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,§
*
Departments of Medicine and Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206;
NeXstar Pharmaceuticals, Inc., Boulder, CO 80301; and Departments of
Medicine and
§
Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain gene
(TCRA) and TCRB sequences. Together,
these studies suggest that a significant proportion of synovial
CD4+ T cells has been selected and expanded by conventional
Ag(s) in this disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerns have been expressed with regard to the importance of
CD4+ T cells in the pathogenesis of RA, especially the role
of this T cell subset in the perpetuation of advanced disease (reviewed
in Refs. 7 and 15). These arguments are based primarily on studies
showing that T cell-derived cytokines are much less abundant than other
proinflammatory cytokines such as IL-1 and TNF- in chronically
involved joints (15, 16). Furthermore, therapies directed against T
cells and T cell products have not been remarkably effective (17, 18, 19, 20, 21),
especially when compared with therapies directed against TNF- (16, 22, 23). Finally, studies have yet to identify the stimulating synovial
Ags in patients’ joints.
Since the stimulating synovial Ags in RA are unknown, studies have focused on whether the TCRs expressed by synovial T cells show a pattern characteristic of Ag-stimulated populations. The results to date, however, have not been clear-cut. Without question, the repertoire of synovial T cells is markedly heterogeneous (reviewed in 24 . Several studies have shown an increased expression of particular TCR variable (V) regions. However, there has been little consistency between reports, and different TCR V genes have shown increased utilization in different studies. Other studies have noted particular TCR ß-chain gene (TCRB) clonotypes, suggesting clonal expansions within the synovial T cell population. Certain amino acid motifs within the TCR complementarity-determining region 3 (CDR3) have been described in individual patients and among different patients, and these motifs have been used to suggest selection by a similar Ag (24, 25, 26). However, these studies have not found highly homologous receptors as found after peptide immunization in animals, and the significance of these motifs in terms of reflecting recognition of the same Ag remains unclear. Finally, despite a paradigm that involves the same presenting HLA-DR4 molecules, no single TCR or clearly related TCR has been identified in different patients.
Here we report our analysis of the CD4+ T cell repertoire
in synovial fluid from different joints and peripheral blood in
patients with advanced RA. The results show that despite the marked
heterogeneity of synovial CD4+ T cells, a large fraction of
the TCR repertoire is identical in different joints but not in blood of
individual patients. Within the TCRBV regions studied, increased
expression of particular TCRB clonotypes was found in
synovial fluid, which is indicative of in vivo clonal expansions.
Furthermore, we found separate synovial CD4+ T cell clones
that use highly homologous TCR -chain (TCRA) and
TCRB genes. In several instances, these clones used
different CDR3 nucleotide sequences to encode identical TCRs. This
coselection of both TCRA and TCRB genes expressed
by particular T cell clones in the synovial fluid of individual
patients strongly suggests that these cells have been selectively
stimulated and expanded by a conventional Ag(s) in this disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All patients met the American College of Rheumatology criteria
for the classification of RA (27) and were studied under the guidelines
of the human subjects institutional review board at the National Jewish
Medical and Research Center (Denver, CO). Various characteristics of
these patients are shown in Table I
. All
patients had active inflammatory disease at the time of participation.
Arthrocentesis of two joints (both knees) and phlebotomy were performed
on the same day. Mononuclear cells from the peripheral blood and
synovial fluid were isolated by Ficoll-Hypaque density gradient
centrifugation (Pharmacia, Piscataway, NJ). EBV-transformed B cell
lines were prepared by infecting PBL with supernatant from the
EBV-secreting marmoset cell line B95-8 (28) in the presence of 0.5
µg/ml cyclosporin A (Sandoz, East Hanover, NJ). These lines were used
for HLA-DR typing, which was performed using standard serologic and
molecular techniques at the Clinical Laboratories of Denver Immunologic
Associates (Denver, CO).
