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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3008-3016
Characterization of T-Cell Repertoire of the Bone Marrow in
Immune-Mediated Aplastic Anemia: Evidence for the Involvement of
Antigen-Driven T-Cell Response in Cyclosporine-Dependent Aplastic
Anemia
By
Weihua Zeng,
Shinji Nakao,
Hideyuki Takamatsu,
Akihiro Yachie,
Akiyoshi Takami,
Yukio Kondo,
Naomi Sugimori,
Hirohito Yamazaki,
Yuji Miura,
Shintaro Shiobara, and
Tamotsu Matsuda
From the Third Department of Medicine, the Department of Pediatrics,
and Blood Transfusion Section, Kanazawa University School of Medicine,
Kanazawa, Japan.
 |
ABSTRACT |
To determine whether the antigen-driven T-cell response is involved
in the pathogenesis of aplastic anemia (AA), we examined the
complementarity-determining region 3 (CDR3) size distribution of T-cell
receptor (TCR)  -chain (BV) subfamilies in the bone marrow (BM) of
untreated AA patients. AA patients who did not respond to
immunosuppressive therapy and those who obtained unmaintained remission
early after cyclosporine (CyA) or antithymocyte globulin (ATG) therapy
exhibited essentially a normal CDR3 size pattern. In contrast, five
patients who needed continuous administration of CyA to maintain
remission exhibited a skewed CDR3 size pattern in a number (>40%) of
BV subfamilies suggestive of clonal predominance. The skewing of CDR3
size distribution became less pronounced in one of the CyA-dependent
patients when the patient achieved unmaintained remission after a
4-year therapy with CyA, whereas it persisted longer than 7 years in
the other patient requiring maintenance therapy. Sequencing of BV15
cDNA for which the CDR3 size pattern exhibited apparent clonal
predominance in all CyA-dependent patients showed high homology of the
amino acid sequence of the CDR3 between two different patients. These
findings indicate that antigen-driven expansion of T cells is involved
in the pathogenesis of AA characterized by CyA-dependent recovery of hematopoiesis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
APLASTIC ANEMIA (AA) is a syndrome
characterized by pancytopenia and bone marrow (BM) hypoplasia. Although
the etiology is unknown, clinical observations such as the high rate of
response to immunosuppressive therapy in patients with AA suggest the
importance of immune mechanisms in the development of
AA.1,2 Among several immune mechanisms, T-cell-mediated
suppression of hematopoiesis has been considered the most important in
the development of AA.3 This hypothesis has been supported
by in vitro findings such as suppression of hematopoietic progenitor
cell growth4-6 and increased production of myelosuppressive
cytokines by the patients' T cells,7-10 as well as an
increased proportion of activated T cells in the peripheral
blood11,12 and BM.13 Recent studies
demonstrated that T-cell clones capable of inhibiting autologous
hematopoietic progenitor cells can be generated by culturing T cells of
AA patients with autologous hematopoietic progenitor
cells.14,15 These findings suggest that AA is a type of
autoimmune disease that involves a T-cell attack against hematopoietic
progenitor cells. However, there has been no convincing evidence for
involvement of the antigen-driven T-cell response in the development of
AA, not to mention the causal antigen that elicits the T-cell attack against hematopoietic progenitor cells.
In well-known autoimmune diseases such as multiple
sclerosis16-18 and rheumatoid arthritis,19,20
the T-cell repertoire in the involved organ has been extensively
studied to characterize an antigen-driven response that may induce the
disease process. It was revealed that a limited number of T cells using
a restricted diversity of the T-cell receptor (TCR) chain (BV)
dominantly proliferated in the involved organ.21 Such
oligoclonal proliferation of T cells is generally thought to reflect
the specific response of autoreactive T cells to certain antigens,
since T cells bearing similar BV subfamilies have been demonstrated to
proliferate dominantly in different patients and the TCR repertoire of
these patients is distinct from that of patients with other
inflammatory diseases due to a definite pathogen.16,18,22
If specific recognition of hematopoietic cells by T cells plays a role
in the development of AA, clonal expansion of a limited number of T
cells should be observed in the BM of AA patients as well. Such
clonally expanding T cells in the BM of different patients may share a
clonotype if a common antigen is responsible for inciting T cells to
attack hematopoietic cells.
To test these hypotheses, we analyzed the T-cell repertoire in the BM
of AA patients. Since AA is a mixture of illnesses with BM hypoplasia,
studying unselected patients is expected to produce varying results
that may be hard to interpret. We thus limited the study subjects to
patients who were treated with cyclosporine (CyA) or antithymocyte
globulin (ATG) after a failure to respond to CyA.
