Cartilage-reactive T cells in rheumatoid synovium

Qiong Fang1, Yan-Yang Sun1, Wei Cai1, George R. Dodge4, Paul A. Lotke2 and William V. Williams1,3

1 Department of Medicine, Rheumatology Division and
2 Department of Orthopedic Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
3 SmithKline Beecham Pharmaceuticals, Philadelphia, PA 19104, USA
4 Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA 19107, USA

Correspondence to: W. V. Williams, Department of Clinical Pharmacology, SmithKline Beecham, 51 North 39th Street, Philadelphia, PA 19104, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Rheumatoid arthritis (RA) is an inflammatory polyarthritis genetically linked to HLA-DR4 and related haplotypes. RA synovial tissue is characterized by T cell infiltration and activation of macrophage-like cells, strongly implicating a T cell–antigen-presenting cell (APC) interaction in RA pathogenesis. To investigate the nature of the antigens driving the T cell response, synovial tissue was obtained from a patient with chronic RA and T cells were enriched. These T cells were stimulated by endogenous APC from the same synovial tissue. The T cell lines were subsequently evaluated for responsiveness to autologous APC and cartilage antigens. Specific proliferative responses to autologous APC which were enhanced by cartilage extract were seen. Immunomagnetic bead selection and RT-PCR was used to identify TCR {alpha}ß pairs which appeared to respond to antigen(s) in the cartilage extract. T cell clones derived from the same joint were shown to release IL-2 in response to the cartilage extract and expressed a related TCR. With these experiments we have shown direct evidence that autoreactive T cells are found within the inflamed rheumatoid synovium and, further, that the antigens driving these T cells are cartilage derived. Since the antigens recognized by these populations of T cells are found within cartilage our data provides evidence that RA pathology could be related to a self-driven autoimmune response to cartilage proteins.

Keywords: autoreactivity, cartilage, rheumatoid arthritis, single-stranded conformational polymorphism, synovial tissue, T lymphocytes, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Rheumatoid arthritis (RA) is a human polyarthritis characterized by intense inflammation and proliferation of synovial tissue (1). The synovial tissue, which normally functions to supply nutrition to cartilage, becomes hypertrophic and locally invasive. This hypertrophic tissue, termed the pannus, erodes into cartilage and bone causing joint destruction. The pannus is infiltrated with lymphocytes, of which CD4+ T cells typically predominate. There is also proliferation of type A and B synoviocytes, macrophages and B cells. These cells express high levels of HLA-DR (class II MHC) antigens. The immune response becomes so severe in some cases that germinal centers similar to those seen in lymph nodes are observed. RA synovial tissue thus displays all of the characteristics of a localized immune response, but the antigen(s) driving the response remain unknown. The proximity of the synovial pannus to articular cartilage has led to the suggestion that cartilage antigen(s) may drive the immune response, but definitive evidence for this is lacking.

RA has long been considered and autoimmune disease, but the lack of delineation of autoantigen(s) which trigger the inflammatory response leaves this classification in doubt. Reactivity to autoantigens has been sought in RA, with many studies investigating potential responses to type II collagen (26), proteoglycans (7) and other antigens (e.g. synovial fluid antigen-reactive T cell lines and clones) (8,9). Some of these studies have explored serum antibody responses. While such responses may indicate an underlying autoreactive process, they do not take into account the role of T cells nor do they investigate responses at the site of erosive pathology. In experimental models such as collagen-induced arthritis, autoantibodies appear to play a role in disease pathogenesis, but this seems to be to augment the inflammation produced by autoreactive T cell responses (1012). In addition, studies have shown that antibodies to native type II collagen do not precede the onset of RA during early disease (5). Thus, while joint-specific autoantibodies may be an important feature of RA pathogenesis, they do not appear to be a major pivotal contributing factor to the destructive process in the joint.

A large number of recent reports have investigated the T cell repertoire in RA using molecular means (reviewed in 13,14). These studies in general have shown evidence of a restricted repertoire of TCR with oligoclonal expansions, which is consistent with an antigen-driven response. Such oligoclonal expansions are present in inflamed rheumatoid synovial tissue, further supporting a role for antigen-reactive T cells in the pathogenesis of the joint lesion. While these prior reports have provided important evidence in favor of potentially autoreactive T cells in the rheumatoid joint, they have not generally characterized T cells with known functional activity nor have they established what the autoantigen(s) might be. There have been some recent studies suggesting the presence of T cells which respond to synovial fluid antigens in RA (8,9). These studies examined T cell clones derived from synovial effussions and found evidence for reactivity against unspecified synovial fluid antigen(s). This provides additional evidence for autoreactive T cells in the rheumatoid joint, but does not elucidate how common such autoreactive cells might be or the source of the synovial fluid autoantigens.

