is included. (A) Spectratype gel. The actin
control amplification is shown as a separate subpanel. (B)
Hybridization of the DNA from the gel shown in A with a probe for
clonotype 2. (C) Rehybridization of the membrane with a TCR constant region probe after stripping of the previous probe.
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None of the tested probes hybridized with bands of the same size or
other sizes identified in control cultures except for very weak
interaction of Probe 1b with a band 9 bp larger from cells cultured
with PF4 alone and one 6 bp larger from noncultured cells (Fig 5B). No
additional bands were identified even after prolonged autoradiograph
exposures (7 days). As a check on the integrity of the spectratype
gels, the blots were stripped of probe and were rehybridized with a
radiolabeled oligonucleotide specific for the -chain constant region
present in each PCR product. All bands visible in the original gels
(Figs 5A and 6A) were recognized by this probe (Figs 5D and 6C), which
indicates that all were present when blotting with the
clonotype-specific probes was performed. An exception is the faint band
in Lane 3 (PF4 only) of Fig 6A, which was not recognized in 6C because
the amount of DNA present was below the threshold level for detection
in the electroblot/stringency hybridization protocol employed. Since,
within the limits of detection, the clonotype-specific probes exhibited
little or no binding to the bands identified in spectratypes from
control cultures (heparin alone, PF4 alone, medium alone), these
results argue strongly that the high-frequency clonotypes observed in
patients 1 and 2 after 14 days of culture were the result of specific
stimulation by complexes of heparin and PF4.
| |
DISCUSSION |
It is now clear that heparin-induced thrombocytopenia/thrombosis is
almost invariably associated with antibodies that recognize epitopes on
heparin:PF4 complexes.5,13,14 The heparin molecule itself
appears not to be targeted, because polyanionic macromolecules like
polyvinyl phosphate that have no structural relationship to heparin
also form complexes with PF4 that contain the full spectrum of target
antigens.15 On the basis of these and other recent
observations,16 it seems almost certain that HITT
antibodies recognize conformational determinants that are created on
PF4 when it binds to a linear, polyanionic molecule of length
sufficient to span about half the circumference of the PF4
tetramer.15 Although studies of the humoral immune response
have yielded important information about the nature of the B-cell
involvement in HITT, studies to characterize the contribution of T
cells have not yet been reported. It is likely that the antibody
response associated with HITT is dependent on T-cell help, because PF4
is a protein antigen, antibodies associated with clinical disease are
usually of high titer,8 and the most abundant antibody
species identified in patients are usually of the IgG or IgA
isotypes.8,17 The purpose of our investigation was to
determine whether a T-cell response to heparin:PF4 complexes could be
demonstrated in vitro, and if so, whether the components of the T-cell
repertoire involved in this response could be identified.
Our approach to identification of T cells responding to heparin:PF4
complexes was to restimulate T cells from the peripheral blood of
patients with recent HITT. The responding repertoire was analyzed by
CDR3 length analysis of the expressed TCR -chain genes using
spectratyping.9,11 A typical spectratype shows a Gaussian
distribution of bands with a 3-bp spacing characteristic of a complex
repertoire of in-frame TCR rearrangements. Upon culture, the repertoire
will shift depending on the loss, persistence, or expansion of T cells
upon contact with antigen. We have used this approach to define
responses to major histocompatability complex (MHC) class II
alloantigens18 to class I-restricted cytotoxic
responses,19 and to platelet glycoprotein (GP)IIIa polymorphisms associated with neonatal alloimmune
thrombocytopenia.20 However, the experiments reported here
are the first in which a complex antigen has been used for stimulation
of T cells in culture.
As expected from what is known about the diversity of the TCR
repertoire, TCR spectratypes from unstimulated PBMC of the HITT patients studied did not differ significantly from those of normal subjects (Fig 1A, B, and C). However, after stimulation in culture for
14 days in the presence of heparin:PF4 complexes, spectratypes prepared
from the T cells of both patients demonstrated persistence of TCR band
belonging to the BV 5.1 family (Fig 2). These bands appeared to reflect
a specific T-cell response to heparin:PF4 complexes, since they were
not observed in cultures containing PF4, heparin, or medium alone.
