The aims of our experiments are to understand the requirements for growth and differentiation of B lymphocytes. We will define the B lymphocyte surface molecules that receive signals for induction of proliferation and maturation of resting B cells. We will prepare monoclonal antibodies against such functionally important cell surface molecules. Antibodies that inhibit or augment B-cell proliferation or antibody secretion will be identified and characterized by immunofluorescence and flow cytometry. These studies will test the hypothesis that B-lymphocyte heterogeneity is due to expression of subset specific receptors for B cell growth and maturation. Important cell surface receptors for growth or maturation inducing factors will be purified by affinity chromatography on columns containing monoclonal antibodies and will be employed to isolate their specific ligands. We will evaluate the role of Lyb 2, a B cell differentiation antigen, in B cell activation. Our experiments are aimed at defining the activation state of B lymphocytes that respond to monoclonal anti-Lyb 2 antibody by proliferation: The fraction of B cells that are induced into cell division by monoclonal anti-Lyb 2 antibody will be determined by cell cycle analysis on a fluorescence activated cell sorter. Experiments will be performed to see if the inhibitory effect of anti-Lyb 2 on antigen specific responses is a result of its ability to induce polyclonal B cell proliferation. We will evaluate the hypothesis that Lyb 2 molecules are physiological receptors for a B cell growth factor and will attempt to isolate such a molecule on affinity columns of Lyb 2. Cell lines that express Lyb 2 in large quantities will be selected by flow cytometry techniques to build sources useful for its analysis at biochemical and genetic levels. Experiments will be performed in serum free defined tissue culture medium with purified B lymphocytes obtained from inbred strains of mice. B cell activation will be measured by proliferation assay, by detection of antibody secretion in the plaque forming cell assay and by measuring the changes in RNA and DNA content on cell sorter after staining with acrydine orange. Cell surface molecules will be isolated by immunoprecipitation of radiolabelled membrane molecules and characterized by 2-dimensional gel electrophoresis. The long-term goals of the project are to understand the events that lead to antibody response and its regulation. Such information is relevant to the understanding and therapy of malignancies of lymphocytes, allergic disorders and autoimmune disorders.
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