Divergence in the degree of clonal expansions in inflammatory T cell subpopulations mirrors HLA-associated risk alleles in genetically and clinically distinct subtypes of childhood arthritis

Lucy R. Wedderburn, Alka Patel, Hemlata Varsani and Patricia Woo1

Rheumatology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH and
1 Department of Molecular Pathology and Immunology, University College London, 46 Cleveland Street, London W1T 4JF, UK

Correspondence to: L. R. Wedderburn; E-mail: L.Wedderburn{at}ich.ucl.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Clinically distinct forms of childhood arthritis are associated with different risk alleles of polymorphic loci within the MHC, which code for the antigen-presenting class I or class II molecules. We have compared the TCR diversity of synovial T cells from children with enthesitis-related (HLA-B27+) arthritis and oligoarticular arthritis (with class II MHC risk allele associations) in parallel with peripheral blood T cells from each child, using a high-resolution heteroduplex TCR analysis. We demonstrate that multiple clonal T cell expansions are present and persistent within the joint in both groups, but that there is disease-specific divergence in the dominant T cell subset containing these expansions. Thus, the largest clonotypes within the inflamed joints of children with class II-associated arthritis are within the CD4+ synovial T cell population, while the dominant clones from children with enthesitis-related arthritis (associated with a class I allele) are within the CD8+ synovial T cell population. These data provide powerful data to support the concept that recognition of MHC–peptide complexes by T cells plays a role in the pathogenesis of juvenile arthritis.

Keywords: autoimmunity, clonotype, expansion, heteroduplex, HLA-B27, HLA-DRB1, human, juvenile idiopathic arthritis, memory, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One in 1000 children suffers from some form of chronic arthritis, which may become severe and debilitating. Collectively known as juvenile idiopathic arthritis (JIA) (1), these conditions include clinically distinct forms of arthritis, which have genetic associations with different alleles of HLA class I or class II loci. The association of these arthritides with HLA alleles remains the most compelling evidence for the role of T cells in the aetiopathogenesis of JIA. The most common form of JIA, oligoarticular arthritis, has strong associations with alleles in the class II region, i.e. HLA-DRB*0801, 1101, 1301 (2), now confirmed by transmission disequilibrium test analysis (3). This type of arthritis affects four or less joints at presentation and carries a risk of anterior uveitis which, if untreated, may lead to blindness. In contrast, enthesitis-related arthritis (previously known as juvenile spondyloarthropathy), like adult ankylosing spondylitis, is strongly associated with the class I allele HLA-B*2705 (2,4). Although many studies have addressed the issue of T cell recognition in arthritis, few have directly addressed the question of why HLA alleles at different loci (class I or class II) should be associated with diseases with differing clinical distributions and complications. Given that the function of class I and class II proteins is to present peptide antigens to CD8+ and CD4+ T cells respectively, we hypothesized that these genetic associations would be reflected in the biology of the two subsets of T cells found at the sites of active synovitis. In this study we have tested this hypothesis by directly comparing the extent of T cell clonal expansion in the CD4+ and CD8+ T cell populations from the inflamed joints of both groups of children.

We and others have previously shown that the T cells found within the inflamed joints of children with JIA are restricted in their TCR expression, compared to those of peripheral blood mononuclear cells (PBMC) (59). These data suggest that only a very limited subset of T cells is able either to enter or survive within the inflamed site. However, the majority of previous studies did not consider the TCR expression of CD4 and CD8 cells separately. It is clear that the TCR repertoires of these two T cell populations are distinct. Thus, in peripheral blood lymphocytes, CD8+ cells form large clonal expansions, which may persist over many years (10,11). Such expansions are rarely seen in the peripheral blood CD4+ subset, either during acute antigenic challenge or in the memory population (12,13). In the study presented here we demonstrate differences in the degree of oligoclonality of TCR expression for the CD4+ and CD8+ T cell populations found within an inflammatory site, in two clinically distinct groups of patients who carry differing risk HLA alleles. In addition, by analysis of the oligoclonality of inflammatory T cells at different time points, we show that differences between CD4 and CD8+ T cell populations in the two types of JIA are maintained over long periods of time.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Samples and cells
This study had approval from the Great Ormond Street Research Ethics Committee. Samples from six children with JIA were obtained with informed parental consent. All fulfilled the revised international criteria of JIA (1). HLA typing was performed by allele-specific PCR. Three children had oligoarticular arthritis, each of these carrying one risk class II HLA allele, and three had B27+ enthesitis-related arthritis (Table 1Go). Paired samples of venous blood and synovial fluid (SF) were obtained from each child, in some cases at repeated time points over 2 years. SF was aspirated from inflamed joints at the time of clinically indicated arthrocentesis. SF was treated with hyaluronidase (Sigma, Poole UK) 10 U/ml for 30 min at 37°C, before processing. PBMC and SF mononuclear cells (SFMC) were isolated by density centrifugation. CD4+ and CD8+ populations were separated using magnetic beads (Milteyni Biotec, Bisley, UK).


