The application proposes to test an implication of our central hypothesis that B-cell expression of CD5 results from activation via sIg cross-linking: If CD5 B-cells are generated by antigen-mediated activation, then autoreactive CD5 B-cells must result from ligation by autoantigen. This is thought to be important because of the role that CD5 B-cells play in generating protective antibodies in fetal/neonatal life, their contribution to """"""""natural autoantibody"""""""" and their possible contribution to pathogenic autoantibodies in lupus-like syndromes. The hypothesis will test whether autoantigen can stimulate B-cells so that they produce autoantibody and express a CD5 + (B-1a) phenotype. Using a transgene system in which B-cells produce antibody specific for H-2K*, the application will determine if CD5 B-cells can arise in vivo or in vitro as the result of binding with autoantigen. We will attempt to define the conditions in which responding cells are deleted or are activated and the biochemical basis for these responses. The application also proposes to explore why the x-linked immune deficiency (xid) mutation that alters Bruton's tyrosine kinase (BTK) blocks production of autoantibodies and the generation of CD5 B-cells, yet permits responses to T-dependent antigens. Preliminary data in this system indicate that xid alters the B-cell response to autoantigen such that potential autoreactive cells are deleted. We will now determine how wild type BTK function functions in the production of antibody and the generation of CD5 B-cells. Two consequences of xid have already been identified, an inability to produce bcl-XL in response to B-cell receptor (BCR)-mediated activation and deficiencies in the nuclear expression of NF-kappa B components, c-rel in particular. Based on these preliminary data, the investigator will test the hypotheses that activation of B-cells induces an inhibition of l-kappa-B synthesis and that normal BTK function is needed to induce this inhibition. To understand the normal function of BTK in B-cells, the application proposes to study the interactions of wild type and mutant BTK with the protein Cbl, and SHIP and the subcellular localization of BTK following activation. To study mutant forms of BTK, the application proposes to exploit a transient tranfection system that the investigators have developed to study primary B-cell activation responses. Alternatively, stable transfection of transformed xid B-cells will be used. BTK loss variants of wild type mature murine B-cells will be created. This will enable them to approach the question as to whether BTK is important solely because it is essential at a critical point in B-cell development or whether it serves important functions in mature B-cells.
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