Molecular fingerprinting reveals non-overlapping T cell oligoclonality between an inflamed site and peripheral blood
Lucy R. Wedderburn,
Mala K. Maini1,2,
Alka Patel,
Peter C. L. Beverley1,3 and
Patricia Woo
Department of Molecular Pathology, University College, 46 Cleveland Street, London W1P 6DB, UK
1 ICRF Tumour Immunology Unit, University College, London W1P 8BT, UK
Correspondence to:
L. R. Wedderburn
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Abstract
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We have demonstrated a stable expansion of CD8+ T cells in the peripheral blood of a child with chronic arthritis. The expanded TCRBV family (TCRBV14) was enriched for CD57hiCD28 T cells. Sequencing of the TCRBV14 amplification products showed a TCR sequence which contributed 32% of the total TCR in the CD8+TCRBV14 population. Using the modified heteroduplex technique, the CD8+TCRBV14 cells showed a clonal pattern and these bands were restricted to the CD28 population. This method also detected multiple other clones within the CD8+ population but few in the CD4+ cells. The dominant TCRBV14+ clone was not detectable in synovial fluid T cells from two inflamed joints by CDR3 length analysis or heteroduplex probing, suggesting that this long-lived clone is excluded from inflammatory sites. Synovial fluid T cells showed an unexpected discordance of the CD28 and CD57 phenotype compared to peripheral blood mononuclear cells. T cells from both inflamed joints both showed marked oligoclonality in all TCR families and had almost identical heteroduplex patterns. Taken together these data suggest that some clones are actively excluded from inflamed sites in juvenile chronic arthritis, yet the pattern of restricted T cell expansion is shared between sites of inflammation.
Keywords: autoimmunity, clonal expansion, human, juvenile chronic arthritis, TCR
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Introduction
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The demonstration of oligoclonality of T cells within synovial fluid (SF) or tissue from children with chronic arthritis is often cited as evidence for specific proliferation, possibly antigen driven, of T cells within the inflamed site (13). It is now clear that the TCR repertoire of functionally distinct T cell populations (CD4 and CD8, CD45) is different both in terms of TCR V gene usage as well as in the size of clonal populations (47). Many studies have shown that T cells within inflamed joints show an activated or memory phenotype, expressing CD45RO, CD29, CD69 and HLA-DR (8,9). This could therefore cause an intrinsic bias in the expressed TCR at these sites.
Oligoclonal expansions within the CD8 T cell population were initially reported in the elderly (10) but subsequently in healthy young adults (11,12). These expansions frequently have the phenotype CD57hiCD28. The presence of clonal expansions in rheumatoid arthritis (13) has been used to suggest that these clones may have a pathogenic role in autoimmune disease. Little is known about the developing TCR repertoire in children and whether this differs in children with autoimmune phenomena. In order to dissect the role of T cells in the pathogenesis of arthritis, sensitive methods are required which allow tracking of T cells at a clonal level from different samples and within different T cell populations.
Using the modified heteroduplex technique, CDR3 length analysis and phenotyping, we have characterized the TCRBV repertoire in blood and SF T cells from a child with juvenile chronic arthritis (JCA) and have shown a long-lived T cell clone within the CD8+CD57hiCD28 population. In addition we have shown that this child has considerable oligoclonality in the peripheral blood mononuclear cells (PBMC) CD8 but not the CD4 compartment. In contrast to PBMC, the SF T cells show a highly activated phenotype, yet a discordance between the CD57hi and CD28 phenotypes. The expanded TCRBV14+ clone appears to be excluded from the inflamed joints. At these sites we have demonstrated a large number of expanded clones which are different from the oligoclonality of peripheral blood lymphocytes. We suggest that this non-overlapping oligoclonality of the TCR repertoire in inflammatory T cells compared to PBMC may reflect an intrinsic restriction which is a consequence of the particular subpopulation of cells which survives within such sites.
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Methods
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Peripheral blood and SF samples
Patient PA7, a female Caucasian, was attending Great Ormond Street Hospital, London with chronic arthritis. All samples were obtained with informed parental consent. SF was removed at clinically indicated arthrocentesis. Peripheral blood samples were obtained from PA7 at time 0, and at 3, 12, 15 and 18 month follow-up. SF was aspirated from two inflamed joints (right and left knees) at several time points. Data included in this paper arise from SF taken at the beginning of the study, and PBMC taken at times 0 and 3 months. The HLA-DR type of PA7 was HLA-DRB1*1301, 1601 and she was positive for the antinuclear antibody.
