The origin and consequences of receptor editing and allelic-inclusion.

描述(由申请人提供)：我们最近证明，令人惊讶的是，10%的B淋巴细胞表达不止一个B细胞受体，从而违反了获得性免疫的基本原理。更糟糕的是，这是由于受体编辑造成的。进一步重排免疫球蛋白kappa(Ig-kappa)基因以去除自身反应受体，反而会导致第二kappa等位基因的表达。最终，“等位包含的”B细胞被产生，表达一种自身反应性的B细胞受体，这可能是发生不适当甚至危险的免疫反应的一种手段。以Ig-kappa等位基因包涵体的出现作为受体编辑后B细胞的标志，我们做了一些重要的观察。我们有新的初步数据显示，在未成熟B细胞发育的T2阶段，三分之一的细胞同时表达Ig-kappa等位基因。相反，T1、T3或成熟B细胞仅罕见地表达这两个Ig-kappa等位基因。受体编辑被定义为在已经表达自身反应性bcr的细胞中表达新的抗体可变基因。我们将我们的新数据解释为T2过渡性B细胞中正在进行或最近正在编辑的Ig-kappa受体的证据。已知脾T2 B细胞在B细胞选择中起作用，并产生被认为是无能的前MZ和T3B细胞。总之，我们解释了这些不同的观察结果，以表明未成熟B细胞发育的T2阶段是初级B细胞选择的纽带。这个项目的基础是在受体编辑的背景下表征B细胞的发展以及由此产生的潜在危险的等位基因包含。建议进行一系列高度集中的实验，以最终确定T2和所有过渡性B细胞在受体编辑和初级B细胞选择中的作用。在特定的目标1中，我们将使用分子分析来确定T2或任何一组过渡性B细胞是否确实发生了受体编辑。我们还将探索过渡性B细胞发育过程中受体编辑的动力学。在具体目标2中，我们将使用经典的抗鸡蛋溶菌酶(anti-HEL)小鼠自身反应性B细胞选择模型的改进来分析受体编辑和B细胞选择。这些实验将在受体编辑的背景下提供过渡性B细胞选择的体内评估。我们是强大的技术专家，该技术允许从通过流式细胞仪分离的任何B细胞中表达大量的单克隆抗体。在特定的目标3中，我们将通过分析正常小鼠的BCR特异性，直接和决定性地识别过渡性B细胞发育中的选择性检查点。在整个过程中，我们还将确定等位基因包含的B细胞的命运和自身免疫潜力。随着实验的提出，我们的目标是确定介导初级免疫耐受的特定B细胞的特征。这些细胞的特征将为治疗提供靶点，从而可能彻底改变自身免疫性疾病的治疗方法。

DESCRIPTION (provided by applicant): We recently demonstrated that a surprising 10% of B lymphocytes break a fundamental principal of adaptive immunity by expressing more than one B cell receptor. Making matters worse, this occurs due to receptor editing. Further rearrangement of the immunoglobulin kappa (Ig-kappa) genes to remove autoreactive receptors instead results in expression of the second kappa allele. In the end, "allelically included" B cells are generated that express an autoreactive B cell receptor and may be a means for inappropriate and even dangerous immune responses to occur. Using the occurrence of Ig-kappa allelic-inclusion as a marker of B cells that had been subjected to receptor editing, we made some important observations. We have new preliminary data showing that one third of cells at the T2 stage of immature B cell development express both Ig-kappa alleles. In contrast, T1, T3 or mature B cells only infrequently expressed both Ig-kappa alleles. Receptor editing is defined as expression of new antibody variable genes in cells that already express an autoreactive BCR. We interpreted our new data as evidence of ongoing or recent Ig-kappa receptor editing in the T2 transitional B cells. It is known that splenic T2 B cells play a role in B cell selection and give rise to preMZ and T3 B cells that are believed to be anergic. Together we interpret these various observations to suggest that the T2 stage of immature B cell development is the nexus of primary B cell selection. This project is based on characterizing the development of B cells in the context of receptor editing and the potentially dangerous allelic-inclusion that results. A set of highly focused experiments are proposed to conclusively determine the role of T2 and all transitional B cells in receptor editing and primary B cell selection. In Specific Aim 1, we will determine if indeed receptor editing occurs in T2 or any population of transitional B cells using molecular assays. We will also explore the kinetics of receptor editing during transitional B cell development. In specific aim 2, we will analyze receptor editing and B cell selection using a modification of the classic anti-Hen Egg Lysozyme (anti-HEL) mouse model of autoreactive B cell selection. These experiments will provide an in vivo assessment of transitional B cell selection in the context of receptor editing. We are experts at powerful technology that allows expression of numerous monoclonal antibodies from any B cell isolated by flow cytometry. In Specific Aim 3 we will directly and conclusively identify the selective checkpoints in transitional B cell development by analyzing the BCR specificities in normal mice. Throughout, we will also determine the fate and autoimmune potential of allelically-included B cells. With the experiments proposed our goal is to characterize the particular B cells mediating primary immune tolerance. Characterizing these cells would provide the targets for therapy that could revolutionize treatment of autoimmune diseases.