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The percentages of CD4+ T cells in peripheral blood and synovial fluid that express different TCR Vß-chains were determined by two-color immunofluorescence staining and cytofluorographic analysis. CD4 expression was determined using a fluorescein-conjugated mAb to CD4 (Becton Dickinson, San Jose, CA). TCR Vß expression was determined with biotinylated mAbs directed against Vß2 (clone E22E7.2, Immunotech, Westbrook, ME) (29), Vß3.1 (clone 8F10, T Cell Diagnostics, Cambridge, MA) (30, 31), Vß8.1/8.2 (clone MX-6, T Cell Diagnostics) (32), Vß14 (clone CAS1.1.3, Immunotech) (33, 34), and Vß17 (clone E17.5F3, Immunotech) (29). The biotinylated anti-Vß mAbs were detected with either Cy-Chrome-streptavidin (PharMingen, San Diego, CA) or phycoerythrin-streptavidin (Southern Biotechnology Associates, Birmingham, AL). Fluorescence intensity was analyzed on a FACScan (Becton Dickinson), and the percentage of CD4+ T cells bearing each Vß was determined.
Analysis of TCRB gene sequences
Peripheral blood and synovial fluid mononuclear cells were double stained with fluorescein-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8 mAbs (Becton Dickinson). CD4+/CD8- cells were sorted using an EPICS 751 or Elite cytofluorograph (Coulter, Hialeah, FL). The cell pellet (>100,000 cells) was immediately frozen, and total cellular RNA was isolated using a commercially available kit (Ultraspec RNA Isolation System, Biotecx Laboratories, Houston, TX) according to the manufacturer’s directions. This RNA was used for the synthesis of first-strand cDNA using reverse transcriptase (Superscript RT, Life Technologies, Grand Island, NY) and random hexamers (Pharmacia). One-twentieth of the cDNA from each sample was added to a 50-µl reaction mixture containing forward TCR Vß gene segment (TCRBV) and reverse TCR constant region gene segment (TCRBC) primers (each at a final concentration of 0.3 µM), and PCR amplification (using AmpliTaq or AmpliTaq Gold, Perkin-Elmer, Branchburg, NJ) was performed for 35 cycles under the following conditions: 95°C melting, 55°C annealing, and 72°C extension, each for 1 min, followed by a final extension step at 72°C for 10 min. The primer sequences were: TCRBV2, 5'-TCATCAACCATGCAAGCCTGACCT-3'; TCRBV3, 5'-GTCTCTAGAGAGAAGAAGGAGCGC-3'; TCRBV8.1, 5'-ATTTACTTTAACAACAACGTTCCG-3'; TCRBV14, 5'-GTCTCTCGAAAAGAGAAGAGGAAT-3'; TCRBV17, 5'-CAGATAGTAAATGACTTTCAG-3'; and TCRBC, 5'-TTCTGATGGCTCAAACAC-3'. Each PCR product was ligated into the pCRII TA cloning vector as specified by the manufacturer (Invitrogen, San Diego, CA), and the ligation product was transformed into Epicurian coli XL-1 Blue supercompetent Escherichia coli cells (Stratagene, La Jolla, CA). Colonies containing appropriate inserts were chosen randomly for sequencing. Cycle sequencing was performed using the Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Perkin-Elmer, Foster City, CA) and automated sequencer (Applied Biosystems/Perkin-Elmer). Sequencing primers included an M13 reverse primer, 5'-CAGGAAACAGCTATGAC-3' (Promega, Madison, WI), and a M13 forward primer, 5'-CCCAGTCACGACGTTGTAAAACG-3' (Life Technologies).
Cloning of synovial fluid CD4+ T cells and analysis of expressed TCRB gene sequences
To further analyze the TCRB repertoire and to identify the TCRA coexpressed in particular T cell clones, synovial fluid mononuclear cells were stained for CD4 and the TCR Vß of interest as described above, and double-positive cells were sorted and cloned by limiting dilution. Variable numbers (1, 3, or 10/well) of sorted T cells and 5 x 104 irradiated (9000 rad) TK6 feeder cells/well were cultured in 96-well microtiter plates (Falcon, Becton Dickinson Labware, Bedford, MA) in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (HyClone, Logan, UT), 20% HL-1 culture medium (BioWhittaker), 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Life Technologies), 0.25 µg/ml PHA (Murex Diagnostics, Dartford, U.K.), and 10% T cell growth factor supernatant (a gift from R. J. Albertini, University of Vermont, Burlington, VT) (35). After 12 to 14 days of culture, T cell colonies were transferred to 1-ml cultures. Cells from confluent cultures were harvested, and total cellular RNA was isolated using a commercially available kit (RNaid PLUS, BIO 101, La Jolla, CA). cDNA was prepared, and the TCRB gene fragments were amplified using the TCRBV primer corresponding to the Vß sorted and TCRBC primer as described above. Each PCR product was purified using a DNA binding membrane spin column (QIAquick PCR Purification Kit, Qiagen, Chatsworth, CA) and sequenced using a TCRBC sequencing primer (5'-CGACCTCGGGTGGGAACA-3').
