MOLECULAR GENETICS OF AUTOANTIBODIES IN HUMANS

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The long term goal of this study is to delineate the molecular and genetic basis for sustained production of potentially pathogenic autoantibodies in autoimmune diseases. These "disease specific" autoantibodies are high affinity autoantibodies of IgM and IgG isotypes, such as IgG rheumatoid factors (RFs) in rheumatoid arthritis (RA) and IgG anti-DNA antibodies in systemic lupus erythematosus (SLE). During the last funding period, our analysis of IgG RFs from RA patients has revealed that the RFs in each patient are clonally related, and arise from natural autoantibodies. Recent studies of immunological tolerance show that self-reactive B cells either are deleted by apoptosis or are functionally inactivated (anergy). However, some self-reactive B cells are not tolerized in autoimmune MRL- lpr/lpr mice, which have a defective Fas-mediated apoptosis pathway. It is known that, under selection pressure, a variant cell with a survival advantage can expand in a clonal manner. We hypothesize that some disease specific autoantibody-secreting B cells in RA and SLE may escape tolerance regulation because of: 1) loss-of-function mutations in apoptosis-related genes or gain-of-function mutations in apoptosis suppressor genes (such as Bcl-2) at the clonal B cell level, resulting i tolerance-resistant variants; or 2) gain-of-function mutations in stimulation-related genes (such as B7), resulting in constitutively activated anergic variants. To examine these hypotheses, the specific aims are: 1) Generation and characterization of disease specific autoantibody- secreting B cell lines and isotype-specific control Ig-secreting cell lines from RA and SLE patients. 2) Comparative analysis of the Fas-mediated apoptosis pathway in these cell lines. Cell lines from each patient will be analyzed for Fas expression on the cell surface, secretion of soluble Fas, and apoptosis induced separately by either Fas ligand (FasL) or cytotoxic anti-Fas antibodies. 3) Comparative analysis of Bcl-2 expression in these cell lines. 4) Comparative analysis of the Fas-independent, surface Ig-mediated apoptosis pathway in these cell lines; 5) Comparative analysis of the B7 costimulator expression in these cell lines. 6) Determination if the defects identified in the above studies are also present in freshly isolated autoreactive B cells from corresponding patients.