British Journal of Rheumatology 1992;31:55-57
BRIEF REPORT
POLYCLONAL ORIGIN OF RHEUMATOID SYNOVIAL T-LYMPHOCYTES BY W. HYLTON*, C. SMITH-BURCHNELL*, B. K. PELTON*, R. G. PALMERf, A. M. DENMAN* AND M. MALKOVSKY* *MRC Clinical Research Centre and Northwick Park Hospital, Watford Road, Harrow, Middlesex
SUMMARY Nineteen T-cell clones from seven patients with RA were obtained by cloning infiltrating lymphocytes from needle synovial biopsies. Southern blot analysis of the T-cell receptor (TCR) |3-chain genes in these clones revealed that there were no T-cell clones with an identical rearrangement of the TCRfJ gene. These results do not support the idea that the infiltrating T-lymphocytes in RA are of monoclonal or oligoclonal origin. KEY WORDS: T-cell
receptor gene, Rheumatoid synovitis, Clonality.
phocytes migrating from biopsy fragments over a 24 h period were cloned by limiting dilution and cultured in medium containing PHA, recombinant IL-2, and irradiated feeder cells [7]. The expression of the T-cell receptor (TCR complex) was confirmed using antiCD3 monoclonal antibodies (MoAb) on a fluorescence-activated cell sorter [8]. All the 19 expanded lymphocyte clones were CD3-positive. DNA of high molecular weight isolated from cloned cells [9] was digested with Eco RI, Hind III or Bg/II, size fractionated on a 0.8% agarose gel and transferred to a Hybond-N nylon filter (Amersham) by Southern blotting [10]. Filters were then hybridized to the TCR(3 constant region gene probe, M131B10BB1 [11] labelled with [a-32/>]dCTP [12]. Autoradiography was carried out at — 70°C with an intensifying screen for 2-4 days. TABLE I GENETIC ANALYSIS OF T-CELL CLONES
PATIENTS AND METHODS Samples for analysis were obtained from seven patients (one male and six females) with rheumatoid arthritis (RA) [6] of less than 3 years' duration, who were receiving non-steroidal anti-inflammatory drugs, and who had not been treated with systemic steroids or cytotoxic drugs. The synovial samples were obtained by closed transcutaneous biopsy from affected knee joints using a Vim-Silverman needle under local anaesthetic. LymSubmitted 11 September 1990; revised version accepted 25 January 1991. tPresent address: Solihull Hospital, Lode Lane, Solihull, West Midlands. +Present address: Department of Medical Microbiology and Immunology and Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA. Correspondence to Dr W. Hylton, Division of Immunological Medicine, Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ.
Clone Eco RI
Hind III
GL1 8.1,7.0,3 .5 GL2 9.0, 6.0, 4.2 GL3 8.0, 3.5 GL4 10.0, 4.0 GL5 10.0,4.0 GL6 9.0, 7.0, 3 .5 GL7 11.0, 9.0, 4.0 GL8 9.0,4.0 GL9 12.0, 10.0 ,4.5 GA1 9.0,4.51 DO1 11.0,4.0 DO2 4.0 DO3 4.0 PA1 9.5,4.0 HA1 11.0, 10.0 ,4.0 KP1 9.0, 3.0 WH1 5.0,4.5 WH2 11.0,8.5, 4.5 WH3 11.0, 10.0 ,4.0 Germline configuration
7.0,5 .8,4.5 7.8,5 .8,3.5 7.8,4 .0, 3.5 7.8, 7.0, 5.8, 3.0 7.8,6 .0, 5.0, 3.0
fig'11
NT1 NT NT NT NT NT NT NT 10.5, 7.0,5.8 10.5, 7.0, 5.8 ,3.5 NT NT 12.0, 10.0, 1.3 NT 7.8,7 .0,3.2 10.0, 7.0,5.8 ,3.5 NT NT 4.5,4.0,2.0 NT 4.8,4.5,2.0 NT NT NT 10.0,9.5, 1.3 NT NT NT 11.0,8.0, 1.3 NT 11.0,1.3 NT 9.0,2.2,1.3
12.0, 4.0 7.8,5 .8, 3.5 10.5,0.8 NT, not tested. Figures are size of DNA fragments (kb) calibrated with molecular weight ladder on Southern blot gel hybridizing with a TCRfJ gene probe. 55
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
infiltration is a prominent feature of rheumatoid synovitis [1]. It is usually considered to represent part of a local immune response to unknown persistent antigen [2, 3]. One might therefore predict that the infiltrate would predominantly consist of one or several clonal T-cell populations with a specific reactivity for this putative antigen. T-cell receptor (TCR)specific probes have been commonly used to detect the dominant T-cell clone in malignant lymphoproliferative diseases of T-cells [4], and the same approach has been used to study the nature of the T-cell infiltrate in rheumatoid arthritis (RA). On the basis of this technique, it has been claimed that there is clonal dominance in the rheumatoid synovial membrane [5]. This claim has therapeutic as well as aetiological implications, since if a dominant clone with the same receptor sequences was shown to be shared by patients with the disease, antibody directed at this sequence might prove generally effective in therapeutic immunomanipulation. However, the experiments reported in this paper suggest that the T-lymphocyte infiltrate is polyclonal in nature. T-LYMPHOCYTE
