Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology. Synovium in patients with active RA often contains structures resembling germinal centers seen in normal secondary lymphoid organs such as spleen or lymph node. Plasma cells in RA synovium may secrete high levels of immunoglobulin. Because of strong circumstantial evidence that immune complexes containing rheumatoid factor (RF) are involved in the pathogenesis of RA, previous studies of synovial antibodies have generally focused on these autoantibodies that react against the Fc portion of IgG. RFs typically comprise only 5-10% of the RA synovial repertoire. The antigen specificity of the majority of the synovial antibody repertoire remains largely unknown. Our previous studies of a synovial cDNA library from a patient with longstanding RA have provided evidence for oligoclonal B cell expansion as a mechanism of immunoglobulin secretion in RA synovium. Both heavy (H) and light (L) chain sequences in this library were found to contain somatic mutation patterns typical of antigen-driven responses, with high replacement/silent substitution ratios in the antigen binding sites (CDRs). Furthermore, approximately 5% of the kappa light chain and gamma heavy chain repertoires were noted to be clonally related. Surprisingly, the antibodies generated in this synovial tissue were enriched for kappa light chains with unusually long Vkappa-Jkappa junctions (CDR3s) that are predicted to yield unusual antigen binding sites. Because inflammation in RA may be initiated or sustained as a result an antigen-driven response, we propose to express in vitro VH and VL domains from early and late RA synovium. This will allow us to assess the reactivity of the resultant antibody fragments against antigens for which there is substantial evidence of involvement in the initiation or propagation of RA. Such antigens include the Fc portion of IgG (the target antigen of RF), type II collagen, the E. coli heat shock protein dnaJ, and mycoplasmal and Chlamydiae antigens. Initially, we will generate a combinatorial library from VH and Vkappa domains cloned from our well- characterized synovial cDNA library. The phagemid expression system pComb3 will be used to express Fab fragments that contain clonally expanded or kappa chains or those with unusually long CDR3 regions. ELISA assays will be used to screen Fab fragments for reactivity to the antigens of interest. Subsequently, we will assess the antigen specificity of Fab fragments containing paired H and L chain V domains cloned by single cell PCR from individual plasma cells in early RA synovium. By studying this population of terminally differentiated, presumably antigen-stimulated B lineage cells, we hope to gain further insight into antigens that may be initiating or propagating the immune response in RA. These studies should not only shed significant light into a poorly understood manifestation of rheumatic disease, but also serve as reference for future studies of abnormal immune responses.
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