Analysis of TCRA genes expressed in T cell clones
RNA from T cell clones that expressed the TCRB of interest was then used for anchored PCR amplification of TCRA cDNA using the rapid amplification of 5' cDNA end system (Life Technologies). Briefly, TCRA first strand cDNA was synthesized using a TCRAC-specific primer (5'-TTCAGTCTAAACAACGAGGTC-3') and SuperScript II reverse transcriptase (Life Technologies). RNA was then digested with a RNase mixture, and the cDNA was purified using a silica-based membrane column (GlassMax DNA isolation spin cartridge, Life Technologies). A homopolymeric dC tail was then added to the cDNA using dCTP and terminal deoxynucleotidyl transferase. This dC-tailed cDNA was amplified for 35 cycles of PCR, using the abridged anchor primer provided by the manufacturer and a second nested TCRAC primer, 5'-GAACTTCAGGTATCTGGAGTA-3'. A second round of 35 cycles of nested PCR was then performed using the abridged universal amplification primer provided, a third nested TCRAC primer, 5'-TCAGAGAGTCGACCATGTGCC-3', and 5 µl of first round product.
The anchored PCR product was ligated into pCRII and cloned as described above. The insert was sequenced as described above using a TCRAC sequencing primer (5'-TGGTACACGGCAGGGTCAGG-3'). The CDR3 sequence of each clone’s expressed TCRA was verified by performing a TCRAV-specific PCR using the appropriate TCRAV primer (TCRAV1, 5'-CTGAGGTGCAACTACTCA-3'; TCRAV5, 5'-GGCCCTGAACATTCAGGA-3'; TCRAV8, 5'-GGAGAGAATGTGGAGCAGCATC-3'; TCRAV11, 5'-AGAAAGCAAGGACCAAGTGTT-3'; or TCRAV16, 5'-CATCACAGGGGATAACCTGGT-3') and the nested TCRAC primer described above. The subsequent PCR products were then sequenced using the TCRAC sequencing primer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Paired synovial fluid (knee) samples as well as peripheral blood
from three individual patients with long-standing, active RA were
studied (Table I
). We first determined the percentage of
CD4+ T cells that expressed particular TCR Vßs using
immunofluorescence staining and cytofluorographic analysis. For these
studies, we used a limited panel of anti-Vß mAbs directed to
certain V regions suggested to be important in RA, including Vß2,
3.1, 8.1/8.2, 14, and 17 (24, 36, 37, 38, 39, 40, 41, 42). As shown in Figure 1, the synovial CD4+ Vß
repertoire was generally similar to that of PBL, although there were a
few exceptions. For example, in patient CS-2, there was a greater than
twofold increase in the percentage of cells expressing Vß3.1 in the
synovial fluid compared with peripheral blood. A similar finding was
noted in patient CS-3, who demonstrated an increased percentage of
Vß8.1/8.2+ cells in both joints compared with that in
blood. There was one example of a larger Vß8.1/8.2+
subset in blood compared with synovial fluid lymphocytes in patient
CS-2. None of the patients demonstrated a consistent decrease in the
synovial fluid Vß percentages compared with those in blood. The
absence of such reciprocal changes suggests that another synovial
subset expressing a Vß not studied in this analysis was unlikely to
be greatly expanded and dominate the synovial population.
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We sorted freshly isolated synovial fluid and peripheral blood
CD4+ T cells from each patient to analyze the
TCRB gene repertoire expressed in these lymphocyte
populations. cDNAs were PCR amplified with TCRBV-specific primers that
corresponded to the five Vß subsets studied above. The PCR products
from each sample were then cloned, and randomly selected bacterial
isolates containing the appropriate insert were sequenced to determine
the nucleotide and amino acid sequences of the CDR3. Sequences of
interest for each patient are ordered by TCRBV and TCRBJ gene segment
usage in Figure 2. This figure includes
identical nucleotide sequences found at least three times, sequences of
a clone closely related to or identical with another sequence in the
same fluid, and sequences matching in the other fluid or in blood
(indicated by bold type). When the same CDR3 amino acid sequence is
shown more than once (such as the TCRBV14-LGTEG-BJ1S2 (BJ1.2) sequences
in Fig. 2A), a different CDR3 nucleotide sequence (i.e.,
expressed by a different clone) was used to encode the identical amino
acid sequence (see below).
