T-cell Receptor β Chain Variability in Bone Marrow and Peripheral Blood in Severe Acquired Aplastic Anemia

Blood Cells, Molecules, and Diseases (1997) 23(7) April 15: 110–122 Article No. MD970127

Chantal Y. Manz, et al.

T-cell Receptor b Chain Variability in Bone Marrow and Peripheral Blood in Severe Acquired Aplastic Anemia Submitted 03/13/97 (communicated by Ernest Beutler, M.D., 04/09/97)

Chantal Y. Manz1, Pierre-Yves Dietrich2, Vale´rie Schnuriger2, Catherine Nissen1, Aleksandra Wodnar-Filipowicz1 ABSTRACT: Aplastic anemia (AA) is characterized by multilineage bone marrow failure of unknown etiology. In order to assess the role of immune-mediated mechanisms in hematopoietic suppression, we examined the diversity of T lymphocyte repertoire in terms of variable (V) gene segment usage of the T cell receptor (TCR) b chain in bone marrow and peripheral blood of six patients with severe untreated AA. Expression of transcripts encoding Vb1-Vb24 subfamilies was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that T lymphocytes in AA utilize highly diverse segments of the b chain loci. Over the heterogenous Vb expression background, transcripts encoding Vb3, Vb20, Vb21, and Vb22 subfamilies were enhanced by at least threefold in 5 of 6 patients as compared to normal samples, but a different transcript species was over expressed in each patient. To evaluate clonality of T cells, size diversity within the complementarity determining region 3 (CDR3) and usage of TCRb joining (J) gene segments were analyzed in PCR products specific for each of the 24 Vb subfamilies. We found that the majority of transcripts display normal CDR3 size patterns, as is characteristic of polyclonal populations. Nevertheless, one or two predominating junctional rearrangements were observed in each patient. They were identified in Vb5, Vb7, Vb8, Vb13, Vb15, Vb16, and Vb23 transcripts, which differed from patient to patient and did not correspond to transcripts with an abnormally high expression level. Our results demonstrate that T cell repertoire in AA is random with respect to the TCR b chain. Unique rearrangements detected in the CDR3 region are suggestive of a limited process of an antigen-driven (oligo)clonal T cell expansion which may take place over the overwhelmingly polyclonal repertoire of T lymphocytes at the onset of severe AA. Keywords: aplastic anemia, T cell receptor, Vb chain, complementarity determining region 3

DR1 T cells have been found to infiltrate aplastic bone marrow and to decrease in parallel with hematopoietic improvement after immunosuppressive treatment (3). Overexpression and overproduction of IFNg and other hematotoxic cytokines by T cells has been described in some patients (4-6) and found to be predictive of responsiveness to immunosuppression (7). T lymphocytes capable of inhibiting growth of hemato-

INTRODUCTION Immune phenomena are implicated in bone marrow failure in aplastic anemia (AA). Clinical responsiveness to immunosuppressive therapy (1, 2) as the most convincing argument, has been supported by numerous in vitro data incriminating T lymphocytes as offenders destroying hematopoietic precursors. Activated cytotoxic CD81HLA-

1 Department

of Research, University Hospital Basel, Hebelstrasse 20, 4031 Basel Switzerland; of Oncology, Cantonal University Hospital, Rue Micheli-du-Crest 24, 1211 Geneva, Switzerland. Reprint request to: Aleksandra Wodnar-Filipowicz, Department of Research, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland, phone 41-61-2652333, fax 41-61-2652350, e-mail: [email protected] 2 Division

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1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.