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Table 1. Clinical characteristics and HLA typing data for the six patients in this study
 
Heteroduplex (HD) assay
Total RNA was extracted from 2x106 cells (either unseparated mononuclear cells or purified T cell populations) and all of the total RNA used for first-strand cDNA synthesis using MMLV-RT (Gibco, Paisley, UK) and oligo(dT) (Boehringer Mannheim, Lewes, UK). RT-PCR-HD analysis was performed as described (9). In brief, 1/60 of cDNA was used in each of 26 PCR reactions for TCR, using 26 TCR ß chain variable (TCRBV)-specific primers and an internal TCR constant region (TCRBC) primer (14). In parallel 26 HD carrier PCR reactions were performed using an external TCRBC primer. Aliquots of 20 µl of sample PCR products were mixed with 200 ng of appropriate TCRBV carrier DNA, denatured at 95°C for 5min and then annealed at 50°C for 1 h. HD products were analysed on a 12% non-denaturing polyacrylamide/0.5% TBE gel, blotted onto Hybond N+ membrane and probed using a carrier-specific TCRBC probe as described (9). Short (2-h)- and long (12-h)-exposure autoradiographs were processed for each blot. For analysis with CDR3-specific oligonucleotides, membranes were stripped in 0.5% SDS/distilled H2O at 72°C and then re-probed.

TCRBV sequence analysis
For sequence analysis of rearranged TCRBV sequences, PCR amplification products were cloned into the TA pCR2.1 cloning vector (Invitrogen, Groningen, The Netherlands) according to the manufacturer's instructions. Plasmids containing inserts were identified by restriction digestion and sequenced by dideoxy-chain termination using Sequenase (Amersham, Little Chalfont, UK) or through MGW-Biotech (Milton Keynes, UK). Where individual clonotypes were found to represent >10% of an individual TCRBV family from one sample, the unique N-region sequence was used to prepare oligonucleotides of 17–20 bases for re-probing of HD membranes.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Disease divergent oligoclonality in different clinical types of juvenile arthritis
We have previously shown that the T cells from the joints of children with oligoarticular JIA express a highly oligoclonal set of TCR compared to those expressed in PBMC and that the same set of TCR clonotypes is expanded in two different joints at one time point (9). We have now tested the hypothesis that genetic associations between MHC alleles and clinically distinct forms of arthritis would be reflected in the features of the synovial T cell infiltrate. To do this, we analysed the TCR expression in paired samples of PBMC and SFMC from children with two different clinical types of JIA, which have different associated HLA risk alleles: oligoarticular arthritis and enthesitis-related arthritis, associated with class II and class I HLA alleles respectively. We used a high-resolution HD technique, whose sensitivity is ~1 cell in 50,000 and which, like single-strand conformational polymorphism analysis, is based upon CDR3 sequence differences (15), to compare the TCR expression of CD4+ and CD8+ T cells in these samples.

A summary of the clinical features of the six children studied is shown in Table 1Go. For each child, SF T cells were shown by HD to include clonal expansions in every TCRBV family (detected as bands above the `carrier ' DNA in the HD assay). Parallel HD assays were then performed on separated CD4+ and CD8+ T cells which had been normalized for cell numbers. In all three children with oligoarticular JIA analysed, the most prominent expanded clones (those seen even on a short exposure film) were seen in the CD4+ T cells. Representative reactions for several TCRBV families analysed on CD4+ and CD8+ cells from peripheral blood and SF (Fig. 1AGo, PA13) or SF alone (Fig. 1BGo, PA10) are shown in Fig. 1Go. In no case were these same clonotypes detectable by HD in the peripheral blood CD4+ T cells, indicating that they were present in blood at low frequency which was below the threshold of detection by this method. In marked contrast, in the children with B27+ enthesitis-related arthritis, the largest (and therefore most readily detectable) T cell clones were always in the CD8+ T cell population from the SF (Fig. 2A and BGo). Again the dominant CD8+ clones seen in synovial T cells from HLA-B27+ children were not detectable in the peripheral blood CD8+ T cells (Fig. 2AGo, JAS2). In a minority of children, other T cell clonotypes giving rise to HD clonal bands were detectable in peripheral blood CD8+ T cells, but these were different from the dominant clones within the synovial T cells. The mean number of HD bands in peripheral blood CD8+ T cells was no greater for patients with JIA than those in age-matched healthy children (16 and unpublished data).