Here, we have taken a straightforward approach to characterizing the T cell component of the immune response in rheumatoid synovium, based on the rationale that synovium contains professional antigen-presenting cells (APC) which present the relevant antigen(s) in vivo. Synovial tissue was obtained from a patient with chronic RA undergoing joint replacement. T cells were enriched by nylon wool chromatography and the non-T cells were used as APC to stimulate the T cells for two cycles of stimulation/rest. These `autoreactive' T cell lines were subsequently evaluated for responsiveness to autologous APC with and without cartilage extract. Proliferative responses were seen in response to the APC which was augmented by the cartilage extracts tested, which contained a variety of potential antigens. In contrast, an IL-2-stimulated T cell line grown from the same knee did not respond to either APC or cartilage extract. Molecular analysis of the TCR repertoire of the `autoreactive' T cell lines revealed oligoclonal expansions. T cell clones derived from the knee synovium were shown to release IL-2 in response to cartilage antigen. These studies indicate that autoreactive and cartilage-reactive T cells are present in RA synovium. In addition to these autoreactive T cells, APC capable of presenting antigen to these T cells also appear to be present.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
Preparation and characterization of cartilage extract
The articular cartilage remaining on the femoral chondyles of three RA patients undergoing knee arthoplasty was removed asceptically, minced into small pieces (3–5 mm3) and rinsed with PBS. The minced cartilage was kept on ice throughout the preparation procedure. In a volume of 5 ml of ice-cold PBS the tissue was homogenized using a Virtis tissue homogenizer (Virtis, Gardiner, NY) for a 60 s pulse, after each the supernatant was removed and an additional 5 ml of cold PBS added for another 60 s pulse. This was repeated a total of 3 times and the homogenates were combined. After the particulate was removed by centrifugation (5000 g for 15 min) the supernatant was filter sterilized using a 0.45 µm filter (Millipore, Bedford, MA) and stored until used at –80°C. Total protein concentration was determined spectrophotometrically and was determined to be 2.65 mg/ml. The resultant solution represented the soluble fraction of the articular cartilage obtained from these RA joints.

Biochemical and immunochemical characterization of cartilage extract
This extract was partially characterized by SDS–PAGE and parallel Western blotting using specific antibodies to collagen type II (Chemicon, Temecula, CA), fibronectin (Cappel, Durham, NC), human Ig (Roche Molecular Biochemicals, Indianapolis, IN) and albumin (Cappel). Aliquots (15 µl) of the crude extract were precipitated with 9 volumes of ethanol. SDS–PAGE sample buffer (10 µl) containing ß-mercaptoethanol was added to the air-dried pellet and standard SDS–PAGE was performed as previously described (15). For the purposes of identifying putative proteins in the extract, duplicate gels were prepared so that one was stained with 0.5% (w/v) solution of Coomassie brilliant blue R-250 (BioRad, Hercules, CA) in 40% methanol/10% acetic acid. The other gel was transblotted and prepared for successive incubations with the aforementioned antibodies. Between incubations the Western blot was stripped using a solution of 0.2 M glycine, 0.05% (v/v) Tween 20, pH 2.5. Western blots placed in this solution pre-heated to 75°C were then allowed to cool to 30 min at room temperature, which successfully removes the bound antibodies and permits re-incubation. Bound antibodies were identified using peroxidase-labeled secondary antibodies and the chemiluminescence reagent ECL (Amersham, Arlington Heights, IL), and exposed for 1–5 min to X-ray film.

Generation of T cell lines
Patient.
The patient, a 31-year-old woman, with seronegative RA for 10 years, was taking only ibuprofen at the time of surgery. Prior medications included prednisone, gold, sulfasalazine and hydroxychloroquine. HLA-DR sequencing as previously described (14) revealed homozygosity for HLA-DRß1*0101 (both alleles expressing the shared epitope) (16).

Digestion.
Synovial tissue obtained at the time of joint replacement was placed in a Petri dish, and the superficial layer of synovium was snipped off and minced with sterile forceps and scissors. The minced tissue was placed in 20–100 ml of PBS with 3 µg/ml collagenase, 0.1 µg/ml DNase I, 1 µg/ml hyaluronidase and 1% human AB serum (all from Sigma, St Louis, MO). The tissue was continuously stirred at 37°C for 90 min, then the undigested tissue was removed by passing it through a tissue sieve, and the cells collected and washed twice with 4% human AB serum in RPMI complete medium [CM; RPMI 1640 with penicillin/streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids, HEPES buffer, 5x10–5 M ß-mercaptoethanol (all from Gibco, Grand Island, MI)]. Before the second wash, hypotonic lysis in Gey's solution (7.0 g/l NH4Cl, 0.37 g/l KCl , 0.3 g/l Na2HPO4.12H2O, 0.024 g/l KH2PO4, 1.0 g/l glucose, 10.0 mg/l phenol red, 8.4 mg/l MgCl2.6H2O, 7.0 mg/l MgSO4.7H2O, 6.8 mg/l CaCl2 and 45 mg/l NaHCO3) was used to remove red blood cells and the remaining leukocytes were counted.

Cell separation.
T cells were separated from the non-T cells using a nylon wool column (17). The effluent (containing the T cells) was washed in 4% human AB serum-containing CM (4% HS medium). The nylon wool-adherent cells were obtained by pushing ice-cold medium through the column 3–4 times with a plunger. The T cells and non-T cells were frozen in 90% human AB serum with 10% DMSO (Sigma) initially in –80°C and then in liquid nitrogen until use.

Epstein–Barr virus (EBV) transformation.
To obtain autologous APC lines, EBV transformation of B cells was performed on the nylon wool-adherent cells by mixing the adherent cells with 0.45 µm filtered culture supernatant from the EBV-producing B95-8 cell line (kindly provided by J. Boyer, University of Pennsylvania). The B cell lines were expanded and used as APC (EBV-APC).

