IgM Fc receptor in CLL

描述(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)的疾病亚型，从而推动改进的预后工具的开发和治疗靶点的确定。本应用的目的是确定CLL B细胞增强表达IgM的Fc受体(Fc？R)的临床意义，并探讨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细胞过度表达Fc？R，但缺乏直接检测一直是明确分析的主要障碍。我们最近对FCMR基因的功能性克隆和随后产生的受体特异性单抗使快速阐明Fc？R的表达及其功能作用成为可能。根据我们的初步数据和现有的有关CLL的文献，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和CLL细胞上的Ig M B细胞受体(BCR)共同连接，从而提供生存信号。我们的方法是在目标1中，确定膜结合和可溶性形式的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水平都可以预测免疫球蛋白重链V区基因的突变状态和CLL的临床进展；以及(Ii)在CLL患者血清中检测到的可溶性Fc？R是由CLL B细胞产生的，通过与膜IgM相互作用或作为诱饵受体来调节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用于流式细胞仪分析的单抗和一种新开发的酶联免疫吸附试验进行量化。同时，转录水平将使用实时定量聚合酶链式反应进行分析。在目标2中，将探讨Fc？R在CLL B细胞存活中的作用。Fc？R和表面IgM共连接对CLL细胞存活的影响，以及高表达的Fc？R是否与同一CLL细胞上的膜IgM相互作用将被确定。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与Fc？R阻断抗体进行交叉连接，而且在概念上也是创新的，因为它有望首次证明由于与IgM/抗原免疫复合物的相互作用，Fc？R对CLL B细胞具有生存功能。其临床意义在于预期将表面和可溶性Fc？R定义为预测CLL预后和疾病活动性的可靠标志物，这将为随后快速开发一种可靠的、经济有效的CLL患者预后检测方法奠定基础。支持中心假说的证据将极大地提高对CLL发病机制的理解，并证明未来靶向Fc？R在CLL中的有效性的探索。 公共卫生相关性：拟议的研究与公共卫生相关，因为新发现的IgM抗体受体在慢性淋巴细胞白血病(CLL)细胞中的作用最终有望增加对这种最常见的不可治愈白血病的发病机制的了解。此外，分析该受体在CLL细胞上的增强表达有望有助于预后和预测疾病活动性。因此，拟议的研究与美国国立卫生研究院的使命相关，即促进基本的创造性发现、创新的研究战略及其应用，作为最终保护和改善健康的基础。

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.