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Fig. 1. HD analysis of CD4+ and CD8+ T cells from children with oligoarticular JIA reveals that the largest clonal expansions are within the CD4+ synovial T cells. PBMC and SF T cells were separated into CD4+ and CD8+ populations before HD analysis. After RT-PCR with 26 different TCRBV primers and HD reactions with a TCRBV-specific carrier DNA, HD reactions were run on 12% non-denaturing acrylamide gel, blotted and probed with a TCRBC probe which detects any HD containing carrier DNA. (A) HD analysis on PBMC and SF T cells for five representative TCRBV families, on samples from patient PA13 (B). HD analysis for six representative TCRBV families on CD4+ and CD8+ synovial T cells (patient PA10).

 



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Fig. 2. HD analysis of SF T cells from children with B27+ JIA reveals that the largest clonal expansions are within the CD8+ synovial T cells. (A) HD analysis on PBMC and SF T cells for five representative TCRBV families on samples from patient JAS2. (B) HD analysis for four representative TCRBV families on CD4+ and CD8+ synovial T cells (patient JAS3).

 
For both oligoarticular and enthesitis-related arthritis, smaller but detectable T cell clones were also seen in the synovial cells in the reciprocal (CD8+ and CD4+ respectively) T cell populations on longer exposure films (e.g. JAS3 in Fig. 2BGo). Previous data suggest that in a mixture of T cells, each clone gives rise to either one or two clonal bands in the HD assay (14). By counting all of the major bands visible on an early exposure film, we estimated that each SF exudate contains between 50 and 90 T cell expansions which are large enough to produce discrete clonal bands on the HD assay. Flow cytometric analysis of T cell subsets showed that the absolute numbers of CD4+ and CD8+ T cells within the joint were not significantly different between the two types of JIA (data not shown). As shown previously, the ratio of CD4:CD8+ cells within the joint was lower than in peripheral blood, and synovial T cells had high expression of HLA-DR, CD45RO and CD25 (17,18 and unpublished data). This was observed for both oligoarticular and enthesitis-related JIA, with no significant difference in the expression of these markers on synovial T cells between the two groups (data not shown). In addition, staining for the marker of cell division, Ki67, showed no significant differences in Ki67+ cells between CD4+ and CD8+ cells from the two clinical types of JIA (data not shown).

T cell oligoclonality of both disease groups is persistent
In order to ask whether expanded clones detected in any one sample of SF might be the result of random survival of clones within the joint, children undergoing repeat arthrocentesis were analysed on several time points. In a total of three children (two oligoarticular and one enthesitis-related arthritis) from whom samples of several time points were run in parallel, the majority of expanded clonotypes were present at each time point, giving rise to identical banding patterns (Fig. 3A and BGo). In all three of these, the predominance of clones in either the CD4+ or CD8+ synovial cells was preserved over time. This persistence of the expanded clonotypes was maintained even when the involved joint had been clinically improved between aspirations. Figure 3(A)Go shows HD analysis on synovial T cells from right and left knees (patient PA7), each analysed from samples obtained 1 year apart. In this patient some new HD bands, indicating expansion of additional clonotypes, are seen at the later time point (e.g. TCRBV13.2, Fig. 3AGo, marked by an asterisk), suggesting a `spreading ' of the T cell response. In Fig. 3(B)Go, CD4+ and CD8+ synovial T cells from samples taken 3 months apart (patient PA13) have been processed in parallel and again show the persistence of clones on of the dominance of restricted clonality, in this case in CD4+ synovial T cells.




