The long term goal of this proposal is to gain insight into the mechanisms underlying the generation of autoantibodies in two major autoimmune diseases, systemic lupus erythematosus (SLE) and rheumatoid arthritis. The predominant and possibly pathogenic anti-self Ag responses in these systemic autoimmune conditions include autoantibodies to chromatin components, such as histones and DNA, and the Fc fragment of homologous IgG (rheumatoid factors, RFs). The origin of these autoantibodies remains enigmatic, but their structural features, as suggested by experiments from the first funding period of this grant, resemble those of high affinity antibodies induced by exogenous antigens (Ags). This resemblance includes the choice of VH, D, JH, VL and/or JL genes, and the pattern of somatic point-mutation in the Ig VHDJH and VLJL gene sequences. We hypothesize that, when appropriately presented, cell nuclear Ags or IgG can recruit virgin B lymphocytes expressing surface receptors for Ag encoded by unmutated Ig V gene segments, and drive these antibody-producing cell precursors through a process of somatic mutation and selection (affinity maturation), in a fashion not dissimilar from that shown by us to occur in anti-insulin IgG auto-antibodies arising in patients with type 1 diabetes mellitus, a prototypic organ-specific autoimmune disease. We formally argue that evidence for selection by self Ag of an autoantibody-producing cell precursor entails the demonstration that the germline template Ig progenitor of the somatically mutated autoantibody is capable of binding the relevant self Ag, and, that at least some of the point-mutations observed in the """"""""affinity mature"""""""" autoantibody are crucial in enhancing this binding. We further argue that such an evidence also requires the demonstration that affinity mature autoantibodies are directed mainly to the inducing self Ag, and they recognize more than one epitope on that molecule. To test our hypothesis, we propose to: (i) determine the Ag- specificity, the primary structure, and the nature and distribution of somatic point-mutations in the H and L chain V segments of anti-chromatin components and anti-IgG monoclonal antibodies (mAbs) that we will generate at different disease stages in SLE and rheumatoid patients; (ii) construct, using the human Ig Y gene recombinant, mutagenesis, and in vitro Ig V gene expression technology, """"""""full germline"""""""" revertants (template Ig progenitors) of these somatically point-mutated, high affinity mAbs, and analyze their ability to bind the relative self Ags; (iii) verify that equivalent anti-self B clonotypes expressing unmutated surface receptor for Ag naturally, occur in healthy humans; (iv) analyze the ability of the somatic point mutations detected in given """"""""affinity mature"""""""" lupus or rheumatoid autoantibodies to enhance the affinity for the relevant self Ag of the respective """"""""full germ-line"""""""" revertant ig templates; and, finally, (v) validate the above studies performed at the level of selected B cell clones, and extend them to multiple elements of individual clonotypes as well as multiple clonotypes, by Ig """"""""repertoire cloning"""""""" in combinatorial phage display libraries. The proposed experiments should further our understanding of the events that lead to the generation of autoantibodies in human autoimmune diseases, and may, thereby, help design specific means of therapeutic intervention.
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