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 1
56
1 2
4 5
6
7 8
9 10 II 12
13 14 15 (to 17
18 19
10 — 5—
3_
cloned T-lymphocytes from synovial fragments. Possibly clonal dominance resulted from artefactual in vitro selection. Their experiments were performed on surgical synovectomy samples of patients with advanced disease. The design of our study (cloning first and then expanding the individual clones) excludes the risk of artefactual in vitro development of clonal dominance, which can occur in prolonged in vitro culture systems of uncloned lymphocytes. ACKNOWLEDGEMENTS
0-5—
RESULTS Rearranged patterns were detected in all the clones (Table I) (Fig. 1). There was no consistent pattern of rearrangement in the T-lymphocyte populations propagated from inflammatory synovia, nor were the patterns substantially dissimilar from those obtained with clones of peripheral blood T-lymphocytes from healthy volunteers (data not shown). Out of 19 clones, an identical pattern was observed in no more than two clones (10.5%); 95% confidence limits for this proportion are 1.3% and 33.1 %. If there were clonal dominance, one would expect there to be at least 50% of identical patterns in the population of samples. DISCUSSION These results do not support the possibility that dominant clones with a unique TCR gene arrangement play a major role in the pathogenesis of RA. Thus in this pilot study we have failed to identify a unique predominant rearrangement pattern of the TCR gene. Our findings corroborate the results of Feldmann's gToup [13,14], who used the MX9 and 42/1 Cl MoAbs (recognizing the TCR V|58 and V05 gene families, respectively) and showed that there was no preferential or dominant use of a single TCR V family in the T-cell infiltrate in rheumatoid synovitis. A similar conclusion—a lack of a predominating T-cell clone in the majority of RA patients—can be drawn from the previous study with uncloned T-lymphocytes isolated from rheumatoid synovial effusions [15]. These findings conflict with those of Stamenkovic and colleagues [5], who
REFERENCES
1. Harris ED Jr. pathogenesis of rheumatoid arthritis. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology. 3rd ed. Philadelphia: W.B. Saunders, 1989. 2. Res PCM, Schaar CG, Breedveld FC, et al. Synovial fluid T-cell reactivity against 65 kD heat shock protein of mycobacteria in early chronic arthritis. Lancet 1988;ii:478-80. 3. Londei M, Savill CM, Verhoef A, etal. Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis. Proc Natl Acad Sci USA 1989;86:636-40. 4. Flug F, Pelicci PG, Bonetti F, Knowles DM 2d, DallaFavera R. T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms. Proc Natl Acad Sci USA 1985;82:346XM. 5. Stamenkovic I, Stegagno M, Wright KA, etal. Clonal dominance among T-lymphocyte infiltrates in arthritis. Proc Natl Acad Sci USA 1988;85:1179-83. 6. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24. 7. Palmer RG, Smith-Burchnell CA, Pelton BK, Hylton W, Denman AM. Use of T-cell cloning to detect in vivo mutations induced by cyclophosphamide. Arthritis Rheum 1988;31:757-61. 8. Fisch P, Malkovsky M, Braakman E, et al. Gamma/ delta T-cell clones and natural killer cell clones mediate distinct patterns of non-major histocompatibility complex-restricted cytolysis. J Exp Med 1990;171:1567-79. 9. Davis LG, Dibner MD, Battey JF. Basic methods in molecular biology. New York: Elsevier, 1986:44. 10. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Molec Biol 1975;98:503-17. 11. Sims JE, Tunnacliffe A, Smith WJ, Rabbitts TH. Complexity of human T-cell antigen receptor betachain constant and variable-region genes. Nature 1984;312:541-5. 12. Rigby PWJ, Dieckmann M, Rhode C, Berg P. Labelling deoxyribonucleic acid to high specific activity in
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
FIG. 1.—Agarose gel. Electrophoresis analysis of RE digests of DNA from T-cell clones. Southern blots were hybridized to TCRy probe. Lanes 1 and 13 represent molecular weight markers; lanes 2 and 5,4 and 6 represent DNA from T-cell clones from donor 1 digested with Eco RI and Hind III respectively. Lanes 7, 9 and 11, 8, 10 and 12, represent Eco RI and Hindlll DNA digests from donor 2 T-cell clones. Lanes 14,16, 18andl5,17,19 are Eco RI and Bgl II digests from donor 3. Molecular weights given in kilobase.
