Biomedisch Onderzoeksinstituut DWI, Limburgs Universitair Centrum, Diepenbeek, Belgium
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Abstract |
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Results. Over-represented TCR V genes were observed in the synovium, but not in the blood of all RA patients (n = 38). The number of over-represented V genes was higher in the synovium of chronic RA patients (n = 31) than in that of early RA patients (n = 7). The V-gene profile was different among patients, and similar in the two knees for patients with bilateral synovitis (n = 5). The clonal composition of over-represented TCR BV genes in a patient with early RA and a patient with chronic RA was further studied by CDR3 region sequence analysis. A high level of clonal diversity was found in the joints and the blood of the early RA patient, suggesting a polyclonal T-cell expansion. In the chronic RA patient, predominant clonal expansions were observed in the blood and synovium, and some expanded clones were still present 2 yr later.
Conclusions. The observation of similar T-cell populations in both joints in patients with bilateral synovitis and the persistence of clonally expanded T cells for more than 2 yr in the joints of a chronic RA patient may indicate a pathogenic role for these cells in the disease process.
KEY WORDS: Rheumatoid arthritis, Autoimmunity, T-cell receptor usage, Clonal expansion, Synovial T cells.
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Introduction |
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On the assumption that disease-relevant T lymphocytes may undergo expansion in the synovium, several groups have studied T-cell receptor (TCR) gene expression in the joints and blood of RA patients [8, 10]. If biased TCR expression is associated with the pathogenic T-cell populations, the corresponding TCR elements could be targeted by TCR-specific immunotherapies, such as TCR peptide vaccination or T-cell vaccination [11, 12]. Unfortunately, many contradictory findings have been reported. Paliard et al. [13] observed elevated expression of BV14 genes in synovial fluid (SF), while Howell et al. [6] found increased expression of BV3, BV14 and BV17 in interleukin 2 receptor-positive T cells in the joints. Sottini et al. [14] reported preferential use of BV7 genes in the SF, and Struyk et al. [15] observed heterogeneous use of TCR V genes in the SF of 12 RA patients, with increased expression of AV10 in most patients. Some authors described oligoclonal expansion of CD4+ T cells in the joints, while others found oligoclonal expansion of the CD8+ subset [1621]. Some of the contradictory findings may be related to differences in patient populations, such as the HLA background and disease duration, or to the use of unstimulated vs in vitro-expanded cells from the SF. In addition, various techniques, such as Southern blot analysis [6], polymerase chain reaction (PCR) amplification [14, 15, 22] and staining with TCR V-gene-specific antibodies have been applied [23]. Some of these methods may provide limited quantitative data only, while flow cytometry depends on the availability of V-gene-specific antibodies.
We used a sensitive and powerful PCRenzyme-linked immunosorbent assay (ELISA) [24] to identify over-represented TCR V-gene elements in the synovium of two groups of RA patients, a group with short disease duration (n = 7) (less than 1 yr) and a group with chronic disease (n = 31) (mean duration 14 yr). To avoid in vitro bias, fresh unstimulated and unseparated SF cells and paired peripheral blood mononuclear cells (PBMC) were used. The relative levels of expression of the BV and AV genes in the blood of healthy control subjects (n = 10) were used to calculate cut-off values for each V gene. Over-represented AV and BV genes in patient samples were defined as having an expression level above the cut-off value for a particular V gene family. Over-represented TCR V genes were found in the joints of patients with RA, but not in their blood, which is in line with previous observations. None of the BV genes was, however, consistently over-expressed in the joints of all patients or a subgroup of patients. Interestingly, fewer over-represented BV genes were identified in the joints of patients with early RA than in those of chronic RA patients. Over-represented TCR BV gene families of a patient with early RA and a chronic RA patient were further characterized by CDR3 sequence analysis. Polyclonally expanded T cells were observed in the synovium of the early RA patient, while major clonal expansions were observed in the joints of the chronic RA patient. Identical CDR3 sequences were identified in both knees of the early and chronic RA patients. In the chronic patient, clonally expanded TCR sequences persisted for 2 yr, suggesting that the corresponding T-cell population plays a role in the progression of the synovial inflammation in this patient.