To investigate the character of the BV 5.1 TCR in responding T cells,
the cDNA templates derived from patient cells cultured in the presence
of antigen were amplified with primers specific for BV 5.1, and the PCR
products were cloned and sequenced. In patient 1, dominant PCR products
that encoded a CDR3 10 amino acids in length (86% frequency) and two
others that encoded CDR3 nine amino acids in length (each 50% in
frequency) were identified. In patient 2, two dominant clonotypes were
identified, each encoding a CDR3 10 amino acids in length at
frequencies of 70% and 20%, respectively. A nine-amino acid CDR3
from patient 1 and a 10-amino acid CDR3 from patient 2 were of
particular interest because they shared peptide motif (PGTG) in their
predicted amino acid sequences. By analyzing the identified nucleotide
sequences and comparing them with germ-line sequences, it was
determined that the hexanucleotide encoding the first and second amino
acids (PG) of each tetrapeptide resulted from random introduction of
nucleotides during V(D)J recombination of the TCR chain, whereas
the next two amino acids (TG) were the result of a D region (D1.1)
contribution (Fig 4). Identification of a common CDR3 motif in the TCR
of responding T cells is generally accepted as an indication of a
common antigenic stimulus. There have been multiple reports of the
conservation of CDR3 motifs in T cells stimulated with known antigens
or antigenic peptides.21-30 We have demonstrated motif
conservation in T cells stimulated with peptides derived from platelet
GPIIIa (3 integrin)20 and with influenza
M1 peptide.19 We interpret our finding of selection for the
same CDR motif, constructed from different gene elements in patients
with HITT, as an indication that at least one portion of the processed
heparin:PF4 complex is recognized by T cells by both patients. The
10-amino acid CDR3 from patient 1, which did not display the motif,
could be viewed as a private response specific for that
individual.31
Additional evidence that the TCR motifs identified in responding T
cells of the patients were antigen-specific was obtained by preparing
radiolabeled oligonucleotides complementary to the dominant clonotypes
and using these to probe BV 5.1 spectratypes obtained from cultured and
noncultured PBMCs. These probes hybridized only with bands of the
expected size derived from PBMC cultured in the presence of heparin:PF4
complexes and not with bands derived from uncultured PBMC or from PBMC
cultured with PF4, heparin, or medium alone. This finding argues
strongly that the high frequency clonotypes observed in both patients
after 14 days of culture with heparin:PF4 complexes were the result of
specific antigen stimulation. Together, our observations indicate that
the helper T-cell response associated with humoral immunity to
heparin:PF4 complexes, a hallmark of HITT, can be reproduced in in
vitro culture and suggest that further studies in patients with HITT
may permit the patterns of T-cell reactivity associated with this
condition to be identified.
A question closely related to the specific T-cell response in HITT is
the nature of the immunogenic stimulus. Helper T cells are triggered by
peptides presented in the context of class II human leukocyte antigen
(HLA) molecules on the surface of antigen-presenting cells.32 PF4 released from platelets is presumably
processed into peptides, which are presented regularly to T cells in
the body, yet antibodies reactive with PF4 are almost never found in
normal subjects.2 When heparin is infused, it forms
complexes with a reservoir of PF4 normally associated with
glycosaminoglycans (GAG) molecules displayed on the luminal surface of
endothelial cells.33 How these complexes are metabolized is
not known, but it can be speculated that when macrophages process PF4
complexed with heparin, peptides not ordinarily seen by the immune
system are generated and presented to helper T cells (cryptic
epitopes). Sensitization to "cryptic" epitopes has been
documented in murine models of autoimmunity,34,35 in which
species the response to one peptide can diversify over time, leading to
recognition of other domains on an autoantigen to which there was
previous T-cell tolerance.36-39 Our model for studying
T-cell responsiveness in HITT should lend itself to future studies of
the mechanisms by which peptides derived from PF4 elicit specific
T-cell responses.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted March 5, 1999.
Supported by Grants No. HL-13629 and HL-44612 from the National Heart,
Lung, and Blood Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to R.H. Aster, MD, Blood Research Institute,
8727 Watertown Plank Rd, Milwaukee, WI 53226-3548.
| |
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