The authors would like to thank Dr Letizia Foroni of the Royal Postgraduate Medical School, Hammersmith Hospital, for the generous gift of TCR|3 receptor gene probe [16]. This work was supported in part by NIH grant RR-00167 to WRPRC. This is publication no. 29-033 of WRPRC. We are grateful to Dr David Hill for statistical advice.
HYLTON ETAL.: POLYCLONAL ORIGIN OF RHEUMATOID SYNOVIAL T-LYMPHOCYTES vitro by nick translation with DNA polymerase I. J Molec Biol 1977;113:237-51. 13. Brennan FM, Allard S, Londei M, et al. Heterogeneity of T-cell receptor idiotypes in rheumatoid arthritis. Clin Exp Immunol 1988;73:417-23. 14. Feldmann M, Londei M, Leech Z, Brennan F, Savill C, Maini RN. Analysis of T-cell clones in rheumatoid arthritis. Springer Semin Immunopathol 1988;10:157-67.
57
15. Savill CM, Delves PJ, Kioussis D, et al. A minority of patients with rheumatoid arthritis show a dominant rearrangement of T-cell receptor beta chain genes in synovial lymphocytes. Scand J Immunol 1987; 25:629-35. 16. Gascoigne NRJ, Chien YH, Becker DM, Kavaler J, Davis MM. Genomic organization and sequence of T-cell receptor P-chain constant and joining region genes. Nature 1984;310:387-91.
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
BRIEF REPORT
POLYCLONAL ORIGIN OF RHEUMATOID SYNOVIAL T-LYMPHOCYTES BY W. HYLTON*, C. SMITH-BURCHNELL*, B. K. PELTON*, R. G. PALMERf, A. M. DENMAN* AND M. MALKOVSKY* *MRC Clinical Research Centre and Northwick Park Hospital, Watford Road, Harrow, Middlesex
SUMMARY Nineteen T-cell clones from seven patients with RA were obtained by cloning infiltrating lymphocytes from needle synovial biopsies. Southern blot analysis of the T-cell receptor (TCR) |3-chain genes in these clones revealed that there were no T-cell clones with an identical rearrangement of the TCRfJ gene. These results do not support the idea that the infiltrating T-lymphocytes in RA are of monoclonal or oligoclonal origin. KEY WORDS: T-cell
receptor gene, Rheumatoid synovitis, Clonality.
phocytes migrating from biopsy fragments over a 24 h period were cloned by limiting dilution and cultured in medium containing PHA, recombinant IL-2, and irradiated feeder cells [7]. The expression of the T-cell receptor (TCR complex) was confirmed using antiCD3 monoclonal antibodies (MoAb) on a fluorescence-activated cell sorter [8]. All the 19 expanded lymphocyte clones were CD3-positive. DNA of high molecular weight isolated from cloned cells [9] was digested with Eco RI, Hind III or Bg/II, size fractionated on a 0.8% agarose gel and transferred to a Hybond-N nylon filter (Amersham) by Southern blotting [10]. Filters were then hybridized to the TCR(3 constant region gene probe, M131B10BB1 [11] labelled with [a-32/>]dCTP [12]. Autoradiography was carried out at — 70°C with an intensifying screen for 2-4 days. TABLE I GENETIC ANALYSIS OF T-CELL CLONES
PATIENTS AND METHODS Samples for analysis were obtained from seven patients (one male and six females) with rheumatoid arthritis (RA) [6] of less than 3 years' duration, who were receiving non-steroidal anti-inflammatory drugs, and who had not been treated with systemic steroids or cytotoxic drugs. The synovial samples were obtained by closed transcutaneous biopsy from affected knee joints using a Vim-Silverman needle under local anaesthetic. LymSubmitted 11 September 1990; revised version accepted 25 January 1991. tPresent address: Solihull Hospital, Lode Lane, Solihull, West Midlands. +Present address: Department of Medical Microbiology and Immunology and Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA. Correspondence to Dr W. Hylton, Division of Immunological Medicine, Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ.