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experimentally induced autoimmune diseases (19, 20) and prompted the search for non-random TCRVb distrubution in clinical samples. Selective usage of Vb gene segments or recurrence of unique TCR sequences was found in insulindependent diabetes mellitus type I (21), multiple sclerosis (22-24), rheumatoid arthritis (25, 26), graft-versus-host disease (27), and other disorders with an immune or autoimmune background (2830), and have been attributed to the pathogenesis of these diseases. The purpose of this study was to characterize TCRVb gene usage in AA as indirect means of identifying potentially pathogenic T cell responses associated with bone marrow failure. The analysis was performed using bone marrow and peripheral blood cells from patients with acute untreated severe disease and healthy donors as controls. We determined the expression level of 24 Vb subfamilies and assessed clonality of T cells by analysis of the CDR3 size diversity in TCRVb transcripts, applying the newly established PCRbased high resolution method, termed ‘‘Immunoscope’’ technique (31). Results are suggestive of a limited degree of (oligo)clonal T cell expansion in an overwhelmingly polyclonal T cell population in AA.

poietic progenitor cells have been identified in peripheral blood and bone marrow of some patients with AA (8-11). Furthermore, the established association of AA with the HLA-DR2 allele (12, 13) suggests that T cell responses in AA are MHC-restricted. The etiology of AA remains unknown (14). Potential target antigens of viral or bacterial origin, or autoantigens expressed by hematopoietic precursors remain unidentified. T cells infiltrating aplastic bone marrow are expected to be enriched in the antigen-specific invasive cells. However, the phenotypic and functional abnormalities of T cells is not common to all patients, their frequency varying widely in individual studies, and evidence for an antigen-driven expansion of potentially autoaggressive T cell populations as a general mechanism in the pathogenesis of AA has yet to be provided. The T cell receptor (TCR) is responsible for antigen recognition by T cells. Most mature T cells interact with antigens through the TCR heterodimer consisting of a and b chains. Diversity of TCR repertoire is the result of somatic recombination events and random nucleotide additions that occur during the joining of variable (V), diverse (D) and joining (J) segments comprising the b-chain, and of V and J segments comprising the a chain. The fine specificity of antigen recognition is carried by the hypervariable junctional regions of V-D-J segments called the complementarity determining region 3 (CDR3) (15, 16). During differentiation, T cells are exposed to a large number of antigens which results in a polyclonal TCR profile. Within pathogenic lesions, the presence of offending foreign- or autoantigens as T cell targets may lead to biased expression of TCR gene segments reflecting preferential expansion of antigen-specific T cells. Characterization of the TCR repertoire is facilitated by PCR technology which is particularly well established for analyzing the expression of the 24 gene segment subfamilies encoding the V region of TCRb chain (17, 18). Restricted usage of TCR Vb genes was initially observed in animal models of

MATERIAL AND METHODS Patients and Controls Six patients and three bone marrow donors were included in the study and gave informed consent. The characteristics of the disease at the time of study (at presentation) and a current status of the patients are summarized in Table 1. All six patients presented with acute previously untreated severe disease and were transfusion-dependent. The disease was idiopathic in five patients and involvement of drug-exposure was suspected in one case. None of the patients showed signs of clonal disorders of hematopoiesis, as demon-

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strated by a negative Ham test and the absence of cytogenetic abnormalities. Patients 3, 4, and 5 underwent allogeneic bone marrow transplantation (BMT) and patients 1, 2, and 6 were treated with antilymphocyte globulin (ALG) and achieved partial or complete remission under continuing immunosuppressive treatment with cyclosporin A (see Table 1).

K] and Cb 38- primers amplifying a 190 bp-long fragment within the C region were used. In reactions serving as negative controls, cDNA was omitted. 30 PCR cycles were performed. Conditions for the first 5 cycles: denaturation at 94°C for 10 seconds, annealing at 60°C and extension at 72°C each for 1 minute. Conditions for the following 25 cycles: denaturation at 92°C for 10 seconds, annealing at 60°C and extension at 72°C each for 30 seconds. One half of each PCR product was separated by 2% agarose gel electrophoresis and transferred onto nylon membranes. The amplified products were detected by hybridization with an internal Cb oligonucleotide probe [(18), primer D] 32P-labeled using (g32P)ATP (10.0 mCi/mL; sp.act. 3000 Ci/mmol; Du Pont NEN, Boston, MA). Blots were exposed to a Phosphor Imager Screen (Bio-Rad, Hercules, CA) for 2 hours. The relative intensity of each Vb signal was determined in Pixel Density Units (PDUnits) with the Phosphor Analyst Software. Autoradiography was performed for not longer than 1 hour at 270°C using Kodak X-ray film and amplifying screens. Each RNA sample has been analyzed at least twice to confirm the reproducibility of the PCR amplification reactions.