Stimulation with autologous synovial APC.
The prepared T cells underwent two cycles of stimulation and rest. For stimulation, nylon wool-adherent cells (see above) were used as autologous synovial APC. T cells at 106/ml were incubated with irradiated (10,000 rad) nylon wool-adherent cells at a 1:1 ratio in 10% HS medium containing 10 U/ml IL-2 and 10 U/ml IL-4 (both from Sigma, hereafter referred to as IL-2/4). After 7 days of stimulation, the T cells were rested for 7 days in 10% HS medium/10 U/ml IL-2/4 with EBV-APC (105/ml 10,000 rad irradiated from the same patient). A second cycle of stimulation and rest was performed prior to analysis of the `autoreactive' T cell lines for antigen-specific proliferative responses.

Stimulation with phytohemagglutinin (PHA).
The PHA-stimulated T cell line was developed by expanding the nylon wool non-adherent cells with PHA (Sigma), 7.8 µg/ml for 2 days, and then with 50 U/ml IL-2 + IL-4 for ~1 week prior to freezing cell pellets.

Stimulation with IL-2 + IL-4.
The IL-2 + IL-4-stimulated T cell line was developed by growing the nylon wool-purified T cells in 50 U/ml IL-2 + IL-4 for 17 days without feeder cells. This was designated the `IL-2 + IL-4-stimulated' T cell line.

Stimulation with cartilage extract.
The `cartilage-stimulated' T cell line was developed by two cycles of stimulation and rest with cartilage extract. First, 7.5x106/ml EBV-APC from the same patient were pulsed with cartilage extract (100 µg/ml) for 4–6 h at 37°C. The pulsed EBV-APC were then irradiated (10,000 rad) and diluted to 1–5x105/ml prior to addition of T cells (~5x105/ml initially) in 10% HS medium with cartilage extract (100 µg/ml) and 10 U/ml IL-2 + 10 U/ml IL-4. The stimulation with cartilage extract was continued for 1 week followed by a rest cycle without cartilage extract for 1 week [T cells in 10% HS medium with 10 U/ml IL-2 + 10 U/ml IL-4 with irradiated (10,000 rad) EBV-APC diluted to 1–5x105/ml]. The T cell line was evaluated following two cycles of stimulation and rest,.

T cell subpopulation selection.
Prior to molecular characterization, T cell subpopulations were selected from some of the T cell lines developed. We also selected T cells with specific anti-Vß antibodies. The T cell lines were thawed and then CD4+ T cells selected using Dynabeads M-450 CD4 (Dynal, Oslo, Norway) according to the instructions provided. For selection with specific anti-Vß mAb, the T cells were first bound by the anti-Vß mAb (see flow cytometry below), the cells washed twice and then selected with Dynabeads M-450 goat anti-mouse IgG as per the instructions provided.

Development of T cell clones
T cells were stimulated non-specifically with PHA and then grown in the presence of 6-thioguanine (6-TG) to select from T cells which had mutations in hypoxanthine guanosine phosphoribosyltransferase (HGPRT). This method selects cells which have been passing through multiple cell cycles in vivo and thereby accumulating mutations in HGPRT. Briefly, T cells were thawed, washed twice and resuspended in RPMI CM with 10% FCS (Hyclone, Logan, UT) and 7.8 µg/ml PHA for 2 days. The cells were then washed and expanded in CM with 10% FCS and 50 U/ml IL-2 and IL-4 until sufficient cells were available for cloning (at least 107 cells). The T cells were then washed and resuspended in CM with 10% FCS and 20% AIM-V medium (Gibco/BRL, Gaithersburg, MD) at 2x105/ml.

TK6 cells (an allogeneic EBV-transformed B cell line, the kind gift of D. Monos, Childrens Hospital, Philadelphia, PA) were either irradiated (10,000 rad) or treated with mitomycin C (50 µg/ml with 3.75x106 cells/ml for 2 h at 37°C and washed 3–4 times) prior to their use as feeder cells. The TK6 feeder cells were resuspended in CM with 100 U/ml IL-2, 100 U/ml IL-4 and 0.25 µg/ml PHA at 105/ml.

To determine the cloning efficiency, the T cells were serially diluted in CM with 10% FCS and 20% AIM-V medium, and mixed 1:1 with the TK6 cells (105/ml). The cell mixture was plated in 96-well round-bottom plates (Falcon, Lincoln Park, NJ) at 200 µl/well. These cloning efficiency plates were set-up with 5, 2, and 1 cells/well. In these experiments, there was 100% growth in all of the plates, indicating a high (but indeterminate) cloning efficiency.

To select HGPRT mutants, 6-TG (Sigma) was added to the TK6 cells to a final concentration of 20 µM. The TK6 cells (105/ml) were mixed 1:1 with the T cells (2x105/ml) and 200 µl/well plated into five 96-well round-bottom plates. Following a 10–12 day incubation, individual colonies were clearly visible, with a total of 46 clones growing (~9.5% of cells with growth). This indicates a >98% probability that these cells were clones. The clones were expanded into 24-well plates in CM with 5% FCS, 10% AIM-V, 50 U/ml IL-2 + IL-4 and 0.125 µg/ml PHA, and expanded until ~1–2x106 cells/well and frozen viable for later testing.

