The long-term goals of our studies are to develop an improved understanding of the disease sub-sets in chronic lymphocytic leukemia (CLL) and thereby propel the development of improved prognostic tools and identification of therapeutic targets. The objectives of this application are to determine the clinical relevance of the enhanced expression of the Fc receptor for IgM (Fc?R) by CLL B cells and to explore the role of Fc?R signaling in promoting CLL-cell survival. The over-expression of Fc?R by CLL cells has been suspected for some time, but the lack of direct assays has been a major barrier to unambiguous analysis. Our recent functional cloning of the FCMR cDNA and subsequent generation of receptor-specific mAbs are enabling rapid clarification of the expression of the Fc?R and its functional roles. Based on our preliminary data and the available literature concerning CLL, we have formulated the central hypothesis that: the enhanced Fc?R expression by CLL B cells is a result of chronic antigenic stimulation and the ensuing IgM/antigen immune complexes lead to co-ligation of Fc?R and the IgM B cell receptor (BCR) on CLL cells, thereby providing a survival signal. Our approach is in Aim 1, to define the potential clinical relevance of the enhanced expression of both membrane-bound and soluble forms of Fc?R in patients with CLL. The working hypotheses are: (i) Both cell surface and serum levels of Fc?R predict the mutation status of the Ig heavy chain V region gene and the clinical progression in CLL;and (ii) The soluble form of Fc?R detected in CLL patients'sera, which is encoded by an alternatively spliced transcript, is produced by CLL B cells and modulates CLL B-cell function as a decoy receptor or by interacting with the membrane IgM. The cell surface and serum levels of Fc?R in patients with CLL and normal individuals will be quantified using a unique panel of monoclonal antibodies for flow cytometric analyses and a newly developed enzyme-linked immunosorbent assay. In parallel, transcript levels will be analyzed using real time quantitative PCR.
In Aim 2, the role of Fc?R in survival of CLL B cells will be explored. The effects of co-ligation of Fc?R and surface IgM on CLL cell survival will be determined, as well as whether the highly-expressed Fc?R interacts with membrane IgM on the same CLL cells. The study is technically innovative as it utilizes IgM antibodies for cross-linkage of the B-cell receptor and the Fc?R along with Fc?R -blocking antibodies, and is conceptually innovative as it is expected to provide the first demonstration of a survival function of Fc?R for CLL B cells as a consequence of interaction with IgM/antigen immune complexes. The clinical significance lies in the expected definition of surface and soluble Fc?R as reliable markers for predicting the prognosis and disease activity of CLL, which will form the basis for subsequent rapid development of a robust cost-effective prognostic test for CLL patients. Evidence supporting the central hypothesis will greatly improve the understanding of the pathogenesis of CLL and justify the future exploration of the effectiveness of targeting Fc?R in CLL.
The proposed research is relevant to public health because the demonstration of a role for the newly identified IgM antibody receptor in chronic lymphocytic leukemia (CLL) cells is ultimately expected to increase understanding of the pathogenesis of this most common incurable leukemia. Moreover, the analysis of enhanced expression of this receptor on CLL cells is expected to aid in the prognosis and in predicting disease activity. Thus, the proposed research is relevant to the mission of NIH, i.e., to foster fundamental creative discoveries, innovative research strategies and their applications as a basis for ultimately protecting and improving health.
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