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) β-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.

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,19,20the 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) β 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.16,18,22If 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.

Table 1.

Patient Characteristics

Patient No.  Age (yr)/ Sex  Diagnosis Response to Therapy  CyA Dependency  HLA-DRB1 Allele  
1  60/F  MAA  CyA  −  1302/1502  
46/M  SAA  CyA  −  0101405  
3  58/M  SAA CyA  −  1502801  
4  78/M  SAA  CyA  − 0405601  
5  65/M  MAA  CyA  +  1501405  
17/M  SAA  CyA  +  1501/1405  
7  43/M  SAA CyA  +  1501/1302  
8  32/M  MAA  CyA  1501/1501  
9  60/F  MAA  CyA  +  1501/1201  
10 49/F  SAA  ATG  −  1401/1502  
11  65/F  MAA ATG  −  1502/1402  
12  63/F  SAA  ATG  − 1502802  
13  20/M  SAA  ATG  −  0801405 
14  23/M  SAA  —  −  1301803  
15 61/F  SAA  —  −  0405405  
16  24/F MAA  —  −  1502/1301  
17  19/M  MAA —  −  1501/1201  
18  15/F  SAA —  −  1502405 
Patient No.  Age (yr)/ Sex  Diagnosis Response to Therapy  CyA Dependency  HLA-DRB1 Allele  
1  60/F  MAA  CyA  −  1302/1502  
46/M  SAA  CyA  −  0101405  
3  58/M  SAA CyA  −  1502801  
4  78/M  SAA  CyA  − 0405601  
5  65/M  MAA  CyA  +  1501405  
17/M  SAA  CyA  +  1501/1405  
7  43/M  SAA CyA  +  1501/1302  
8  32/M  MAA  CyA  1501/1501  
9  60/F  MAA  CyA  +  1501/1201  
10 49/F  SAA  ATG  −  1401/1502  
11  65/F  MAA ATG  −  1502/1402  
12  63/F  SAA  ATG  − 1502802  
13  20/M  SAA  ATG  −  0801405 
14  23/M  SAA  —  −  1301803  
15 61/F  SAA  —  −  0405405  
16  24/F MAA  —  −  1502/1301  
17  19/M  MAA —  −  1501/1201  
18  15/F  SAA —  −  1502405 

Abbreviations: SAA, severe AA; MAA, moderate AA.

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’st-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). Figure3 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). Table2 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.

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.

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.

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.

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.

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.

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.

Table 2.

BV Subfamilies Showing Abnormal CDR3 Size Pattern

Patient No.  BV No. With Abnormal CDR3 Size Pattern  BV Subfamilies With CDR3 Abnormalities (%)  
1  —  
2  8  4.2  
3  —  0  
4  15, 22, 24  12.5 
5  1, 4, 5S1, 6, 8, 13S1, 13S2, 14, 15, 16, 17, 21  50.0 
6  5S2, 7, 11, 12, 13S1, 13S2, 14, 15, 20, 21, 22  45.8  
1, 6, 7, 13S1, 13S2, 14, 15, 21, 22, 23, 24  45.8  
8  1, 2, 3, 4, 5S2, 7, 8, 9, 13S1, 14, 15, 16, 17, 18, 21, 22, 23, 24 75.0  
9  1, 2, 5S2, 11, 12, 13S1, 15, 16, 20, 24  41.7 
10  14, 22, 23  12.5  
11  3, 7, 21  12.5  
12 9, 11, 22, 23, 24  20.8  
13  3, 5S1, 7, 14, 21  20.8 
14  —  0  
15  8, 9, 24  12.5  
16  5S2  4.2 
17  7, 21, 23, 24  16.7  
18  6, 7, 15, 23 16.7 
Patient No.  BV No. With Abnormal CDR3 Size Pattern  BV Subfamilies With CDR3 Abnormalities (%)  
1  —  
2  8  4.2  
3  —  0  
4  15, 22, 24  12.5 
5  1, 4, 5S1, 6, 8, 13S1, 13S2, 14, 15, 16, 17, 21  50.0 
6  5S2, 7, 11, 12, 13S1, 13S2, 14, 15, 20, 21, 22  45.8  
1, 6, 7, 13S1, 13S2, 14, 15, 21, 22, 23, 24  45.8  
8  1, 2, 3, 4, 5S2, 7, 8, 9, 13S1, 14, 15, 16, 17, 18, 21, 22, 23, 24 75.0  
9  1, 2, 5S2, 11, 12, 13S1, 15, 16, 20, 24  41.7 
10  14, 22, 23  12.5  
11  3, 7, 21  12.5  
12 9, 11, 22, 23, 24  20.8  
13  3, 5S1, 7, 14, 21  20.8 
14  —  0  
15  8, 9, 24  12.5  
16  5S2  4.2 
17  7, 21, 23, 24  16.7  
18  6, 7, 15, 23 16.7 
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.