Complementarity-determining region 3 (CDR3) size distribution analysis,
single-strand conformation polymorphism (SSCP) analysis, and sequencing
of TCR cDNA were used to determine whether clonal expansion of a
limited number of T cells occurs in the BM. Clonal predominance was not
evident in AA patients who obtained unmaintained remission early after
CyA or ATG therapy after failing to respond to CyA and those who did
not respond to CyA therapy, whereas it was present in a number of BV
families in AA patients with HLA-DRB1*1501 whose hematopoietic function
depended on continuous administration of CyA. Sequencing of BV15 cDNA
for which the CDR3 size profile exhibited apparent clonal predominance
in all CyA-dependent patients showed a high homology of the amino acid
sequence of the CDR3 between two different patients with CyA-dependent AA.
| |
MATERIALS AND METHODS |
Patients.
Eighteen patients with idiopathic AA were included in the analysis of
the T-cell repertoire in the BM. These patients were first treated with
CyA 4 to 6 mg/kg daily for at least 4 months, and nonresponders were
subsequently treated with ATG 15 mg/kg daily for 5 days. The BM of 14 normal subjects aged 16 to 51 years was studied as a control. All but
two patients (patients no. 8 and 9) were untransfused at the time of
sampling. Three of 14 normals (normals no. 1, 2, and 3) possessed
HLA-DRB1*1501. Table 1 summarizes clinical
characteristics of the patients. Patients no. 1 to 9 improved with CyA
therapy. Patients no. 1 to 4 achieved unmaintained remission after CyA
therapy for 10 to 20 months, whereas CyA could not be withdrawn within
3 years due to the recurrence of pancytopenia after dose reduction or
cessation of CyA in patients no. 5 to 9; in these patients, AA was thus
designated CyA-dependent. All patients with CyA-dependent AA possessed
HLA-DRB1*1501, which is strongly associated with this type of
AA.23,24 Patients no. 10 to 13 improved with ATG after
failure with CyA therapy. Patients no. 14 to 18 did not respond to CyA
therapy; three (no. 14, 16, and 18) underwent BM transplantation from
HLA-identical siblings after treatment failure with CyA. BM was
obtained before treatment from all but one patient (no. 5), who
underwent sampling at the time of exacerbation of AA in association
with the dose reduction of CyA. All patients provided informed consent
prior to sampling, and the study was approved by the institutional
human research committee.
RNA extraction and cDNA preparation.
BM mononuclear cells (BMMCs) were isolated using density gradient
centrifugation. For the BMMCs of patient no. 5, CD4+ and
CD8+ cells were sorted using monoclonal antibodies
(Becton-Dickinson, Mountain View, CA) and an Epics C cell sorter
(Coulter Electronic Inc, Hialeah, FL). Total RNA was extracted from
BMMCs and the T-cell subset using a technique described
elsewhere,25 and then reverse-transcribed into cDNA in a
reaction primed with oligo(dT)12-18 using SuperScript II reverse
transcriptase as recommended by the manufacturer (GIBCO-BRL, Bethesda, MD).
CDR3 size distribution analysis.
Conditions for the CDR3 size distribution analysis have been reported
elsewhere.21,26,27 Briefly, cDNA was polymerase chain
reaction (PCR)-amplified through 35 cycles (94°C for 1 minute, 55°C
for 1 minute, and 72°C for 1 minute) with a primer specific to 24 different BV subfamilies (BVs1-2028 and
BVs21-2429) and a fluorescent BC primer.28 The
analysis of BV10 and BV19 was excluded from this study because these
are pseudogenes.30 One microliter of amplified products was
mixed with 1.5 µL 100% formamide and 0.5 µL size standard
(Genescan-500 ROX,ABI 373; Perkin-Elmer, Urayasu, Japan), heated at
90°C for 3 minutes, and electrophoresed in a 6.75%
denaturing polyacrylamide gel. The distribution of CDR3 size within the
amplified product of each BV subfamily was analyzed using an automatic
sequencer (Applied Biosystems, Foster City, CA) equipped with a
computer program allowing determination of the fluorescence intensity
of each band. The results are depicted as peaks corresponding to the
intensity of the fluorescence. CDR3 size patterns that failed to
exhibit a bell-shaped distribution due to the appearance of prominent peaks with or without a reduced peak number (< five peaks) were judged to be abnormal. This judgment was made by three different investigators to minimize interindividual differences. The frequency of
BV subfamilies displaying an abnormal CDR3 size profile was determined
for each subject.