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Patients and methods |
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Isolation of T cells
PBMC were isolated from heparinized blood by standard FicollHypaque centrifugation (Pharmacia Biotec, Roosendaal, The Netherlands), washed with RPMI 1640 (Life Technologies, Merelbeke, Belgium) and aliquoted into fractions of 2 x 106 cells in cold phosphate-buffered saline (PBS). SF was diluted in RPMI 1640 (1:15) and centrifuged for 10 min at 1800 r.p.m. at 4°C. The cells were resuspended in 5 ml RPMI 1640 and counted. If more than 14 x 106 cells were obtained, an additional purification of the mononuclear cells was performed using FicollHypaque as described above. The cells were washed twice with RPMI 1640 and resuspended in PBS in fractions of 2 x 106 cells. Fresh synovial tissue specimens were collected in RPMI 1640 at 4°C and processed within 3 h after surgery. The tissue was cut into small fragments and digested enzymatically overnight at room temperature in RPMI 1640 containing 0.01% (w/v) hyaluronidase, 0.02% (w/v) DNase I (Life Technologies), 0.1% (w/v) collagenase (Sigma, St Louis, MO, USA), 50 µg/ml gentamicin (Life Technologies) and 250 ng/ml fungizone (Life Technologies) dissolved in RPMI 1640. The single-cell suspension was filtered through a sterile coarse wire grid and washed four times in RPMI 1640. The cells were resuspended in cold PBS.
PCRELISA of TCR AV and BV genes
PCRELISA was performed as described [24]. Briefly, total RNA was extracted from 2 x 106 PBMC, SF and/or synovial tissue by the Rneasy method and reverse-transcribed into first-strand cDNA using oligo-dT. cDNA was PCR-amplified using 19 TCR AV and 21 BV gene-specific primers as the forward primer and a digoxigenin (DIG)-labelled TCR C region-specific primer as the reverse primer. Primer sequences were as described [25]. The PCR amplification cycle consisted of 35 cycles of 20 s at 94°C, 20 s at 55°C and 40 s at 72°C. DIG-labelled PCR amplicons were analysed by ELISA. Therefore, the amplicons were hybridized with fluorescein isothiocyanate (FITC)-labelled TCR AC or TCR BC region-specific probes. The DNA hybrids were captured on anti-FITC-coated ELISA plates and visualized by an anti-DIG peroxidase-labelled antibody.
To enable semiquantitative analysis of the TCR AV and BV PCR amplicons, it was important to perform the amplification with comparable amounts of starting cDNA copies. The amount of TCR-specific cDNA was estimated by PCR amplification of the TCR C region. The PCR products were serially diluted from 1/25 to 1/400 and quantified by ELISA as described earlier. Equal amounts of cDNA from the PB and SF were then used in the TCR V-gene repertoire analysis with the same PCR conditions.
Relative levels of expression of TCR AV and BV genes in the total TCR V-gene repertoire were calculated as percentages of the total TCR V-gene expression using the formula
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Sequence analysis of TCR rearrangements
CDR3 region sequences were determined by subcloning the TCR BV gene amplification products into a TA cloning vector, following the manufacturer's instructions (Invitrogen, Leek, The Netherlands). cDNA was amplified using the TCR BV region-specific primer and a TCR C region-specific primer using the conditions as described in the preceding section (PCRELISA of TCR AV and BV genes). PCR amplicons were ligated in the pCR2.1 cloning vector and transformed by heat shock in Escherichia coli. Plasmid DNA was prepared from 1015 recombinant plasmids and inserts were amplified by PCR using BV and BC region-specific primers as described above. The amplicons were sequenced using the dye terminator cycle sequencing reaction mix (Perkin-Elmer) with a TCR C-region-specific primer. The PCR conditions were: 30 s at 96°C followed by 10 s at 96°C, 5 s at 50°C and 4 min at 60°C for 25 cycles. Fluorescently labelled PCR amplicons were purified on a Sephadex-G50 M column, vacuum-dried and resuspended in 5 µl 1:50 25 mM EDTA/formamide. Sequencing was performed on an ABI 373A DNA sequencer using the related data analysis software.
