Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement

The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.


Complete T- and B-Cell Receptor Repertoire Analysis in Rheumatoid Arthritis Using Massive Parallel Sequencing

Klarenbeek1,  P.L., Doorenspleet1,  M.E., van Schaik1,  B.D.C., Herenius1,  M.M., Jakobs1,  M.E., Cantaert2,  Tineke, Baeten1,  D.L.P.

Academic Medical Center/Univ. of Amsterdam, Amsterdam, Netherlands,
Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands,
Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands

Purpose:

T-cells and B-cells are likely to play important roles in the pathogenesis of rheumatoid arthritis (RA). Previous attempts to investigate the roles of T- and B-cell clones in RA by screening the T-/B-cell receptor (TCR/BCR) repertoires were hampered by the sheer size and complexity of the repertoires. Current techniques are unable to analyse the whole repertoire in sufficient detail, are vulnerable to artefacts, and do not provide quantitative data. Here, we used our newly developed protocol based on massive parallel sequencing which overcomes current limitations and produces the DNA-sequence of >100.000 receptors in a single experiment. Using this technique we performed the first quantitative, high- resolution analysis of the complete TCR and BCR repertoires in an RA patient.

Objectives:

Describe the complete BCR and TCR repertoires in synovial tissue (ST) and peripheral blood (PB) samples of an RA-patient and screen for dominant T- and B-cell clones.

Method:

mRNA was isolated from paired PB and ST samples from an ACPA+ RA-patient with active disease despite treatment with methotrexate. A linear amplification with multiplex primers for all V(ariable)-families of the receptor beta-chain (TCR) or heavy-chain (BCR) was performed. The samples were analyzed on a Genome Sequencer FLX (Roche) resulting in 14000 reads/samples for TCR and 35000 for BCR analysis respectively, each containing the full CDR3 sequence. Bioinformatic algorithms were used to identify gene segments and correct for sequencing errors.

Results:

TCR-repertoire: in ST most TCRs contained a Vbeta6(46%), 10(19%), and 27(13%) gene segment, while in PB V29(32%) and 7(26%) were most frequent. The TCR repertoire was dominated by low-frequency clones (>95%), both in the PB and ST. Several clones were clearly expanded (up to 217 and 121 copies/clone for PB and ST). However, the dominant clones in ST were different from those in PB, as compared by V-segment and CDR3 sequence. BCR-repertoire: the ST sample showed preferential usage of the small V2,5,6,7 families (total 50%) when compared to published data in PB1(20%). Several clearly expanded clones (up to 1892 copies) were found against a background of low-frequency clones.

Conclusion:

This is the first high-resolution analysis of the TCR and BCR repertoire in RA, providing detailed insight into the presence of T- and B-cell clones. We found clear differences between the TCR-repertoire in ST compared to PB in an RA-patient. Several expanded clones were found only in ST, suggesting proliferation or local retention of T-cells. The BCR-repertoire also showed expanded clones within the ST. Further studies will elucidate the role of these clones in RA.

1van Dongen, JJ, Leukemia 2003;17:2257–317.

To cite this abstract, please use the following information:
Klarenbeek, P.L., Doorenspleet, M.E., van Schaik, B.D.C., Herenius, M.M., Jakobs, M.E., Cantaert, Tineke, et al; Complete T- and B-Cell Receptor Repertoire Analysis in Rheumatoid Arthritis Using Massive Parallel Sequencing [abstract]. Arthritis Rheum 2009;60 Suppl 10 :1228
DOI: 10.1002/art.26302

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