T cell proliferation and IL-2 assays
T cell proliferation assays were done using autologous EBV-APC as APC in all cases. EBV-APC at 7.5x106/ml were pulsed with cartilage extract (100 µg/ml) for 4–6 h, or left without antigen, and irradiated (10,000 rad) prior to addition of T cells. The T cells were suspended at a concentration of 106/ml, 100 µl/well (in duplicates) and aliquoted into a 96-well flat-bottom plate. As a negative control, medium only (no T cells) was used. EBV-APC (either pulsed with antigen or not pulsed) 106/ml were aliquoted 100 µl/well into 96-well flat-bottom plates and were diluted as indicated in the figure legends. Cartilage extract at a final concentration of 100 µg/ml (or dilutions as noted in the figure legends) was added. For negative and positive controls, 100 µl/well of medium or 20 U/ml IL-2 + IL-4 was added. After 3 days, 100 µl of supernatant was removed for IL-2 assay and [3H]thymidine (1 µCi/well) was added for 16–18 h, and then the cells were harvested and c.p.m. incorporated determined in a standard liquid scintillation system.

IL-2 production was determined using the IL-2-dependent T cell line CTLL under assay conditions previously described (18), although we also used the MTT assay to evaluate CTLL growth in response to IL-2 (19). IL-2 production was considered significantly stimulated by cartilage extract if the P value comparing cartilage extract-stimulated T cells with EBV-APC alone was <0.05 (Student's t-test). Two assays on culture supernatants were analyzed to determine IL-2 production by the T cell clones.

Flow cytometry analysis
T cells were washed and resuspended in FACS buffer (PBS with 1% BSA and 0.1% NaN3 from Sigma), aliquoted at 105/100 µl and incubated 30 min on ice with primary antibody at the concentration recommended by the supplier. The cells were washed twice with FACS buffer, and a recommended dilution of secondary antibody conjugated with phycoerythrin was added and incubated on ice for 30 min. The cells were resuspended in 100 µl of 1% paraformaldehyde after washing twice. In the case of double staining, a fluorescein-labeled antibody was applied as for the primary antibody. Following staining, the cells were resuspended in 100 µl 1% paraformaldehyde. The percentage of staining was determined by the Wistar Institute Flow Cytometry Facility (Jeffrey Faust; Wistar Institute, Philadelphia, PA). For double staining, controls included: no antibody; secondary antibody only and tertiary antibody only. For single staining, no antibody was the control for directly conjugated antibodies and secondary antibody only was the control for primary antibody followed by secondary antibody staining.

The antibodies used included: anti-Vß1, -Vß9 (PharMingen, San Diego, CA); anti-Vß2, -Vß11, -Vß14, -Vß16, -Vß17, -Vß18, -Vß20, -Vß21.3, -Vß22, -Vß23 (Immunotech, Westbrook, ME); anti-Vß3, -Vß5a, -Vß5b, -Vß5c, -Vß6, -Vß8, -Vß12, -Vß13a TCR {alpha}ß (T Cell Diagnostics, Woburn, MA); anti-Vß13.2, -Vß13.2 (Brian Kotzin, University of Colorado, Denver, CO); anti-Vß17 (Dr Steve Friedman, Cornell University, New York, NY); OKT3.1, OKT8 (ATCC, Rockville, MD); and anti-CD4–FITC and anti-CD8–FITC (Sigma). Anti-TCR Vß1, 2, 3, 5, 6, 8, 9, 11, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, OKT3 and anti-TCR{alpha}ß were used as primary antibody, anti-mouse IgG-PE was the secondary antibody, and anti-CD4–FITC or anti-CD8–FITC was used as tertiary antibody.

T cell subset selection
T cell subsets were selected from the T cell lines developed using immunomagnetic bead selection. The T cell lines were washed and resuspended in PBS with 1% FCS. Initially, CD4+ T cells were selected using Dynabeads M-450 CD4 beads. The selection procedure followed the manufacturer's instructions (Dynal). The cells that bound the Dynabeads M-450 CD4 beads were placed directly in Triazol RNA extraction buffer (Gibco/BRL) and the RNA isolated. The unbound cells were then sequentially selected with different Vß-specific mAb, selecting those Vß comprising the greatest proportion of the T cell line, then the second greatest, etc. Vß-specific selection was performed with commercially available mAb and Dynabeads M-450 goat anti-mouse IgG according to the manufacturer's instructions. Selected populations were lysed with Triazol reagent, and the RNA extracted and used for RT-PCR. This selection was effective, as the selected Vß families dominated on RT-PCR analysis (see below).

RT-PCR
RT-PCR was performed using a nested set of primers for specific Vß amplification as previously described (20). For TCR V{alpha} amplification, we used a similar nested PCR technique [same number of cycles and other conditions as in (20)] using previously published primers (22). Generally, the primers described in (21) were used in an equimolar mixture in a preamplification step using a 3' C{alpha} primer. The preamplified reaction mixture was split into seperate tubes and reamplified in a family-specific fashion with primers described in (21,22) as the inner primers with a more 5' C{alpha} primer. Gel electrophoresis and Southern blotting with 5' Cß or C{alpha} primers were as described (2022).

Single-stranded conformational polymorphism (SSCP) analysis
The SSCP denaturing buffer was: 1xTBE (90 mM Tris, 92 mM boric acid and 2.5 mM EDTA) with 26.67 mM methylmercury hydroxide/2% Ficoll 400/0.03% bromphenol blue/0.03% xylene cyanole. A Novex ThermoFlow Mini-Cell was employed to keep the gel temperature constant (Novex, San Diego, CA). Novex precast 4–20% TBE gels were used for SSCP. A Novex Western transfer apparatus was used to transfer DNA from gel to nylon membranes for Southern blotting. The ThermoFlow unit was turned on to cool gel(s) down to 10°C prior to electrophoresis. Then 4 µl of PCR product was mixed with 12 µl of SSCP denaturing buffer, heated at 95°C for 4 min, followed by immediately placing samples in ice water. The samples were loaded between 2 and 10 min on precooled 4–20% TBE gel(s). The gel(s) were run in 1.25xTBE buffer at 300 V for 2–3 h at 10°C. After running, the gels were stained in ethidium bromide water for 20 min and destained in 0.5xTBE before photography. The gels were then transferred to Amersham Hybond-N+ membranes by following the instructions (30 V for 1–2 h). The membranes were dried at room temperature and probed with appropriate probe by Southern blotting as previously described (2022).