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and
data not shown). However, repeated sequences of individual clonotypes
were clearly apparent. In these three subjects studied for five
different TCRBV subsets, >40 clones occupied >5% (three or more
repeated sequences) of the total sequences within a TCRBV subset in a
single joint. The probability for one set of three repeated sequences
to be found by chance alone (assuming 
5,000 cells within a
particular TCRBV subset from >100,000 CD4+ cells sorted)
was calculated to be p = 1.6 x 10-7.
A few synovial clones were quite large and comprised nearly 50% of a
particular TCRBV family, such as the BV3-SLLQQAGKA-BJ2.3,
BV14-LFLAGG-BJ2.2, and BV17-SPAGV-BJ2.2 clones in patient CS-2. In
general, repeated sequences were not present in peripheral blood,
although one clonal expansion was found only in the blood BV17 subset
of patient CS-2. We obtained synovial fluid from one patient 6 mo after the initial analysis to determine whether large clones persist in patients with continued evidence of inflammatory arthritis. In patient CS-2, a CD4+ clone that initially occupied 45% of the TCRBV3 subset was noted to actually increase in frequency to 71% (44 of 62 sequences) of the left knee TCRBV3 sequences at the later time point. Similarly, the large Vß17+ clone in this individual persisted at about 50% (31 of 64 sequences) of the left knee TCRBV17 sequences. These data from one individual suggest that the synovial fluid TCR repertoire may remain relatively constant over time in patients with continued arthritis.
Different TCR sequences were considered to be related if they had
identical TCRBV and BJ gene segments and highly homologous CDR3 amino
acid sequences. We also required the length of the CDR3 be identical,
since this has been shown to be important in TCR recognition of Ag (43, 44). Related sets of sequences were identified within the BV2, BV3, and
BV14 subsets of CS-1, within the BV8 and BV14 subsets of CS-2, and
within the BV3, BV8, and BV17 subsets of CS-3 (Fig. 2). More related
sets of sequences were also probably present but had other slight
variations in BJ usage or length of the CDR3. The ultimate forms of
related TCRs were those that expressed different TCRB genes
(defined as having at least two nucleotide differences in the CDR3) but
identical TCR ß-chain amino acid sequences. These are shown in Figure 2 as repeats of the same amino acid sequence. Examples were found in
each patient. Based on the enormous potential sequence diversity within
a TCRBV subset (45, 46), related sequences, as defined above, are
predicted to occur very rarely by chance alone (p
<< 10-6) and almost certainly represent selection by Ag.
No related clones were found in the peripheral blood of these three
patients.
The most striking set of related synovial clones was apparent in the
TCRBV14 subset of patient CS-1 (Figs. 2A and
3). Overall, there were 10 different
related clones that had the BV14-LGTEG-BJ1.2 related motif, and half
were found in both joints studied. We identified four different
nucleotide sequences that encoded the identical LGTEG junctional amino
acids and identical TCR ß-chain, and three different nucleotide
sequences that encoded the identical PGTEG-containing TCR ß-chain.
These receptors used differing aliphatic leucine (L), proline (P),
alanine (A), or aliphatic hydroxyl (serine; S) amino acids at position
94 and either glycine (G) or asparagine (N) at position 98 of the TCR
ß-chain sequence, but maintained a core CDR3 sequence of glycine (G),
threonine (T), and glutamate (E) as well as CDR3 length. It is
important to note that PCR sequence errors did not account for the
variation in the CDR3 nucleotide sequence. Thus, in these 28 different
cDNA clones, analysis of the invariant TCRB segments (i.e,
BV, BJ, and BC) showed only one sequence error in over 10,000
nucleotides sequenced (data not shown). As shown in Figure 2A, the set of related BV14 clones comprised approximately
25% of the BV14 subset in the two joints of patient CS-1. Considering
that Vß14+ cells were 3 to 4% of the synovial
CD4+ T cell population (Fig. 1), this set of related clones
comprised <1% of the total CD4+ T cells within the knee
joints of this patient.