Preparation of mononuclear cells and purification of T cell populations
PBMC were isolated by Ficoll-Hypaque density centrifugation. For the preparation of mononuclear cells from SF, samples were treated with Hyaluronidase (Sigma, Poole, UK) 10 U/ml for 30 min at 37°C before isolation by Ficoll-Hypaque. CD4+ and CD8+ T cells were separated using directly conjugated anti-CD4 magnetic beads (Miltenyi Biotech), producing populations which were >93% CD4. Negatively selected cells were used as the CD8+ fraction. For CD28 separations, cells were incubated with anti-CD28 antibody (L293; Becton Dickinson) at 10 µg/ml for 25 min at 4°C, washed and then separated using anti-mouse IgG beads (Miltenyi Biotech), producing a CD28+ population which was 97% CD28+. Negatively selected cells were used as the CD28 fraction.
Immunofluorescence analysis of T cells
The following mAb were used in this study: anti-CD3FITC (Leu4), anti-CD4FITC (Leu3a), anti-CD8FITC (Leu2a), anti-CD57biotin (HNK1) and anti-CD28phycoerythrin (PE) (L293; Becton Dickinson, Mountain View, CA); anti-CD4PE (MT310; Dako, Carpinteria, CA); and anti-CD8Quantum Red (UCHT4), anti-HLA-DRPE (HK14) and anti-CD45ROQuantum Red (UCHL1; Sigma). StreptavidinQuantum Red (Sigma) or streptavidinPE (Becton Dickinson) were used to detect cells labelled with biotinylated antibodies. The anti-TCRBV mAb used were either FITC conjugated [TCRBV2 (E22E7.2), 3 (LE89), 5.1 (IMMU157), 6.1 (CR1304.3), 8 (56C5.2), 11 (C21), 12 (VER231.1), 13.1 (IMMU222), 13.6 (JU74.3), 14 CAS1.1.3), 16 (TAMAYA1.2), 17 (E17.5F3), 20 (ELL1.4), 21.3 (IG125) and 22 (IMMU546)] or unconjugated [TCRBV 9 (FIN9), 13.2 (H132), 18 (BA62.6) and 23.1 (AF23)] reagents (all from Immunotech, Marseilles, France). Anti-mouse IgGFITC (Sigma) was used to detect unconjugated anti-TCR antibodies. Cells were analyzed on a FACScan cytometer (Becton Dickinson) equipped with an argon laser exciting at 488 nm and using the CellQuest software. After live gating on forward and side scatter, between 10,000 and 20,000 events were collected per condition. Data is shown as logarithmic dot-plots.
RNA and cDNA synthesis
Total RNA was extracted from cells using RNAzolB (Biogenesis, UK). Then 5 µg of total RNA was used for first-strand cDNA synthesis using MMLV reverse transcriptase Superscript II (Gibco, Paisley, UK) and oligo(dT) (Boehringer Mannheim, Lewes, UK).
Heteroduplex analysis of TCRBV
Twenty-six PCR reactions, each using 1/60 of the cDNA, were performed for every sample. TCRBV family- or subfamily-specific primers were as described (14,15). DNA carriers consisting of cDNAs encoding TCRBV124 cloned from T cell clones or lines as described previously (15) were generously donated by G. Casorati and P. Dellabona (Milan, Italy). The 3' primer for patient samples was the internal TCRBC primer: 5'-CACCCACGAGCTCAGCTCCCGTGGTC. For carrier PCR reactions, an external TCRBC primer, 5'-TGCTGACCCCACTGTCGACCTCCTTCCCATT, which is 30 bp 3' to the internal primer, was used. PCR conditions were: 94°C for 5 min, 30 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1 min with a final 10 min extension period at 72°C.