Cell Preparation Bone marrow was aspirated in 1-2 mL aliquots to minimize contamination with peripheral blood. Mononuclear cells from bone marrow and peripheral blood were isolated by Ficoll-Hypaque (d51.077; Pharmacia, Uppsala, Sweden) density gradient centrifugation and were processed for RNA without prior freezing.

RNA Isolation and Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) Total cellular RNA was extracted using the guanidine-isothiocyanate method (32). RT-PCR analysis was performed as described previously (18, 33). One µg of RNA was reverse-transcribed into complementary DNA (cDNA) in 20 µL reactions using oligo-dT primer [p(dT)15, 1 A260U; Boehringer Mannheim, Germany] and 200 units Superscripty II RNase H--reverse transcriptase (200 U/µL; GIBCO, Gaithersburg, MD). The RT product was diluted 1:3 in water. One µL of cDNA was amplified with each of the 24 different Vb 58-oligonucleotide primers [(18); for Vb6 (17)] and a common downstream Cb 38-primer [(18), primer F], 0.25 µM each, in PCR reactions containing AmpliTaqy DNA polymerase (0.025 U/µL; Perkin Elmer, Branchburg, New Jersey) and dNTPs (0.2 mM each; Perkin Elmer, ibid) in a final volume of 10 µL. As a control of RNA integrity and concentration, seminested Cb 58- [(18), primer

CDR3 Size Analysis The analysis has been performed according to Pannetier et al. (31, 34). Briefly, cDNA (1 µL) was amplified using 24 different Vb primers and a downstream Cb 38- primer as described above, except that 40 amplification cycles were used and PCR reactions were performed in a total volume of 25 µL. Subsequently, 2 µL aliquots were subjected to 3 cycles of run-off reactions (in a volume of 10 µL) primed with fluorescent-labeled (Applied Biosystems; fluorophor Fam, Foster City, CA) oligonucleotide primers specific for Cb or for each of the 13 Jb regions (35). The run-off products were subjected to electro-phoresis on a ABI sequencer in the pres-

112

113

Platelets (x

no

no

R at 1y

G-CSF

G-CSF R at 2y

BMT, CyA,

6

16

0.12

8.8

SAA / d

Patient 3 (521) 32y / M

ALG, CyA,

36

8

0.30

9.1

SAA / i

Patient 2 (499) 66y / M

no

R at 8m

BMT, CyA

11

45

0.29

7.3

SAA / i

Patient 4 (553) 20y / F

RP

no

at 1y

G-CSF

BMT, CyA, Ig,

3

28

0.17

6.8

SAA / i

Patient 5 (561) 5y / M

no

RP at 6m

G-CSF

ALG, CyA,

28

29

0.98

9.2

SAA / i

Patient 6 (569) 38y / M

UPN, unique patient number; SAA, severe aplastic anemia; y, years; m, months; M, male; F, female; i, idiopathic; d, drug-induced; ALG, antilymphocyte globulin; CyA, cyclosporin A; G-CSF, granulocyte colony-stimulating factor; R, remission; RP, remission with pancytopenia; BMT, bone marrow transplantation; Ig, immunoglobulin; lab PNH, laboratory paroxysmal nocturnal hemoglobinuria.

lab PNH

Development of clonal disorders

at 5y

Current clinical condition

ALG, CyA

71

19

0.72

RP

Treatment and medication

Current hematological status:

Reticulocytes (x 109/L)

109/L)

Neutrophils (x

9.0

Hb (g%)

109/L)

