The production of highly specific autoantibodies to nuclear antigens is one of the hallmarks of systemic autoimmune diseases. Antibodies reacting with histones and chromatin epitopes represent an important component of the antinuclear antibody population. These autoantibodies have been less studied than other antinuclear specificities, appear at an early stage during the autoimmune process and may play an important role in the pathogenesis of autoimmune diseases. Using spontaneously autoimmune mice, we propose to study the specificities and variable region genes of these autoantibodies, the mechanisms responsible for tolerance to chromatin antigens and the ontogeny of these autoreactive B cells. (1) We will generate, from single autoimmune mice, panels of hybridomas that will be selected by various methods for reactivity with chromatin or nuclei. These antibodies will be characterized with respect to their specificities and variable region genes. This approach will not be biased toward a single specificity and will shed light on the relative importance (and overlap) among subpopulations of autoantibodies to chromatin. New or rarely studied autoantibody specificities will be analyzed with respect to their epitopes and their variable region genes. (2) Using heavy and light chain recombination techniques and site directed mutagenesis, we will assess the respective importance of the heavy and light chains and the role of certain critical residues in antibody binding to histones and chromatin. This will allow us to assess the relationships among various sets of antinuclear antibodies and the role of certain specific somatic mutations. One point of interest is to determine whether some anti-DNA antibodies originate through specific somatic mutations from autoantibodies to the histone H2A-H2B-DNA complex. (3) Mice transgenic for an antiH2A-H2B-DNA autoantibody will be generated. This model is particularly fitting since loss of tolerance to this self-antigen occurs at the onset of systemic autoimmunity. The characterization of these mice will provide insight into the mechanisms responsible for tolerance to chromatin antigens and other multimolecular complexes. Other antichromatin transgenic or V gene replacement models will be developed later during this project. (4) The heavy chain CDR3s of antibodies to histone-DNA complexes and to DNA contain many cationic residues that result from unusual VH-D-JH rearrangements. We will investigate whether these rearrangements occur more frequently during B cell ontogeny in autoimmune mice. The detection of these rearrangements early in the ontogeny of autoimmune B cells before antigen selection would point to an intrinsic defect in autoimmune B cells. This last step of the project will be accomplished by generating PCR-amplified libraries of VH-D- JH junctions obtained from lymphoid organs of auto immune or control mice at various stages of development.
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