Given the BV-NDN-BJ sequence of the most dominant clone characterized
in patient no. 5, a more specific primer covering both CDR3 and BJ2.2
(5'-TGTTCGGCCCGCTAGTCAGGTCACT-3') was designed specifically to amplify
cDNA of the BV15-positive (BV15+) T-cell clone in different
T-cell subsets. The BV15+ amplified products from patient
no. 5 were submitted to five cycles of primer extension using the
fluorescent clonotypic primer under the same PCR conditions and
analyzed in the same way as before.31
SSCP analysis.
cDNA amplified by PCR with a combination of BC and BV primers after 35 cycles of PCR was diluted in the SSCP loading buffer (95% formamide,
10 mmol/L EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol),
heated at 90°C for 2 minutes to denature, and then subjected to
nondenaturing 5% polyacrylamide gel electrophoresis at constant power
and temperature.32 The DNA was then transferred to
Immobilon-S (Millipore Intertech, Bedford, MA) and hybridized with a
biotinylated BC probe (5'-AACAAGCGTGTTCCCGAGGTCGCTGTGTT-3') at 42°C
overnight. The membrane was washed with 0.2X SSPE and 0.5% sodium
dodecyl sulfate for 10 minutes at 55°C and visualized by subsequent
incubation of streptoavidin, biotinylated alkaline phosphatase, and a
chemiluminescent substrate system (Plex Luminescence kit, Millipore).
Cloning and sequencing of PCR-amplified cDNA.
PCR products of BV15 cDNA were electrophoresed on an agarose gel. The
amplified fragment of expected size was purified using DEAE paper and
cloned into a pGEM-T Vector system (Promega Corp, Madison, WI).
Eighteen to 40 colonies containing the insert fragment were randomly
selected and sequenced using an ABI pRISM cycle Sequencing Kit
(Perkin-Elmer) and an automatic DNA sequencer (ABI 373; Perkin-Elmer).
The amino acid sequence of CDR3 was deduced using DNASIS-Mac Version
3.6 software (Hitachi Software, Yokohama, Japan).
Statistical methods.
Differences in the frequency of BV subfamilies displaying an abnormal
CDR3 size profile and in the frequency of clones exhibiting the
identical CDR3 sequence among all cDNA clones sequenced between control
and patient groups were tested using the unpaired Student's t-test and Mann-Whitney U test, respectively.
| |
RESULTS |
CDR3 size distribution of TCR BV cDNA of BM T cells.
The cDNA of 24 different BV subfamilies was amplified using a
fluorescent BC primer, and the CDR3 size distribution of each BV
subfamily was compared among normal individuals and AA patients responsive to immunosuppressive therapy. CDR3 patterns of two normal
individuals possessing HLA-DRB1*1501 (normals no. 1 and 2) and four AA
patients (no. 1 to 4) who obtained unmaintained remission early after
CyA therapy are shown in Fig 1. In normal individuals, some BV subfamilies exhibited skewed CDR3 size
distribution; the frequency of BV subfamilies displaying an abnormal
CDR3 size profile in relation to all BV subfamilies was 11.0% ± 6.4% (mean ± SD). The majority of the amplified products of AA
patients (4.2% ± 5.9%, P = .1) displayed a bell-shaped
size pattern, with more than five peaks as well, indicating the
predominance of polyclonal T cells. In contrast, a number of BV
subfamilies in patients with CyA-dependent AA (54.2% ± 14.0%,
P < .0001) displayed an apparently skewed distribution of
CDR3 size, indicating clonal or oligoclonal proliferation of T cells
(Fig 2). Figure
3 illustrates CDR3 size profiles of four
patients who did not respond to CyA but improved with subsequent ATG
therapy. Although several BV families exhibited skewed CDR3 patterns,
the frequency of BV families displaying abnormal CDR3 size patterns was
comparable to the frequency for normal controls (16.7% ± 4.8%,
P = .1). Its frequency in the other five patients refractory
to CyA therapy (no. 14 to 18) was also comparable to the frequency for
normal controls (10.1% ± 7.6%, P = .8). Table
2 summarizes the number of BV subfamilies
and the proportion of abnormal BV families in all patients. These results suggest that among AA patients responsive to immunosuppressive therapy, clonal proliferation of a limited number of T cells in the BM
might occur primarily in those characterized by the CyA-dependent response.
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| Fig 1.
CDR3 size distribution of TCR BV derived from BM T cells
of AA patients who obtained unmaintained remission early after CyA
therapy. cDNA that was amplified using primers specific for 24 different BV subfamilies and coupled with a fluorescent BC primer was
analyzed for size with the gene scan program. N1 and N2, normals no. 1 and 2 possessing HLA-DRB1*1501; P1-P4, patients no. 1-4. Most BV
subfamilies of the patients and normal controls displayed a bell-shaped
size pattern with >5 peaks, indicating the predominance of polyclonal
T cells.
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| Fig 2.
CDR3 size distribution of TCR BV derived from BM T cells
of CyA-dependent AA patients. TCR cDNA of another normal individual
with HLA-DRB1*1501 (N3, normal no. 3) and patients no. 5-9 (P5-P9)
whose hematopoietic function depended on continuous administration of
CyA was analyzed. A large number of BV subfamilies (> 40%) of the
patients displayed a skewed or collapsed pattern with a reduced peak
number, indicating clonal or oligoclonal proliferation of T cells.
