CLINICAL
IDEOLOGY
AND
I~~NO~ATHOL~Y
Vol. 63, No. 1, April, pp. 28-33, 1992
Clonal Heterogeneity of Synovial Fluid T Lymphocytes Inflammatory Synovitis YANG Lu,* *Department
BYUNGS.KIM,~
of Medicine, Division of Arthritis-Connective University, 303 East Chicago Avenue, Chicago,
AND RICHARD M. POPE+
Tissue Diseases and i%)epartment of Microbiology-Immunology, Illinois 60611; and the $VA Lakeside Medical Center, Chicago,
Several studies have examined patients with rheumatoid arthritis for the presence of oligoclonal populations of synovial T lymphocytes. The results of these studies have been conflicting. In this study one patient with rheumatoid arthritis and two with other forms of inflammatory synovitis were examined by Southern blot analysis of T cell clones generated from synovial fluid by primary limiting dilution. Evidence of oligocionality was documented only in a patient with psoriatic arthritis. The distin~ishing ~h~acteristics of this patient, in addition to the diagnosis, included the fact that only onejoint was involved, the synovitis in the atIected joint was of recent onset, and the synovial fluid lymphocytes from which the T cells were cloned responded strongly to soluble antigens. Because of the association with the strong responseto soluble antigens, synovial fluid T lymphocytes from another patient with rheumatoid arthritis were cloned in response to a crude mycoba~teria1 antigenie mixture. Three of the seven clones examined were identical by Southern blot analysis. These observations suggestthat the presence of oligoclonality is limited in patients with inflammatory arthritis. The relationship of a specific antigen-driven responsewithin the joint to the detection of oligoclonal T cells within that joint remains to be determined. Q1992 AWdemic Press,
Inc.
INTRODUCTION Numerous studies have examined T cells obtained from the synovial fluid of patients with rheumatoid arthritis in order to detect the presence of oligoclonal populations of T lymphocytes. If present, such populations may indicate responsiveness to a unique antigen that might lead to new forms of therapy. Studies which examined clonal heterogeneity of synovial T lymphocytes employing whole populations of T cells which were unactivated or expanded nonspecifically with mitogen and IL-2 have provided inconsistent results (l4). Other studies employed the use of T cells grown in the presence of IL-2 without mitogen, in order to expand T cells that had been preactivated in. uiuo (5). Dominant T cell rearrangements were observed (5). In order to more precisely define rearrangements expressed by individual T cells within the rheumatoid
Northwestern Illinois
joint, primary limiting dilution analysis was employed, nonspeci~cally stimulating T cells, obtained directly from the joints of patients with rheumatoid arthritis, with mitogen, IL-2, and feeder cells. Initial studies have suggested a very limited degree of oligoclonality (6,7). When clones were expanded employing only feeder cells and IL-2 without mitogen, in order to expand in viva-activated T cells, a more restricted repertoire of T cells has been noted (8, 9). The current studies were performed in order to examine the degree of clonal heterogeneity in patients with different forms of inflammatory synovitis and to relate any observed oligoclonality with the degree of synovial proliferation generated in response to soluble antigens by the synovial fluid lymphocytes from which the T cells were cloned. We observed no oligoclonality in a patient with seropositive rheumatoid arthritis or in a patient with a HLA-B27 -I- chronic polyarthritis. The synovial fluid lymphocytes of these two patients responded only weakly to soluble antigens. In contrast. the T cells obtained from the synovial fluid of a patient with psoriatic arthritis, whose synovial fluid lymphocytes responded strongly to all antigens tested, demonstrated evidence of oligoclonality. In addition, the T cell clones from a patient with rheumatoid arthritis, generated in response to a crude mycobacterial antigenie complex, also demonstrated oligoclonality. These observations suggest that synovial T lymphocyte oligoclonality may occur in some individuals and that it may be associated with an antigen-driven response of recent onset.
Patients examined. Synovial fluid was obtained from four patients with inflammatory synovitis. The clinical characteristics of these patients are detailed in Table 1. Synovial fluid was obtained from Patient 1 on two separate occasions 2 weeks apart. T cell clones were generated on each occasion. Each patient was receiving a nonsteroidal anti-inflammatory drug and methotrexate at the time of ~~ocentesis. The two patients with rheumatoid arthritis fulfilled the 1987 28
0090-1229192 $1.50 Copyright 0 1992 by Academic Press, k. All rights of reproduction in any form reserved.
in
SYNOVIAL
TABLE 1 Characteristics of Patients Patient 1”
2 3 4
Dx HLA-B27 + polyarthritis Psoriatic arthritis Rheumatoid arthritis Rheumatoid arthritis (HLA-DRl + 1
Age (years old) 41 45 66 47
29
FLUID T LYMPHOCYTES
RF& + -
Examined
Erosive disease Yes’ Yes Yes Yes
Disease duration (years) 18 4 12 11
No. swollen joints l/2 1 a 5
Duration flare
of
4/6 weeked 2 weeks 27 weeks 2-3 weeks
-DPatient 1 also demonstrated sacroiilitis on X ray. Synovial fluid was obtained on two occasions 2 weeks apart. The number of joints swollen and the duration of the synovial fluid present in the joint aspirated, by history, are indicated for each aspiration separately. ’ The presence ( + 1 or absence ( - 1 of rheumatoid factor (RF) in patient serum. ’ The presence of erosive disease as evidenced on X ray of peripheral joints. d The duration of the synovial effusion in weeks prior to aspiration as determined by patient history.
College of Rheumatolo~ agnosis (10).