Our analysis of TCRB gene sequences also showed that a
relatively large fraction of the TCRs is identical in different joints,
but there is little overlap with T cells in the circulating pool (Fig. 2 and Table II). For example, in patient
CS-1, 29 of 159 clones (18%) in the right knee joint were also present
in the left knee, but none was present in the peripheral blood. The
data in Table II are expressed as the fraction of total sequences
(rather than clones) that were shared among different joints and
peripheral blood lymphocytes. For example, in patient CS-2, 61 of 189
sequences (32%) in the right knee matched sequences in the left knee
compared with none in the blood (p < 5 x
10-10, by Fisher’s exact test). Overall, the data showed
that the synovial T cell repertoire is not random and is not a passive
reflection of the circulating T cells.
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) failed to
accurately predict the presence of large clones identified by sequence
analysis. For example, large clones were present in the left knee
TCRBV3 and BV17 subsets in patient CS-2, but only the percentage of
Vß3+ cells was increased in the synovial fluid compared
with the blood. Also, the percentage of synovial Vß8+
cells was increased in synovial fluid relative to that in blood in
patient CS-3, but no large clones were apparent by sequence analysis. Analysis of TCRA sequences coexpressed in T cell clones
Selection by the same conventional Ag should be reflected in both
the TCRA and TCRB gene sequences coexpressed by
related T cell clones. Therefore, we cloned synovial CD4+ T
cells by limiting dilution, identified those clones that expressed
related TCRB sequences, and sequenced the coexpressed
TCRA gene segments. We first focused on the large set of
related BV14-LGTEG-BJ1.2 clones in patient CS-1 by cloning synovial
fluid CD4+ Vß14+ T cells from this patient.
Figure 4 shows the sequences of the
TCRA genes coexpressed in six clones that have this related
CDR3 motif. Two of the six (no. 34 and 337) expressed identical
TCRB and TCRA gene sequences, confirming their
derivation from the same precursor clone. The relatedness of the
TCRA sequences expressed by the five unique clones was
clearly evident (Fig. 4). Thus, each TCRA used AJ22 of about
60 TCRAJ gene segments to choose from in the genomic complex (47).
Although three different TCRAV gene segments were used by these clones,
all were closely related. All but one of the clones expressed TCRAV1S2
or TCRAV1S3, which are closely related subfamily members, with 75%
nucleotide sequence identity. One clone (no. 314) expressed TCRAV16S1,
which has about 72% identity with the TCRAV1 subfamily at the
nucleotide level (48). Finally, the TCR -chain CDR3s of these clones
were remarkably similar, with conserved length and only slight
variation in sequence. All shared the CDR3 motif CAVRXSGSARQL, with
only one variable (X) position. Interestingly, in both cases where an
arginine (R) was used in the variable position of the -chain CDR3
(clones 109 and 34/337), an asparagine (N) was used at position 98 of
the ß-chain CDR3.
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). Two of the sequences were identical
with those expressed by T cell clones characterized above (clones
34/337 and 325). In addition, three unique sequences were found, which
maintained the same CDR3 length and invariant CAVRX sequence. One used
TCRAJ44 rather than TCRAJ22, but the amino acid sequences encoded by
these AJ gene segments were similar, with mostly conservative
substitutions.
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. The first set of related clones
all expressed TCRBJ2S3 in conjunction with TCRBV3 (Fig. 6, upper). The amino acid, but not nucleotide, sequence of the
CDR3 in the first two clones were identical, and the third clone showed
two conservative amino acid changes, leucine (L) to serine (S) and
serine (S) to threonine (T). The TCRA sequences also showed
conservation of CDR3 length, used TCRAJ10, and used either TCRAV5S1 or
11S1. There was one nonconservative change in clone 103 (glycine (G) to
glutamate (E)), but other changes were conservative (leucine (L) to
valine (V) or threonine (T) to valine (V)).
