Recent observations in a number of systems have suggested a model of autoimmune disease initiation and epitope spreading in which an autoreactive B cell plays a key role in presentation of self-determinants to normal T cells. The theory postulates that some autoreactive antigen- specific B cells are able to process their cognate self antigen and present cryptic determinants to normal T cells, which, in genetically susceptible individuals, initiate the autoimmune cascade. By the same mechanism, additional rounds of antigen uptake and processing by newly arising autoantibody-producing B cells is hypothesized to contribute to epitope spreading in the T cell compartment. The induction of IDDM would, according to this theory, involve the uptake of antigens associated with IDDM by such B cells, and presentation of the derived peptides to disease-causing T cells. A key prediction, and the central hypothesis to be tested in this proposal, is that different B cells displaying antibodies having different epitope specificities, will differ with respect to the predominant peptides that are presented in the context of specific MHC class II molecules. In this proposal, a series of B cell lines which express recombinant surface Ig, differing with respect to epitope-binding specificity and V gene utilization, will be constructed and used to study peptide presentation following specific antibody- mediated uptake of antigen. Construction of recombinant lines will employ LCL selected for class II expression and ability to be transfected. In the first specific aim, antibody genes will be cloned from currently available anti-insulin antibody-producing B cell lines, as well as from additional anti-GAD and anti-insulin producing cell lines to be generated from GAD autoantibody-, and IAA-positive subjects. Cloned V/H and V/L genes will be inserted into appropriate vectors for transfection and expression. In the second aim, B cell lines will be transfected and will be characterized for level of surface Ig expression and antigen binding specificity prior to use m antigen processing assays.
The third aim will analyze the peptide products generated from processing of insulin or GAD by this panel of B cell lines in order to ascertain the extent to which different B cells differentially process antigen. The last aim will use a functional assay to examine the capacity of the B cell lines to elicit specific T cell responses and to determine parameters of selection of T cell specificity by B cells.
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