American
criteria
for the di-
Cell isolation and culture. Synovial fluid mononuclear cells were obtained following hi&opaque gradient centrifugation as previously described (11, 12). In order to determine the responsiveness of synovial fluid mononuclear cells to soluble antigens, lo5 mononuclear cells were added to round-bottomed microtiter wells. Cultures were pe~ormed in medium containing 10% heat-inactivated human AB serum (11, 12). Optimal concentrations of antigens were added to the wells as previously determined (11, 12). These antigens included a crude water soluble extract from Mycobucterium t~~e~uZos~s H37Ra which was acetone precipitated CAP-MT), a purified 65kDa heat shock protein obtained from the H37Ra, and tetanus toxoid. Cultures were incubated with antigens for 6 days. Tritiated thymidine was added during the final 6 hr of culture (11, 12). T cell clones were generated by primary limiting dilution (6). T cells were added to microtiter wells containing lo5 irradiated autologous peripheral blood feeder cells, phytohemagglutinin (PHA) (1 kg/ml), and recombinant IL-2 (10 units/ml) in medium containing 10% heat-inactivated fetal bovine serum. On one occasion, autologous synovial fluid mononuclear cells were used as feeder cells. T cells were added at 0.7 cells/well. Clones were fed three times weekly with fresh medium containing IL-2. Clones were expanded every 2 weeks with allogeneic feeder cells, PHA, and IL-2. The cloning efficiency for the nonspecifically propagated clones was 23 +- ll%, with a range of 13 to 35%. This cloning efhciency was similar to that previously described for synovial fluid (6) and was substantially lower than the cloning efficiency observed with peripheral blood by us and others [(6), unpublished observations]. Synovial fluid T lymphocytes from Patient 1 were cloned on two occasions 2 weeks apart. On the first occasion, the cloning efficiency was 35%, employing autologous peripheral blood cells as feeders. On the second occasion, em-
ploying synovial fluid mononuclear cells as feeders, a cloning efficiency of 13% was noted. Following expansion, T cell clones were washed in phosphate-buffered saline, counted, and snap frozen in liquid nitrogen until DNA extraction. Antigen-specific T cell clones were generated from Patient 4, as previously described (111, in response to the crude AP-MT which contained multiple protein bands when analyzed by SDS-PAGE [Fig. 1 in Ref. flZ>]. Following initial activation with AP-MT, without IL-2, for 1 week, lymphoblasts were isolated by histopaque gradient centrifugation and cloned at limiting dilution employing autologous irradiated peripheral blood mononuclear cells as feeder cells, Al?-MT, and IL-2 in medium containing 10% heat-inactivated human AB serum. The clones were fed three times a week with fresh medium and IL-2. Growing clones were expanded every 2 weeks with irradiated autologous peripheral blood feeder cells, AP-MT, and IL-2. T cell clones from Patient 3 and Patient 4 were tested for antigen specificity. T cell clones, 10 to 14 days after the prior round of stimulation, were washed with fresh medium and added a lo4 cells per well together with irradiated, autologous peripheral blood feeder cells alone or feeder cells plus specific antigen. Testing for antigen specificity was performed in medium containing 10% heat-inactivated human AB sereum. The wells were cultured for 72 hr and tritiated thymidine was added for the final 6 hr of culture prior to harvesting as previously described (11, 12). A total of 43 clones were expanded from Patient 1. Sufficient DNA was available for Southern blot analysis on 24 of these clones. Twenty-two clones were obtained from Patient 2 and 14 were examined by Southern blot analysis. Seventeen clones were generated from Patient 3 and 11 were examined by Southern blot analysis. From Patient 4, 22 of 32 clones generated in response to AP-MT recognized this antigen (stimulation index >4 and ~2000 cpm). Of the 33 clones that responded to the AP-MT, 19 responded to the 65-kDa heat shock protein. These clones did not respond to the
30
LU, KIM, AND POPE
human 60-kDa heat shock protein or to tetanus toxoid. Seven of these clones were selected for Southern blot analysis because sufficient DNA was available. Immunophenotyping. The clones from Patient 4 were immunophenotyped employing anti-CD3, -CD4, -CD& and -WT31 monoclonal antibodies as previously described (14). All the clones were CD3 + , CD4 + , and WT31+. The synovial fluid mononuclear cells from which the clones were generated were examined in Patients 1 and 2 employing anti-CD3.
blots were perSouthern blot analysis. Southern formed by standard techniques (13). Briefly, cells were incubated in lysis buffer and the aqueous phase was extracted with phenol and chloroform. The DNA was precipitated with ethanol and incubated with DNAsefree RNAse. After reextraction, ethanol precipitation, and washing, the DNA was redissolved in buffer and the concentration was determined by optical density at 260 nm and the purity was determined by the optical density ratio 260:280. The DNA (10 kg) was digested with EcoRI for 16 hr at 37”C, electrophoresed through 7.5% agarose gels, transferred to nylon membranes (Nytran, Schleicher & Schuell, Keene, NH), and hybridized with a 32P C, cDNA probe. Commercially obtained 32P-labeled C, and J,2 probes were employed (Oncor, Inc., Gaithersburg, MD). Hybridization was performed for 24 hr in 6x SSC, 10x Denhardts, 100 pg/ml denatured salmon sperm DNA, 1% SDS, and 50% formamide at 45°C. Placental DNA was the germline control employed for each blot. Following autoradiography, employing the C, probe, the nylon membranes were stripped and rehybridized with the Js2 probe. Those samples with one or two rearranged bands following hybridization with both probes were considered clonal (both chromosomes may rearrange). With EcoRI digestion, germline bands at 11 kb (Cell and 4 kb (Cs2) were seen employing the C, probe (Fig.
1). Rearrangements using J,l appeared as unique bands and those rearranging through J,2 were not seen because of an EcoRI restriction site between J,2 and $2. The germline fragment hybridizing with J,2 was seen as a 4.4-kb band (Fig. 11, and rearrangements using J,2 were seen as unique bands with this probe. Samples were considered clonal if one or two rearranged bands were observed following digestion with EcoRI and hybridization with both the C, and Jo2 probes. Of all the samples examined, five were nonclonal as evidenced by the presence of more than two rearranged bands. One example is presented in Fig. 2. Those samples that appeared identical employing EcoRI were examined with additional restriction endonucleases. Also, those clones with bands that appeared identical, even though the clones were not identical, were examined with additional restriction endonucleases whenever sufficient DNA was available. Hind111 generated an 8-kb germline DNA fragment containing J,2 (Fig. 1) that was altered by rearrangements employing J,2. Hybridization of the C, probe with the 3’ end of the C, (a 6.5kb fragment) and with the C,l (a 3.5-kb fragment) will not be effected by these rearrangements. In order to examine common rearrangements noted through J,l, BamHI was employed. A 24-kb germline fragment was generated which hybridized with both the $1 and the J,2 probes. RESULTS
Examination of the clones obtained from Patients 1 and 3 generated by nonspecific cloning failed to reveal any instances of identical rearrangements. Examples of the clones from Patient 1 are presented in Fig. 2. One example of a nonclonal sample, with three rearranged bands (lane E), is also presented. Although no identical rearrangements were noted, individual bands of similar molecular weights were apparent with both
DE2 J8l t
B
9
t
E
J3
=2
t
t t
t
H
HE
E
t
tt
H
EB
t
H
EcoRI:
53 11.6
5
I kb
4.0 I
III:
i
Jn2 4.4
Hind
kb
kb
JB2 8.0
kb
1. Schematic representation of the T cell receptor B gene and the fragments generated by the restriction endonucleases employed in this study. The locations of the EcoRI (E), the Hid11 (H), and BumHI (B) are indicated by the arrows. The size of the germline DNA fragments generated with EcoRI and HindIII, hybridizing with the Ce and the J,2 probes, are indicated. FIG.