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, lower). The
TCRB sequences again showed conserved CDR3 length, and
whereas all used the TCRBV3 gene segment, they each used a different
TCRBJ gene segment. The ß-chain junctional region amino acid sequence
was identical in two of the clones, and two others showed only single
residue changes. The corresponding -chain sequences were also
strikingly similar, and two junctional regions were identical. All
clones used TCRAV8S1 and showed conservation of CDR3 length, and three
of the four used TCRAJ9. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and were
chosen based on past studies that suggested their possible importance
in this disease (24, 36, 37, 38, 39, 40, 41, 42). We also addressed aspects of TCR
repertoire analysis that would be least subject to PCR amplification
variation, including the search for related (rather than identical)
clones in synovial fluid, the matching of TCR sequences in the synovial
fluid from a separate joint and in blood, and the analysis of
coexpressed TCRA sequences for relatedness. The results
clearly show that a significant fraction of the synovial fluid
CD4+ TCR repertoire is distinct from that of the
circulating resting pool. Furthermore, the results demonstrate the
accumulation in synovial fluid of clonal expansions that persist over
time and document sets of related synovial CD4+ T cell
clones with highly homologous TCRB and TCRA
genes. In the first stage of our analysis of the synovial CD4+ T cell repertoire in RA, anti-TCR Vß specific mAbs were used to accurately quantitate Vß expression. The results showed a synovial repertoire not so different from that found in blood T cells, which is consistent with the overall heterogeneous TCR expression in synovial fluid. The absence of reciprocal decreases in Vß percentages in synovial fluid vs blood also suggested that a large expansion within a different Vß subset is unlikely. For example, if 30% of the synovial T cells expressed a Vß not analyzed, we would have expected to find, on the average, a 30% decrease in the percentages of T cells expressing Vßs 2, 3.1, 8.1/8.2, 14, and 17 relative to peripheral blood T cells. Although immunofluoresence staining has the potential advantages of being relatively rapid, easily performed, and accurate for determining the size of a Vß subset, these data appeared to be somewhat misleading. Most importantly, immunofluorescence analysis of TCR expression did not accurately predict the presence of large clones or sets of related clones in synovial fluid.
We matched the expressed TCRB sequences from one joint with those in a second synovial fluid from the same patient and with those in blood. Remarkably, 20 to 30% of the sequences from the two joints matched. The absence of any matches in blood, concomitantly analyzed, provided a convincing control for the possibility of PCR contamination. Although no matches in blood were found, previous studies, including those from our laboratory, have suggested that synovial CD4+ clones may rarely be present in the circulating pool (36, 49). This may explain how T cell clones travel to different joints, although the original source of the distributed synovial clones is unknown. The extent of TCR matching in different joints is surprising. In studies of experimental autoimmune encephalomyelitis, pathogenic T cells appear to comprise only a small fraction of the total T cell repertoire in involved central nervous system tissue (50, 51, 52). Nonspecific influx of T cells into the areas of inflammation has been postulated to account for these findings in experimental autoimmune encephalomyelitis. Based on the extensive amount of inflammation occurring in rheumatoid synovium, a similar nonspecific influx should be occurring, which would result in dilution of the relevant clones.
Within two synovial compartments of an individual patient, the accumulation of related CD4+ T cell clones was apparent. We found multiple examples of different T cell clones, i.e., with different TCRB nucleotide sequences, encoding the same TCR ß-chain amino acid sequence. Sets of related TCRBsequences with the same BV and BJ gene segments as well as highly homologous CDR3s were also found. Furthermore, a number of these related clones could be isolated by limiting dilution culture after sorting for CD4+ Vß+ synovial T cells. The coexpressed TCRA gene sequences were also shown to be related to each other. We believe that these results provide the most convincing evidence that a subset of synovial CD4+ T cells have accumulated in synovium based on the recognition of a common Ag. The apparent importance of the TCRB CDR3 as well as the selection for particular coexpressed TCRA sequences indicates that the selecting Ag is a conventional protein (peptide) Ag/MHC complex rather than a superantigen (43, 53). Of course, the identity of this stimulating Ag (or Ags) is currently unknown as is its role in the disease process. The TCRs expressed by these in vivo expanded synovial clones do not appear to be unusual compared with other TCR, and it is currently not possible to predict a particular type of Ag based on TCR structure.