Cloning and sequencing
PCR products were cloned using the TA cloning kit (Invitrogen, San Diego, CA) according to the manufacturers instructions. Individual clones were restriction digested with appropriate restriction endonucleases to release the inserts and analyzed by SSCP to determine if identical clones were present. At least one of each identical clones seen on SSCP was sequenced using the ABI Prism dye terminator cycle sequencing core kit according to the manufacturer's instructions (Perkin-Elmer, Norwalk, CT) with primers derived from the vector sequence as specified by the supplier.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
The aim of these studies was to determine if we could detect T cell populations within the synovial tissue of a patient with chronic RA which displayed autoreactivity to cartilage antigens. To determine this, we isolated cell populations from the synovial membrane of a patient with chronic RA, stimulated them with endogenous APC isolated from the same synovial membrane and determined their reactivity with a partially characterized soluble cartilage extract derived from three rheumatoid knees. An extract was used, containing a mixture of many different soluble cartilage proteins, as we were uncertain which specific cartilage antigens might be recognized. On Western blot analysis, the extract did contain albumin, type II collagen, fibronectin fragments and antibodies among other proteins (Fig. 1Go).



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Fig. 1. Cartilage extract. The cartilage extract used in these studies is shown here in a Coomassie brilliant blue-stained SDS–PAGE gel (A). Parallel lanes of cartilage extract transblotted to supported nitrocellulose were successively incubated with antibodies to human Ig, fibronectin, type II collagen and albumin (not shown) (B). Bands in the Coomassie-stained gel were identified by specific antibodies as described in Methods and comparison to the Mr of standards (left side arrows, A).

 
T cell lines
Following digestion of the synovium, this tissue yielded ~8x108 cells. Most of the cells were frozen, although some were separated by nylon wool chromatography, and used to develop T cell clones and an EBV-transformed B cell line (EBV-APC). Initially, two T cell lines were grown: one in response to endogenous APC (which we term the `autoreactive' T cell line), and one in response to IL-2 and IL-4 (IL-2 + IL-4-stimulated T cell line). Later, a third cell line was developed stimulated with cartilage-derived antigens (cartilage-stimulated cell line) presented by the EBV-APC. The growth characteristics of these T cell lines are shown in Fig. 2Go. All three cell lines showed an initial decline in cell counts, then an expansion of cell number, although the timing and extent of the changes differed for the three cell lines.



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Fig. 2. Growth of cell lines. The autoreactive T cells were stimulated with endogenous APC on days 0–8 and 14–21, and were rested on autologous EBV-APC on days 8–14 and 21–28. The IL-2 + IL-4-stimulated T cells were grown continuously in 50 U/ml IL-2 + IL-4 for 17 days. The cartilage-stimulated T cells were stimulated with autologous EBV-APC plus cartilage antigen on days 0–7 and 14–22, and were rested on autologous EBV-APC on days 7–14 and 22–28. The absolute cell numbers versus time are shown.

 
The characteristics of these T cell lines were analyzed by flow cytometry (Table 1Go). For comparison, the unstimulated nylon wool non-adherent cells isolated from the synovial membrane were also analyzed. The unstimulated cells, which were only ~50% CD3+ cells, were predominately CD4+ and did not show dominant Vß expansions on this analysis. The autoreactive T cell line was >95% CD3+, and was predominately CD8+. Specific Vß expansions were also not seen in this cell line. In contrast, both the IL-2 + IL-4-stimulated and cartilage-stimulated T cell lines showed evidence of specific Vß expansions. While most of the expansions were CD4 (Vß8 and Vß14 for the IL-2 + IL-4-stimulated T cell line and Vß8 for the cartilage-stimulated T cell line), CD4+ expansions were also seen (Vß2+ in the IL-2 + IL-4-stimulated and Vß12+ in the cartilage-stimulated T cell lines).


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Table 1. Characterization of T cell lines
 
Proliferative responses
The proliferative responses of these T cell lines were evaluated, with a typical experiment shown in Fig. 3Go. Both the IL-2 + IL-4-stimulated T cell line and the `autoreactive' T cell line proliferated in response to the IL-2 + IL-4 mixture (Fig. 3AGo), while the unstimulated cells did not proliferate in response to IL-2/4. When evaluated against the autologous EBV-APC (Fig. 3BGo), the autoreactive T cell line showed a definite proliferative response, while the IL-2 + IL-4-stimulated T cell line did not. In the presence of cartilage extract (Fig. 3CGo), the proliferative response of the autoreactive T cell line was significantly higher compared with the EBV-APC alone (3–4 times increase in c.p.m. incorporated, P < 0.005 at all three concentrations via Student's t-test). Thus, the autoreactive T cell line, which had not been exposed to cartilage extract, showed a significant proliferative response to the cartilage extract. This was not a non-specific effect of the cartilage extract, as the IL-2 + IL-4-stimulated T cell line, which was able to proliferate to IL-2 + IL-4, did not proliferate in response to the cartilage extract. We also attempted to block the cartilage extract-specific response with antibodies to class I and class II MHC antigens, but at this point the cell lines developed had lost proliferative capacity.