SAA / i

Patient 1 (410) 35y / M

Diagnosis

Hematological status at time of study (at presentation):

Patient No. (UPN) Age / Sex

Table 1. Patient Characteristics

Chantal Y. Manz, et al. Blood Cells, Molecules, and Diseases (1997) 23(7) April 15: 110–122 Article No. MD970127

Blood Cells, Molecules, and Diseases (1997) 23(7) April 15: 110–122 Article No. MD970127

Chantal Y. Manz, et al.

that the expression levels of transcripts in these two compartments were similar. The pattern of Vb expression in severe AA was not significantly different from normal. Nevertheless, individual patients displayed some selectivity in Vb gene usage. For example, bone marrow and peripheral blood from patient 3 contained an increased proportion of Vb3 mRNA, as compared to mean values of normal donors (18.6% vs 4.960.4% and 21.1% vs 6.561.5%, respectively). Overexpression of Vb20, 21, and 22 was observed in other patients, but unlike Vb3, these transcripts belong to the poorly expressed ones and an increase in their levels is difficult to interprete. There were also several transcripts of which the expression level appeared decreased. For example, in the bone marrow of patient 3, the normally abundant Vb8, Vb12, and Vb13 transcripts were significantly reduced (see also Figure1), probably as a direct consequence of the high expression of Vb3. In patient 4, several bone marrow-derived transcripts (Vb7, Vb10, Vb12, Vb18, and Vb24) were barely detectable (see also Figure1). All the differences in Vb expression pattern in samples from all analyzed AA patients are summarized in Table 2, where transcripts are subdivided into two groups according to their increased or decreased expression level differing from normal by at least 3-fold.

ence of fluorescent size markers and analyzed by Gene Scan Analysis Software (Applied Biosystems). Statistical Analysis Expression levels of the Vb1-Vb24 gene subfamilies are presented as the mean 6 SEM of results obtained from multiple separate experiments. Statistical significance of the relative frequency of Vb transcripts in AA as compared to normal samples was determined using the unpaired Student’s t-test. RESULTS Variability of TCR Vb Gene Expression The TCRVb repertoire was examined in peripheral blood and bone marrow cells from 6 patients with newly diagnosed severe AA (Table 1) and from 3 normal controls. RNA samples were isolated at onset of the disease, prior to BMT or immunosuppressive treatment with ALG. RT-PCR was carried out in the presence of oligonucleotide primers specific for 24 subfamilies of Vb transcripts. The panel of primers and the PCR conditions have been previously experimentally validated for the analysis of expression of Vb transcripts in human specimens (18). As a control of RNA integrity, amplification of a Cb-specific fragment was performed (not shown). Vb-specific PCR products varying in size from 250 to 535 bp were separated by gel electrophoresis and detected by hybridization with a 32P-labeled internal oligonucleotide probe. Examples of auto-radiograms are shown in Figure 1. The signal intensities were quantitated by phosphorimaging and the relative rate of expression of each Vb subfamily was determined as a percentage of the total amount of all 24 Vb transcripts (Figure 2). Results of the analysis demonstrated that virtually all Vb gene segments were expressed in RNA isolated from peripheral blood and bone marrow samples and

Variability of CDR3 Size To assess the putative clonality of T cells in AA patients, we examined the size variability within CDR3 region of 24 Vb-specific Vb-Cb PCR amplicons. PCR products were copied with nested fluorescent Cb or Jb primers and the sizes of the fluorescent run-off products were determined by electrophoresis on an automated sequencer. In peripheral blood lymphocytes of healthy donors, Vb-Cb and Vb-Jb amplified transcripts display a bell-shaped profile with an average of eight peaks spaced by three nucleotides corresponding to in-frame sequences (31). Size

