Longhurst, Celia Margaret; (1995) Sequence analysis of human monoclonal autoantibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Autoimmune diseases are thought to result from the inappropriate activation of the immune system resulting in damage to tissues in the affected individuals. The presence of circulating autoantibodies is associated with most autoimmune diseases. Systemic lupus erythematosus is characterised by the presence of high titre autoantibodies which bind to a variety of nuclear antigens including DNA. Anti-DNA antibodies have been found deposited in the disease lesions of SLE patients, thus suggesting that these autoantibodies are involved in disease pathogenesis. Investigation of the genetic origin of autoantibodies is required to see if they are derived from different immunoglobulin genes than those utilised in the production of antibodies to exogenous antigens in healthy adults. The immunoglobulin variable region genes utilised in a panel of monoclonal autoantibodies derived from three SLE patients with active disease were investigated and compared with immunoglobulin gene usage in autoantibodies derived from vasculitis and polymyositis Northern blotting analysis utilising cDNA probes specific for six human VH gene families revealed a VH4 gene family bias had occurred in the generation of these autoantibodies. The VH4 gene family bias usage was further investigated by sequence analysis. Anti-DNA antibodies of the IgM isotype were found to be expressing immunoglobulin variable genes with close identity to known germline genes. These data support the idea that autoantibodies can be generated with little or no somatic mutation. For example, the VH4.21 germline gene was utilised by three anti-ssDNA antibodies. The distinctive feature of these autoantibodies was the presence of arginine residues within the heavy chain variable gene CDR3 region. Positively charged amino acid residues such as arginine and lysine have been suggested as important elements in conferring DNA binding ability in murine anti-DNA autoantibodies. These results show that these amino acids could be involved in DNA binding in human anti-DNA antibodies. The VH 4.21 gene was also found to encode an anti-myeloperoxidase autoantibody derived from a patient with vasculitis. The anti-MPO antibody E3-MP0 did not bind DNA and did not express the arginine residues seen in the VH4.21 encoded anti-DNA autoantibodies. Anti-DNA antibodies encoded by other variable region germline genes such as VH4.22 did not contain arginine residues suggesting that other factors apart from the presence of positively charged amino acid residues are involved in generating DNA binding ability. The studies described here show strong evidence that human monoclonal anti-DNA autoantibodies resemble those found in murine models of SLE.

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