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Results |
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PCRELISA was performed with PB and paired SF samples of seven early and 31 chronic RA patients. Five patients with OA and four with PA were used as controls (Table 1). Figure 2
shows the typical TCR V-gene expression profiles of a patient with early RA (RA-5) and a patient with chronic RA (RA-11). The TCR AV5 and AV22 genes and the TCR BV2, BV6 and BV20 genes were over-represented (above the cut-off values) in the synovial fluid of patient RA-5, while AV3 and AV15 and BV10, BV13.1 and BV18 were over-represented in the synovium of the chronic RA patient RA-11. Thus, while TCR V-gene use in the blood of these patients was rather heterogeneous, restricted TCR V-gene use was observed in their SF. Similar heterogeneous use of all TCR V-gene elements was observed in the PBMC of the 38 RA patients and nine control patients (data not shown). In contrast, over-represented TCR V-gene segments were identified in the synovium of all RA and control patients tested (Table 2
). The number of over-represented TCR AV and BV genes in the joints ranged from 1 to 5, with a mean number of 2.2 AV and 3.5 BV genes in the RA population and 2.6 AV and 4.0 BV genes in the joints of the control patients (OA and PA) (Table 3
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Over-represented TCR V genes vary significantly among the RA patients
The TCR AV and BV genes which were over-represented in the SF varied significantly among RA patients (Table 2). No particular TCR V gene was over-represented in more than 50% of the RA-derived SF samples (n = 49) or in the HLA-DR1/DR4+ subgroup (n = 26) (Fig. 3
). However, some V genes, including AV5 and BV2, BV7 and BV13.1, were over-represented in more than 25% of the RA-derived synovial samples. Other V genes, including AV9, AV13, AV14 and AV16 and BV3, BV4 and BV11, were less frequently over-represented in the DR1/DR4+ RA patients compared with the total RA group. No V-gene element was commonly over-represented in the SF of the three DR1/DR4+ early RA patients. Thus, our data indicate a lack of TCR BV-gene expression bias in RA patients, and do not confirm the previously reported over-representation of the BV3, BV14 and BV17 genes [6, 16]. In the non-RA control group (n = 9), nearly all V-gene segments were over-represented in at least one patient studied. The small number of samples analysed (nine) does not allow us to draw conclusions on preferentially expressed V genes in this group.
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The TCR V-gene expression profile is similar in synovial fluid and tissues of both affected knees of RA patients with bilateral synovitis
We studied SF and synovial tissue from the same diseased joint in four RA patients (RA-33, RA-35, RA-36, RA-38) and paired SF samples of both knees in five patients with bilateral synovitis (RA-7, RA-32, RA-34, RA-37, RA-38). In these patients, the TCR V-gene profile was very similar at the two disease sites (Table 2). For example, in patient RA-7, AV5 and BV16 were over-represented in the SF of both knees. In patient RA-38, two separate samples from the same cavity (fluid and tissue of the right knee) and fluid of the opposite knee were analysed. In the right knee, AV9 and BV1, BV4 and BV10 were over-represented in both synovial tissue and fluid. The AV9, BV1, BV4 and BV18 genes that were expressed in the synovial fluid of the right knee (SFR) were also over-represented in the synovial fluid of the left knee (SFL) of this patient. These data suggest that similar T-cell populations are present at different sites of the same cavity, and in both knees of patients with bilateral synovitis.