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Fig. 3. Proliferative response of T cell lines. Two T cell lines were developed: autoreactive and IL-2 + IL-4 stimulated. These were compared with unstimulated T cells from the same synovial tissue (`Original' T cells in Table 1Go) and with the control (no T cells, EBV-APC only). A typical experiment using these T cell lines is shown. (A) Proliferative response of T cells to IL-2 and IL-4 at the doses shown or human serum (HS) media alone. No EBV-APC were added to this culture. (B) Proliferative response to EBV-APC alone. The autologous EBV-APC were irradiated and mixed with the T cell lines at the ratios noted. The number of T cells was kept constant (105/well) while the number of APC were diluted 2-fold starting at 105/well to produce APC:T cell ratios as shown. (C) The autologous EBV-APC were pulsed with cartilage extract (Fig. 1Go) and diluted as in (B) (100 µg/ml cartilage extract corresponds to a 1:1 ratio of APC:T cells, 50 µg/ml corresponds to 1:2 and 25 µg/ml corresponds to 1:4). For (A–C), the mean ± SD c.p.m. incorporated of triplicate wells is shown.

 
Based on this result, as noted above, we developed a T cell line by stimulating with the cartilage extract in the presence of autologous EBV-APC. This cartilage-stimulated T cell line expanded during culture with the cartilage extract during the second cycle of stimulation (Fig. 2Go), and had clear expansion of Vß8+/CD4 and Vß12+/CD4+ T cells (Table 1Go). However, this cartilage-stimulated T cell line did not exhibit a specific proliferative response to the cartilage extract (data not shown).

TCR heterogeneity
We analyzed the TCR heterogeneity of these T cell lines using an RT-PCR/SSCP protocol we previously developed (20). SSCP separates different molecular species of PCR products by mobility under denaturing conditions and is capable of detecting single nucleotide substitutions. Single bands seen on SSCP analysis usually correspond to individual molecular clones. Prior to this analysis, we separated T cell subpopulations from the T cell lines, using immunomagnetic bead selection. The protocol was as follows. The T cell line was thawed, washed and CD4+ T cells selected using Dynabeads M-450 CD4 beads. The CD4+ selected cells and the CD4-depleted residual cells were used directly for RNA extraction and RT-PCR/SSCP analysis with Vß-specific primers. The results of this analysis are shown in Fig. 4Go for three cell lines: IL-2 + IL-4 stimulated, autoreactive and cartilage stimulated. For comparison, polyclonal PHA-stimulated cells are shown (the PHA-stimulated cells were not separated into T cell subsets).



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Fig. 4. TCR heterogeneity analysis. The T cell lines developed were positively selected with anti-CD4+ mAb and magnetic beads. The CD4-selected and CD4-depleted cells were analyzed by a novel nested RT-PCR protocol, followed by SSCP and Southern blotting. Each band defines an individual TCR and identical clones have identical migration on the gels. The Vß specificity of the primers used for amplification are shown above each blot. The lanes are as follows: (A) unselected PHA-stimulated T cells (polyclonal control), (B) CD4-selected IL-2 + IL-4-stimulated T cells, (C) CD4-selected `autoreactive' T cells, (D) CD4-selected cartilage-stimulated T cells, (E) CD4-depleted IL-2 + IL-4-stimulated T cells, (F) CD4-depleted `autoreactive' T cells and (G) CD4-depleted cartilage-stimulated T cells.

 
The polyclonal PHA-stimulated population usually presented a smear on SSCP analysis (e.g. for Vß2 or Vß23), and this was often quite faint due to the diffuse nature of the smear (e.g. Vß8, Vß5.2). The bead selection did seem to separate T cell populations, as the bands seen for the CD4-selected populations usually differed from those seen for the CD4-depleted populations (e.g. Fig. 4Go, cf. lanes B and D, C and F, and D and G for Vß2 or Vß23), although this was not always the case (e.g. for Vß12). It was of interest to compare the bands seen for the different T cell lines developed. Interestingly, the IL-2/4-stimulated T cell line (Fig. 4Go, lanes B and E, corresponding to CD4-selected and CD4-depleted respectively) appeared molecularly distinct from the autoreactive and cartilage-stimulated T cell lines. This correlates with the different proliferative responses seen for these T cell lines. For most of the Vß families examined, the `autoreactive' and cartilage-stimulated T cell lines also were molecularly distinct (Fig. 4Go, cf. lanes C and D for CD4-selected autoreactive and cartilage-stimulated T cell lines, lanes F and G for the corresponding CD4-depleted T cell lines). The exception to this was Vß12. For Vß12, a dominant band was seen for both the `autoreactive' and the cartilage-stimulated T cell lines, and this was present in both the CD4-selected and CD4-depleted populations. On flow cytometry analysis, these Vß12+ T cells were a single population and gated as CD4+. The same band seen for the CD4-selected and CD4-depleted populations is likely due to incomplete depletion of the CD4+ T cells with the bead selection procedure used. This indicates that these Vß12+ autoreactive and cartilage-reactive T cells populations are dominated by a single clone.