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.

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.

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 (Fig5A). In contrast, skewed CDR3 size patterns of several BV families of patient no. 8 persisted after 7 years (Fig5B) despite the fact that the patient was in remission with 2 mg/kg/d CyA.

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.

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.

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 and4 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.

Table 3.

Junctional Amino Acid Sequences of TCR BV15 From Normal Individuals With HLA-DRB1*1501

Subject No. 
BV15  N-D-N BJ   Frequency  
Normal 1   
 CATS  DAGGD QPQHFGDGTRLSIL  1S5  4/30  
 CATS  DLAGFH NEQFFGPGTRLTV  2S1  2/30  
 CATS  DSKRTS GETYEQYFGPGTRLTVT  2S5  2/30  
 CATS  DYRS GEQYFGPGTRLTVT  2S5  1/30  
 CATS  GLAGS QETQYFGPGTRLLVL  2S5  1/30  
 CATS  DMTGPA YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DFKGD NEQFFGPGTRLTVL  2S1  1/30  
 CATS  VAGGD EQFFGPGTRLTVL  2S1  1/30  
 CATS  TDRGH GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  VRVG DEQFFGPGTRLTVL  2S1  1/30  
 CAT  LELAGS YNEQFFGPGTRLTVL  2S1  1/30  
 CAT  RR TGLDFGAGTRLSVL   1/30  
 CATS  DPTSGI YNEQFFGPGTRLTVL  2S1  1/30  
 CAT  RGTGS NTEAFFGQGTRLTVV  1S1  1/30  
 CATS  DRTS GANVLTFGAGSRLTVL  2S6  1/30  
 CATS  TRHAG TGELFFGEGSRLTVL  2S2  1/30  
 CATS  DGHVG QETQYFGPGTRLLVL  2S5  1/30  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/30  
 CATS  DGVS GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  VHAGS QETQYFGPGTRLLVL  2S5  1/30  
 CATS  GPH NTEAFFGQGTRLTVV  1S1  1/30  
 CATS  RSVG NEQFFGPGTRLTVL  2S1  1/30  
 CATS  DHVSLE GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  QYLAGS TGELFFGEGSRLTVL  2S2  1/30  
 CATS  APSHG YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  EWKDHSA YNEQFFGPGTRLTVL  2S1  1/30  
Normal 2  
 CATS RAKG  ETQYFGPGTRKKVL  2S5  2/26  
 CATS  AEGSG TQYFGPGTRLTVL  2S3  2/26  
 CATS  AEAGSPS GANVLTFGAGSRLTVL  2S6  2/26  
 CATS  DPDR YQETQYFGPGTRLLVL  2S5  2/26  
 CATS  EAGID GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/26  
 CATS  IPNQ ALQPHFGDGTRLSIL  1S5  1/26  