SYNOVIAL PROBE:
31
FLUID T LYMPHOCYTES A
A~CDEFEHIJKLMNOP
6
C
0
E
F
G
H
IJ
K
L
M
PROBE:
- 11.0
J&J c
_ 4.4
- 4.0
- 11.0
Je*
ce
- 4.0
FIG. 2. Southern blots of 14 different T cell preparations from the synovial fluid of patients with inflammatory synovitis. The DNA was digested with EcoRI and hybridized with C, and J,2 probes as indicated. The molecular weight markers are indicated on the right. The placental germline control is in lane P. Lane N contains a specimen from Patient 2 and the remainder of the samples are from Patient 1. The samples in lanes C and E were obtained from the second arthrocentesis and the remainder from the first. Lanes I and L contain an identical sample which was run at different times. The specimen in lane E demonstrated three rearranged bands and was not considered clonal.
the C, probe (Fig. 2, lanes A, K, and I/L at 6.6 kb) and with the J,2 probe (Fig. 2, lanes D, F, and J at 5.4 kb). In order to better define whether or not identical rearrangements of individual chromosomes might be represented by bands of similar molecular weight, additional Southern blot hybridization was performed on selected clones following Hind111 endonuclease digestion employing the J,2 probe. If identical rearrangements were used, the rearrangements hybridizing with J,2, which appeared similar following EcoRI digestion, should also possess similar molecular weights following Hind111 digestion. Three sets of clones were examined, including those presented in Fig. 2. No identical rearrangements were observed (data not shown). The three clones that appeared to have similar rearrangements through J,l following EcoRI digestion (Fig. 2, lanes A, K, and I/L) were also examined following BamHI digestion to confirm that these bands might represent similar rearrangements through J,l. Unfortunately, adequate digestion employing this restriction endonuclease did not occur with the DNA available from these three clones. The results obtained with clones generated from Patient 2, with psoriatic arthritis, were different. Five of the 11 clones examined demonstrated identical rearrangements (Fig. 3, lanes B, F, G, H, and I). Each of these clones possessed a rearrangement of one chromosome utilizing J,l and the other employing Js2. The rearrangements through J,2 resulted in identical bands employing the Hind111 restriction endonuclease (Fig. 4). The synovial fluid T cell population from which these T cell clones were generated responded strongly to all of the soluble antigens tested (Table 2). Unfortunately, the individual clones of this patient,
FIG. 3. Southern blots of 11 T cell clones from Patient 2 with psoriatic arthritis. The DNA was digested withEcoRI and hybridized with C, and J,2 probes. The position of the molecular weight markers is indicated on the right. The placental germline control is presented in lanes A and M. Lanes B, F, G, H, and I possess rearrangements that appear identical.
which were generated nonspecifically, were not analyzed for their antigen specificity. In contrast, the whole synovial fluid mononuclear cells of Patients 1 and 3, which demonstrated no oligoclonality, responded weakly to the soluble antigens tested (Table 2). Seventeen clones from Patient 3 were examined for antigen specificity. None of the 17 T cell clones examined from this patient responded to any of the antigens tested, including AP-MT, tetanus toxoid, and bovine type II collagen. Because of the suggestion that responsiveness to soluble antigens might be associated with the ability to detect clonal rearrangements in the inflammatory joint, T cell clones were generated in response to a very crude preparation of mycobacterial antigens CAP-MT). We reasoned that if oligoclonality was associated with an antigen-driven response, in vitro responsiveness to a crude antigen might enrich a limited number of clones. Similar rearrangements were observed through J,l for three of the seven clones examined following EcoRI digestion (Fig. 5 lanes B, C, and E). Similar rearrangements were noted following Bar&II endonuclease digestion with each of the clones which demonstrated similar rearrangements following EcoRI digestion (data not shown).
ABCDEFG PROBE:
J2
- 8.0
FIG. 4. Southern blots of the five T cell clones from the synovial fluid of Patient 2 with psoriatic arthritis which appeared identical following EcoRI digestion. The DNA was digested with HindI and hybridized with the J,2 probe. The position of the molecular weight markers is indicated on the right. The placental germline control is presented in lanes A and G.
32
LU,
KIM,
TABLE 2 Phenotype and Proliferative Response by Uncloned Synovial Fluid Mononuclear Cells Patient
Medium”
1
183'
2 3 4
390
AP-MT” 2.070 21,132
6%kDa
(11)” (641
hsp”
915 (5) 15,525
(40)
Tetanus
toxoid*
1,619 (91 47,454 (122) 438(l) 8,328 (21
CD3 + b 76% 71%
468 NDd ND 4,199 (9) 4005 11,879 (3) 25,722 (6) ND ____~_~.._. .._~~~___ .. ,.~ _ ._~.. ~~(1Cells were cultured in medium alone or medium pbns an optimal concentration of a soluble antigen. The antigens inchaded AP-MT, the mycobacterial 65-kDa heat shock protein (hsp), and tetanus toxoid. bThe percentage of CD3t cells contained in the original synovial fluid mononuclear cell preparation. c The results are presented as mean counts per minute. The values in parentheses represent the stimulation index obtained by dividing the counts per minute for cells plus antigen by the counts per minute for cells in medium alone. The results presented for Patient 1 represent those obtained following the initial arthrocentesis. d Not done.
DISCUSSION
Earlier studies examining T cells isolated freshly from the synovial fluid as well as from those nonspecifically propagated with mitogens have produced conflicting results. Some studies have noted a minor to a substantial degree of oligoclonality (l-9). The differences between these studies, in part, may be technical since overdevelopment of Southern blots employing polyclonal T cells may result in the detection of a limited number of Dsl-Jsl, DslJs2, and Ds2-Js2 rearrangements that might be confused with oligoclonality (6). The use of primary limiting dilution analysis avoids this potential problem. Employing primary limiting dilution with mitogen and IL-2, olig~lonality was not observed in two of our patients, one with seropositive rheumatoid arthritis, the other with HLA-B27-associated chronic polyarthriAI(CDEFGH PROBE: -
11.0
5
- 4.0
J2 - 4.4 5. Southern blots of seven T cell clones generated in response to AP-MT from the synovial fluid of Patient 4 with rheumatoid arthritis. The DNA was digested with EcoRI and hybridized with C, and J,2 probes, The position of the molecular weight markers is indicated on the right. The germline control is presented in lane A. Identical rearrangements were observed in lanes B (the upper band was faint but present), C, and E. FIG.