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Fig. 3. Dominant clonotypes demonstrated by HD analysis persist over long periods of time. (A) HD analysis on SF T cells from both right (R) and left (L) knees at two time points as shown, for four representative TCRBV families on samples from patient PA7. An asterisk marks bands which are visible at 1 year but not at time = 0. (B) HD analysis on CD4+ and CD8+ synovial T cells for three representative TCRBV families on samples taken 3 months apart from patient PA13.

 
Estimate of the sizes of expanded clonotypes from CD4+ or CD8+ synovial T cells.
To investigate the size of synovial T cell expansions seen on HD analysis, synovial T cells were used to amplify specific TCRBV families of interest. For this analysis, samples from patients PA13 and JAS3 were selected, and two TCRBV families which showed prominent HD bands on early exposure films were amplified for each patient. These were then analysed by subcloning and sequencing. Nucleotide and predicted protein sequences of a total of 104 the amplified TCR sequences for four TCRBV families analysed are shown in Table 2Go. Expanded clonotypes were confirmed in each case, as evidenced by multiple identical copies of TCR with specific CDR3 sequences, identical at the nucleotide level. In the sequences from TCRBV3 of CD8+ synovial T cells from patient JAS3, six of 37 (16.2%) of the whole family were represented by one sequence and five of 37 (13.5%) by another. Similarly, in the TCRBV14 amplified TCR from CD8+ synovial cells of JAS3, 17 of 41 (41.5%) of the TCRBV14 family was occupied by one clone, with several smaller clones also present (Table 2Go). The largest clonotypes of the CD4+ cells from patient PA13 represented four of 12 (33.3%) and five of 14 (35.7%) of TCRBV6 and TCRBV13.2 respectively. In each case, further smaller clones were also present. No identical protein sequences coded for by different VDJ nucleotide sequences, either within one TCRBV family or in different families, were demonstrated. Furthermore, we could not demonstrate conserved motifs across the CDR3 regions of the TCR analysed.


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Table 2. Nucleotide and predicted protein sequences across the CDR3 regions of TCR amplified from SF T cells of two patients with JIA
 
Expanded synovial clonotypes are below the threshold of detection in PBMC
Inflammatory T cells within the joint originate from peripheral blood by crossing the endothelium. To look for the specific clones which we had demonstrated in the synovial T cell population, within the PBMC of children with JIA, N-region probes for the four largest synovial T cell clones (one for each of the TCRBV analysed) were designed. HD membranes from the two patients who were analysed at the sequence level (PA13 and JAS3) were stripped and re-probed using these CDR3 specific probes. In each case, the N-region oligonucleotide hybridized specifically to the lane representing the SF T cell population (CD4 in the case of PA13 and CD8 for JAS3) of the appropriate TCRBV family, only. No hybridization of N-region oligonucleotides was detected in the corresponding peripheral blood T cell population. This indicated that within the PBMC these clones are at a frequency of <1 cell in 50,000 (15). In addition, no non-specific bands were seen in other TCRBV families on the same membranes or in the reciprocal T cell populations. In Fig. 4Go, a representative result using the HD membrane for patient JAS3 is shown probed with the standard TCRBC probe and also after reprobing with the N-region sequence of the largest TCRBV3 clone. Interestingly, the HD banding pattern seen after N-region sequence probing is different from that seen with the TCRBC probe. The TCRBC probe detects only HD which contain one strand of carrier DNA since the PCR is designed by a nested approach. In many of the SF T cell populations there are several TCR species at high frequency. In this situation, the largest clones themselves act as `co-carriers' for each other in the HD assay (14) giving rise to different dominant bands when probing is performed using an N-region oligonucleotide. The dense band at the bottom of Fig. 4(B)Go represents homoduplex TCR of the expanded clonotype. Homoduplex bands consistently run below the carrier DNA bands.