 CATS  DYGTGG YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  YFWRIP YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  DHGQGE ETQYFGPGTRLLVL  2S5  1/26  
 CATS  DYRS GEQYFGPGTRLTV  2S5  1/26  
 CATS  DRAG TDTQYFGPGTRLTVL  2S3  1/26  
 CATS  DRAG TDTQYFGPGTRLTVL  2S3  1/26  
 CATS  DSGTSIG TQYFGPGTRLLVL  2S3  1/26  
 CATS  DGGTA EQFFGPGTRLTVL  2S1  1/26  
 CATS  AKGTV ALQPHFGDGTRLSIL  1S5  1/26  
 CATS  RGDTA GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  DKQSG ETQYFGPGTRKKVL  2S5  1/26  
 CATS  VGKAP EQFFGPGTRLTVL  2S1  1/26  
 CATS  DAGSTWS YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  DGSHK GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  SGPG TQYFGPGTRLLVL  2S3  1/26  
 CATS  DLHK TQYFGPGTRLLVL  2S3  1/26  
Normal 3   
 CATS  DGHQ ETQYFGPGTRLLVL  2S5  1/20  
 CAT  GATG TDTQYFGPGTRLTVL  2S3  1/20  
 CATS  DNRAS DTQYFGPGTRLTVL  2S3  1/20  
 CATS  EGSY YGYTFGSGTRLTVL   1/20  
 CATS  DPRPGG TDTQYFGPGTRLTVL  2S3  1/20  
 CATS  DSVSS YNEQFFGPGTRL  2S1  1/20  
 CATS  DPYH  EQFFGPGTRLTVL 2S1  1/20  
 CATS  DIGT  TDTQYFGPGTRLTVL  2S3 1/20  
 CATS  LGASTS  YNEQFFGPGTRLTVL  2S1  1/20 
 CATS  DWVGMRSGKP  NEQFFGPGTRLTVL  2S1  1/20 
 CATS  DLF  EAFFGQGTRLTVV  1S1  1/20  
 CATS DPPG  NEQFFGPGTRLTVL  2S1  1/20  
 CAT  RARQGGS EQFFGPGTRLTVL  2S1  1/20  
 CAT  PRTSGAPT EQFFGPGTRLTVL  2S1  1/20  
 CATS  DSRHTSGS EQFFGPGTRLTVL  2S1  1/20  
 CATS  DGQGAHDA EAFFGQGTRLTVV  1S1  1/20  
 CATS  DAGGD QPQHFGDGTRLSIL  1S5  1/20  
 CATS  EPTGNPN QPQGFGDGTRLSIL  1S5  1/20  
 CATS  DTGTG TDTQYGPGTRLTVL  2S3  1/20  
 CATS  GRDWSS YNEQFFGPGTRLTVL  2S1  1/20 
Subject No. 
BV15  N-D-N BJ   Frequency  
Normal 1   
 CATS  DAGGD QPQHFGDGTRLSIL  1S5  4/30  
 CATS  DLAGFH NEQFFGPGTRLTV  2S1  2/30  
 CATS  DSKRTS GETYEQYFGPGTRLTVT  2S5  2/30  
 CATS  DYRS GEQYFGPGTRLTVT  2S5  1/30  
 CATS  GLAGS QETQYFGPGTRLLVL  2S5  1/30  
 CATS  DMTGPA YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DFKGD NEQFFGPGTRLTVL  2S1  1/30  
 CATS  VAGGD EQFFGPGTRLTVL  2S1  1/30  
 CATS  TDRGH GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  VRVG DEQFFGPGTRLTVL  2S1  1/30  
 CAT  LELAGS YNEQFFGPGTRLTVL  2S1  1/30  
 CAT  RR TGLDFGAGTRLSVL   1/30  
 CATS  DPTSGI YNEQFFGPGTRLTVL  2S1  1/30  
 CAT  RGTGS NTEAFFGQGTRLTVV  1S1  1/30  
 CATS  DRTS GANVLTFGAGSRLTVL  2S6  1/30  
 CATS  TRHAG TGELFFGEGSRLTVL  2S2  1/30  