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Figure 1. Southern blot analysis of PCR-amplified TCRVb gene segments in peripheral blood (PB) and bone marrow (BM) of three patients with severe AA and one control. RNA was subjected to RT-PCR using Vb1-Vb24-specific primers in 30 amplification cycles. Vb gene segments varying in size from 250 to 535 bp were detected by hybridization with 32P-labeled internal probe and autoradiography, as described in Materials and Methods. Weakly expressed transcripts which were barely or not visible in autoradiograms, could be detected after exposure of blots to Phosphor Imager Screen (for quantitation see Figure 2).

variations of the run-off products are strictly due to different CDR3 lengths reflecting the imprecise V-D-J joining mechanism. In all AA patients studied, the majority of Vb-Cb PCR products displayed a normal bellshaped size pattern, as illustrated by representative examples in Figure 3A. However, skewing of CDR3 profiles was observed in a few Vb-Cb amplified transcripts, such as in Vb8 from patient

5 or Vb16 and Vb23 from patient 6 (Figure 3B). Appearance of prominent peaks in excentric positions reflected an expansion of T cells with CDR3 size shorter or longer than the normally predominating centrally located peak and was highly suggestive of (oligo)clonality of cells. This result was further refined using primers specific for the 13 Jb human genes. The high resolution of this run-off method enabled us to deduce from the

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Figure 2. TCRVb repertoire in patients with severe AA. Analysis was performed in peripheral blood (PB) and bone marrow (BM) of six patients with severe AA (white bars, arranged in the order corresponding to patient numbers in Table 1) and compared with normal controls (black bars 6 SEM) obtained with PB [n511, (50)] and BM (n53) cells. RT-PCR reactions were carried out as described in legend to Figure 1. The rate of expression of each of the Vb1-Vb24 transcripts is presented as a percentage of the sum of all Vb signals detected by phosphorimaging analysis. According to Student’s t-test, the statistical significance of Vb expression level in AA versus normal PB and BM samples was p . 0.05 for each transcript. PB from patient 2 was not available for the analysis.

primer positions on the V, J and C gene segments that the 202 nucleotide-long Vb8-Jb2.1 and the 219 nucleotide-long Vb8-Jb2.7 rearrangements actually corresponded to the prominent 261 nucleotide-sized peak observed in the Vb8-Cb profile of patient 5. Similarly, the 121 nucleotide-long Vb16-Jb1.3 and the 243 nucleotide-long Vb23Jb2.5 rearrangements matched the overwhelming 154 and 281 nucleotide-sized peaks observed in

the Vb16-Cb and Vb23-Cb profiles of patient 6. Interestingly, the same Vb-Jb transcripts were found in bone marrow and peripheral blood samples. It is, however, noteworthy that in the bone marrow of patient 6 the prominent peaks were more obvious than in the peripheral blood, at least at the first level of CDR3 analysis by Vb-Cb size screening. Similarly, skewing of CDR3 profile in Vb transcripts from patient 3 was more

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pronounced in bone marrow than in peripheral blood (not shown). A summary of the results on CDR3 size distribution in AA is presented in Table 2. Analysis of the Vb-Cb amplification products demonstrated that one or two predominating peaks standing out from the usual bell-shape CDR3 profiles of Vb5, Vb7, Vb8, Vb13, Vb15, Vb16, and Vb23 transcripts could be identified in each patient, although the prevailing junctional rearrangements differed from individual to individual. There was no correlation between transcripts characterized by (oligo)clonal CDR3 size distribution pattern and transcripts expressed at enhanced levels. For example, Vb3 which was significantly overexpressed in patient 3 displayed a normal CDR3 size profile (data not shown).

