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Synovial tissue in rheumatoid arthritis displays a complex infiltration of many cell types like T and B lymphocytes, plasma cells, folicular dendritic cells, macrophages etc. Presence of B and plasma cells results in secretion of large amounts of multiple pathologic autoantibodies.
To analyze immunoglobulin VH and VL gene usage on the level of mRNA from a single B or plasma cell isolated from synovium and peripheral blood of patients with RA, in order to determine a clonality and a molecular structure of produced antibodies.
RA synovial tissue obtained during synovectomy was enzymatically digested and single synovial B/plasma cells were sorted using immunofluorescent staining with anti-CD19 or anti-CD138. Peripheral B cells were isolated in the same way without enzyme treatment. cDNA library from each single B and plasma cell was generated. Two stage polymerase chain reaction to analyse VH and VL genes was performed. VH, DH and JH or VL and JL gene segments were assigned and somatic mutations determined by comparison with germline sequences on the V BASE/Genbank data base. As a control peripheral B lymphocytes from healthy donors were screened.
Analysis of RA synovial mRNA transcripts revealed prevalence of C gamma recombinants that contained rearranged VH1, VH3, VH4, VH5 and VH6 genes. Utilization 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. We also found preferential utilization of a limited number of VH and DH gene segments. There was increased frequency of kappa light chains containing unusually long CDR3 when compared to normal peripheral B cells.
Our findings are consistent with hypothesis that B cell response RA synovium is probably antigen driven and oligoclonal.
The project was supported by grant VS96129 from Ministry of Education, Youth and Sport in the Czech Republic.