Molecular Basis for Overcoming Tolerance to Sm

描述（申请人提供）：系统性红斑狼疮（SLE）涉及自身反应性B细胞失调。当B细胞耐受性被克服并且对核抗原特异性的细胞通过BCR更新信号转导导致自身抗体分泌时，疾病发生。了解诱导和维持B细胞无反应状态的机制对于了解SLE的分子基础至关重要。在这个应用程序中，我们将测试的假设，即耐受Sm被克服时，抗原表位显示在凋亡细胞的膜泡。我们提出，这种独特的显示高度有序的膜结合表位克服了部分无反应性的状态，在抗Sm B细胞通过更新的BCR信号转导。 Sm特异性B细胞（2-12/Vk 8 Db 1）发育成成熟的B2细胞，其表现出活化的细胞表面表型，并且不能响应于LPS分泌抗Sm抗体。有趣的是，与无反应性B细胞相关的其他表型变化在2- 12 Nk 8 Db 1 B细胞中不存在，表明部分无反应性状态。在目标1和2a中，我们将评估部分无反应性表型是否与BCR的不完全脱敏相关，以及显示较高效价表位的抗原（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，当脱敏受体重新分配到称为脂筏的膜微区时，可能会发生更新的信号转导。在目标2B中，我们将评估2-12/Vk 8 Dbl B细胞上的BCR是否被排除在脂筏之外，以及这些受体是否能够在通过高价抗原连接后易位至脂筏。 在目标3中，我们将使用体内模型评估凋亡细胞对Sm耐受性的影响。首先，我们将凋亡细胞注射到2-12/Vk 8 Dbl小鼠中，并评估是否可以克服对Sm的耐受性。在第二种方法中，我们将在2-12/Vk 8 Dbl免疫球蛋白转基因模型中引入有缺陷的mer基因，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.