A number of previous studies of RA patients have presented evidence for expanded CD4+ clones in synovial tissue or synovial fluid based on finding repeated sequences (6, 24, 25, 26, 36, 37, 38, 54) or discrete bands after separation of TCRB fragments by single-stranded conformational polymorphism (55). Also consistent with our findings, Alam et al. (54) studied two RA patients and found examples of the same clone in two different synovial tissues. These authors also presented evidence for common CDR3 motifs among the different synovial clones found. However, related clones were allowed to have significantly different CDR3 amino acid substitutions and length, and they also frequently differed in BV and BJ usage. The effect of such differences on Ag recognition is likely to be profound (43, 44), and therefore their conclusions regarding a common antigenic stimulus seem limited. Another study (25) concluded relatedness based on the presence of two hydrophobic amino acid residues within the CDR3. Again, other components of the TCR important for recognition, such as CDR3 sequence and length, and BV and BJ gene segment usage, were allowed to vary, raising serious questions about the relatedness of such clones. None of the above studies analyzed coexpressed TCRA sequences for relatedness. Li et al. (26) focused on two potentially related TCRBV17 sequences in synovial tissue that persisted in synovial fluid after synovectomy. These clones varied in CDR3 sequence, CDR3 length, and BJ usage. More importantly, Vß17+ clones derived from this joint were considered to be related to the original synovial sequences, yet the TCRs being compared varied in CDR3 sequence, length, and/or BJ usage and the two clones showed major differences in the coexpressed TCRA sequences. Finally, no previous studies of synovial T cell repertoire have documented the same TCR ß-chain to be encoded by different T cell clones, which we found in all three patients studied.
We found one example of an expanded CD4+ Vß17+ clone in peripheral blood, which did not match any of the synovial clones in this patient. Dominant clonotypes in peripheral blood CD4+ T cells have been described previously, and a preference for T cells expressing BV3, BV14, and BV17 was noted (49). It may be of interest that the CDR3 region of one of our synovial BV3 clonotypes matched a previously reported blood clone in an RA patient (49).
The specificity of the related synovial T cell clones in these RA patients remains unknown. Preliminary studies indicate that these sets of expanded clones from CS-1 and CS-2 do not respond to autologous EBV-transformed B lymphoblastoid cells (M. T. Falta, C. C. Striebich, J. Bill, and B. L. Kotzin, unpublished observations). Thus, it is unlikely that they are directed to EBV trans-activator proteins as recently shown for a subset of CD8+ synovial T cells in RA (56, 57). Our TCR analysis also suggests that many different Ags are involved in the stimulation and selection of different clonal sets, especially in patients with advanced disease. It is emphasized that our TCR analysis and studies of other investigators have not been able to document related T cell clones among different patients. Furthermore, in a comparison of our TCRB sequences to those previously reported to be expressed in RA synovium, no matches were found. This is particularly troublesome considering the association of RA with HLA-DR4 and the hypothesis that DR4 functions as the presenting element for unknown arthritogenic peptide(s). All patients in the current study expressed HLA-DR4, and one patient carried two DR4 alleles. This hypothesis, however, may not explain recent studies that suggest a gene dosage effect in which the presence of two HLA-DR4 alleles confers greater risk of disease and increased severity of disease compared with those in individuals with one copy of the gene (12, 14, 58, 59), and other models for the role of DR4 in Ag presentation and T cell selection have been proposed (7, 60). Thus, the heterogeneity of expanded synovial clones in different RA patients may be explained by responses to multiple Ags, different antigenic determinants, and different presenting class II MHC molecules.
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Acknowledgments |
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Footnotes |
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2 The following sequences discussed in this report have been deposited in GenBank: accession numbers AF043747–AF043878, AF043884-AF043895, and AF043982–AF043995.
3 Current address: Alexion Pharmaceuticals, Inc., New Haven, CT 06511.
4 Address correspondence and reprint requests to Dr. Brian L. Kotzin, Division of Allergy and Clinical Immunology (B-164), University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail address:
5 Abbreviations used in this paper: RA, rheumatoid arthritis; TCRB, TCR ß-chain gene; CDR, complementarity-determining region; TCRA, TCR -chain gene; TCRAV or TCRBV, TCR - or ß-chain variable region gene segment.