AND
POPE
tis. These results are similar to those previously reported by others in rheumatoid arthritis employing a similar technique (6-9). Oligoclonality has been more frequently observed when synovial T cells were expanded in IL-2 without the addition of mitogen (5,8,91; suggesting that T cells which have been activated i,z uiuo, which possess IL-2 receptor, are more likely to demonstrate evidence of oligoclonality. In uiuoactivated cells were not selected by our method of cloning by primary limiting dilution employing mitogen and IL-2. Recent studies of whole synovial fluid and peripheral blood mononuclear cells, employing the polymerase chain reaction, have suggested the presence of oligoclonality in the joints of patients with rheumatoid arthritis (15, 161. Both of our patients who demonstrated no evidence of oligoclonality had erosive disease and the synovial effusions from which the T cells were cloned were chronic in one and subacute in the other (Table 111.The synovial fluid mononuclear cells of these two patients failed to respond strongly to any souble antigen when tested. Additionally, the clones of one of these patients (Patient 3) failed to respond to any soluble antigen examined. In contrast, the synovial fluid mononuclear cells of the patient with psoriatic arthritis, from which the T cells were cloned, responded strongly to soluble antigens. Clonal rearrangements were noted in 5 out of 11 T cell clones examined. Unfortunately, the antigen specificity of these clones was not examined. Additional features of this patient, besides the diagnosis and the ability to respond to soluble antigens, that were distinct from the other two patients examined, included the fact that only a single joint was effected and that this synovial effusion was of recent onset, present for only 2 weeks at the time of joint aspiration. It may be that a limited repertoire of cells was attracted to the joint acutely. Alternatively, upon entering the joint a subset of cells may have become activated and expanded. The duration of the arthritis in the joint studied and the ability to respond to soluble antigens have not been consistently analyzed in previous reports. While the cloning efficiency in our study was not as high as can be obtained with normal peripheral blood. it does not appear to account for the differences between the patients and is comparable to the efficiency noted with synovial fluid by others (6). The cloning efficiency for Patient 2 was 22%, which was similar to the others, and the method of cloning employed for this patient was the same as that for the others who did not demonstrate oligoclonality. The fact that three of the seven clones generated in response to the AP-MT demonstrated similar rearrangements was somewhat surprising, given the large number of distinct antigenic bands present in the APMT [Fig. 1 in Ref. t’12)l. However, since the 65kDa
SYNOVIAL
FLUID T LYMPHOCYTES
heat shock protein is an immunodominant antigen contained within this complex, these results suggest a dominant response by a limited number of T cell clones, perhaps to a restricted epitope. Consistent with this possibility, the synovial fluid T cell clones of a patient with a self-limiting arthritis generated in response to the mycobacterial65-kDa heat shock protein showed a very restricted response which recognized a single HLA-DR3-restricted epitope (17). We have not determined whether these T cell clones were responding to a single epitope or whether they were restricted through DRl. Any association of oligoclonality with response to soluble antigens will require specificity testing of the clones generated by primary limiting dilution. In summary, these observations suggest that oligoclonality may be present in some patients with inflammatory synovitis. Overall, oligoclonality or restricted heterogeneity of rearranged T cell receptor genes appears to be more readily detected in compartments, such as the spinal fluid and brains of patients with multiple sclerosis (16-20). The ability to detect oligoclonality in patients with arthritis may be related to factors not previously recognized, such as the duration of the synovial effusion, and the ability of the synovial fluid T cells to respond to soluble antigens. Other factors, such as the precipitating mechanism responsible for the flare, might also be important. Such precipitating factors might include mechanical trauma, synovial-derived cytokines, or synovial or exogenous antigens. ACKNOWLEDGMENTS The authors thank Drs. Frank Schmid and Rowland Chang for allowing us to examine their patients. This work was supported in part by a Multipurpose Arthritis Center Grant (AR306921, The VA Research Service, and the Illinois Chapter of the Arthritis Foundation. REFERENCES Savill, C. M., Delves, P. J., Kioussis, D., Walker, P., Lydyard, P. M., Colaco, B., Shipley, M., and Roitt, I. M., A minority of patients with rheumatoid arthritis show a dominant rearrangement of T-cell receptor 9 chain genes in synovial lymphocytes. &and.
J. Zmmunol.
25, 629-635,
1987.
Miltenburg, A. M. M., Van Laar, J. M., Daha, M. R., De Vries, R. R. P., Van Den Elsen, P. J., and Breedveld, F. C., Dominant T-cell receptor P-chain gene rearrangements indicate clonal expansion in the rheumatoid joint. Stand. J. Zmmunol. 31, 121125,199O. Keystone, E. C., Minden, M., Klock, R., Poplonski, L., Zalcberg, J., Takadera, T., and Mak, T. W., Structure of T cell antigen receptor 9 chain in synovial fluid cells from patients with rheumatoid arthritis. Arthritis Rheum. 31, 1555-1557, 1988. van Laar, J. M., Miltenburg, A. M. M., Verdonk, M. J. A., BernReceived May 31, 1991; accepted with revision December 16, 1991
5.
6.
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18.
19.
20.