Then 20 µl of each sample PCR product was mixed with 200 ng of the appropriate TCRBV carrier DNA, denatured at 95°C for 5 min and allowed to re-anneal at 50°C for 1 h. The mixtures were separated on a 12% polyacrylamide/0.5% TBE gel for 16 h at 10 mA and 4°C. Heteroduplex gels were blotted onto Hybond N+ (Amersham, Amersham, UK) in 20xSSC. The DNA was denatured and fixed in 0.4 M NaOH. DNA probes were end-labelled with [
-32P]ATP using polynucleotide kinase (Gibco). Membranes were pre-hybridized for 2 h in 6xSSC, 5xDenhart's, 0.1% SDS, 100 µg/ml blocking DNA at 42°C and hybridized overnight at 42°C in 6xSSC, 5xDenhart's. Washes were 5xSSC, 0.1% SDS for 20 min at 42°C, followed by 20 min in 1xSSC, 0.1% SDS at 42°C for the external TCRBC probe and at 50°C for the N-region probe. Filters were exposed to film at 70°C for 6 or 16 h. For reprobing, membranes were stripped by washing twice in 0.5% SDS at 75°C for 20 min.
CDR3 length analysis of TCRBV amplification products.
The same cDNA was also used as template for TCR amplifications using the following labelled TCRBV primers: TCRBV13.1, 5'-GACCAAGGAGAAGTCCCCAAT-HEX; TCRBV14, TCTCGAAAAGAGAAGAGGAAT-FAM; TCRBV17, CACAGATAGTAAATGACTTTCAG-FAM (Oswell, Southampton, UK) and the TCRBC primer: 5'-CTTCTGATGGCTCAAACAC. PCR conditions were: 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 45s. PCR products were separated on 6% acrylamide gels run at a constant 1000 V in ABI373 sequencers and the data analysed using GENESCAN software (Applied Biosystems, UK).
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Results
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A stable TCRBV expansion detected in PBMC was not demonstrated in SF T cells
Child PA7 presented at age 8 years with a pauciarticular form of arthritis and eye disease. Peripheral blood and SF from two inflamed joints were obtained from this patient on several occasions over 18 months. After separation over Ficoll, the SF samples contained 23x106 (right knee) and 5x106 (left knee) mononuclear cells. The SF samples PBMC and SF cells were initially analysed by flow cytometry after staining with CD4PE, CD8Quantum Red and a panel of 19 TCRBVFITC antibodies. An expansion was defined as expression of a particular TCRBV product greater than the mean + 2 SD of the values obtained from healthy children using the same antibodies (L. R. Wedderburn and A. Patel unpublished data). The PBMC were 81% CD3+, with a normal CD4+:CD8+ ratio of 1.7. For the SF samples these figures were left knee 94% CD3+, CD4+:CD8+ ratio of 1.0, and right knee 93% CD3+, CD4+:CD8+ ratio of 1.1. The PBMC of PA7 showed an expansion of TCRBV14-expressing cells within the CD8 population (15.3%; control range 6.10 ± 2.08, Fig. 1A
). TCRBV14 was not expanded in any SF samples (Fig. 1B and 1C
). The CD8+TCRBV14 expansion was stable in PBMC for 18 months and the overall TCR repertoire showed no significant changes over this time (data not shown).

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Fig. 1. Expression of TCRBV proteins on peripheral blood and SF T cells. Three-colour flow cytometry was performed on cells isolated from child PA7 using mAb to CD4, CD8 and a panel of anti-TCRBV antibodies. The percent positive for a particular TCRBV family or subfamily was calculated for CD4+ cells (white bars) and CD8+ cells (hashed bars) in each sample. (A) PBMC. (B and C) SF cells from left (SFL) and right (SFR) knees.
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Peripheral blood CD8 cells showed multiple clones by heteroduplex analysis
We used two molecular techniques, one based on the formation of heteroduplices due to junctional diversity and the other a CDR3 length assay, to analyse the TCR repertoires of PBMC and SF T cells. We have previously shown that the modified heteroduplex technique can detect clonal T cell expansions which are at a frequency of ~1:10,000 cells and at least an order of magnitude smaller that those detected by a CDR3 length-based assay (16). After RT-PCR using 26 different TCRBV primers and heteroduplex analysis, PA7 CD4+ and CD8+ PBMC showed divergent patterns. For CD8+ cells 15 out of 26 reactions showed clear heteroduplex bands suggestive of clonal expansions, while in the CD4+ reactions the majority of TCR families were polyclonal, with clear heteroduplex bands in only two families, TCRBV9 and 18 (Fig. 2
). Analysis of CD4 and CD8 reactions for selected TCRBV families on the same gel showed clear segregation of bands in these subpopulations (Fig. 3
). Because the pairing of each DNA strand with carrier DNA will produce two different heteroduplices, a single clone is generally detected as two bands on hybridization with the carrier specific probe, with a characteristic migration pattern. For these and subsequent heteroduplex data, the gels shown are one of two or three experiments on the same cDNA samples: in each case the band patterns obtained were highly reproducible.

