IgM Fc receptor in CLL

描述（由申请人提供）：我们研究的长期目标是更好地了解慢性淋巴细胞白血病（CLL）的疾病亚群，从而推动改进预后工具的发展和治疗靶点的确定。本应用的目的是确定CLL B细胞中IgM Fc受体（Fc¿R）表达增强的临床相关性，并探索Fc¿R信号在促进CLL细胞存活中的作用。一段时间以来，人们一直怀疑CLL细胞中Fc¿R的过表达，但缺乏直接的检测方法一直是进行明确分析的主要障碍。我们最近对FCMR cDNA的功能克隆和随后一代的受体特异性单克隆抗体能够快速澄清Fc¿R的表达及其功能作用。基于我们的初步数据和有关CLL的现有文献，我们提出了一个中心假设：CLL B细胞Fc¿R表达的增强是慢性抗原刺激的结果，随后的IgM/抗原免疫复合物导致Fc¿R与CLL细胞上的IgM B细胞受体（BCR）共同结合，从而提供生存信号。我们的方法是在目的1中，确定膜结合和可溶性Fc¿R在CLL患者中表达增强的潜在临床相关性。工作假设是：(i)细胞表面和血清Fc¿R水平预测Ig重链V区基因的突变状态和CLL的临床进展；（ii） CLL患者血清中检测到的Fc¿R的可溶性形式，由一个选择性剪接转录物编码，由CLL B细胞产生，并作为诱饵受体或通过与膜IgM相互作用调节CLL B细胞的功能。CLL患者和正常人的细胞表面和血清Fc¿R水平将使用一组独特的单克隆抗体进行流式细胞分析和新开发的酶联免疫吸附测定。同时，转录水平将使用实时定量PCR进行分析。在Aim 2中，将探讨Fc¿R在CLL B细胞存活中的作用。将确定Fc¿R和表面IgM共同连接对CLL细胞存活的影响，以及高表达的Fc¿R是否与相同CLL细胞上的膜IgM相互作用。这项研究在技术上是创新的，因为它利用IgM抗体将B细胞受体和Fc¿R以及Fc¿R阻断抗体交叉连锁，并且在概念上是创新的，因为它有望首次证明Fc¿R由于与IgM/抗原免疫复合物相互作用而对CLL B细胞具有存活功能。临床意义在于表面和可溶性Fc¿R作为预测CLL预后和疾病活动性的可靠标记物的预期定义，这将为随后快速开发一种强大的具有成本效益的CLL患者预后检测奠定基础。支持中心假设的证据将极大地提高对CLL发病机制的理解，并证明未来探索靶向Fc¿R在CLL中的有效性。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.