Molecular Basis for Overcoming Tolerance to Sm

描述(申请人提供)：自身反应性B细胞调节失调与系统性红斑狼疮(SLE)有关。当B细胞耐受被克服，核抗原特异性细胞通过BCR更新信号转导，导致自身抗体分泌时，疾病就会发生。了解B细胞诱导和维持无反应状态的机制对于了解SLE的分子基础至关重要。在这个应用中，我们将检验这样的假设，即当抗原表位显示在凋亡细胞的膜泡上时，对Sm的耐受性被克服。我们认为，这种高度有序的膜结合表位的独特展示，通过更新BCR的信号转导，克服了抗Sm B细胞的部分无能状态。 Sm特异性B细胞(2-12/Vk8 DBL)发育成成熟的B2细胞，表现为活化的细胞表面表型，不能分泌抗Sm抗体。有趣的是，与无能B细胞相关的其他表型变化在2-12Nk8 DBL B细胞中缺失，表明部分无能状态。在目标1和2a中，我们将评估部分无能表型是否与BCR的不完全脱敏有关，以及显示更高价位表位的抗原，如剪接体、Sm四聚体或凋亡膜，是否能够更新信号转导。从机制上讲，当不敏感的受体重新分配到称为脂筏的膜微域时，可能会发生新的信号转导。在目标2b中，我们将评估2-12/Vk8 DBL B细胞上的BCR是否被脂筏排除，以及这些受体在被高价抗原连接后是否能够移位到脂筏上。 在目标3中，我们将使用活体模型评估凋亡细胞对Sm耐受性的影响。首先，我们将把凋亡细胞注射到2-12/Vk8 DBL小鼠中，并评估是否可以克服对Sm的耐受。在第二种方法中，我们将把有缺陷的mer基因引入2-12/Vk8 DBL免疫球蛋白转基因模型中，并评估凋亡细胞水平的增加是否通过诱导Sm特异性自身抗体来克服耐受性。

DESCRIPTION (provided by applicant): Dysregulation of autoreactive B cells has been implicated in systemic lupus erythematosus (SLE). Disease occurs when B cell tolerance is overcome and cells specific for nuclear antigens renew signal transduction through the BCR leading to autoantibody secretion. Understanding the mechanisms that induce and maintain an unresponsive state in B cells is crucial to understanding the molecular basis of SLE. In this application we will test the hypothesis that tolerance to Sm is overcome when antigenic epitopes are displayed on the membrane blebs of apoptotic cells. We propose that this unique display of highly ordered membrane-bound epitopes overcomes a state of partial anergy in anti-Sm B cells by renewing signal transduction through the BCR. Sm-specific B cells (2-12/Vk8 Dbl) develop into mature B2 cells that exhibit an activated cell surface phenotype and fail to secrete anti-Sm antibodies in response to LPS. Interestingly, other phenotypic changes associated with anergic B cells are absent in the 2-12Nk8 Dbl B cells suggesting a state of partial anergy. In aims 1 and 2a, we will assess if the partially anergic phenotype is associated with incomplete desensitization of the BCR and if antigens displaying higher valency epitopes, such as spliceosomes, tetramers of Sm, or apoptotic membranes, are capable of renewing signal transduction. Mechanistically, renewed signal transduction may occur when desensitized receptors repartition to membrane microdomains called lipid rafts. In aim 2b we will assess if the BCR on 2-12/Vk8 Dbl B cells are excluded from lipid rafts and if these receptors are capable of translocating to the rafts upon ligation by high valency antigens. In aim 3 we will assess the effect of apoptotic cells on tolerance to Sm using an in vivo model. First, we will inject apoptotic cells into the 2-12/Vk8 Dbl mice and assess if tolerance to Sm can be overcome. In a second approach, we will introduce a defective mer gene into the 2-12/Vk8 Dbl immunoglobulin transgenic model and assess if increased levels of apoptotic cells overcome tolerance by inducing Sm-specific autoantibodies.