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Fig. 2. Heteroduplex analysis of PBMC isolated from PA7. After RT-PCR using 26 TCRBV primers and heteroduplex reactions of PCR products each mixed with a known carrier PCR product, samples were analysed on 12% polyacrylamide non-denaturing gels for 16 h at 4°C. Gels were blotted and probed with a TCRBC probe. The figure shows reactions for 26 TCRBV families or subfamilies, with samples from CD4+ (upper panel) and CD8+ (lower panel) cells.
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Fig. 3. Comparative heteroduplex reactions using cDNA samples from total PBMC, CD4+ or CD8+ PBMC, and SF T cells. (A and B) TCRBV families 9 and 14 were analysed, as shown above each panel, and were probed with the carrier TCRBC probe. (C) The membrane shown in (B) was stripped and reprobed using a probe designed from the junctional sequence of the expanded T cell clone, (PA7-cl16). In the TCRBV14 reactions, the clonal homoduplex band (**) runs below the carrier homoduplex (*), and was strongly detectable in total PBMC and CD8+ PBMC, very weakly detectable in SF T cells, but absent in CD4+ PBMC.
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The clonal expansion contained a dominant sequence which was under-represented in SF
In the TCRBV14 reaction on CD8 cells, several strong heteroduplex bands were observed (Figs 2 and 3
). To confirm oligoclonality of the CD8+TCRBV14+ expansion, TCRBV14 amplification products from CD8+ PBMC were subcloned and a total of 25 inserts sequenced. Eight of these had an identical sequence across the VDJ junction while all other 17 transcripts were different (data not shown). This sequence is available from GenBank under accession no. AF100783. This sequence represented eight of 25, i.e. 32%, of expressed CD8+TCRBV14 TCR. Since at the single cell level by FACS analysis the BV14 expansion was 15% of CD8+ cells, this clone was ~5% of all CD8+ T cells. As for other systems based on PCR analysis of the T cell repertoire, this calculation makes the assumption of equal amounts of mRNA from different T cell clones; this may introduce a degree of error, since activation of T cells may result in increased transcription from the TCR loci (17).
A 29 bp N-region probe was designed from the dominant TCR clone. Probing of the membranes shown in Fig. 2
with this oligonucleotide showed hybridization in a single lane, that of the TCRBV14 in the CD8+ PBMC, indicating that this method allows specific identification of a single T cell clone from the whole repertoire (data not shown). N-region probing of the membrane shown in Fig. 3(B)
produced a strong lower homoduplex band, as well as one dominant heteroduplex band, running in the same position as that seen by external TCRBC probing, in the total PBMC and CD8 TCRBV14 reactions (Fig. 3C
). In SF, the homoduplex band was just detectable with the N-region probe, but any heteroduplices containing this N-region sequence were below the detection limit of the assay (Fig. 3C
). In addition, several new bands were seen in PBMC CD8 cells which had not been detected with the TCRBC probe. This suggests that the dominant clone may itself act as `carrier' in the heteroduplex reaction for other, smaller clones within the CD8 population. Such heteroduplices would not have been detectable on initial probing with the TCRBC external probe, which hybridizes with the 3' end of the carrier TCRBC sequence.