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Fig. 4. Dominant SF T cell TCR sequences are below the level of detection by HD in PBMC. HD analysis of PBMC and SF T cells from a child with HLA-B27+ arthritis, JAS3. (A) HD reactions for four TCRBV families probed with the standard TCRBC probe. After subcloning and sequencing of TCR from TCRBV3, an N-region oligonucleotide (gacagggaatttatgggc) specific for the largest clone was designed. (B) After stripping the same membrane and reprobing with the N-region probe, specific hybridization was seen only in the SF TCRBV3 lane. The gels have been precisely aligned by the top and corners. The dark band seen at the bottom of the gel (B, TCRBV3 SF) represents homoduplex of the clonal TCR which runs just below the main carrier band for the TCRBV3 product.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The major novel finding of this study is that we have shown a differential degree of clonal expansion for the CD4 and CD8 T cell populations in the inflamed joint. These expansions correlated with the different clinical forms of JIA under investigation, as well as the HLA risk alleles associated with the two types of disease. The HD assay was used to assess the overall receptor diversity across the TCR repertoire, initially in CD3+ cells as a whole, and then in T cells separated into CD4 and CD8 populations. Parallel HD analysis of T cells from different compartments or subsets allows confident identification of identical clonotypes. Using this approach, we have shown that the largest expanded clonotypes among the inflammatory T cells from children who carry the class I HLA risk allele HLA-B*2705 were within the CD8+ T cells. Other, distinct CD8+ HD bands were seen in some peripheral blood samples, although the number of these was not significantly greater than in healthy children. However, the hierarchy of dominant clones in the joint was in each case different from that seen in peripheral blood, indicating that the restriction of number of clones surviving within the joint is not simply due to a `filtering' of the largest clones present in peripheral blood. In those children with oligoarticular disease (each of whom was positive for at least one class II, HLA-DRB1 risk allele), we detected CD4+ T cell expansions in multiple TCRBV families in each case.

For both HLA-DRB1- and HLA-B27-associated disease, clonal expansions were confirmed by sequencing of a set of synovial TCR, and expanded clones used to design oligonucleotides with which to probe the whole TCR repertoire. We have previously observed that use of an N-region probe instead of the TCRBC probe increases the sensitivity of the HD assay ~10-fold for a specific T cell clone (L. R. Wedderburn and M. Maini, unpublished observations). Use of N-region oligonucleotide sequences derived from the TCR expressed by synovial T cell expansions, to re-probe the concurrent PBMC T cell repertoire of the same children, showed that the T cells expanded within the joint were below the level of detection of HD in blood T cells. This indicated that they reside at very low frequency in the periphery. This demonstration of significant enrichment of T cell clones within the joint compared to blood corroborates our earlier study in JIA in which we showed that the corollary can also be true: a large, CD8+CD57+ peripheral blood T cell expansion was shown to be under-represented in the inflamed joint (9).

It is interesting that we have seen either CD4+ or CD8+ synovial T cell expansions, depending on the clinical type of arthritis. It is known that the T cell expansions in peripheral blood, which accumulate with age but can also be detected in healthy young children, are predominantly within the CD8 population (10,11,16). These clones are thought to include memory/effector cells, typically specific for viruses including cytomegalovirus and Epstein–Barr virus (19,20). In contrast expansions of sufficient size to be detectable by spectratyping or HD in the CD4 population are rare, especially in children. There is now evidence that control of clone size, both during an acute immune response and chronically, is under different mechanisms for these two T cell populations (12,21).

Previous studies of juvenile arthritis have also shown that the synovial T cells contain expanded clones, but the majority of these used analysed unseparated T cells (57,22,23). The detailed reports by Thompson et al. focused on two particular families, TCRBV8 and 20, in SF and PBMC from JIA patients, and demonstrated expanded clonotypes in both CD4+ and CD4- T cell subsets. In these studies, children with oligoarticular and B27-related arthritis are not separated, and it is therefore difficult to compare these with our data (8,24).

While interpreting the findings presented here it is informative to compare studies of TCR clonality in the adult arthritides which have distinct HLA associations. Several previous reports in adult rheumatoid arthritis (RA) (strongly associated with HLA-DRB1 alleles) show CD4+ T cell clones in the SF (2527). The majority of these studies have not made a comparison of CD4 and CD8 populations simultaneously. There is also accumulating evidence for a systemic alteration of the CD4+ T cell repertoire in RA. Thus patients with RA and even their unaffected family members show clonal expansions of CD4+ peripheral blood T cells (28,29), and have evidence for impaired thymic function (30,31). We have not observed peripheral blood expansions of CD4+ cells in children with oligoarticular JIA, but have not as yet undertaken analysis of thymic function in these children.