TCR V-gene expression is more restricted in the joints of early vs chronic RA patients
Next, we studied TCR V-gene restriction in relation to the disease duration of the patients. We compared the mean number of over-represented V-gene elements in early RA patients (disease duration <1 yr) and chronic RA patients (disease duration > 1 yr; mean disease duration 14 yr). The mean number of over-represented BV genes (3.7) was significantly higher (Student's t-test, P < 0.05) in chronic RA patients than in early RA patients (2.4) (Table 3). The mean number of TCR AV and BV genes over-represented in the joints of early RA patients (1.8 and 2.4 respectively) was also lower than in the non-RA control population (OA and PA patients) (2.6 and 4.0), although these differences were not statistically significant.
Similar TCR V genes are over-represented in the joints of a chronic RA patient for at least 2 yr
In one RA patient (RA-36) we evaluated the TCR repertoire in the joints on two occasions at an interval of 2 yr. An SF sample from both inflamed knees was obtained at a disease duration of 7 yr, and a sample of SF and synovial tissue was obtained from the right knee 2 yr later. The patient was being treated with DMARDs before and during the observation period. The disease activity was similar at the two time points. Interestingly, out of five TCR AV and five BV genes over-represented in the right knee at the first sampling, one AV gene (AV2) and two BV genes (BV2 and BV6) were still over-represented 2 yr later (Table 4). The total number of over-represented TCR V genes was decreased after 2 yr in this patient. It is unclear whether the administration of DMARDs influenced the TCR V-gene expression profile in the joints of this patient.
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CDR3 sequence analysis of over-represented V-gene elements in the synovium of two RA patients
To provide information on the clonal composition of an over-represented V gene family, we cloned the PCR products in a plasmid vector and sequenced 1015 randomly selected recombinant clones. For this experiment we selected a representative patient of the early RA group (RA-7) and one from the chronic RA group (RA-36) (Tables 5 and 6
respectively).
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We further studied whether shared amino acid motifs were present among the expanded TCR BV-gene segments. Such shared CDR3 amino acid motifs may indicate T-cell activation induced by structurally related antigenic peptides. One motif, XGGX, was found in 15 of 35 BV2 and four of 35 BV4 VDJ region sequences of the SF and blood (Table 5). The motif was also seen in eight of 37 BV8 sequences of the SF (data not shown). This motif was also present in CDR3 sequences of BV genes that were not over-represented in the SF: in five of 15 and three of 14 CDR3 sequences of the BV13.1 and BV11 genes respectively. Almost all (17 of 19) of the XGGX motifs were encoded by the germ line; 10 of 19 XGGX motifs in the BV2 and BV4 gene family were encoded by the germ-line BD1 gene and seven of 19 motifs were encoded by the BD2 gene segment. The frequent observation of this motif may therefore be caused by the preferential use of the DB1 gene in this patient, and may not relate to antigen-driven activation by a structurally related antigen.
In the chronic patient RA-36 we studied the CDR3 sequences of the BV2 and BV4 families, which were predominantly expressed in the synovium at the first or second sampling. Major clonal expansions were observed in the left- and right-knee SF, but also in the blood (Table 6). In the BV2 family some of these expansions accounted for one-third to one-half of all sequences, while in the BV4 gene family a single CDR3 sequence was observed in more than 80% of sequences from the left and right knees. The two BV4 CDR3-region motifs (DPGPAV and NRDGG) observed among 10 clones from the right knee were all shared with the left knee, and were also observed in eight of 11 CDR3 sequences in the blood of the patient. Furthermore, three BV2-region sequences were shared between the blood and the left knee. The RVDS motif was observed in four independent BV2 CDR3 sequences, and was present in 10 of 21 sequences in the blood and in 10 of 15 sequences in the left knee SF.
