The immature B cell represents an important window in B lymphocyte development, for it is at this stage that cells expressing B cell antigen receptors (BCRs) specific for endogenous or """"""""self antigens can be identified and eliminated, inactivated, or undergo alterations in their receptor specificity, in processes collectively referred to as B cell tolerance. The long-term objective of this project is to define the molecular basis for differences in the responsiveness of immature and follicular mature B cells to BCR signaling. Although multiple mechanisms of tolerance affect the ultimate fate of the B cell, we have determined that intrinsic or """"""""hard-wired"""""""" differences in BCR-induced signal transduction dictate B cell fate decisions, specifically when isolated from specific sources of B cell extrinsic signals that both immature B cells from the bone marrow and transitional immature B cells from the periphery undergo an abortive entry into the cell cycle followed by apoptosis while mature B cells are induced to proliferate following BCR engagement. Transitional B cells fail to sustain BCR triggered pathways linked to survival, proliferation, and cytoskeletal re-organization and maintain lower levels of unesterified cholesterol in their plasma membrane relative to mature B cells. We have recently shown that this latter phenotype accounts for their inability to localize their BCR into cholesterol-enriched membrane (CEM) compartments following BCR aggregation. As repletion of membrane cholesterol in transitional B cells reverses proximal signaling differences, we hypothesize that developmental differences in cholesterol levels represent the defining difference between immature and mature B cells that dictates their cell fate following BCR engagement. Therefore, we propose to 1) use a genetic approach to specifically upregulate cholesterol biosynthesis in transitional B cells and evaluate potential links between cholesterol content and differential BCR-induced signal transduction and cell fate, 2) use co-crosslinking and genetic systems to determine whether the transient BCR-induced signal transduction in transitional B cells reflects an inability to engage a PIP3-dependent PIP2 regenerating amplification loop and 3) to determine if transient BCR-induced signaling in transitional B cells impacts on """"""""inside-out"""""""" integrin activation. In autoimmune diseases, B lymphocytes that are normally inactive become activated and secrete autoantibodies that contribute to the disease process. We propose to determine if alterations in cholesterol levels can contribute to the activation of these potentially dangerous B lymphocytes and thereby suggest targets for future treatment of B cell-dependent autoimmune diseases. ? ? ?
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