Analysis of CDR3 length of the TCR was performed on the same cDNA samples. Two PBMC samples taken 3 months apart and the CD8+ PBMC showed a highly skewed distribution in the TCRBV14 reactions, while CD4+ PBMC showed a normal distribution (Fig. 4
). The SF (SFR and SFL) distributions differed from a typical Gaussian curve, but did not show a peak at the length of the expansion seen in the CD8+ PBMC. This corresponded to a unique pattern of clonal heteroduplex bands seen in SF samples which was different from those in PBMC. The CDR3 length distributions in two control TCRBV families, 13.1 and 17, were Gaussian for both CD4+ and CD8+ PBMC (data not shown). The CDR3 length, 12 amino acids, of the dominant peak seen in CD8+ cells corresponded exactly to the predicted CDR3 length, as calculated according to Chothia et al. (18), of the CD8+TCRBV14 clonal sequence. Since this clone represented 5% of peripheral blood CD8+ T cells and the CD8+ cells were 33.8% of all CD3+ T cells, this clone was ~1.7% of peripheral blood T cells. This would be expected to give a clear peak in the CDR3 length assay. We have previously shown that in our hands the limit for detection of a clone within a polyclonal T cell population is ~1:1000 cells (16). The absence of a peak at the clonal CDR3 length within the SF spectratypes suggested a clonotype frequency of <1:1000 in SF, that is at least an order of magnitude lower than in peripheral blood.

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Fig. 4. CDR3 length analysis of PBMC and SF samples. TCRBV14 amplification products were run on a 6% acrylamide gel and analysed using GENESCAN software. A skew of the normal distribution was seen in PBMC from two time points (PBMC1 and PBMC2) and in separated CD8+ PBMC. There was no distortion of the Gaussian distribution of CDR3 lengths in CD4+ PBMC.
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The TCRBV14 expansion was CD57hiCD28
PBMC and SF T cells from three time points were stained with several markers of activation or differentiation, including CD45RO, HLA-DR, CD28 and CD57. In PBMC, the CD8+TCRBV14+ cells showed an increased number of HLA-DRhi, CD57hi and CD28 cells compared to other CD8 cells, while CD45RO expression was not significantly different between CD8 subpopulations (Fig. 5
). In addition the expression of CD45RA was not significantly different between these populations (data not shown). Analysis of cells gated on CD8 and CD57hi showed that TCRBV14 represented 42.9% of the CD8+CD57hi population; 96.3% of the TCRBV14+CD57hi PBMC were also CD28. To confirm that the clonal expansion of CD8+TCRBV14+ cells had the CD28 phenotype, cells were separated on magnetic beads into CD4 and CD8 populations or CD28+ and CD28 populations. After PCR and heteroduplex for TCRBV14, the clonal bands seen in the CD8 PBMC sample were also seen in the CD28 lane but were absent from the CD28+ sample (Fig. 6
). Reprobing of these membranes with the N-region probe as described confirmed that the clone was within the CD28 population (data not shown).

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Fig. 5. Enrichment of the CD8+TCRBV14 population for CD57+CD28. PMBC were analysed by flow cytometry using mAb to CD8, TCRBV2 or 14 and a panel of activation or differentiation markers as shown. For each sample, data were gated on CD8+ cells. The percent expression of each marker was then calculated for CD8 cells as a whole (white bars), CD8+TCRBV2+ cells (hashed bars) and CD8+TCRBV14+ cells (grey bars).
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Fig. 6. Confirmation that the expanded clone is of the CD28 phenotype. After separation on magnetic beads, heteroduplex analysis of TCRBV14 amplification products was performed on CD4, CD8, CD28+ and CD28 populations of PBMC as shown. The membranes were probed with the external TCRBC probe.
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SF T cells show dissociation of the CD57hi and CD28 phenotypes
By cytofluorometric analysis the majority of SF T cells were CD45RO+ and HLA-DRhi, and a high proportion were also CD25+ (data not shown). In addition the overall expression of the CD28 marker within CD4+ and CD8 cells was not significantly different between PBMC and SF. However the expression of CD57 was no longer consistently associated with a CD28 phenotype. Thus of those SF CD8 T cells which were CD57hi, only 26.6% were also CD28, while in PBMC CD8 cells, 91.9% of CD57hi cells were also CD28 (Fig. 7
). These differences may account in part for the apparent `exclusion' of the TCRBV14-expressing clone from the joint, since the CD57hiCD28 phenotype was rare in SF CD8 T cells.

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Fig. 7. SF CD8+ T cells show a discordance of CD57 and CD28 expression. PBMC and SF were stained with CD57biotin followed by avidincychrome, and then CD8FITC and CD28PE mAb. Both panels show cells gated on CD8. The left panel is of PBMC, the right of SF. Percentages of events are as shown in each quadrant.