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stein, B. H., de Vries, R. R. P., van den Elsen, P. J., and Breedveld, F. C., Analysis to T cell receptor p-chain gene rearrangements in patients with rheumatoid arthritis (RA). Arth~itk Rheum. 33, S56, 1990. Stamenkovic, I., Stegagno, M., Wright, K. A., Krane, S. M., Amento, E. P., Colvin, R. B., Duquesnoy, R. J., and Kurnick, J. T., Clonal dominance among T-lymphocyte infiltrates in arthritis. Proc. Natl. Acad. Sci. USA 85, 1179-1183, 1988. Duby, A. D., Sinclair, A. K., Osborne-Lawrence, S. L., Zeldes, W., Kan, L., and Fox, D. A., Clonal heterogeneity of synovial fluid T lymphocytes from patients with rheumatoid arthritis. PFOC. Natl. J. Acad. Sci. USA 86, 62066210,1989. Cush, J. J., Duby, A. D., Lightfoot, E., and Lipsky, P. E., The search for oligoclonal T cells in rheumatoid synovium. Arthritis Rheum. 33, S16, 1990. Cooper, S. M., Dier, D. L., Roessner, K. D., Budd, R. C., and Nicklas, J. A., Diversity of rheumatoid synovial tissue T cells by T cell receptor analysis: Oligoclonal expansion in IL-2 responsive cells. Arthritis Rheum. 34, 537-546, 1991. Kroczek, R. A., Hennerkes, B., Menninger, H., Zacher, J., and Emmrich, F., T-cell clonality in joints of rheumatoid arthritis patients. Arthritis Rheum. 33, S16, 1990. Arnett, F. C., Revised criteria for the classification of rheumatoid arthritis. Bull. Rheum. Dis. 38, l-6, 1989. Pope, R. M., Pahlavani, M. A., LaCour, E., Sambol, S., and Desai, B. V., Antigenic specificity of rheumatoid synovial fluid lymphocytes. Arthritis Rheum. 32, 1371-1380, 1989. Pope, R. M., Wallis, R. S., Sailer, D., Buchanan, R. M., and Pahlavani, M. A., T cell activation by mycobacterial antigens in inflammatory synovitis. Cell. Zmmunol. 133, 95-108, 1991. Maniatis, T., Fritsch, E. F., and Sambrook, J., “Molecular cloning: A Laboratory Manual,” pp. 382-389, Cold Spring Harbor Laboratory, New York, 1982. Bray, R. A., Pope, R. M., and Landay, A. L., Identification of a population of large granular lymphocytes obtained from the rheumatoid joint coexpressing the CD3 and CD16 antigens. Clin. Zmmunol. Zmmunopathol. 58, 409-418, 1991. Sottini, A., Imberti, L., Gorla, R., Cattaneo, R., and Primi, D., Restricted expression of T cell receptor V, but not V, genes in patients with rheumatoid arthritis. EUF. J. Zmmunol. 21, 461466, 1991. Paliard, X., West, S. G., Lafferty, J. A., Clements, J. R., Kappler, J. W., Mar-rack, P., and Kotzin, B. L., Evidence for the effects of superantigen in rheumatoid arthritis. Science 253, 325-329, 1991. Hill Gaston, J. S., Life, P. F., Jenner, P. J., Colston, M. J., and Bacon, P. A., Recognition of a mycobacterial specific epitope in the 65kD heat shock protein by synovial fluid-derived T cell clones. J. Exp. Med. 171, 831, 1990. Hafler, D. A., Duby, A. D., Lee, S. J., Benjamin, D., Seidman, J. G., and Weiner, H. L., Oligoclonal T lymphocytes in the cerebrospinal fluid of patients with mulitple sclerosis. J. Exp. Med. 167, 1313-1322, 1988. Wucherpfenning, K. W., Ota, K., Endo, N., Seidman, J. G., Rosenzweig, A., Weiner, H. L., and Hafler, D. A., Shared human T cell receptor V, usage to immunodominant regions of myelin basic protein. Science 248, 10161019, 1990. Oksenberg, J. R., Stuart, S., Begovich, A. B., Bell, R. B., Erlich, H. A., Steinman, L., and Bernard, C. C. A., Limited heterogeneity of rearranged T-cell receptor V, transcripts in brains of multiple sclerosis patients. Nature 345, 344-346, 1990.
IDEOLOGY
AND
I~~NO~ATHOL~Y
Vol. 63, No. 1, April, pp. 28-33, 1992
Clonal Heterogeneity of Synovial Fluid T Lymphocytes Inflammatory Synovitis YANG Lu,* *Department
BYUNGS.KIM,~
of Medicine, Division of Arthritis-Connective University, 303 East Chicago Avenue, Chicago,
AND RICHARD M. POPE+
Tissue Diseases and i%)epartment of Microbiology-Immunology, Illinois 60611; and the $VA Lakeside Medical Center, Chicago,
Several studies have examined patients with rheumatoid arthritis for the presence of oligoclonal populations of synovial T lymphocytes. The results of these studies have been conflicting. In this study one patient with rheumatoid arthritis and two with other forms of inflammatory synovitis were examined by Southern blot analysis of T cell clones generated from synovial fluid by primary limiting dilution. Evidence of oligocionality was documented only in a patient with psoriatic arthritis. The distin~ishing ~h~acteristics of this patient, in addition to the diagnosis, included the fact that only onejoint was involved, the synovitis in the atIected joint was of recent onset, and the synovial fluid lymphocytes from which the T cells were cloned responded strongly to soluble antigens. Because of the association with the strong responseto soluble antigens, synovial fluid T lymphocytes from another patient with rheumatoid arthritis were cloned in response to a crude mycoba~teria1 antigenie mixture. Three of the seven clones examined were identical by Southern blot analysis. These observations suggestthat the presence of oligoclonality is limited in patients with inflammatory arthritis. The relationship of a specific antigen-driven responsewithin the joint to the detection of oligoclonal T cells within that joint remains to be determined. Q1992 AWdemic Press,
Inc.
INTRODUCTION Numerous studies have examined T cells obtained from the synovial fluid of patients with rheumatoid arthritis in order to detect the presence of oligoclonal populations of T lymphocytes. If present, such populations may indicate responsiveness to a unique antigen that might lead to new forms of therapy. Studies which examined clonal heterogeneity of synovial T lymphocytes employing whole populations of T cells which were unactivated or expanded nonspecifically with mitogen and IL-2 have provided inconsistent results (l4). Other studies employed the use of T cells grown in the presence of IL-2 without mitogen, in order to expand T cells that had been preactivated in. uiuo (5). Dominant T cell rearrangements were observed (5). In order to more precisely define rearrangements expressed by individual T cells within the rheumatoid
Northwestern Illinois
joint, primary limiting dilution analysis was employed, nonspeci~cally stimulating T cells, obtained directly from the joints of patients with rheumatoid arthritis, with mitogen, IL-2, and feeder cells. Initial studies have suggested a very limited degree of oligoclonality (6,7). When clones were expanded employing only feeder cells and IL-2 without mitogen, in order to expand in viva-activated T cells, a more restricted repertoire of T cells has been noted (8, 9). The current studies were performed in order to examine the degree of clonal heterogeneity in patients with different forms of inflammatory synovitis and to relate any observed oligoclonality with the degree of synovial proliferation generated in response to soluble antigens by the synovial fluid lymphocytes from which the T cells were cloned. We observed no oligoclonality in a patient with seropositive rheumatoid arthritis or in a patient with a HLA-B27 -I- chronic polyarthritis. The synovial fluid lymphocytes of these two patients responded only weakly to soluble antigens. In contrast. the T cells obtained from the synovial fluid of a patient with psoriatic arthritis, whose synovial fluid lymphocytes responded strongly to all antigens tested, demonstrated evidence of oligoclonality. In addition, the T cell clones from a patient with rheumatoid arthritis, generated in response to a crude mycobacterial antigenie complex, also demonstrated oligoclonality. These observations suggest that synovial T lymphocyte oligoclonality may occur in some individuals and that it may be associated with an antigen-driven response of recent onset.