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| Fig 3.
CDR3 size distribution of TCR BV derived from BM T cells
of AA patients who responded to ATG after treatment failure with CyA.
TCR cDNA of patients no. 10-13 (P10-P13) was analyzed. Although several
BV families exhibited skewed CDR3 patterns, the frequency of BV
families displaying abnormal CDR3 size patterns was comparable to
normal controls.
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SSCP analysis of amplified BV cDNA of BM T cells.
The skewed CDR3 size distribution does not necessarily indicate the
presence of clonally proliferating T cells, since a prominent peak in
the histogram may represent the presence of polyclonal T cells with the
same CDR3 size. To confirm clonal proliferation of T cells with several
BVs in the BM of AA patients, amplified BV cDNA products were subjected
to SSCP analysis. Figure 4 shows the
results for selected BV families. The amplified products of BV cDNA of
a normal individual exhibited a smear, indicating a predominance of
polyclonal T cells. In contrast, the amplified products derived from
three CyA-dependent patients (no. 5, 6, and 9) exhibited distinct bands
in these BVs, indicating clonal expansion of a limited number of T
cells. Although the amplified products of BV14 and BV15 of patient no.
11 produced discernible bands, the intensity of the bands was much
lower than for patients no. 5, 6, and 9.
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| Fig 4.
SSCP analysis of TCR BV cDNA derived from BM of AA
patients. Amplified BV cDNA from a normal individual (N3, normal no.
3), P5 (patient no. 5), P6, P9, and P11 was subjected to SSCP
analysis.
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Changes in CDR3 size pattern associated with achievement of stable
remission off CyA therapy.
CyA could be withdrawn from patient no. 5 without aggravation of the
pancytopenia 4 years after initiating the therapy. The CDR3 size
distribution in BM T cells at the time of unmaintained remission, as
well as that in patient no. 8, who required low-dose CyA to maintain
stable hematopoiesis for more than 7 years after therapy, was analyzed
and compared with the distribution obtained at the time the disease was
active. Figure 5 illustrates the changes in the CDR3 size pattern of
representative BV subfamilies. Although some showed a slightly skewed
pattern, most BVs of patient no. 5 obtained at the time of remission
exhibited more than five peaks, suggesting the recovery of polyclonal
predominance associated with resolution of CyA-dependent AA (Fig
5A). In contrast, skewed CDR3 size patterns
of several BV families of patient no. 8 persisted after 7 years (Fig
5B) despite the fact that the patient was in remission with 2 mg/kg/d
CyA.
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| Fig 5.
Changes in CDR3 size patterns associated with
achievement of stable remission off CyA therapy. CDR3 size patterns of
patient no. 5 (P5) at the time of unmaintained remission after 4 years
of CyA therapy and patient no. 8 (P8) in CyA-dependent remission after
7 years of therapy were compared with patterns at the time the disease
was active. A, before CyA therapy; B, after CyA therapy.
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Deduced amino acid sequence of CDR3 of BV15 cDNA.
These findings indicate that oligoclonal expansion of a limited number
of T cells was a feature common to the BM of CyA-dependent AA patients
possessing HLA-DRB1*1501. To demonstrate directly the clonal expansion
of a limited number of T cells, we cloned the amplified products of
BV15 cDNA derived from the BM of three normal individuals with
HLA-DRB1*1501 and five CyA-dependent patients (no. 5 to 9) and
determined the nucleotide sequence of CDR3 of cDNA clones that were
randomly selected. BV15 was chosen because the clonal predominance of
this BV family was detected in all CyA-dependent AA patients (Table 2),
and the intensity of the band in the SSCP gel appeared to be the
strongest (Fig 4). Tables 3 and
4 summarize the deduced amino acid
sequence of each clone from three normal individuals and from
CyA-dependent AA patients, respectively. In normals no. 1 to 3, some
sequences were detected repeatedly, although their frequency was four
in 30 at most. In contrast, a large number of clones proved identical
in each AA patient. The most frequent amino acid (nucleotide) sequence
of the N-D-N region and its frequency in the total clones of each patient was DLTSGP (GACCTGACTAGCGGGCCG, 21 of 30) in patient no. 5, GSP
(GGCTCCCCC, 14 of 38) in patient no. 6, PRDRR (CCTAGAGACAGAAGG, 18 of 30) in patient no. 7, DLTNGP (GACCTGACTAACGGGCCG, seven of 40) in
patient no. 8, and DESY (GATGAGTCGTAT, seven of 18) in patient no. 9. The frequency of identical N-D-N sequences was significantly higher in
CyA-dependent AA patients versus normal individuals with HLA-DRB1*1501
(P = .025, Mann-Whitney U test). Such high
frequencies of certain clones were compatible with the results of the
CDR3 size distribution and SSCP analysis showing clonal predominance in
BV15+ T cells. Furthermore, N-D-N sequences of patients no.