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SF T cells consist of a restricted set of clones which are present in two distinct sites of inflammation
In contrast to the heteroduplex patterns seen in PBMC, both SF samples showed strong bands in the majority of TCRBV families (Fig. 8A
). In addition, analysis of the heteroduplex reactions from each SF sample (right and left knees) showed a striking degree of identity of clonal patterns in many TCRBV families. However, for the majority of TCRBV families, these were different from the bands seen in PBMC (Fig. 8B
). This demonstration of identical clones at a high frequency in the SF T cell population suggests that cells which can enter and/or survive in the inflamed joint are a highly selected subpopulation of the peripheral blood T cell pool.

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Fig. 8. Heteroduplex analysis of SF T cells. Gels were probed with a TCRBC probe. (A) Global analysis of SFL TCR showed marked oligoclonality in a majority of families. RT-PCR and heteroduplex reactions were performed for all 26 TCRBV families or subfamilies as shown. (B) SF T cells consist of a restricted set of clones which are present in two distinct sites of inflammation. Heteroduplex reactions from PBMC and SF from left (SFL) and right (SFR) knees were analysed simultaneously. Seven typical TCRBV families are shown. In each family, all of the heteroduplex bands present in one SF heteroduplex reaction are detectable in the other SF sample, but a minority of these bands are present in PBMC.
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Discussion
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In this study we have demonstrated a stable T cell expansion in the peripheral blood CD8 population of a child with JCA. This clone constituted ~5% of the total CD8+ cells. By cytofluorometric analysis the phenotype of the CD8+TCRBV14+ population differed significantly from other CD8+ peripheral T cells. Thus, in the CD8+TCRBV14+ population there was a higher expression of HLA-DR and a large number of CD57hiCD28 cells. This is in keeping with previous reports of long-lived stable CD8 expansions which have the phenotype CD57hiCD28 (19,20). Some of these expansions have been associated with viral infection (21) or bone marrow transplantation (22) and they have also been reported in healthy individuals (10,12). Recent data using MHCpeptide tetramer staining support the idea that chronically stimulated antigen-specific T cells acquire a CD28CD57hi phenotype (23).
In the case of our patient, a CD8 expansion was present in peripheral blood at the time of active autoimmune disease. Oligoclonal expansions have been described in adult patients with rheumatoid arthritis (13,24,25) as well as in patients with reactive arthritis (26) and JCA (2). The expansions associated with Felty's syndrome in adults with rheumatoid arthritis are of the CD57hi `large granular lymphocyte' phenotype and these have been suggested to have a pathogenic role in this condition (27). By cytofluorometric analysis the TCRBV14 family was not expanded in the SF CD8 T cells of this patient. We therefore characterized the TCR expression and repertoire in PBMC and SF at a molecular level. Heteroduplex analysis of PBMC showed that several TCRBV families contained strong bands within the CD8 cells which were absent in the CD4 population. Only two TCRBV families had clear clonal bands in CD4 PBMC: these were not due to `carry over' of CD8 cells during the cell separation, since they were not observed in the CD8+ population when analysed simultaneously. These data confirm previous observations that the CD8 TCR repertoire differs considerably from that of CD4 cells (5,6). However, in our hands it is unusual to see so many clonal bands on heteroduplex analysis of fresh PBMC from children (L. R. Wedderburn, unpublished observations).
CDR3 length analysis of TCRBV14 showed a clear skewing of the normal distribution in CD8 PBMC, which was also demonstrable in the total PBMC, but not CD4 cells or SF T cells. Sequencing of TCRBV14 amplification products from CD8 PBMC showed a TCR which comprised 32% of the CD8+TCRBV14+ population and the CDR3 length of this sequence corresponded to the length predicted from the peak seen in the CDR3 length assay. The clonal heteroduplex patterns seen in TCRBV14 within the peripheral blood were initially not detectable in SF. On reprobing using the more sensitive N-region clonotypic probe, a weak homoduplex band was demonstrated in SF samples, though heteroduplex bands were still not seen. Together, these data suggested that the clone is at much lower frequency in SF than in peripheral blood and that it is either selectively excluded from, or unable to survive within, two sites of active inflammation.