DISCUSSION Responsiveness of AA to immunosuppressive treatment and the presence of local T cell infiltrates in hypoplastic bone marrow provide arguments for an immune-mediated mechanism of destruction of hematopoietic precursors. However, neither the recognized epitope on target cells nor the specificity of the effector cells are known. Since pathogenic T cell responses may be limited in terms of TCR diversity, we searched for restricted or preferential Vb gene segment usage in TCR repertoire in AA patients with severe untreated disease. Expression of 24 distinct TCRVb subfamilies and the junctional diversity in the CDR3 region of TCRb chain in each of these subfamilies were analyzed with the use of PCRbased approaches including the sensitive ‘‘Immunoscope’’ technique, which allows to detect low numbers of in vivo clonally expanded T cells in a complex mixture of uncultured T lymphocytes (18, 31). Results demonstrated that T cells in AA utilize diverse TCRVb gene segments and the frequency of individual Vb transcripts is comparable to normal. Although no typical pattern of skewed Vb repertoire could be identified, subtle

Figure 3. CDR3 size distribution pattern of Vb-Cb and Vb-Jb gene segments in peripheral blood (PB) and bone marrow (BM) of two patients with severe AA. Analysis was performed with 40 amplification cycles followed by 3 run-off cycles with fluorescent Cb or Jb-specific primers, as described in Materials and Methods. Fluorescent amplification products were separated on an automated sequencer. (A) examples of Vb transcripts with Gaussian-like separation profile indicating a normal diversity in CDR3 size; (B) examples of Vb transcripts with skewed separation profile, confirmed by (oligo)clonality of the Vb-Jb regions.

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Table 2. Summary of the Differences in TCR Vb Gene Expression and CDR3 Size Profile in AA Vb expression*

CDR3 size profile**

Patient No.

Source of RNA

increased at least 3-fold

decreased at least 3-fold

Vb-Cb

Vb-Jb

1

PB BM BM‡ PB BM PB BM PB

22

21; 22 20; 21

17; 18 18 15 1;9;10;12;15;18;22 8; 12; 13 10 7; 10; 12; 18; 24

n.d.† n.d.† Vb13 Vb5 / Vb15 Vb5 / Vb15 Vb7 Vb7 Vb8

BM

20; 21

PB

22

BM

22

n.d.† n.d. n.d. n.d. n.d. n.d. n.d. Vb8-Jb2.1 Vb8-Jb2.7 Vb8-Jb2.1 Vb8-Jb2.7 Vb16-Jb1.3 Vb23-Jb2.5 Vb16-Jb1.3 Vb23-Jb2.5

2 3 4 5

6

3 3

Vb8 3; 9

Vb16 Vb23 Vb16 Vb23

* Vb expression in AA was compared with mean values in normal peripheral blood [PB, n511 (50)] and bone marrow (BM, n53) samples. ** Listed are transcripts displaying prominent peaks, standing out the usual bell-shaped CDR3 profiles. † There was not enough RNA available from patient 1 to perform the CDR3 size analysis. ‡ There was no RNA from PB available from patient 2. n.d., not done

differences in TCRVb usage in AA were noted. In 5 of 6 patients, expression of one or two subfamilies was enhanced by at least 3-fold above normal levels. In one single patient, Vb3 was abundant and constituted about 20% of the TCRVb repertoire; other overexpressed transcripts amounted to less than 5% of total Vb’s. CDR3 size analysis of Vb-Cb and Vb-Jb PCR products identified abnormalities in Vb5, Vb7, Vb8, Vb13, Vb15, Vb16, and Vb23 gene segment usage, differing from patient to patient. Among these transcripts, most were expressed at low levels and only Vb8 and Vb13 represented 10-15% of the Vb transcript population. The pathogenic significance of subtle abnormalities in the level and size diversity of transcripts should be interpreted with caution. Sensitization by allo-antigens due to inevitable transfusions, although only few of them preceeded cell sampling in the analysed group of AA

patients, may have influenced the T cell profile. Furthermore, a skewed CDR3 size profile with one or two dominating peaks and recurrent clonal rearrangements can be found also in normal peripheral blood samples and in enriched normal T cell subsets analyzed by the ‘‘Immunoscope’’ technique and other methods (35-38) Similarly, among three control bone marrow samples included in this study, a clonal expansion of Vb15 subfamily utilizing Jb2.7 gene segment was detected in one donor (results not shown). Due to clonal T cell expansions observed even in healthy adults and probably related to environmental exposure, the involvement of T cells in disease-related immune responses is more convincing if clonal TCR rearrangements were detected in T cells isolated directly from pathogenic lesions, such as inflamed joint or skin, demyelinating brain plaques or tumor tissue (27, 39, 40). In AA patients, the pattern of Vb gene segment