 CATS  DGHVG QETQYFGPGTRLLVL  2S5  1/30  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/30  
 CATS  DGVS GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  VHAGS QETQYFGPGTRLLVL  2S5  1/30  
 CATS  GPH NTEAFFGQGTRLTVV  1S1  1/30  
 CATS  RSVG NEQFFGPGTRLTVL  2S1  1/30  
 CATS  DHVSLE GDTQYFGPGTRLTVL  2S3  1/30  
 CATS  QYLAGS TGELFFGEGSRLTVL  2S2  1/30  
 CATS  APSHG YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  EWKDHSA YNEQFFGPGTRLTVL  2S1  1/30  
Normal 2  
 CATS RAKG  ETQYFGPGTRKKVL  2S5  2/26  
 CATS  AEGSG TQYFGPGTRLTVL  2S3  2/26  
 CATS  AEAGSPS GANVLTFGAGSRLTVL  2S6  2/26  
 CATS  DPDR YQETQYFGPGTRLLVL  2S5  2/26  
 CATS  EAGID GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/26  
 CATS  IPNQ ALQPHFGDGTRLSIL  1S5  1/26  
 CATS  DYGTGG YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  YFWRIP YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  DHGQGE ETQYFGPGTRLLVL  2S5  1/26  
 CATS  DYRS GEQYFGPGTRLTV  2S5  1/26  
 CATS  DRAG TDTQYFGPGTRLTVL  2S3  1/26  
 CATS  DRAG TDTQYFGPGTRLTVL  2S3  1/26  
 CATS  DSGTSIG TQYFGPGTRLLVL  2S3  1/26  
 CATS  DGGTA EQFFGPGTRLTVL  2S1  1/26  
 CATS  AKGTV ALQPHFGDGTRLSIL  1S5  1/26  
 CATS  RGDTA GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  DKQSG ETQYFGPGTRKKVL  2S5  1/26  
 CATS  VGKAP EQFFGPGTRLTVL  2S1  1/26  
 CATS  DAGSTWS YNEQFFGPGTRLTVL  2S1  1/26  
 CATS  DGSHK GANVLTFGAGSRLTVL  2S6  1/26  
 CATS  SGPG TQYFGPGTRLLVL  2S3  1/26  
 CATS  DLHK TQYFGPGTRLLVL  2S3  1/26  
Normal 3   
 CATS  DGHQ ETQYFGPGTRLLVL  2S5  1/20  
 CAT  GATG TDTQYFGPGTRLTVL  2S3  1/20  
 CATS  DNRAS DTQYFGPGTRLTVL  2S3  1/20  
 CATS  EGSY YGYTFGSGTRLTVL   1/20  
 CATS  DPRPGG TDTQYFGPGTRLTVL  2S3  1/20  
 CATS  DSVSS YNEQFFGPGTRL  2S1  1/20  
 CATS  DPYH  EQFFGPGTRLTVL 2S1  1/20  
 CATS  DIGT  TDTQYFGPGTRLTVL  2S3 1/20  
 CATS  LGASTS  YNEQFFGPGTRLTVL  2S1  1/20 
 CATS  DWVGMRSGKP  NEQFFGPGTRLTVL  2S1  1/20 
 CATS  DLF  EAFFGQGTRLTVV  1S1  1/20  
 CATS DPPG  NEQFFGPGTRLTVL  2S1  1/20  
 CAT  RARQGGS EQFFGPGTRLTVL  2S1  1/20  
 CAT  PRTSGAPT EQFFGPGTRLTVL  2S1  1/20  
 CATS  DSRHTSGS EQFFGPGTRLTVL  2S1  1/20  
 CATS  DGQGAHDA EAFFGQGTRLTVV  1S1  1/20  
 CATS  DAGGD QPQHFGDGTRLSIL  1S5  1/20  
 CATS  EPTGNPN QPQGFGDGTRLSIL  1S5  1/20  
 CATS  DTGTG TDTQYGPGTRLTVL  2S3  1/20  
 CATS  GRDWSS YNEQFFGPGTRLTVL  2S1  1/20 
Table 4.

