The activation and differentiation of B cells from the resting state to terminal maturation is regulated, in large part, by T cells through a series of cognate and non-cognate interactions. Over the past three years, we have characterized many of these interactions and have now developed a model system to explore mechanisms of B cell differentiation. This will be accomplished by the purification, to homogeneity, of a non IL-6 B cell differentiation factor (BCDF) which we have isolated from anti-CD3 stimulated T cells. We will utilize this factor as well as monoclonal antibody (mAb) technology to isolate the BCDF receptor using strategies derived from the isolation of both the low and high affinity IL-2R (homobifunctional crosslinkers). Having purified factor and isolated receptor will allow us to study, in a pure system (B cell lines, factor, receptor), signal transduction across the membrane (i.e. does the signal activate a PI linked pathway, Ca++ mobilization, a tyrosine kinase or PKC translocation). We have identified the end result of such activation, reversal of membrane/secreted Ig mRNA ratio, and will explore such events relating to the regulation of this process (i.e. phosphorylation of perinuclear or nuclear proteins. Lastly, we will attempt to generate T cell clones which display distinct patterns of lymphokine secretion (analogous to Th1 and Th2 clones in mouse). Two approaches will be used: 1) an allergy desensitization protocol (to honeybee venom) harvesting and activating T cells with specific antigens at various stages of desensitization and 2) stimulation of T cells with anti-CD3 mAbs 147, 446 and 454 which result in distinct patterns of lymphokine secretion. Clones of such activated cells will be generated and regulation of lymphokine production established. Such an integrated approach (regulation of factor production, factor isolation, receptor identification, receptor/factor interaction, signal transduction, and nuclear differentiation events) will allow us to clearly define distinct stages of B cell maturation (with their associated changes) and aid in the understanding of disorders of B cell maturation such as common variable immunodeficiency (CVI), chronic lymphocytic leukemia (CLL) and autoimmunity.
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