Sequence analysis
The PCR products shown in Fig. 4Go were cloned and sequenced. In addition, specific Vß-expressing T cells were selected from the CD4-depleted population using specific anti-Vß mAb and Dynabeads M-450 goat anti-mouse IgG. These were sequenced as well, with the sequences shown in Fig. 5Go. As we noted previously (20), the Vß families seen on sequence analysis did not always correspond to the Vß-specific primers used for amplification. For example, the Vß21 primer amplified Vß6 sequences, the Vß10 primer amplified Vß2 sequences and the Vß13 primer amplified Vß12 sequences. This appeared to be due to cross-amplification of dominant clones by other PCR Vß primers. Thus, the clonal heterogeneity of the T cell lines was more restricted than initially thought based on SSCP and Southern blot analysis.



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Fig. 5. Sequence analysis of T cell lines. The PCR products shown in Fig. 4Go were cloned and sequenced. The Vß family and Jß family are shown, along with the proportion of molecular clones which the sequence represents. Only recurrent clones were sequenced in this analysis, with single clones (as analyzed by SSCP analysis of inserts) not analyzed.

 
With the exception of a dominant Vß12.4/Jß2.5 sequence seen in both the autoreactive and the cartilage-stimulated T cell lines, none of the other Vß sequences were present in more than one cell line. Similarly, the Vß sequences seen in CD4-selected and CD4-depleted populations also differed, indicating that the selection was effective. The Vß12 sequences were indeed identical between the `autoreactive' and the cartilage-stimulated T cell lines. These and other dominant T cell populations were selected using immunomagnetic beads. The same Vß12 sequence was seen following immunomagnetic bead selection for both the autoreactive and cartilage-stimulated T cell lines. For the selected populations, {alpha} chain sequence was also determined by nested RT-PCR and cloning. The {alpha} chains detected differed for these two T cell lines, with the autoreactive Vß12-selected T cells expressing V{alpha}8/J{alpha}11, V{alpha}7/J{alpha}43 and V{alpha}1/J{alpha}14, while the cartilage-stimulated Vß12-selected T cells expressed V{alpha}8/J{alpha}40 and V{alpha}17/J{alpha}23. While the reason for this difference is unclear, we suspect that the immunomagnetic bead selection was not clean and that Vß12 T cells contaminated the `autoreactive' population, (as in this population, Vß12+ cells were a smaller proportion of the T cell line). The cartilage-stimulated T cell line, which showed a marked expansion of the Vß12 population, was more likely cleanly selected and the {alpha} chain pairing more accurately detected.

Development of T cell clones
To further investigate the potential for cartilage reactivity in this patient's synovial T cells, we developed T cell clones by a method that selects for chronically stimulated T cells. This method is based on the accumulation of mutations in somatic cells as they proceed through multiple cell cycles. The method is to stimulate T cells non-specifically, then to select the growing T cells in 6-TG. Cells with a normal HGPRT gene metabolize the 6-TG to toxic metabolites which kill the cells. Only cells with mutations of HGPRT survive and the appearance of such mutations implies that these cells were chronically cycling in vivo. 6-TG-resistant T cell clones were developed from the nylon wool non-adherent cell population following polyclonal stimulation, and evaluated for their response to cartilage antigen (presented by autologous EBV-APC) and TCR expression by flow cytometry, RT-PCR/SSCP and sequencing.

A total of 46 clones was developed, out of a total of 480 wells, indicating a >99% probability that these arose from single progenitors. Of these clones, 30 grew sufficiently for evaluation of responses to cartilage antigen; 17 for TCR expression by RT-PCR/SSCP and four for evaluation by flow cytometry. Three clones also had sequencing performed. These results are shown in Table 2Go. As can be seen, only one clone proliferated in response to cartilage antigen (clone 11), while nine produced IL-2 in response to cartilage antigen. The remaining clones did not respond to cartilage antigen by proliferation or IL-2 production. Four clones (none of which responded to cartilage antigen) were shown to express Vß12 on flow cytometry and this confirmed the expression of Vß12 on RT-PCR analysis. Interestingly, all of the 17 clones evaluated by RT-PCR expressed Vß12. One of these (clone 11) also demonstrated expression of Vß17 by RT-PCR. On SSCP analysis, the Vß12-expressed by all of these clones migrated identically (data not shown). While some of these Vß12 expressing clones responded to cartilage extract, some did not. V{alpha} expression was evaluated in three clones, two of which responded to cartilage extract. Interestingly, both clones which responded to cartilage extract expressed V{alpha}4, while the clone that did not respond to cartilage extract expressed V{alpha}19. The sequences of the {alpha} and ß chains expressed by these clones is shown in Fig. 6Go. Clones 11 and 21, both of which responded to cartilage extract, expressed the same two V{alpha}4 chains. Clone 4 expressed a single V{alpha}19 chain. This suggests that cartilage reactivity by these clones with identical ß chains is a property of the {alpha} chains expressed.


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Table 2. Chronically stimulated T cell clones
 


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Fig. 6. Sequences of the CDR3 regions of the {alpha} and ß chains of T cell clones. These clones are described in Table 2Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 
The approach taken in the study presented in this report was based on basic observations of RA pathogenesis. T cell responses were examined based on the known linkage of RA to HLA-DR haplotypes, which represents part of the complex which serves as the TCR ligand. The presence of abundant T cell infiltrates in rheumatoid synovium, which is the active site of inflammation, further suggests that autoreactive T cells might be found in this site. It also has been observed that RA can `burn out' and that patients who have total joint replacements (eliminating the cartilage and synovium) only infrequently will subsequently develop a new synovial pannus. These observations suggest that cartilage constituent(s) may function as autoantigens driving immune responses by synovial T cells. We attempted to examine this possibility directly by developing T cell lines in response to endogenous synovial APC and evaluating their responses to cartilage antigen(s). This approach (stimulation with tissue-derived APC and examination of responses to tissue extract) could be applied to T cell infiltrates from other inflammatory diseases.