Junctional Amino Acid Sequences of TCR BV15 From CyA-Dependent AA Patients

Subject No. 
BV15 N-D-N BJ   Frequency  
Patient 5   
 CATS  DLTSGP NTGELFFGEGSRLTVL  2S2  21/30  
 CATS  DLRLAG YTDTQYEGPGTRLTVL  2S3  3/30  
 CATS  DPDR YQETQYFGPGTRLLVL  2S5  3/30  
 CATS  AAGGT YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DRPDS EQFFGPGTRLTVL  2S1  1/30  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/30  
Patient 6   
 CATG  GSP QETQYFGPGTRLLVL  2S5  14/38  
 CATS  EPTGAN TGELFFGEGSRLTVL  2S2  3/38  
 CATS  EAGID GANVLTFGAGSRLTVL  2S6  3/38  
 CATS  DAGTSG ETQYFGPGTRLLVL  2S5  2/38  
 CAA  QGAPH QPQHFGDGTRLSIL  1S5  2/38  
 CATT  ESRSG TDTQYFGPGTRLTVL  2S3  2/38  
 CATS  DRQ TGELFFGEGSRLTVL  2S2  2/38  
 CATS  DLELY TGELFFGEGSRLTVL  2S2  2/38  
 CATS  DQRDL GELFFGEGSRLTVL  2S2  1/38  
 CATS  DPGIL YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  DPSGA YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  DRLAEA YNEQFFGPGTRLTVL  2S1  1/38  
 CATR  QGHN EQFFGPGTRLTVL  2S1  1/38  
 CATS  DPGIL YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  AQTGS YNEQFFGPGSRLTVL  2S1  1/38  
 CATS  GPLS QETQYFGPGTRLLVL  2S5  1/38  
Patient 7   
 CATS PRDRR  NTGELFLGEGSRLTVL  2S2  18/30  
 CATS  RGGTSV NTGELFFGEGSRLTVL  2S2  2/30  
 CATS  DTTSG YNEQFFGPGTRLTVL  2S1  2/30  
 CA  TRGRGP TGELFFGEGSRLTVL  2S2  2/30  
 CATS  DPGRP YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  GPGG QETQYFGPGTRLLVL  2S5  1/30  
 CATS  NPGA TDTQYFGPGTRLTVL  2S3  1/30  
 CATS  DSPGLGR YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DSQGSA DTQYFGPGTRLTVL  2S3  1/30  
 CATS  SPPS YNEQFFGPGTRLTVL  2S1  1/30  
Patient 8   
 CATS DLTNGP  NTGELFFGEGSRLTVL  2S2  7/40  
 CATS  DRTWS EQFFGPGTRLTVL  2S1  6/40  
 CAT  D  SYNEQFFGPGTRLTVL 2S1  5/40  
 CATS  DPS  STDTQYFGPGTRLTVL  2S3 5/40  
 CATS  DFSA  STDTQYFGPGTRLTVL  2S3  4/40 
 CATS  PGG  SYEQYFGPGTRLTVT  2S7  3/40  
 CATS TG  SYEQYFGPGTRLTVT  2S7  2/40  
 CATS  DFTGRGH NEQFFGPGTRLTVL  2S1  1/40  
 CATS  DSW NEQFFGPGTRLTVL  2S1  1/40  
 CATS  DMDTRTG NTEAFFGQGTRLTVV  1S1  1/40  
 CATS  DMDTG NTEAFFGQGTRLTVV  1S1  1/40  
 CATS  DLQFG QTETQYFGPGTRLLVL  2S5  1/40  
 CATS  DPGTGY NQPQHFGPGTRLSIL  1S5  1/40  
 CATS  DAGVE EQFFGPGTRLTVL  2S1  1/40  
 CATS  DRPRTG NEQFFGPGTRLTVL  2S1  1/40  
Patient 9   
 CATS DESY  EQFFGPGTRLTVL  2S1  7/18  
 CATS  DLRES NEQFFGPGTRLTVL  2S1  4/18  
 CATS  VAGSG ETQYFGPGTRLLVL  2S5  3/18  
 CATS  DAGTSG ETQYFGPGTRLLVL  2S5  2/18  