Our data from children who carry the HLA-B27 allele are supported by previous reports of CD8+ expansions in the joints of adults with ankylosing spondylitis (32) and other B27-associated arthritides such as reactive arthritis (33). In a recent study of psoriatic arthritis, synovial T cell clones were seen in both CD8+ and CD4+ T cells, but again the largest clones were within the CD8+ T cells (34). There is evidence that patients with ankylosing spondylitis may also have a general alteration of TCR repertoire (35). Taken together these data strongly suggest that the MHC alleles associated with different forms of arthritis in both adults and children are exerting their effect through a T cell-specific mechanism, presumably involving interaction with the TCR.

An explanation for the demonstration of restricted oligoclonal TCR expression in the joint that is frequently suggested is that specific antigens (referred to as arthritogenic peptides) are being recognized by T cells within the synovium, leading to clonal expansion and T cell survival (33,36,37). In HLA-B27-related disease a range of candidate antigens including bacterial products and autoantigens have been studied, and HLA-B27-restricted persistent CD8+ clones recognizing these peptides have been derived (3840). Candidate antigens which are presented through class II MHC molecules to CD4+ T cells have also been described in RA (41). In support of candidate antigens which drive T cell clonotypes within the joint, several reports have demonstrated TCR sequences with shared CDR3 sequences at the protein level, but have arisen from different VDJ rearrangement events, suggesting antigenic selection in situ. In our study, sequence analysis did not reveal any common motifs of protein CDR3 sequences, although further sequencing of more clones may have revealed this.

While the possibility of intra-articular antigenic peptides which either recruit T cells or drive T cell survival in the joint remains, this theory has come under considerable criticism in recent years since the data remain conflicting (42,43). Several studies have suggested that the actual number of antigen-specific cells within an inflamed site is very low (44,45). It is therefore likely that in an inflammatory site there are mechanisms which support the expansion of T cell clones without requiring a TCR-driven signal: one such mediator is IFN-ß (46). In addition, memory T cells have a lower requirement for interaction with MHC than their naive counterparts (47,48). An alternative hypothesis to explain TCR clonality within the joint arises from the observation that cytokine-driven T cells may show a restricted use of TCR (49). For example, IL-15, known to be present in SF in some forms of inflammatory arthritis, induces proliferation and expansion of a restricted number of synovial T cell clones (49,50). Both IL-15 and other cytokines can have differential effects on CD4 and CD8+ T cells (51). Other mechanisms for differences in clone sizes between CD4+ and CD8+ cells may include a differential sensitivity to apoptosis (52) or expression of Fas ligand (53). In addition, the specific effects of particular MHC alleles may be exerted at the level of thymic selection rather that at the site of inflammation, a concept supported for the growing evidence for generalized abnormalities of TCR repertoire in patients with both RA and ankylosing spondylitis. Finally, particular MHC molecules may be able to exert peptide-non-specific effects themselves. The recent finding that the protein coded for by the disease-associated HLA-B*2705 allele can form disulphide-linked heavy chain homodimers without ß2-microglobulin (54) raises the possibility that B27 may interact with a large number of CD8 T cells, or even other cells (NK or CD4 + cells), perhaps in a peptide-non-specific manner (55).

In conclusion, we have shown that in different clinical subtypes of childhood arthritis, the distinct MHC risk alleles are paralleled by a difference in the degree of restriction in clonality of CD4 or CD8+ T cells found within the joint. The dominance of clone size is precisely maintained between different sites of inflammation and over long periods of time. We suggest that these data provide evidence in favour of a role for the specific interaction between MHC–peptide and the TCR, perhaps in driving the generation of the dominant expanded clones within the joint, in the pathogenesis of these arthritides.


    Acknowledgments
 
We thank the patients and their parents for allowing us to collect and use samples. We thank Dr M. Maini and Professor R. Callard for helpful discussions and reading the manuscript. This work was supported by the Wellcome Trust (L. R. W. and A. P.) and the Medical Research Council (H. V. and P. W.), and was undertaken at Great Ormond Street Hospital for Children NHS Trust, which received a proportion of its funding from the NHS Executive. The views expressed in this publication are those of the authors and are not necessarily those of the NHS Executive.


    Abbreviations
 
HD heteroduplex
JIA juvenile idiopathic arthritis
PBMC peripheral blood mononuclear cell
RA rheumatoid arthritis
SF synovial fluid
SFMC synovial fluid mononuclear cells
TCRBC TCR constant region
TCRBV TCR variable ß chain

    Notes
 
Transmitting editor: E. Simpson

Received 25 June 2001, accepted 6 September 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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