The patient described here had abundant synovial proliferation and the large number of cells allowed the generation of several T cell lines in response to distinct stimuli. This allowed the use of some T cell lines as controls for others. In these studies, the T cell line which was stimulated by endogenous synovial APC showed a definite proliferative response to autologous EBV-APC. This may have been due to the presence of the EBV-APC during the rest cycles used in developing this line, with the T cells responding to autoantigens presented by the EBV-APC, although cell lines from the other patients which were not exposed to EBV-APC showed similar responses (data not shown). Similar autoreactive T cells have been described by other investigators who have examined T cell lines and clones derived from rheumatoid synovium (23). The autoreactive T cell lines developed here which responded to EBV-APC were exposed during development either to EBV-APC or to endogenous synovial APC, while the IL-2-stimulated T cell line, which was not exposed to APC, did not show this response. It is likely that such T cells are responding to self-MHC molecules (e.g. HLA-DR) in some manner (presumably with presentation of an endogenous peptide), as was shown for the T cell clones described by Li et al. (23). Others have suggested a role for autoreactive recognition of HLA-DR peptides presented by other class II MHC molecules in RA pathogenesis (24). This is reminiscent of the experimental studies which demonstrated that mice transgenic for a TCR which recognized (after backcrossing) a self MHC class II molecule (postulated to recognize a ubiquitous tissue antigen presented in the context of self MHC) spontaneously developed arthritis similar to RA (25). Thus, autoreactive T cells of this kind may also play a role in disease pathogenesis.

It should be emphasized that TCR rarely recognize MHC molecules devoid of antigen and that the predominant antigenic peptides bound to MHC are often a product of the environment in which the APC find themselves in. In this situation, cartilage antigens may play more of a permissive role in allowing TCR recognition, thereby augmenting T cell responses which otherwise may not produce inflammatory lesions. There also may be cross-reactivity between cartilage antigens and self-MHC peptides, as had been suggested for EBV-gp110 peptides and HLA-DR (26). There exists the possibility that the response seen to EBV-APC in this study could represent a response to EBV antigens expressed by these cells.

It is clear, however, that the proliferative response seen by the autoreactive T cell line was clearly augmented by the addition of cartilage extract (Fig. 3Go). This response was reproducible in several assays and was not seen in T cells grown in response to non-specific stimuli (IL-2/4) or in the unstimulated nylon wool non-adherent cells from the same joint. While the autoreactive T cell line was predominately CD8+, such proliferative responses usually depend on CD4+ T cells. Unfortunately, we were unable to evaluate the MHC restriction of this response due to the limited life span of these T cell lines in vitro. Interestingly, none of the T cell lines developed here proliferated in response to the endogenous synovial APC which were used to stimulate the `autoreactive' T cell line (data not shown). Similarly, the cartilage-stimulated T cell line did not proliferate in response to the cartilage extract with which it had been stimulated. The CD8+ predominance of the autoreactive T cell line could indicate the presence of regulatory T cells which are activated by the endogenous synovial APC and suppress the autoreactive response. Similar regulatory T cells might also have suppressed the response of the cartilage-stimulated T cell line to the cartilage extract. Thus complex regulatory phenomena may be at play in these APC–T cell interactions and the response to the cartilage extract seen in the autoreactive T cell line may represent the unmasking of a cryptic response.

The autoreactive T cell line and a related line grown in response to cartilage extract appeared to have similar Vß12 expansions. In addition, T cell clones grown from the same joint under conditions which select for in vivo cycling cells also expressed Vß12, and a subset of these responded to cartilage extract by proliferation and/or IL-2 production. The clones which responded to cartilage extract differed from a non-responsive clone in its V{alpha} expression. Dual V{alpha} expression by T cells has been clearly demonstrated in a number of studies and a role for such dual expression in autoimmunity has been postulated (27). While we cannot be certain which of the V{alpha} chains were involved in the autoreactivity seen, it seems likely that these T cell clones possess dual antigen specificity, and this may play a role in their appearance and persistence in the joint.

In summary, these studies have demonstrated that T cells can be found in rheumatoid synovium whose proliferative and cytokine responses are augmented by cartilage antigen(s). The ability to generate these T cell lines and clones by stimulating synovial T cells with endogenous synovial APC strongly suggests that cartilage antigens are being presented in the rheumatoid joint in vivo. Identification of the specific antigen(s) which are responsible for this phenomenon may elucidate important steps in the pathogenesis of RA.


    Acknowledgments
 
The authors are grateful to Dr Toshiro Maeda for his excellent critical input, to David Hawkins for expert technical assistance, and to the numerous students who contributed and helped in these studies, and the inspiration of H. S. These studies were supported by NIH grant AR-42417 to G. R. D. and a grant from the Arthritis Foundation to W. V. W.


    Abbreviations
 
6-TG 6-thioguanine
APC antigen-presenting cell
CM complete medium
EBV Epstein–Barr virus
HGPRT hypoxanthine guanosine phosphoribosyltransferase
RA rheumatoid arthritis
SSCP single-stranded conformational polymorphism

    Notes
 
Transmitting editor: M. Feldmann

Received 15 April 1999, accepted 28 January 2000.


    Reference
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 

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