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cells. Earlier studies have shown that both suppression of colony formation in vitro by coculture of bone marrow cells with T cells and the beneficial effect of T cell depletion on colony growth are found in single patients, but not in the majority (47-49). Recently, a Vb17 CD41 T cell clone was isolated from a cyclosporin-dependent AA patient in relapse and was shown to inhibit the colony formation by autologous CD341 precursor cells (11). However, in this particular patient, the frequency of autoreactive clones was below 10%. Also activated CD81HLADR1 cells, reportedly increased in AA bone marrow, were observed in about half of the analyzed patients with severe AA and constituted on average about 10% of the T cell population (3). These results may provide an explanation for the diversity of TCRVb repertoire in AA. In summary, heterogeneity in the pattern of Vb expression and CDR3 size profiles in patients with severe AA argues against overwhelming clonal amplification of TCRb-bearing T cells at the onset of the disease. Nevertheless, determination of CDR3 size allowed to detect subtle abnormalities in the TCR Vb repertoire in every analysed patient. It cannot be excluded that small expansion of potentially autoaggressive T lymphocytes elicits secondary immune responses and thus contributes to suppression of hematopoiesis. Further studies involving purified subpopulations of T cells and analysis of TCR usage complemented by determination of specific cytotoxicity and profiles of lymphokines produced by T cells, should help to establish the link between molecular and functional properties of immune cells in AA.

usage and CDR3 size diversity in T cells from bone marrow, as the tissue of primary injury in this disease, was closely paralleled by the pattern in peripheral blood for the vast majority of Vb transcripts. It cannot, however, be excluded that autoaggressive cells are also present in the circulation or that peripheral blood cells contaminating bone marrow aspirates obscure the putative differences in T cell repertoire between these two compartments. In addition, patchy hematopoiesis in severely hypocellular bone marrow at the onset of AA may prevent detection of all putative abnormalities in TCR repertoire in individual patients. So far, available information on the TCR repertoire in AA is scarce. In two abstracted reports, T cell clonality was analysed by the method of single stranded conformation polymorphism combined with PCR amplification of Vb transcripts (41, 42). Distinct bands indicating selective gene segment usage were observed in very few Vb subfamilies in single cases only. These data, together with the results of our study demonstrating a heterogenous pattern of Vb transcripts and polymorphism of CDR3 size, argue for a low frequency of expansion of T cells bearing uniquely rearranged TCRs in AA. It remains highly speculative whether broad TCR diversity in AA reflects lack of an immunodominant antigen, or whether T cells respond to a well defined epitope with multiple TCR specificities, as observed for example with cytolytic T lymphocytes directed against tumor antigen MAGE.1.A1 in a melanoma patient (43). We also cannot exclude that specific T cell responses in AA are, at least partially, hidden by the bulk population of bystander T cells. In animal models in which the frequency of T cells specifically induced by viral stimuli was analysed, the most intense immune response was generally mediated by no more that 1-2% of the overall T cell population (44-46). In AA, functional abnormalities of T lymphocytes are not common to all patients and are probably localized in a fraction of

ACKNOWLEDGMENTS We thank Y. Uematsu and M.R. Bu¨rk for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (grant 3236278.92) and Stiftung zur Krebsbeka¨mpfung No.66 to C.N. and A.W-F., by Stipendienfonds der Basler Chemischen Industrie, Wolfermann-Na¨geli-Stiftung and Emilia Guggenheim-Schnurr-Stiftung to C.Y.M. and by

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the Swiss National Science Foundation (grant 3140704.94), the Terwindt Foundation, the San Salvatore Foundation and the Spinola Foundation to P-Y.D.

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