This proposal will describe studies aimed at exploring the regulation of Ig isotype switching by murine B cells stimulated through their antigen receptor, using a novel conjugate, dextran-linked anti-immunoglobulin D antibody (alpha/delta-dex). We have demonstrated that alpha/delta-dex induces resting murine B cells to proliferate at 1000-fold lower concentrations of alpha/delta-dex than that required for induction of comparable responses to unconjugated anti-IgD. T cell-derived cytokines induce alpha/delta-dex-activated B cells to secrete immunoglobulin (Ig) and switch to the expression of most Ig isotypes, but fail to stimulate detectable IgE. By contrast, Sepharose-bound anti-IgD (alpha/delta-seph), when cultured with B cells and T cell-derived cytokines stimulates a large IgE response. Furthermore, IFN-gamma selectively induces IgG3 secretion by alpha/delta-dex-activated, membrane (m)IgM+mIgG3-B cells in the presence of a maturation factor, such as IL-5. Alpha/delta-dex also potently and selectively inhibits LPS + ILA-4-mediated IgE secretion. In this proposal we will describe experiments designed to 1) determine the basis for the selective failure of alpha/delta-activated B cells to secrete IgE in order to delineate the non-IL-4-mediated signals necessary for IgE production, 2) explore the mechanism by which IFN-gamma simulates alpha/delta-dex-activated B cells to switch to the expression of IgG3, the major IgG isotype induced by bacterial-derived polysaccharides in vivo, and 3) investigate mechanisms underlying the selective alpha/delta-dex-mediated inhibition of LPS + IL-4-induced IgE production. The effects of cytokines and costimuli (alpha/delta-dex, alpha/delta-seph, LPS) on IgE and IgG3 class switching will be explored using traditional cell biologic methodology in parallel with studies of constant heavy (CH) gene regulation. The latter will include analyses of induction of regulatory DNA-binding proteins specific for CH gene loci, quantitation of CH gene germline transcription, and quantitation of switch recombination at the DNA level. These studies will clarify key parameters which regulate Ig class switching upon antigen receptor crosslinkage.
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