5 and 8 were identical to those of the BV15 cDNA extracted from the
strongest band in the SSCP gel (Fig 4 and data not shown).
Interestingly, the amino acid sequence of the DLTSGP of patient no. 5 differed from the DLTNGP of patient no. 8 by only one amino acid.
Phenotype of the predominant BV15+ T-cell clone in
patient no. 5.
To characterize the BV15+ T-cell clone with the CDR3
sequence of DLTSGP in patient no. 5, CD4+ and
CD8+ cells were sorted from the patient's BMMCs, cDNA
derived from each T-cell population was amplified using a primer
specific to BV15 coupled with a BC primer, and the amplified products
were submitted to primer extension using the fluorescent clonotypic primer specific to the CDR3 sequence. A discernible peak of
fluorescence was detected only in CD4+ cells, indicating
that the T cells bearing BV15 with this sequence were CD4+
(Fig 6).
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| Fig 6.
Phenotype of BV15+ T-cell clone with CDR3
sequence of DLTSGP in patient no. 5. cDNA derived from
CD4+ and CD8+ T cells of BM from patient
no. 5 was amplified using a primer specific to BV15 coupled with a BC
primer, and the amplified products were submitted to primer extension
using the fluorescent clonotypic primer containing the DLTSGP motif.
Amplified BV15 cDNA was analyzed for size with the gene scan program.
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| |
DISCUSSION |
The present analysis of the T-cell repertoire in the BM of AA patients
treated with immunosuppressive therapy reveals several new findings
regarding the role of T cells in the pathophysiology of AA. We expected
that for patients no. 1 to 13 the BM would exhibit more or less
abnormality in the T-cell repertoire, since all of them had shown a
relative lymphocytosis in the BM and eventually improved with CyA or
ATG, both of which selectively inhibit T-cell function. However, in
addition to the patients who were refractory to CyA therapy (patients
no. 14 to 18), neither the patients who obtained unmaintained remission
early after CyA therapy nor those who responded to ATG after treatment
failure with CyA exhibited an apparently high frequency of CDR3 size
abnormalities suggestive of clonal predominance as compared with normal
individuals. The results are in agreement with reports by Melenhorst et
al.33 and Manz et al.34
documenting that BV families of BM T cells from AA patients are
predominantly polyclonal. Although it cannot be excluded that a small
expansion of T cells within limited BV families contributes to
suppression of hematopoiesis, it seems unlikely that antigen-driven
T-cell expansion plays an essential role in the pathophysiology of AA
in these patients. BM aplasia in these patients may have been caused
instead by polyclonal T-cell activation leading to excessive production
of myelosuppressive cytokines such as interferon gamma7,35
and tumor necrosis factor.35 In ATG-responsive patients who
were refractory to CyA, immunostimulatory effects rather than
immunosuppressive effects of ATG may have worked to improve
hematopoietic function.36
In contrast to the above two patient groups that responded to
immunosuppressive therapy, the frequency of BV families with abnormal
CDR3 size patterns was apparently higher in CyA-dependent AA patients
versus normal controls. Forty-one percent to 75% of BV subfamilies
exhibited CDR3 size patterns with a decreased peak number and/or skewed
shape suggestive of clonal predominance. Although all of the
CyA-dependent AA patients shared HLA-DRB1*1501, the abnormal CDR3
patterns were not due to the physiologic skewing associated with the
particular HLA-DRB1 allele, because CDR3 patterns of three normal
individuals possessing this DRB1 allele were not skewed.37
The abnormal CDR3 size pattern of patient no. 5 is not attributable to
his age as previously reported, since it was no longer detected after
the patient achieved unmaintained remission after 4 years of CyA
therapy.38 Moreover, abnormal CDR3 size patterns could not
be corrected by long-term CyA therapy in patient no. 8, who still
depended on CyA more than 7 years after therapy. It is possible that
the skewed T-cell repertoire is not involved in the pathogenesis of
CyA-dependent AA, but is only the consequence of the AA disease
process. However, the disappearance and persistence of clonal
predominance associated with each disease activity of patients no. 5 and 8 strongly suggest that clonally proliferating T cells play a role
in the development of CyA-dependent AA. In this particular subset of AA
patients, some antigens that elicit proliferation of a limited number
of T cells may persist in the BM, and resultant antigen-specific T
cells may inhibit hematopoiesis directly or indirectly via secretion of
myelosuppressive cytokines.
In support of this hypothesis is the high homology between patients no.