 CATS  GEG SDTQYFGPGTRLTVL  2S3  2/18 
Subject No. 
BV15 N-D-N BJ   Frequency  
Patient 5   
 CATS  DLTSGP NTGELFFGEGSRLTVL  2S2  21/30  
 CATS  DLRLAG YTDTQYEGPGTRLTVL  2S3  3/30  
 CATS  DPDR YQETQYFGPGTRLLVL  2S5  3/30  
 CATS  AAGGT YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DRPDS EQFFGPGTRLTVL  2S1  1/30  
 CATS  DASGRI EQFFGPGTRLTVL  2S1  1/30  
Patient 6   
 CATG  GSP QETQYFGPGTRLLVL  2S5  14/38  
 CATS  EPTGAN TGELFFGEGSRLTVL  2S2  3/38  
 CATS  EAGID GANVLTFGAGSRLTVL  2S6  3/38  
 CATS  DAGTSG ETQYFGPGTRLLVL  2S5  2/38  
 CAA  QGAPH QPQHFGDGTRLSIL  1S5  2/38  
 CATT  ESRSG TDTQYFGPGTRLTVL  2S3  2/38  
 CATS  DRQ TGELFFGEGSRLTVL  2S2  2/38  
 CATS  DLELY TGELFFGEGSRLTVL  2S2  2/38  
 CATS  DQRDL GELFFGEGSRLTVL  2S2  1/38  
 CATS  DPGIL YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  DPSGA YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  DRLAEA YNEQFFGPGTRLTVL  2S1  1/38  
 CATR  QGHN EQFFGPGTRLTVL  2S1  1/38  
 CATS  DPGIL YNEQFFGPGTRLTVL  2S1  1/38  
 CATS  AQTGS YNEQFFGPGSRLTVL  2S1  1/38  
 CATS  GPLS QETQYFGPGTRLLVL  2S5  1/38  
Patient 7   
 CATS PRDRR  NTGELFLGEGSRLTVL  2S2  18/30  
 CATS  RGGTSV NTGELFFGEGSRLTVL  2S2  2/30  
 CATS  DTTSG YNEQFFGPGTRLTVL  2S1  2/30  
 CA  TRGRGP TGELFFGEGSRLTVL  2S2  2/30  
 CATS  DPGRP YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  GPGG QETQYFGPGTRLLVL  2S5  1/30  
 CATS  NPGA TDTQYFGPGTRLTVL  2S3  1/30  
 CATS  DSPGLGR YNEQFFGPGTRLTVL  2S1  1/30  
 CATS  DSQGSA DTQYFGPGTRLTVL  2S3  1/30  
 CATS  SPPS YNEQFFGPGTRLTVL  2S1  1/30  
Patient 8   
 CATS DLTNGP  NTGELFFGEGSRLTVL  2S2  7/40  
 CATS  DRTWS EQFFGPGTRLTVL  2S1  6/40  
 CAT  D  SYNEQFFGPGTRLTVL 2S1  5/40  
 CATS  DPS  STDTQYFGPGTRLTVL  2S3 5/40  
 CATS  DFSA  STDTQYFGPGTRLTVL  2S3  4/40 
 CATS  PGG  SYEQYFGPGTRLTVT  2S7  3/40  
 CATS TG  SYEQYFGPGTRLTVT  2S7  2/40  
 CATS  DFTGRGH NEQFFGPGTRLTVL  2S1  1/40  
 CATS  DSW NEQFFGPGTRLTVL  2S1  1/40  
 CATS  DMDTRTG NTEAFFGQGTRLTVV  1S1  1/40  
 CATS  DMDTG NTEAFFGQGTRLTVV  1S1  1/40  
 CATS  DLQFG QTETQYFGPGTRLLVL  2S5  1/40  
 CATS  DPGTGY NQPQHFGPGTRLSIL  1S5  1/40  
 CATS  DAGVE EQFFGPGTRLTVL  2S1  1/40  
 CATS  DRPRTG NEQFFGPGTRLTVL  2S1  1/40  
Patient 9   
 CATS DESY  EQFFGPGTRLTVL  2S1  7/18  
 CATS  DLRES NEQFFGPGTRLTVL  2S1  4/18  
 CATS  VAGSG ETQYFGPGTRLLVL  2S5  3/18  
 CATS  DAGTSG ETQYFGPGTRLLVL  2S5  2/18  
 CATS  GEG SDTQYFGPGTRLTVL  2S3  2/18 
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).

