The goal of these studies is to define the requirements for activating B lymphocytes to proliferate and differentiate into antibody forming cells and the basis of functional heterogeneity of B lymphocytes. Our research will initially focus on two molecules Lyb2 and Ia which are expressed on all B cells and may serve as surface receptors that receive activating signals. Our experiments will test the hypothesis that Lyb2 molecules are receptors for B cell stimulatory factor-1 (BSF-1 or IL4). Also, we will develop strategies to clone the gene for Lyb2 antigen which has been mapped by classical genetic studies to chromosome 4. We will evaluate the hypothesis that a unique B cell subset is involved in antibody responses to polysaccharide antigens. In particular we will analyze antibody responses to TNP-Ficoll, which we find in our preliminary experiments to stimulate B cells from spleen but not from lymph nodes and that such B cells are unusually long lived. The possibility that B cell subsets may differ in the growth or maturation factor receptors they express will be studied by preparing monoclonal antibodies to such B cell surface molecules. Experiments will be performed in tissue culture with purified B lymphocytes obtained from inbred strains of mice. Activation of B lymphocytes will be measured by increase in RNA or DNA synthesis or changes in calcium concentrations. Maturation of B cells into plasma cells will be determined by plaque forming cell assay or by measuring secreted immunoglobulin in an enzyme linked immunoassay. Cell surface expression of important molecules will be analyzed by flow cytometry and gel electrophoresis. To establish the relationship between BSF-1 receptors and Lyb2, cleavable bifunctional crosslinking reagent will be utilized to isolate the receptor using anti BSF-1 antibody. To isolate genes for Lyb2 by cDNA cloning gamma GT11 expression vectors will be utilized. The long term goals of the project are to understand the basis of activation of resting B cells and the mechanism by which antibody synthesis is regulated. These studies should enable us to devise better strategies for controlling such autoimmune states in which some B cell subsets are hyper-represented or lymphoid malignancies and to increase antibody responses in immunodeficient syndromes.
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