Patients examined. Synovial fluid was obtained from four patients with inflammatory synovitis. The clinical characteristics of these patients are detailed in Table 1. Synovial fluid was obtained from Patient 1 on two separate occasions 2 weeks apart. T cell clones were generated on each occasion. Each patient was receiving a nonsteroidal anti-inflammatory drug and methotrexate at the time of ~~ocentesis. The two patients with rheumatoid arthritis fulfilled the 1987 28
0090-1229192 $1.50 Copyright 0 1992 by Academic Press, k. All rights of reproduction in any form reserved.
in
SYNOVIAL
TABLE 1 Characteristics of Patients Patient 1”
2 3 4
Dx HLA-B27 + polyarthritis Psoriatic arthritis Rheumatoid arthritis Rheumatoid arthritis (HLA-DRl + 1
Age (years old) 41 45 66 47
29
FLUID T LYMPHOCYTES
RF& + -
Examined
Erosive disease Yes’ Yes Yes Yes
Disease duration (years) 18 4 12 11
No. swollen joints l/2 1 a 5
Duration flare
of
4/6 weeked 2 weeks 27 weeks 2-3 weeks
-DPatient 1 also demonstrated sacroiilitis on X ray. Synovial fluid was obtained on two occasions 2 weeks apart. The number of joints swollen and the duration of the synovial fluid present in the joint aspirated, by history, are indicated for each aspiration separately. ’ The presence ( + 1 or absence ( - 1 of rheumatoid factor (RF) in patient serum. ’ The presence of erosive disease as evidenced on X ray of peripheral joints. d The duration of the synovial effusion in weeks prior to aspiration as determined by patient history.
College of Rheumatolo~ agnosis (10).
American
criteria
for the di-
Cell isolation and culture. Synovial fluid mononuclear cells were obtained following hi&opaque gradient centrifugation as previously described (11, 12). In order to determine the responsiveness of synovial fluid mononuclear cells to soluble antigens, lo5 mononuclear cells were added to round-bottomed microtiter wells. Cultures were pe~ormed in medium containing 10% heat-inactivated human AB serum (11, 12). Optimal concentrations of antigens were added to the wells as previously determined (11, 12). These antigens included a crude water soluble extract from Mycobucterium t~~e~uZos~s H37Ra which was acetone precipitated CAP-MT), a purified 65kDa heat shock protein obtained from the H37Ra, and tetanus toxoid. Cultures were incubated with antigens for 6 days. Tritiated thymidine was added during the final 6 hr of culture (11, 12). T cell clones were generated by primary limiting dilution (6). T cells were added to microtiter wells containing lo5 irradiated autologous peripheral blood feeder cells, phytohemagglutinin (PHA) (1 kg/ml), and recombinant IL-2 (10 units/ml) in medium containing 10% heat-inactivated fetal bovine serum. On one occasion, autologous synovial fluid mononuclear cells were used as feeder cells. T cells were added at 0.7 cells/well. Clones were fed three times weekly with fresh medium containing IL-2. Clones were expanded every 2 weeks with allogeneic feeder cells, PHA, and IL-2. The cloning efficiency for the nonspecifically propagated clones was 23 +- ll%, with a range of 13 to 35%. This cloning efhciency was similar to that previously described for synovial fluid (6) and was substantially lower than the cloning efficiency observed with peripheral blood by us and others [(6), unpublished observations]. Synovial fluid T lymphocytes from Patient 1 were cloned on two occasions 2 weeks apart. On the first occasion, the cloning efficiency was 35%, employing autologous peripheral blood cells as feeders. On the second occasion, em-
ploying synovial fluid mononuclear cells as feeders, a cloning efficiency of 13% was noted. Following expansion, T cell clones were washed in phosphate-buffered saline, counted, and snap frozen in liquid nitrogen until DNA extraction. Antigen-specific T cell clones were generated from Patient 4, as previously described (111, in response to the crude AP-MT which contained multiple protein bands when analyzed by SDS-PAGE [Fig. 1 in Ref. flZ>]. Following initial activation with AP-MT, without IL-2, for 1 week, lymphoblasts were isolated by histopaque gradient centrifugation and cloned at limiting dilution employing autologous irradiated peripheral blood mononuclear cells as feeder cells, Al?-MT, and IL-2 in medium containing 10% heat-inactivated human AB serum. The clones were fed three times a week with fresh medium and IL-2. Growing clones were expanded every 2 weeks with irradiated autologous peripheral blood feeder cells, AP-MT, and IL-2. T cell clones from Patient 3 and Patient 4 were tested for antigen specificity. T cell clones, 10 to 14 days after the prior round of stimulation, were washed with fresh medium and added a lo4 cells per well together with irradiated, autologous peripheral blood feeder cells alone or feeder cells plus specific antigen. Testing for antigen specificity was performed in medium containing 10% heat-inactivated human AB sereum. The wells were cultured for 72 hr and tritiated thymidine was added for the final 6 hr of culture prior to harvesting as previously described (11, 12). A total of 43 clones were expanded from Patient 1. Sufficient DNA was available for Southern blot analysis on 24 of these clones. Twenty-two clones were obtained from Patient 2 and 14 were examined by Southern blot analysis. Seventeen clones were generated from Patient 3 and 11 were examined by Southern blot analysis. From Patient 4, 22 of 32 clones generated in response to AP-MT recognized this antigen (stimulation index >4 and ~2000 cpm). Of the 33 clones that responded to the AP-MT, 19 responded to the 65-kDa heat shock protein. These clones did not respond to the
30
LU, KIM, AND POPE
human 60-kDa heat shock protein or to tetanus toxoid. Seven of these clones were selected for Southern blot analysis because sufficient DNA was available. Immunophenotyping. The clones from Patient 4 were immunophenotyped employing anti-CD3, -CD4, -CD& and -WT31 monoclonal antibodies as previously described (14). All the clones were CD3 + , CD4 + , and WT31+. The synovial fluid mononuclear cells from which the clones were generated were examined in Patients 1 and 2 employing anti-CD3.
blots were perSouthern blot analysis. Southern formed by standard techniques (13). Briefly, cells were incubated in lysis buffer and the aqueous phase was extracted with phenol and chloroform. The DNA was precipitated with ethanol and incubated with DNAsefree RNAse. After reextraction, ethanol precipitation, and washing, the DNA was redissolved in buffer and the concentration was determined by optical density at 260 nm and the purity was determined by the optical density ratio 260:280. The DNA (10 kg) was digested with EcoRI for 16 hr at 37”C, electrophoresed through 7.5% agarose gels, transferred to nylon membranes (Nytran, Schleicher & Schuell, Keene, NH), and hybridized with a 32P C, cDNA probe. Commercially obtained 32P-labeled C, and J,2 probes were employed (Oncor, Inc., Gaithersburg, MD). Hybridization was performed for 24 hr in 6x SSC, 10x Denhardts, 100 pg/ml denatured salmon sperm DNA, 1% SDS, and 50% formamide at 45°C. Placental DNA was the germline control employed for each blot. Following autoradiography, employing the C, probe, the nylon membranes were stripped and rehybridized with the Js2 probe. Those samples with one or two rearranged bands following hybridization with both probes were considered clonal (both chromosomes may rearrange). With EcoRI digestion, germline bands at 11 kb (Cell and 4 kb (Cs2) were seen employing the C, probe (Fig.