5 and 8 for the CDR3 motif of the most dominant BV15+
T-cell clone in the BM. The deduced amino acid sequence of the N-D-N
region differed for patient no. 5 (DLTSGP) versus patient no. 8 (DLTNGP) by only one amino acid. Such similarities within the
hypervariable region of the chain of T cells in different patients
have been demonstrated in other immune-mediated diseases, including
multiple sclerosis16,18 and sarcoidosis.39 In
multiple sclerosis patients, an LR motif in the VDJ region was shared
by T cells infiltrating the brain tissue of different patients and by T
cells reacting to the myelin basic protein.16 Similarly, an
RJR sequence was detected in T cells in the bronchoalveolar lavage
fluid from different patients with sarcoidosis.39 However, the high homology of the CDR3 motif covering the whole CDR3 sequence as
detected in patients no. 5 and 8 has never been demonstrated in any
T-cell-mediated diseases of undefined etiology. It has been
established that the CDR3 motif of TCR BV corresponds to an epitope
structure of the target peptide. In viral infections, T-cell clones
with the same CDR3 motif have been shown to proliferate and persist in
different patients.40 Hence, the two T-cell clones expressing BV15 with the similar CDR3 motif probably recognize a common
peptide that is possibly related to the pathophysiology of AA.
AA patients possessing HLA-DRB1*1501 form a distinct subset of
immune-mediated AA cases. This subset is likely to improve with CyA but
also to relapse in association with a dose reduction of CyA, and
therefore immune mechanisms through T cells most likely operate in
these patients.23 The frequency of AA patients requiring continuous CyA therapy among all AA patients is estimated to be approximately 15% based on our experience of CyA therapy for 40 patients with AA (S.N., unpublished observation, November
1998). It is tempting to hypothesize that some antigens
that are likely to be presented by HLA-DR15 sensitize CD4+
T cells to attack hematopoietic cells in these patients. The fact that
the PCR using a clonotypic primer complementary to the CDR3 sequence of
DLTSGP only amplified the cDNA of CD4+ T cells in patient
no. 5 supports this hypothesis. We recently isolated the
CD4+ T-cell clone with this CDR3 motif from the BM of
patient no. 5. Although this clone showed a proliferative response to
autologous BMMCs containing antigen-presenting cells, it did not
respond to purified CD34+ cells (data not shown). Since it
is possible that the CD4+ T-cell clone may recognize a
peptide derived from hematopoietic progenitors that can be presented by
antigen-presenting cells, we are currently screening a peptide library
to identify the target molecule of the T cells.
The present study reveals heterogeneity in the immune mechanisms of AA
for the first time. The finding that antigen-driven expansion of T
cells is primarily involved in the pathophysiology of a limited number
of cases characterized by a repetitive response to CyA therapy and
HLA-DRB1*1501 is of significance to basic studies on the immune
mechanisms of AA. This subset of AA is considered a suitable subject of
epidemiologic and immunologic approaches to identify the etiologic
mechanisms of AA. The results of this study also appear to be of
significance in choosing appropriate therapy for AA: patients
displaying abnormal CDR3 size patterns in greater than 40% of BV
families are likely to benefit from CyA therapy but will probably
require long-term treatment with the immunosuppressive agent.
Characterization of the T-cell repertoire in the BM as in the present
study may facilitate individualized therapy depending on the
immune mechanism of each AA patient.
| |
FOOTNOTES |
Submitted August 5, 1998; accepted December 17, 1998.
Supported by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture, Japan (08671223), and a Grant-in-Aid
for Immunologic Research for Intractable Diseases from the Ministry of
Health and Welfare, Japan.
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 Shinji Nakao, MD, Third Department of
Medicine, Kanazawa University School of Medicine, 13-1 Takaramachi,
Kanazawa, Ishikawa 920-8641, Japan; e-mail: snakao{at}med.kanazawa-u.ac.jp.
| |
REFERENCES |
1.
Bacigalupo A, Broccia G, Corda G, Arcese W, Carotenuto M, Gallamini A, Locatelli F, Mori PG, Saracco P, Todeschini G, Cosar P, Lacopino P, van Lint MT, Gluckman E, for the European Group for Blood and Marrow Transplantation (EBMT) Working Party on SAA:
Antilymphocyte globulin, cyclosporin, and granulocyte colony-stimulating factor in patients with acquired severe aplastic anemia (SAA): A pilot study of the EBMT SAA Working Party.
Blood
85:1348, 1995[Abstract/Free Full Text]
2.
Rosenfeld SJ, Kimball J, Vining D, Young NS:
Intensive immunosuppression with antithymocyte globulin and cyclosporine as treatment for severe acquired aplastic anemia.
Blood
85:3058, 1995[Abstract/Free Full Text]
3.
Young NS:
Pathophysiology II: Immune suppression of hematopoiesis, in
Young NS,
Alter BP
(eds):
Aplastic Anemia Acquired and Inherited. Philadelphia, PA, Saunders, 1994, p 68.