Received for publication April 3, 1998. Accepted for publication June 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(cA2) in patients with rheumatoid arthritis. Lancet 344:1125.[Medline]
ß T-cell receptors ‘see’ peptide/MHC complexes. Immunol. Today 14:597.[Medline]
/C
) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19:478.[Medline]
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  C. Wakasa-Morimoto, T. Toyosaki-Maeda, T. Matsutani, R. Yoshida, S. Nakamura-Kikuoka, M. Maeda-Tanimura, H. Yoshitomi, K. Hirota, M. Hashimoto, H. Masaki, et al. Arthritis and pneumonitis produced by the same T cell clones from mice with spontaneous autoimmune arthritis Int. Immunol., October 1, 2008; 20(10): 1331 - 1342. [Abstract] [Full Text] [PDF]
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 A. K. Sullivan, P. L. Simonian, M. T. Falta, J. D. Mitchell, G. P. Cosgrove, K. K. Brown, B. L. Kotzin, N. F. Voelkel, and A. P. Fontenot Oligoclonal CD4+ T Cells in the Lungs of Patients with Severe Emphysema Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 590 - 596. [Abstract] [Full Text] [PDF]
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 S. A. Curran, O. M. FitzGerald, P. J. Costello, J. M. Selby, D. J. Kane, B. Bresnihan, and R. Winchester Nucleotide Sequencing of Psoriatic Arthritis Tissue before and during Methotrexate Administration Reveals a Complex Inflammatory T Cell Infiltrate with Very Few Clones Exhibiting Features That Suggest They Drive the Inflammatory Process by Recognizing Autoantigens J. Immunol., February 1, 2004; 172(3): 1935 - 1944. [Abstract] [Full Text] [PDF]
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U. Bode, A. Sahle, G. Sparmann, F. Weidner, and J. Westermann The Fate of Effector T Cells In Vivo Is Determined During Activation and Differs for CD4+ and CD8+ Cells J. Immunol., December 1, 2002; 169(11): 6085 - 6091. [Abstract] [Full Text] [PDF]
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L. R. Wedderburn, A. Patel, H. Varsani, and P. Woo 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 Int. Immunol., December 1, 2001; 13(12): 1541 - 1550. [Abstract] [Full Text] [PDF]
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 A. W. Langerak, R. van den Beemd, I. L. M. Wolvers-Tettero, P. P. C. Boor, E. G. van Lochem, H. Hooijkaas, and J. J. M. van Dongen Molecular and flow cytometric analysis of the V{beta} repertoire for clonality assessment in mature TCR{alpha}{beta} T-cell proliferations Blood, July 1, 2001; 98(1): 165 - 173. [Abstract] [Full Text] [PDF]
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P. J. Costello, R. J. Winchester, S. A. Curran, K. S. Peterson, D. J. Kane, B. Bresnihan, and O. M. FitzGerald Psoriatic Arthritis Joint Fluids Are Characterized by CD8 and CD4 T Cell Clonal Expansions that Appear Antigen Driven J. Immunol., February 15, 2001; 166(4): 2878 - 2886. [Abstract] [Full Text] [PDF]
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 A. P. Fontenot, M. Torres, W. H. Marshall, L. S. Newman, and B. L. Kotzin Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease PNAS, October 23, 2000; (2000) 220430797. [Abstract] [Full Text]
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E. Boen, A. R. Crownover, M. McIlhaney, A. J. Korman, and J. Bill Identification of T Cell Ligands in a Library of Peptides Covalently Attached to HLA-DR4 J. Immunol., August 15, 2000; 165(4): 2040 - 2047. [Abstract] [Full Text] [PDF]
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 L. R. Wedderburn Tracking T cells in arthritis Rheumatology, May 1, 2000; 39(5): 458 - 462. [Full Text] [PDF]
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B. L. Kotzin, M. T. Falta, F. Crawford, E. F. Rosloniec, J. Bill, P. Marrack, and J. Kappler Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis PNAS, January 4, 2000; 97(1): 291 - 296. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, M. T. Falta, B. M. Freed, L. S. Newman, and B. L. Kotzin Identification of Pathogenic T Cells in Patients with Beryllium-Induced Lung Disease J. Immunol., July 15, 1999; 163(2): 1019 - 1026. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, M. Torres, W. H. Marshall, L. S. Newman, and B. L. Kotzin Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease PNAS, November 7, 2000; 97(23): 12717 - 12722. [Abstract] [Full Text] [PDF]
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