1). Rearrangements using J,l appeared as unique bands and those rearranging through J,2 were not seen because of an EcoRI restriction site between J,2 and $2. The germline fragment hybridizing with J,2 was seen as a 4.4-kb band (Fig. 11, and rearrangements using J,2 were seen as unique bands with this probe. Samples were considered clonal if one or two rearranged bands were observed following digestion with EcoRI and hybridization with both the C, and Jo2 probes. Of all the samples examined, five were nonclonal as evidenced by the presence of more than two rearranged bands. One example is presented in Fig. 2. Those samples that appeared identical employing EcoRI were examined with additional restriction endonucleases. Also, those clones with bands that appeared identical, even though the clones were not identical, were examined with additional restriction endonucleases whenever sufficient DNA was available. Hind111 generated an 8-kb germline DNA fragment containing J,2 (Fig. 1) that was altered by rearrangements employing J,2. Hybridization of the C, probe with the 3’ end of the C, (a 6.5kb fragment) and with the C,l (a 3.5-kb fragment) will not be effected by these rearrangements. In order to examine common rearrangements noted through J,l, BamHI was employed. A 24-kb germline fragment was generated which hybridized with both the $1 and the J,2 probes. RESULTS
Examination of the clones obtained from Patients 1 and 3 generated by nonspecific cloning failed to reveal any instances of identical rearrangements. Examples of the clones from Patient 1 are presented in Fig. 2. One example of a nonclonal sample, with three rearranged bands (lane E), is also presented. Although no identical rearrangements were noted, individual bands of similar molecular weights were apparent with both
DE2 J8l t
B
9
t
E
J3
=2
t
t t
t
H
HE
E
t
tt
H
EB
t
H
EcoRI:
53 11.6
5
I kb
4.0 I
III:
i
Jn2 4.4
Hind
kb
kb
JB2 8.0
kb
1. Schematic representation of the T cell receptor B gene and the fragments generated by the restriction endonucleases employed in this study. The locations of the EcoRI (E), the Hid11 (H), and BumHI (B) are indicated by the arrows. The size of the germline DNA fragments generated with EcoRI and HindIII, hybridizing with the Ce and the J,2 probes, are indicated. FIG.
SYNOVIAL PROBE:
31
FLUID T LYMPHOCYTES A
A~CDEFEHIJKLMNOP
6
C
0
E
F
G
H
IJ
K
L
M
PROBE:
- 11.0
J&J c
_ 4.4
- 4.0
- 11.0
Je*
ce
- 4.0
FIG. 2. Southern blots of 14 different T cell preparations from the synovial fluid of patients with inflammatory synovitis. The DNA was digested with EcoRI and hybridized with C, and J,2 probes as indicated. The molecular weight markers are indicated on the right. The placental germline control is in lane P. Lane N contains a specimen from Patient 2 and the remainder of the samples are from Patient 1. The samples in lanes C and E were obtained from the second arthrocentesis and the remainder from the first. Lanes I and L contain an identical sample which was run at different times. The specimen in lane E demonstrated three rearranged bands and was not considered clonal.
the C, probe (Fig. 2, lanes A, K, and I/L at 6.6 kb) and with the J,2 probe (Fig. 2, lanes D, F, and J at 5.4 kb). In order to better define whether or not identical rearrangements of individual chromosomes might be represented by bands of similar molecular weight, additional Southern blot hybridization was performed on selected clones following Hind111 endonuclease digestion employing the J,2 probe. If identical rearrangements were used, the rearrangements hybridizing with J,2, which appeared similar following EcoRI digestion, should also possess similar molecular weights following Hind111 digestion. Three sets of clones were examined, including those presented in Fig. 2. No identical rearrangements were observed (data not shown). The three clones that appeared to have similar rearrangements through J,l following EcoRI digestion (Fig. 2, lanes A, K, and I/L) were also examined following BamHI digestion to confirm that these bands might represent similar rearrangements through J,l. Unfortunately, adequate digestion employing this restriction endonuclease did not occur with the DNA available from these three clones. The results obtained with clones generated from Patient 2, with psoriatic arthritis, were different. Five of the 11 clones examined demonstrated identical rearrangements (Fig. 3, lanes B, F, G, H, and I). Each of these clones possessed a rearrangement of one chromosome utilizing J,l and the other employing Js2. The rearrangements through J,2 resulted in identical bands employing the Hind111 restriction endonuclease (Fig. 4). The synovial fluid T cell population from which these T cell clones were generated responded strongly to all of the soluble antigens tested (Table 2). Unfortunately, the individual clones of this patient,
FIG. 3. Southern blots of 11 T cell clones from Patient 2 with psoriatic arthritis. The DNA was digested withEcoRI and hybridized with C, and J,2 probes. The position of the molecular weight markers is indicated on the right. The placental germline control is presented in lanes A and M. Lanes B, F, G, H, and I possess rearrangements that appear identical.
which were generated nonspecifically, were not analyzed for their antigen specificity. In contrast, the whole synovial fluid mononuclear cells of Patients 1 and 3, which demonstrated no oligoclonality, responded weakly to the soluble antigens tested (Table 2). Seventeen clones from Patient 3 were examined for antigen specificity. None of the 17 T cell clones examined from this patient responded to any of the antigens tested, including AP-MT, tetanus toxoid, and bovine type II collagen. Because of the suggestion that responsiveness to soluble antigens might be associated with the ability to detect clonal rearrangements in the inflammatory joint, T cell clones were generated in response to a very crude preparation of mycobacterial antigens CAP-MT). We reasoned that if oligoclonality was associated with an antigen-driven response, in vitro responsiveness to a crude antigen might enrich a limited number of clones. Similar rearrangements were observed through J,l for three of the seven clones examined following EcoRI digestion (Fig. 5 lanes B, C, and E). Similar rearrangements were noted following Bar&II endonuclease digestion with each of the clones which demonstrated similar rearrangements following EcoRI digestion (data not shown).
ABCDEFG PROBE:
J2
- 8.0
FIG. 4. Southern blots of the five T cell clones from the synovial fluid of Patient 2 with psoriatic arthritis which appeared identical following EcoRI digestion. The DNA was digested with HindI and hybridized with the J,2 probe. The position of the molecular weight markers is indicated on the right. The placental germline control is presented in lanes A and G.
32
LU,
KIM,
TABLE 2 Phenotype and Proliferative Response by Uncloned Synovial Fluid Mononuclear Cells Patient
Medium”
1
183'
2 3 4
390
AP-MT” 2.070 21,132
6%kDa
(11)” (641
hsp”
915 (5) 15,525
(40)
Tetanus
toxoid*
1,619 (91 47,454 (122) 438(l) 8,328 (21
CD3 + b 76% 71%
468 NDd ND 4,199 (9) 4005 11,879 (3) 25,722 (6) ND ____~_~.._. .._~~~___ .. ,.~ _ ._~.. ~~(1Cells were cultured in medium alone or medium pbns an optimal concentration of a soluble antigen. The antigens inchaded AP-MT, the mycobacterial 65-kDa heat shock protein (hsp), and tetanus toxoid. bThe percentage of CD3t cells contained in the original synovial fluid mononuclear cell preparation. c The results are presented as mean counts per minute. The values in parentheses represent the stimulation index obtained by dividing the counts per minute for cells plus antigen by the counts per minute for cells in medium alone. The results presented for Patient 1 represent those obtained following the initial arthrocentesis. d Not done.