In this patient we evaluated the persistence of these clones 2 yr later. In the BV2 gene family, none of the CDR3 sequences found at the first sampling was observed 2 yr later (Table 6). At the second time point, one CDR3 sequence that was found in three of 17 BV2 clones from the blood was also observed in the paired tissue sample of this patient. Interestingly, the two BV4 CDR3 sequences that accounted for nearly all BV4 sequences seen in the left and right knees at the first sampling were still identified in the synovial membrane and blood after 2 yr. These sequences were, however, found at a lower frequency at the later time, suggesting that other BV4 T cells had also infiltrated the joints of this patient.
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Discussion |
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We observed different patterns of TCR V-gene expression in the joints and peripheral blood of RA patients. No over-represented TCR V genes were seen in the blood, whereas one to five over-represented TCR V-gene elements were observed in the joints of all RA patients. The restricted TCR V-gene expression profile in the joints may have been caused by differential survival of T cells in the joint or by specific recruitment of T cells to the joint. Interestingly, similar TCR V genes were over-represented in both joints of patients with bilateral synovitis. As no expanded TCR V genes were found in the blood and over-represented TCR V genes were seen in patients with a very short disease duration, it is possible that some of these T cells are involved in the disease process. The number of over-represented TCR V-gene elements was significantly higher in the joints of chronic RA patients than in those of early RA patients, indicating that the TCR V-gene expression profile is broader in patients with long-standing disease than in patients with recent disease onset. Although the V-gene profile does not provide information on the clonal diversity of the TCR repertoire, this observation is consistent with the hypothesis that determinant-spreading mechanisms may lead to the activation and infiltration of a larger variety of autoreactive T cells [29].
One striking observation from the TCR repertoire analysis is that similar TCR V genes were over-represented in both knees of five patients with bilateral synovitis. The expansion of T cells with similar V-gene expression suggests that identical T-cell populations are present in the two joints. However, this can be confirmed only by CDR3-region sequence analysis of the over-represented TCR genes in the joints of these patients. Therefore we determined the CDR3 sequences of over-represented BV genes of the SF and blood of two representative patients, one with early RA and one with chronic RA. Clonal analysis of the over-represented V-gene families in these two patients revealed different pictures. In the early RA patient the over-represented TCR BV-gene families in the joints were of polyclonal origin, while some TCR sequences were identified in both affected joints. Interestingly, a short amino acid motif (XGGX) in the CDR3 region was found frequently among the over-represented TCR families in the joints and the peripheral blood. This GG peptide was encoded mainly by the TCR D-region element BD1. Thus, it is possible that the frequent use of this motif by T cells in the SF and blood of this patient is simply due to the preferential rearrangement of this TCR D-region element by the T lymphocytes in the patient. Another possible explanation for the increased frequency of this motif is that the expansion of T cells with this TCR motif is caused by T-cell sensitization induced by a single epitope or a limited number of structurally related epitopes. Shared CDR3 sequence motifs in the joints of RA patients were also observed in previous studies [3032]. Alam et al. [33] observed a related GXXG motif in the CDR3 of 30% of the dominant clones isolated from the synovial tissue of two RA patients. In line with our observations, it was shown that the clones analysed by Alam et al. [33] that expressed the conserved motif were generally not clonally expanded in the synovium. Therefore, despite the frequent use of a common motif in the CDR3 sequences, it is possible that the polyclonal TCR V-gene expansion in the joints of the early RA patient reflects T-cell activation induced by microbial superantigens, leading to deletion or expansion of T cells in a BV-specific manner [34].
