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THU0012 Clonal analysis of vh and vl mrna in single b and plasma cells from rheumatoid arthritis synovium and peripheral blood
  1. S Ruzickova1,2,
  2. O Krystufkova1,
  3. Z Cimburek1,
  4. J Niederlova1,
  5. J Sinkora3,
  6. O Horvath4,
  7. T Doerner2,
  8. J Vencovsky1
  1. 1Institute of Rheumatology and Laboratory of Gene Expression
  2. 2Department of Rheumatology/Immunology, Medical Faculty, Charite, Humboldt University, Berlin, Germany
  3. 3Division of Immunology and Gnotobiology
  4. 4Department of Immunology, Institute of Microbiology CAS, Prague, Czech Republic

Abstract

Background Rheumatoid arthritis (RA) is characterised by a complex infiltration of many cell types including T and B lymphocytes, plasma cells, folicular dendritic cells, macrophages and other cells into synovial tissue of inflamed joints. Presence of B and plasma cells results in a local synthesis of multiple pathologic immunoglobulins contributing to the chronic joint inflammatory processes.

Objectives To analyse mRNA from individual B and plasma cells isolated from synovium and peripheral blood of patients with RA, in order to investigate a clonality, VH and VL pairing and a molecular structure of produced antibodies.

Methods RA synovial tissue after synovectomy was enzymatically digested. Single synovial B/plasma cells were sorted using immunofluorescent staining with anti-CD19, anti-IgM and anti-CD38. RA peripheral B cells were isolated in the same way without enzyme treatment. cDNA library from each single B and plasma cell was generated. Nested polymerase chain reaction was performed to analyse VH and VL gene usage. VH, DH and JH or VL and JL gene segments were assigned and somatic mutations determined by comparison to germline sequences on the V BASE/Genbank database. As a control peripheral B-lymphocytes from healthy donor were screened.

Results Analysis of RA synovial mRNA revealed prevalence of C gamma rearranged transcripts that contained rearranged VH1, VH2, VH3, VH4, VH5 and VH6 genes. Utilisation of VH segments was similar between RA patients and normal subjects, but the accumulation of somatic mutations was elevated in RA synovial and peripheral B cells. Preferential utilisation of a limited number of VH and DH gene segments was found. There was an increased frequency of kappa light chains containing unusually long CDR3 when compared to normal peripheral B cells. mRNAs resulting from transcription of non-productive rearrangements were also detected as well as transcripts corresponding to isotype-switching.

Conclusion Our findings are consistent with hypothesis that B cell response in RA synovium is probably antigen driven and oligoclonal.

The project was supported by grant VS96129 from Czech Ministry of Education, Youth and Sport in the Czech Republic.

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