DISCUSSION
Earlier studies examining T cells isolated freshly from the synovial fluid as well as from those nonspecifically propagated with mitogens have produced conflicting results. Some studies have noted a minor to a substantial degree of oligoclonality (l-9). The differences between these studies, in part, may be technical since overdevelopment of Southern blots employing polyclonal T cells may result in the detection of a limited number of Dsl-Jsl, DslJs2, and Ds2-Js2 rearrangements that might be confused with oligoclonality (6). The use of primary limiting dilution analysis avoids this potential problem. Employing primary limiting dilution with mitogen and IL-2, olig~lonality was not observed in two of our patients, one with seropositive rheumatoid arthritis, the other with HLA-B27-associated chronic polyarthriAI(CDEFGH PROBE: -
11.0
5
- 4.0
J2 - 4.4 5. Southern blots of seven T cell clones generated in response to AP-MT from the synovial fluid of Patient 4 with rheumatoid arthritis. The DNA was digested with EcoRI and hybridized with C, and J,2 probes, The position of the molecular weight markers is indicated on the right. The germline control is presented in lane A. Identical rearrangements were observed in lanes B (the upper band was faint but present), C, and E. FIG.
AND
POPE
tis. These results are similar to those previously reported by others in rheumatoid arthritis employing a similar technique (6-9). Oligoclonality has been more frequently observed when synovial T cells were expanded in IL-2 without the addition of mitogen (5,8,91; suggesting that T cells which have been activated i,z uiuo, which possess IL-2 receptor, are more likely to demonstrate evidence of oligoclonality. In uiuoactivated cells were not selected by our method of cloning by primary limiting dilution employing mitogen and IL-2. Recent studies of whole synovial fluid and peripheral blood mononuclear cells, employing the polymerase chain reaction, have suggested the presence of oligoclonality in the joints of patients with rheumatoid arthritis (15, 161. Both of our patients who demonstrated no evidence of oligoclonality had erosive disease and the synovial effusions from which the T cells were cloned were chronic in one and subacute in the other (Table 111.The synovial fluid mononuclear cells of these two patients failed to respond strongly to any souble antigen when tested. Additionally, the clones of one of these patients (Patient 3) failed to respond to any soluble antigen examined. In contrast, the synovial fluid mononuclear cells of the patient with psoriatic arthritis, from which the T cells were cloned, responded strongly to soluble antigens. Clonal rearrangements were noted in 5 out of 11 T cell clones examined. Unfortunately, the antigen specificity of these clones was not examined. Additional features of this patient, besides the diagnosis and the ability to respond to soluble antigens, that were distinct from the other two patients examined, included the fact that only a single joint was effected and that this synovial effusion was of recent onset, present for only 2 weeks at the time of joint aspiration. It may be that a limited repertoire of cells was attracted to the joint acutely. Alternatively, upon entering the joint a subset of cells may have become activated and expanded. The duration of the arthritis in the joint studied and the ability to respond to soluble antigens have not been consistently analyzed in previous reports. While the cloning efficiency in our study was not as high as can be obtained with normal peripheral blood. it does not appear to account for the differences between the patients and is comparable to the efficiency noted with synovial fluid by others (6). The cloning efficiency for Patient 2 was 22%, which was similar to the others, and the method of cloning employed for this patient was the same as that for the others who did not demonstrate oligoclonality. The fact that three of the seven clones generated in response to the AP-MT demonstrated similar rearrangements was somewhat surprising, given the large number of distinct antigenic bands present in the APMT [Fig. 1 in Ref. t’12)l. However, since the 65kDa
SYNOVIAL
FLUID T LYMPHOCYTES
heat shock protein is an immunodominant antigen contained within this complex, these results suggest a dominant response by a limited number of T cell clones, perhaps to a restricted epitope. Consistent with this possibility, the synovial fluid T cell clones of a patient with a self-limiting arthritis generated in response to the mycobacterial65-kDa heat shock protein showed a very restricted response which recognized a single HLA-DR3-restricted epitope (17). We have not determined whether these T cell clones were responding to a single epitope or whether they were restricted through DRl. Any association of oligoclonality with response to soluble antigens will require specificity testing of the clones generated by primary limiting dilution. In summary, these observations suggest that oligoclonality may be present in some patients with inflammatory synovitis. Overall, oligoclonality or restricted heterogeneity of rearranged T cell receptor genes appears to be more readily detected in compartments, such as the spinal fluid and brains of patients with multiple sclerosis (16-20). The ability to detect oligoclonality in patients with arthritis may be related to factors not previously recognized, such as the duration of the synovial effusion, and the ability of the synovial fluid T cells to respond to soluble antigens. Other factors, such as the precipitating mechanism responsible for the flare, might also be important. Such precipitating factors might include mechanical trauma, synovial-derived cytokines, or synovial or exogenous antigens. ACKNOWLEDGMENTS The authors thank Drs. Frank Schmid and Rowland Chang for allowing us to examine their patients. This work was supported in part by a Multipurpose Arthritis Center Grant (AR306921, The VA Research Service, and the Illinois Chapter of the Arthritis Foundation. REFERENCES Savill, C. M., Delves, P. J., Kioussis, D., Walker, P., Lydyard, P. M., Colaco, B., Shipley, M., and Roitt, I. M., A minority of patients with rheumatoid arthritis show a dominant rearrangement of T-cell receptor 9 chain genes in synovial lymphocytes. &and.
J. Zmmunol.
25, 629-635,
1987.
Miltenburg, A. M. M., Van Laar, J. M., Daha, M. R., De Vries, R. R. P., Van Den Elsen, P. J., and Breedveld, F. C., Dominant T-cell receptor P-chain gene rearrangements indicate clonal expansion in the rheumatoid joint. Stand. J. Zmmunol. 31, 121125,199O. Keystone, E. C., Minden, M., Klock, R., Poplonski, L., Zalcberg, J., Takadera, T., and Mak, T. W., Structure of T cell antigen receptor 9 chain in synovial fluid cells from patients with rheumatoid arthritis. Arthritis Rheum. 31, 1555-1557, 1988. van Laar, J. M., Miltenburg, A. M. M., Verdonk, M. J. A., BernReceived May 31, 1991; accepted with revision December 16, 1991
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