Many studies have suggested that T cells within an inflamed site are able to enter by virtue of their memory or activated phenotype (8,10). In addition, selective expression of receptors for adhesion molecules such as the receptors for P- and E-selectin (28) or chemokine receptors such as CCR5 or CXCR3 (2931) may preferentially recruit particular subsets of T cells, such as Th1-like cells, to inflamed sites. It is possible that such selection will also restrict the expressed TCR repertoire. Certainly, there is evidence to suggest that clones detectable after normal antigenic stimuli are generally within the CD45RO population (6). However, some data also suggest that antigen-specific clones can be detected in both the RO and RA populations (32).
By heteroduplex analysis of separated cells we have shown that the clonal expansion was in the CD28 population. The CD8+CD28 population has been shown to be associated with shortened telomeres compared to CD8+CD28+ cells, suggesting that they may have reached a state of `replicative senescence' (33). In addition, there is evidence that CD8+CD57hi cells proliferate poorly and have different responses to mitogenic and apoptotic stimuli in vitro (34,35). The mechanisms for persistence of these expansions remain unclear. The CD57hi phenotype is generally found on CD28 cells within the CD8 population (20) and an increased number of CD4+CD28 expansions has been demonstrated in rheumatoid arthritis (36). One report of CD4+CD57hi expansions in patients with chronic lymphocyte leukemia refers to maintenance of CD28 expression on these clones (37), but there are few reports of the expression of CD57 and CD28 on T cells from inflammatory sites. We observed a reversal of the CD57hi phenotype and CD28 expression in SF T cells compared to PBMC. It is possible that the continued expression of CD28 provides these cells with a signal which prolongs their survival in the joint, in part by up-regulation of proteins which protect against apoptosis, such as bcl-xL (38).
While this specific clone was difficult to detect in SF, the heteroduplex analysis of the SF T cells was striking in that it showed a large number of other strong clonal bands in every TCR family. In each case the patterns were different from those seen in whole PBMC. However, the two samples of SF T cells, obtained from left and right knees, showed a high degree of identity of clonal bands, indicating that both sites contained a restricted number of clones compared to the peripheral TCR repertoire and these were closely overlapping sets of clones in the two sites. Many studies have previously compared TCR usage of SF T cells and PBMC in the joints of patients with arthritis, in both adults (39,40) and children (1,3). Such data have been suggested to indicate specific antigenic recognition in these sites. Our data could be interpreted in a similar way. However, the demonstration of identical clones at a high frequency in two joints may suggest that cells which can enter and survive in the inflamed joint are simply a highly selected subpopulation of the peripheral blood T cell pool. Whether this is due to the high proportion of CD45RO+, HLA-DR+ cells compared to PBMC, or other differences, such as the reversal of the CD57hi CD28 phenotype, is currently under investigation.
In conclusion, we have demonstrated a clonal expansion in the blood of a child with chronic arthritis which represents ~5% of the peripheral CD8+ T cells, yet <0.1% of T cells from two different inflamed joints, although it is detectable by a highly sensitive heteroduplex method. In addition we have shown a marked restriction of the TCR expressed in these two joints compared to blood. While it is possible that a tendency to produce large and persistent T cell clones may be associated with autoimmunity, our data suggest that at least this clone is unlikely to be directly pathogenic in active joint destruction, since it is either unable to enter the sites of inflammation or does not survive there. We suggest that all studies showing a skewed TCR usage in T cells from an inflammatory site need to be interpreted with caution, since such a skew could be intrinsic to particular subpopulations of T cells, which may express a restricted TCR repertoire for historic reasons, rather than antigen-specific recognition within the sites of inflammation.
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Acknowledgments
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We would like to thank Professor W. Ollier's laboratory for HLA typing, Dr R. Fellowes for reading the manuscript, and finally Dr Frances Hall and Professor P. Wordsworth for help with the spectratyping experiments. L. R. W. is a Wellcome Trust Career Development Fellow. P. W. is supported by the Medical Research Council, UK. M. M. was a Medical Research Council Training Fellow.
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Abbreviations
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JCA | juvenile chronic arthritis |
PBMC | peripheral blood mononuclear cells |
PE | phycoerythrin |
SF | synovial fluid |
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Notes
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Transmitting editor: E. Simpson
2 Present address: Department of Sexually Transmitted Diseases, University College, Mortimer Market Centre, London WC1E 6AU, UK 
3 Present address: The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK 
Received 7 August 1998,
accepted 10 December 1998.
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