In the chronic RA patient, major clonal expansions were observed in the joints among the BV2+ and BV4+ T cells. These expansions accounted for the vast majority of CDR3-region sequences identified among these subsets in the joints. This TCR pattern is compatible with in vivo T-cell activation induced by only a few antigenic determinants. The T-cell activation probably occurred in the blood, because the clonally expanded BV2 and BV4 sequences were also detected in the circulation. One of the clonally expanded CDR3 sequences was still present in the joints and blood after 2 yr, while other new additional CDR3 sequences were found in the joints at the second time point. The persistence of the clonally expanded T-cell population for a period of 2 yr suggests a role for these T cells in the disease process of this patient. Note that the number of over-represented V genes was slightly decreased at the second sampling; however, the patient already had a disease duration of 7 yr at the first sampling and was being treated with DMARDs during the period between the samplings. The use of specific T-cell-targeted immunotherapies, such as TCR or T-cell vaccination, to target clonally expanded cells in this patient could suggest whether the depletion of these T cells has an effect on the progression of disease [35, 36]. Our results are in agreement with previous reports [31, 33]. Kato et al. [31] also described persisting, though gradually decreasing, clonal expansion in a patient with chronic RA.
On the basis of our data, we hypothesize that different T-cell activation pathways may be operating in RA. In the early RA patient, the TCR expression pattern is compatible with a T-cell activation process induced by microbial superantigens. Perhaps the synovial T-cell repertoire in this patient reflects the peripheral pool of superantigen-stimulated T lymphocytes. If some of these cells cross-react with a synovial autoantigen, they might become reactivated and overgrow other synovial T cells that are not reactivated by a cross-reactive epitope. Such a pattern is seen in the chronic RA patient, in whom only a few antigenic epitopes may have been responsible for the persistent expansion of the T cells in the joints. In subsequent stages, the spreading of determinants may lead to activation and expansion of other T-cell clonotypes in the synovium. Intra- and intermolecular antigen spreading (determinant spreading) has been described in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis [37]. Further studies are needed to address whether this hypothetical process also operates in other RA patients. This may, however, explain the contradictory observations reported previously, as the TCR repertoire diversity will change during disease progression in individual patients. The TCR V-gene repertoire seems to be more restricted in the early phase of the disease, suggesting that the random profile found in the peripheral blood of the patients is distorted. This distortion may be caused by increased expression of a specific subset of T cells as a consequence of antigenic stimulation, or it may be due to depletion of other T cells found in the repertoire. Our data suggest that this antigenic stimulation may be caused by superantigens in the early stage of the disease, whereas it may be that only a few antigenic epitopes cause the persistent expansion in the joints of the chronic RA patients. However, further studies are needed to resolve whether the patterns of TCR expression observed in the early and chronic RA patient are representative of larger groups of early and chronic RA patients.
Over-represented TCR V genes were also observed in the joints of patients with OA, which is mainly considered to be a non-inflammatory process. However, TCR V-gene restriction in OA patients was also reported by Zwillich et al. [38]. Further studies are needed to address whether the TCR restriction in OA patients is caused by T-cell sensitization to cartilage antigens released as a consequence of mechanical destruction or whether it is the result of antigen-non-specific signals acting differentially on certain clones. Restricted TCR V-gene expression was also observed in the joints of four patients with PA, suggesting that similar T-cell-mediated immune mechanisms may operate in RA and PA. In this sense PA and OA patients may not be optimal controls for T-cell studies in RA.
In conclusion, the observed skewing of TCR V-gene expression in the synovium of all RA patients, the similar TCR V-gene profiles in both affected joints of patients with bilateral synovitis, and the clonal expansion and persistence of T-cell clonotypes in the joints of a chronic RA patient are consistent with an important role for T cells in the pathogenesis of RA. However, the expanded V genes differ among patients with RA and can therefore not be targeted by a uniform TCR V-gene-directed therapy. Our detailed clonal analysis of over-represented V genes in two RA patients showed possibly different T-cell activation pathways in an early RA patient and a chronic RA patient. Although T-cell-directed therapies might therefore be effective in RA, such therapies may need to be customized for individual patients. Whereas in some patients a CDR3-specific approach may be effective in depleting the majority of expanded synovial T cells, broader BV family-specific approaches may be required for other RA patients.
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Acknowledgments |
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Notes |
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References |
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