This research project is based on the hypothesis that the human idiotype 16/6 (Id 16/6) participates in stimulation of pathogenic autoantibody production in patients with SLE. Our goals will be: to determine the structural requirements for Id 16/6 recognition by anti-idiotype antibodies and by Id 16/6-selected T cell clones; and to test the ability of Id 16/6- reactive T cells to drive autoantibody formation by autologous B cells. We have prepared a plasmid vector in which various H and L chain V regions can be inserted and expressed by bacteria as single chain Fv molecules. This vector will be used to identify protein sites required for idiotope formation in a folded Fv domain. We will test Fv products with varying H and L chains for reaction with anti-idiotype antibodies. Chains with naturally occurring mutations will be compared and site-specific and cassette mutagenesis will be used to alter specific regions of the Fv. All products will be tested for reaction with monoclonal and polyclonal anti-Id 16/6 antibodies. We will identify a patient with Id 16/6-positive Ig and Id 16/6-responsive T cells, and isolate Id 16/6-selected T cell clones from peripheral blood sample. From the same sample we will clone EBV-transformed B cells that have Id 16/6-positive Ig on the cell surface and other B cells that are Id 16/6-negative. the Id 16/6-negative B cells will be used as antigen- processing and presenting cells for soluble antigens. We will identify T cell idiotopes by tests of T cell proliferation in response to bacterially expressed H and L chain V regions, truncated chains and synthetic peptides. We will determine whether Id 16/6-positive B cells can serve as both the source of processed Id 16/6 and antigen presenting cell for stimulation of cloned T cells, and whether stimulated T cell clones can augment the synthesis of IgG autoantibodies by fresh autologous peripheral blood lymphocytes.
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