This proposal will primarily examine the cellular events regulating the production of human allergic (IgE) antibodies utilizing two newly defined in vitro systems of human IgE synthesis. We have developed and in vitro analogue of ongoing IgE synthesis employing mycoplasma free clones of IgE secreting cells recently derived by us from the human IgE plasma cell line, U266. The clones are responsive to both inhibitory (suppressor cells and supernatants) and helper (T cell IgE enhancing factor) activities and thus allow us to assay both positive and negative aspects of IgE regulation. By employing IgA and IgG secreting cells in the same wells, we will assess isotype specific effects. This will be complemented by induction of IgE synthesis from resting human B cells using IgE enhancing factor derived from cultured T cells from patients with high levels of IgE. Thus we will examine both regulation of induction as well as ongoing IgE production. Particular emphasis will be given to the role of IgE in regulation of both B and T cells through use of defined Ige anti-IgE immune complexes. These complexes will be constructed from purified human IgE and mouse monoclonal anti Fc epsilon (FcEpsilon) antibodies which have been produced and well characterized by us. Data with these complexes shows isotype specific effects on both IgE regulatory T cells and producing B cells. Differential effects on T cell expression of IgE regulatory activities induced by the nature of IgE complex binding (i.e. alone or in the presence of antigen) will be tested. Expression of the FcE receptor will be assessed by our newly perfected technique whereby IgE anti-IgE complexes are bound to cells and then detected via immunoperoxidase staining. We will probe the relationship between FcE and Fc gamma receptor expression on T cells and their regulation of IgE. Further functional evaluation of the various cells will be achieved by enriching the cells according to their isotype Fc receptors. Regulatory IgE cells will be expanded for in-depth study by Interleukon II and T cell clones derived using HTLV II transformation. The expression on IgE committed B cells of FcE receptors as potential acceptor sites for complexes of IgE-IgE binding regulatory factors will also be explored. These studies will be extended to the cells from patients already demonstrated to have abberant control of IgE. Seleted molecular studies will be undertaken to extend the cellular results as to the mechanisms of regulatory events at the B cell level.
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