Clone Eco RI
Hind III
GL1 8.1,7.0,3 .5 GL2 9.0, 6.0, 4.2 GL3 8.0, 3.5 GL4 10.0, 4.0 GL5 10.0,4.0 GL6 9.0, 7.0, 3 .5 GL7 11.0, 9.0, 4.0 GL8 9.0,4.0 GL9 12.0, 10.0 ,4.5 GA1 9.0,4.51 DO1 11.0,4.0 DO2 4.0 DO3 4.0 PA1 9.5,4.0 HA1 11.0, 10.0 ,4.0 KP1 9.0, 3.0 WH1 5.0,4.5 WH2 11.0,8.5, 4.5 WH3 11.0, 10.0 ,4.0 Germline configuration
7.0,5 .8,4.5 7.8,5 .8,3.5 7.8,4 .0, 3.5 7.8, 7.0, 5.8, 3.0 7.8,6 .0, 5.0, 3.0
fig'11
NT1 NT NT NT NT NT NT NT 10.5, 7.0,5.8 10.5, 7.0, 5.8 ,3.5 NT NT 12.0, 10.0, 1.3 NT 7.8,7 .0,3.2 10.0, 7.0,5.8 ,3.5 NT NT 4.5,4.0,2.0 NT 4.8,4.5,2.0 NT NT NT 10.0,9.5, 1.3 NT NT NT 11.0,8.0, 1.3 NT 11.0,1.3 NT 9.0,2.2,1.3
12.0, 4.0 7.8,5 .8, 3.5 10.5,0.8 NT, not tested. Figures are size of DNA fragments (kb) calibrated with molecular weight ladder on Southern blot gel hybridizing with a TCRfJ gene probe. 55
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
infiltration is a prominent feature of rheumatoid synovitis [1]. It is usually considered to represent part of a local immune response to unknown persistent antigen [2, 3]. One might therefore predict that the infiltrate would predominantly consist of one or several clonal T-cell populations with a specific reactivity for this putative antigen. T-cell receptor (TCR)specific probes have been commonly used to detect the dominant T-cell clone in malignant lymphoproliferative diseases of T-cells [4], and the same approach has been used to study the nature of the T-cell infiltrate in rheumatoid arthritis (RA). On the basis of this technique, it has been claimed that there is clonal dominance in the rheumatoid synovial membrane [5]. This claim has therapeutic as well as aetiological implications, since if a dominant clone with the same receptor sequences was shown to be shared by patients with the disease, antibody directed at this sequence might prove generally effective in therapeutic immunomanipulation. However, the experiments reported in this paper suggest that the T-lymphocyte infiltrate is polyclonal in nature. T-LYMPHOCYTE
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 1
56
1 2
4 5
6
7 8
9 10 II 12
13 14 15 (to 17
18 19
10 — 5—
3_
cloned T-lymphocytes from synovial fragments. Possibly clonal dominance resulted from artefactual in vitro selection. Their experiments were performed on surgical synovectomy samples of patients with advanced disease. The design of our study (cloning first and then expanding the individual clones) excludes the risk of artefactual in vitro development of clonal dominance, which can occur in prolonged in vitro culture systems of uncloned lymphocytes. ACKNOWLEDGEMENTS
0-5—
RESULTS Rearranged patterns were detected in all the clones (Table I) (Fig. 1). There was no consistent pattern of rearrangement in the T-lymphocyte populations propagated from inflammatory synovia, nor were the patterns substantially dissimilar from those obtained with clones of peripheral blood T-lymphocytes from healthy volunteers (data not shown). Out of 19 clones, an identical pattern was observed in no more than two clones (10.5%); 95% confidence limits for this proportion are 1.3% and 33.1 %. If there were clonal dominance, one would expect there to be at least 50% of identical patterns in the population of samples. DISCUSSION These results do not support the possibility that dominant clones with a unique TCR gene arrangement play a major role in the pathogenesis of RA. Thus in this pilot study we have failed to identify a unique predominant rearrangement pattern of the TCR gene. Our findings corroborate the results of Feldmann's gToup [13,14], who used the MX9 and 42/1 Cl MoAbs (recognizing the TCR V|58 and V05 gene families, respectively) and showed that there was no preferential or dominant use of a single TCR V family in the T-cell infiltrate in rheumatoid synovitis. A similar conclusion—a lack of a predominating T-cell clone in the majority of RA patients—can be drawn from the previous study with uncloned T-lymphocytes isolated from rheumatoid synovial effusions [15]. These findings conflict with those of Stamenkovic and colleagues [5], who