4.
Kagan WA, Ascensao JA, Pahwa RN, Hansen JA, Goldstein G, Valera EB, Incefy GM, Moore MAS, Good RA:
Aplastic anemia: Presence in human bone marrow of cells that suppress myelopoiesis.
Proc Natl Acad Sci USA
73:2890, 1976[Abstract/Free Full Text]
5.
Hoffmann R, Zanjani E, Lutton JD, Zalusky R, Wasserman LR:
Suppression of erythrocyte-colony formation by lymophocytes from patients with aplastic anemia.
N Engl J Med
296:10, 1977[Abstract]
6.
Ascensao J, Pahaw R, Kagan W, Hansen J, Moore MAS, Good R:
Aplastic anemia: Evidence for an immunological mechanism.
Lancet
1:669, 1976[Medline]
[Order article via Infotrieve]
7.
Zoumbos NC, Gascon P, Djeu JY, Young NS:
Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.
Proc Natl Acad Sci USA
82:188, 1985[Abstract/Free Full Text]
8.
Hinterberger W, Adolf G, Aichinger G, Dudczak R, Geissler K, Hocker P, Huber C, Kalhs P, Knapp W, Koller U, Lechner K, Volcplatzer B:
Further evidence for lymphokine overproduction in severe aplastic anemia.
Blood
72:266, 1988[Abstract/Free Full Text]
9.
Nakao S, Yamaguchi M, Shiobara S, Yokoi T, Miyawaki T, Taniguchi T, Matsuda T:
Interferon-gamma gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia.
Blood
79:2532, 1992[Abstract/Free Full Text]
10.
Nistico A, Young NS:
Gamma-interferon gene expression in the bone marrow of patients with aplastic anemia.
Ann Intern Med
120:463, 1994[Abstract/Free Full Text]
11.
Viale M, Merli A, Bacigalupo A:
Analysis at the clonal level of T-cell phenotype and functions in severe aplastic anemia patients.
Blood
78:1268, 1991[Abstract/Free Full Text]
12.
Zoumbos NC, Gascon P, Djeu JY, Trost SR, Young NS:
Circulating activated suppressor T lymphocytes in aplastic anemia.
N Engl J Med
312:257, 1985[Abstract]
13.
Maciejewski JP, Hibbs JR, Anderson S, Katevas P, Young NS:
Bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure.
Exp Hematol
22:1102, 1994[Medline]
[Order article via Infotrieve]
14.
Nakao S, Takamatsu H, Yachie A, Itoh T, Yamaguchi M, Ueda M, Shiobara S, Matsuda T:
Establishment of a CD4+ T cell clone recognizing autologous hematopoietic progenitor cells from a patient with immune-mediated aplastic anemia.
Exp Hematol
23:433, 1995[Medline]
[Order article via Infotrieve]
15.
Nakao S, Takami A, Takamatsu H, Zeng W, Sugimori N, Yamazaki H, Miura M, Ueda M, Shiobara S, Yoshioka T, Kaneshige T, Yasukawa M, Matsuda T:
Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia.
Blood
89:3691, 1997[Abstract/Free Full Text]
16.
Oksenberg JR, Panzara MA, Begovich AB, Mitchell D, Erlich HA, Murray RS, Shimonkevitz R, Sherritt M, Rothbard J, Bernard CC, Steinman L:
Selection for T-cell receptor V -D -J gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis.
Nature
362:68, 1993[Medline]
[Order article via Infotrieve]
17.
Allegretta M, Albertini RJ, Howell MD, Smith LR, Martin R, McFarland HF, Sriram S, Brostoff S, Steinman L:
Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis.
J Clin Invest
94:105, 1994
18.
Musette P, Bequet D, Delarbre C, Gachelin G, Kourilsky P, Dormont D:
Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.
Proc Natl Acad Sci USA
93:12461, 1996[Abstract/Free Full Text]
19.
Goronzy JJ, Bartz BP, Hu W, Jendro MC, Walser KD, Weyand CM:
Dominant clonotypes in the repertoire of peripheral CD4+ T cells in rheumatoid arthritis.
J Clin Invest
94:2068, 1994
20.
Li Y, Sun GR, Tumang JR, Crow MK, Friedman SM:
CDR3 sequence motifs shared by oligoclonal rheumatoid arthritis synovial T cells. Evidence for an antigen-driven response.
J Clin Invest
94:2525, 1994
21.
Pannetier C, Even J, Kourilsky P:
T-cell repertoire diversity and clonal expansions in normal and clinical samples.
Immunol Today
16:176, 1995[Medline]
[Order article via Infotrieve]
22.
