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淋巴细胞通过表达不止一种B细胞受体而打破了适应性免疫的基本原则。更糟糕的是，这是由于受体编辑而发生的。进一步重排免疫球蛋白kappa （Ig-kappa）基因以去除自身反应性受体而导致第二个kappa等位基因的表达。最后，“等位基因包含”的B细胞产生，表达一种自身反应性B细胞受体，可能是发生不适当甚至危险的免疫反应的一种手段。利用igg -kappa等位基因包涵的出现作为B细胞遭受受体编辑的标记，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。相比之下，T1、T3或成熟B细胞仅罕见地同时表达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移行性B细胞中正在进行或最近的Ig-kappa受体编辑的证据。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preMZ和T3 B细胞。我们共同解释了这些不同的观察结果，表明未成熟B细胞发育的T2阶段是原代B细胞选择的联系。这个项目是基于在受体编辑和潜在危险的等位基因包涵的背景下描述B细胞的发育。我们提出了一组高度集中的实验来最终确定T2和所有移行B细胞在受体编辑和原代B细胞选择中的作用。在Specific Aim 1中，我们将使用分子测定来确定受体编辑是否确实发生在T2或任何过渡B细胞群中。我们还将探索过渡B细胞发育过程中受体编辑的动力学。在特定的目标2中，我们将使用经典的抗鸡蛋溶菌酶（anti-HEL）小鼠自反应性B细胞选择模型的修饰来分析受体编辑和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，可以从流式细胞术分离的任何B细胞中表达大量单克隆抗体。在Specific Aim 3中，我们将通过分析正常小鼠的BCR特异性，直接和最终地确定移行性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。通过实验，我们的目标是表征介导原发性免疫耐受的特定B细胞。表征这些细胞将为治疗提供靶点，从而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.