REFERENCES
1. Harris ED Jr. pathogenesis of rheumatoid arthritis. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology. 3rd ed. Philadelphia: W.B. Saunders, 1989. 2. Res PCM, Schaar CG, Breedveld FC, et al. Synovial fluid T-cell reactivity against 65 kD heat shock protein of mycobacteria in early chronic arthritis. Lancet 1988;ii:478-80. 3. Londei M, Savill CM, Verhoef A, etal. Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis. Proc Natl Acad Sci USA 1989;86:636-40. 4. Flug F, Pelicci PG, Bonetti F, Knowles DM 2d, DallaFavera R. T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms. Proc Natl Acad Sci USA 1985;82:346XM. 5. Stamenkovic I, Stegagno M, Wright KA, etal. Clonal dominance among T-lymphocyte infiltrates in arthritis. Proc Natl Acad Sci USA 1988;85:1179-83. 6. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24. 7. Palmer RG, Smith-Burchnell CA, Pelton BK, Hylton W, Denman AM. Use of T-cell cloning to detect in vivo mutations induced by cyclophosphamide. Arthritis Rheum 1988;31:757-61. 8. Fisch P, Malkovsky M, Braakman E, et al. Gamma/ delta T-cell clones and natural killer cell clones mediate distinct patterns of non-major histocompatibility complex-restricted cytolysis. J Exp Med 1990;171:1567-79. 9. Davis LG, Dibner MD, Battey JF. Basic methods in molecular biology. New York: Elsevier, 1986:44. 10. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Molec Biol 1975;98:503-17. 11. Sims JE, Tunnacliffe A, Smith WJ, Rabbitts TH. Complexity of human T-cell antigen receptor betachain constant and variable-region genes. Nature 1984;312:541-5. 12. Rigby PWJ, Dieckmann M, Rhode C, Berg P. Labelling deoxyribonucleic acid to high specific activity in
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
FIG. 1.—Agarose gel. Electrophoresis analysis of RE digests of DNA from T-cell clones. Southern blots were hybridized to TCRy probe. Lanes 1 and 13 represent molecular weight markers; lanes 2 and 5,4 and 6 represent DNA from T-cell clones from donor 1 digested with Eco RI and Hind III respectively. Lanes 7, 9 and 11, 8, 10 and 12, represent Eco RI and Hindlll DNA digests from donor 2 T-cell clones. Lanes 14,16, 18andl5,17,19 are Eco RI and Bgl II digests from donor 3. Molecular weights given in kilobase.
The authors would like to thank Dr Letizia Foroni of the Royal Postgraduate Medical School, Hammersmith Hospital, for the generous gift of TCR|3 receptor gene probe [16]. This work was supported in part by NIH grant RR-00167 to WRPRC. This is publication no. 29-033 of WRPRC. We are grateful to Dr David Hill for statistical advice.
HYLTON ETAL.: POLYCLONAL ORIGIN OF RHEUMATOID SYNOVIAL T-LYMPHOCYTES vitro by nick translation with DNA polymerase I. J Molec Biol 1977;113:237-51. 13. Brennan FM, Allard S, Londei M, et al. Heterogeneity of T-cell receptor idiotypes in rheumatoid arthritis. Clin Exp Immunol 1988;73:417-23. 14. Feldmann M, Londei M, Leech Z, Brennan F, Savill C, Maini RN. Analysis of T-cell clones in rheumatoid arthritis. Springer Semin Immunopathol 1988;10:157-67.
57
15. Savill CM, Delves PJ, Kioussis D, et al. A minority of patients with rheumatoid arthritis show a dominant rearrangement of T-cell receptor beta chain genes in synovial lymphocytes. Scand J Immunol 1987; 25:629-35. 16. Gascoigne NRJ, Chien YH, Becker DM, Kavaler J, Davis MM. Genomic organization and sequence of T-cell receptor P-chain constant and joining region genes. Nature 1984;310:387-91.
Downloaded from http://rheumatology.oxfordjournals.org/ at RMIT University Library on July 21, 2015