Mantegazza R, Andreetta F, Bernasconi P, Baggi F, Oksenberg JR, Simoncini O, Mora M, Cornelio F, Steinman L:
Analysis of T cell receptor repertoire of muscle-infiltrating T lymphocytes in polymyositis. Restricted V  / rearrangements may indicate antigen-driven selection.
J Clin Invest
91:2880, 1993
23.
Nakao S, Takamatsu H, Chuhjo T, Ueda M, Shiobara S, Matsuda T, Kaneshige T, Mizoguchi H:
Identification of a specific HLA class II haplotype strongly associated with susceptibility to cyclosporine-dependent aplastic anemia.
Blood
84:4257, 1994[Abstract/Free Full Text]
24.
Osman I, Meral B, Onder A, Muhit O, Haluk K, Hamdi A, Gunhan G, Nahide K, Akin U:
HLA DR2: A predictive marker in response to cyclosporine therapy in aplastic anemia.
Int J Hematol
66:291, 1997[Medline]
[Order article via Infotrieve]
25.
Chomczynski P, Sacchi N:
Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
26.
Gorski J, Yassai M, Zhu X, Kissela B, Kissella B, Keever C, Flomenberg N:
Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.
J Immunol
152:5109, 1994[Abstract]
27.
Pannetier C, Cochet M, Darche S, Casrouge A, Zoller M, Kourilsky P:
The size of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germline segments.
Proc Natl Acad Sci USA
90:4319, 1993[Abstract/Free Full Text]
28.
Choi Y, Kotzin B, Herron L, Callahan J, Marrack P, Kappler J:
Interaction of Staphylococcus aureus toxin "superantigens" with human T cells.
Proc Natl Acad Sci USA
86:8941, 1989[Abstract/Free Full Text]
29.
Labrecque N, McGrath H, Subramanyam M, Huber BT, Sekaly RP:
Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features.
J Exp Med
177:1735, 1993[Abstract/Free Full Text]
30.
Jeffrey RC, Harold D, Karyl SB, Patricia JK, Mary AR:
Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity.
Hum Immunol
48:39, 1996[Medline]
[Order article via Infotrieve]
31.
Regnault A, Cumano A, Vassalli P, Guy-Grand D, Kourilsky P:
Oligoclonal repertoire of the CD8 and the CD8 TCR-/ murine intestinal intraepithelial T lymphocytes: Evidence for the random emergence of T cells.
J Exp Med
180:1345, 1994[Abstract/Free Full Text]
32.
Yamamoto K, Sakoda H, Nakajima T, Kato T, Okubo M, Dohi M, Mizushima Y, Ito M, Nishioka K:
Accumulation of multiple T cell clonotypes in the synovial lesions of patients with rheumatoid arthritis revealed by a novel clonality analysis.
Int Immunol
4:1219, 1992[Abstract/Free Full Text]
33.
Melenhorst JJ, Fibbe WE, Struyk L, van der Elsen, Willemze R, Landegent JE:
Analysis of T-cell clonality in bone marrow of patients with acquired aplastic anaemia.
Br J Haematol
96:85, 1997[Medline]
[Order article via Infotrieve]
34.
Manz CY, Dietrich PY, Schnuriger V, Nissen C, Wodnar-Filipowicz A:
T-cell receptor chain variability in bone marrow and peripheral blood in severe acquired aplastic anemia.
Blood Cells Mol Dis
23:110, 1997[Medline]
[Order article via Infotrieve]
35.
Binder D, van den Broek MF, Kagi D, Bluethmann H, Fehr J, Hengartner H, Zinkernagel RM:
Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus.
J Exp Med
187:1903, 1998[Abstract/Free Full Text]
36.
Kawano Y, Nissen C, Gratwohl A, Speck B:
Immunostimulatory effects of different antilymphocyte globulin preparations: A possible clue to their clinical effect.
Br J Haematol
68:115, 1988[Medline]
[Order article via Infotrieve]
37.
Morel PA, Livingstone AM, Fathman CG:
Correlation of T-cell receptor V gene family with MHC restriction.
J Exp Med
166:583, 1987[Abstract/Free Full Text]
38.
Schwab R, Szabo P, Manavalan JS, Welksler ME, Posnett DN, Pannetier C, Kourilsky P, Even J:
Expended CD4+ and CD8+ T cell clones in elderly humans.
J Immunol
158:4493, 1997[Abstract]
39.
Forman JD, Klein JT, Silver RF, Liu MC, Greenlee BM, Moller DR:
Selective activation and accumulation of oligoclonal V -specific T cells in active pulmonary sarcoidosis.
J Clin Invest
94:1533, 1994
40.
Silins SL, Cross SM, Elliott SL, Pye SJ, Burrows SR, Burrows JM, Moss DJ, Argaet VP, Misko IS:
Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in infectious mononucleosis.
J Exp Med
184:1815, 1996[Abstract/Free Full Text]
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|
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ABSTRACT
INTRODUCTION