Clonally-related immunoglobulin VH domains and nonrandom use of DH gene segments in rheumatoid arthritis synovium.

BACKGROUND: Synovia of patients with long-standing rheumatoid arthritis (RA) are typically infiltrated with B lymphocytes and plasma cells that secrete large amounts of immunoglobulin. The CDR3 of an immunoglobulin heavy chain is composed of the VH-DH-JH join, with interposed N region addition, and thus defines clonal relatedness. Furthermore, the CDR3 lies at the center of the antigen binding site, so its length and composition influence antigen binding. We sought definitive evidence of an antigen-driven B cell response (i.e., clones derived from the same VH, DH, and JH gene segments with shared somatic mutations) in RA synovial mRNA transcripts, and to characterize CDR3 intervals at the target of inflammation in this autoimmune disease. MATERIALS AND METHODS: We screened a cDNA library generated from unselected cells from the knee joint of a 62-year-old white female with long-standing RA. This technique does not have the potential bias of selecting for antibodies that express a particular reactivity such as rheumatoid factor. C gamma recombinants were sequenced and progenitor VH, DH, and JH gene segments were assigned and somatic mutations determined by comparison to germline sequences. Analyses of DH reading frame utilization and hydropathy characteristics of CDR3s were performed. RESULTS: Two of 67 recombinants were derived from the same VH (V3-11) and JH gene segments, demonstrated shared mutations, and contained nearly identical VH-DH-JH joins, including N region addition. Three other recombinants contained identical sequence throughout the variable domain. We also found preferential utilization of a limited number of VH and DH gene segments and marked preference for a DH reading frame encoding predominantly hydrophilic residues. CONCLUSIONS: Analysis of expressed heavy chain variable domains strongly supports the hypothesis that the B cell response in RA synovium is at least in part antigen driven and oligoclonal.

Somatic mutation and CDR3 lengths of immunoglobulin kappa light chains expressed in patients with rheumatoid arthritis and in normal individuals.

Immunoglobulin secretion by plasma cells infiltrating synovial membranes is a prominent feature of RA. Previous analyses of a cDNA library generated from synovium of RA patient BC revealed immunoglobulin kappa light chain transcripts with extensive somatic mutation, frequent N region addition, and unexpected variation in the lengths of CDR3 regions which form the center of the antigen binding site. To determine if these characteristics are present in other individuals, we performed reverse transcription-polymerase chain reaction amplification and sequenced > or = 10 V kappa-containing amplicons from nine tissue samples: synovia of three individuals with long-standing RA (including patient BC), PBLs of two of these individuals, and PBLs or splenocytes of four normal individuals. Increased levels of somatic mutation in PBLs appeared to correlate with increased age, which may reflect accumulation of circulating memory cells and/or decreased bone marrow production of naive B lymphocytes. Two of three RA synovial samples and both RA PBL samples exhibited increased proportions of clones with unusual CDR3 lengths. Enrichment for these antibody binding sites could be due to abnormal regulation of the emerging repertoire or to selection for B lymphocytes bearing antibodies of unusual specificity, and may play a role in the pathogenesis of RA.