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.

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.

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.34documenting 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 gamma7,35and 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.37The 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 β 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.

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.

REFERENCES

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
1995
1348
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
1995
3058
3
Young
NS
Pathophysiology II: Immune suppression of hematopoiesis
Aplastic Anemia Acquired and Inherited.
Young
NS
Alter
BP
1994
68
Saunders
Philadelphia, PA
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
1976
2890
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
1977
10
6
Ascensao
J
Pahaw
R
Kagan
W
Hansen
J
Moore
MAS
Good
R
Aplastic anemia: Evidence for an immunological mechanism.
Lancet
1
1976
669
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
1985
188
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
1988
266
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
1992
2532
10
Nistico
A
Young
NS
Gamma-interferon gene expression in the bone marrow of patients with aplastic anemia.
Ann Intern Med
120
1994
463
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
1991
1268
12
Zoumbos
NC
Gascon
P
Djeu
JY
Trost
SR
Young
NS
Circulating activated suppressor T lymphocytes in aplastic anemia.
N Engl J Med
312
1985
257
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
1994
1102
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
1995
433
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
1997
3691
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 β-D β-J β gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis.
Nature
362
1993
68
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
1994
105
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
1996
12461
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
1994
2068
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
1994
2525
21
Pannetier
C
Even
J
Kourilsky
P
T-cell repertoire diversity and clonal expansions in normal and clinical samples.
Immunol Today
16
1995
176
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 α/β rearrangements may indicate antigen-driven selection.
J Clin Invest
91
1993
2880
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
1994
4257
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
1997
291
25
Chomczynski
P
Sacchi
N
Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction.
Anal Biochem
162
1987
156
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
1994
5109
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
1993
4319
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
1989
8941
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
1993
1735
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
1996
39
31
Regnault
A
Cumano
A
Vassalli
P
Guy-Grand
D
Kourilsky
P
Oligoclonal repertoire of the CD8αα and the CD8αβ TCR-α/β murine intestinal intraepithelial T lymphocytes: Evidence for the random emergence of T cells.
J Exp Med
180
1994
1345
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
1992
1219
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
1997
85
34
Manz
CY
Dietrich
PY
Schnuriger
V
Nissen
C
Wodnar-Filipowicz
A
T-cell receptor β chain variability in bone marrow and peripheral blood in severe acquired aplastic anemia.
Blood Cells Mol Dis
23
1997
110
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
1998
1903
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
1988
115
37
Morel
PA
Livingstone
AM
Fathman
CG
Correlation of T-cell receptor Vβ gene family with MHC restriction.
J Exp Med
166
1987
583
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
1997
4493
39
Forman
JD
Klein
JT
Silver
RF
Liu
MC
Greenlee
BM
Moller
DR
Selective activation and accumulation of oligoclonal V β-specific T cells in active pulmonary sarcoidosis.
J Clin Invest
94
1994
1533
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
1996
1815

Author notes

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@med.kanazawa-u.ac.jp.