CONTROL OF ADAPTIVE IMMUNITY BY ACTIN-REGULATORY PROTEINS

描述（由申请人提供）：生产长寿命，高亲和力的抗体对抵抗感染至关重要。这个过程需要生发中心的形成。生发中心形成失调导致免疫缺陷和自身免疫。因此，阐明生发中心形成的调控机制对于理解临床免疫缺陷和自身免疫性疾病至关重要。生发中心的形成需要抗原刺激的B细胞在B细胞滤泡内迁移，在那里它们首先与同源T细胞相互作用，然后与滤泡树突状细胞相互作用。在生发中心形成过程中，活化的B细胞的复杂迁移是由趋化和粘附提示引导的，但B细胞运动的分子调控仍然知之甚少。该应用程序旨在定义肌动蛋白结合蛋白l -活蛋白在整合正常B细胞运动所需的趋化和粘附线索以及生发中心形成中的作用。我已经证明了肌动蛋白捆绑蛋白l -活蛋白在B细胞运动和特化边缘区B细胞的发育中是必不可少的。l -活蛋白缺乏会破坏蛋白质水平的维持和整合素相关激酶Pyk2的激活。生发中心的形成和抗原刺激后类转换抗体的产生也需要L- plastin。在目标1中，我们将使用已建立的慢病毒表达系统定义l -活蛋白的结构域，这些结构域对B细胞运动和边缘区B细胞成熟的功能至关重要。首先要测试的候选结构域是n端丝氨酸磷酸化位点、钙结合结构域和肌动蛋白捆绑结构域。本研究将确定l -活蛋白的肌动蛋白捆绑功能是否对其在B细胞运动中的功能至关重要，或者l -活蛋白是否作为趋化因子/整合素信号传导的一个衔接蛋白，独立于肌动蛋白捆绑，并将确定上游介质调节l -活蛋白功能的机制。在Aim 2中，我们将分析Pyk2激活的上游和下游的趋化因子和整合素信号传导，以描述L-plastin在趋化和粘附信号级联中的作用。我们还将确定B细胞中Pyk2蛋白水平维持的机制。在Aim 3中，新验证的表达条件L-活素等位基因的小鼠将用于产生L-活素B细胞特异性缺失的小鼠。分析仅在B细胞中缺乏l -活蛋白的小鼠体液免疫的发展将确定生发中心B细胞形成最依赖趋化线索的步骤。我有进行这些实验所需的专业知识，也有完成手头提案所需的所有试剂。这些目标将确立l -活蛋白作为体液免疫的关键调节因子，并为抗体产生所必需的趋化和粘附信号级联的整合提供见解。明确B细胞运动的分子调控对于理解免疫缺陷和自身免疫的发病机制，以及设计和改进疫苗和免疫调节药物至关重要。

DESCRIPTION (provided by applicant): The production of long-lived, high-affinity antibodies is critical for resistance to infection. This process requires the formation of germinal centers. Dysregulation of germinal center formation results in immunodeficiencies and autoimmunity. Elucidation of the regulatory mechanisms that govern germinal center formation is therefore essential to understanding clinical immunodeficiencies and autoimmune disorders. Germinal center formation requires antigen-stimulated B cells to migrate within the B cell follicle where they interact first with cognate T cells and then with follicular dendritic cells. The complex migration of activated B cells during germinal center formation is guided by chemotactic and adhesive cues, but the molecular regulation of B cell motility remains poorly understood. This application proposes to define the role of the actin-bundling protein L-plastin in integrating the chemotactic and adhesive cues essential for normal B cell motility, and by extension, germinal center formation. I have shown that the actin-bundling protein L-plastin is essential in B cell motility and for development of specialized marginal zone B cells. Deficiency of L-plastin disrupts maintenance of protein levels and activation of the integrin-associated kinase Pyk2. Germinal center formation and the production of class-switched antibodies following antigenic stimulation also require L- plastin. In Aim 1, we will define the structural domains of L-plastin that are essential for function in B cell motility and marginal zone B cell maturation using an established lentiviral expression system. Candidate domains to be tested first are the N-terminal serine phosphorylation site, the calcium-binding domain, and the actin-bundling domains. This Aim will determine if the actin-bundling function of L-plastin is essential to its function in B cel motility, or if L-plastin serves as an adaptor protein in chemokine/integrin signaling independently of actin-bundling, and will define mechanisms by which upstream mediators may modulate the function of L-plastin. In Aim 2, we will analyze chemokine and integrin signaling upstream and downstream of Pyk2 activation to delineate the role of L-plastin in chemotactic and adhesive signaling cascades. We will also determine the mechanism by which Pyk2 protein levels are maintained in B cells. In Aim 3, newly validated mice expressing a conditional L-plastin allele will be used to generate mice with a B cell-specific deletion of L- plastin. Analysi of the development of humoral immunity in mice lacking L-plastin only in B cells will define the steps of germinal center B cell formation most dependent on chemotactic cues. I have the expertise required to conduct these experiments and all the reagents necessary to complete the proposal in hand. These Aims will establish L-plastin as a key regulator of humoral immunity and provide insight into the integration of chemotactic and adhesive signaling cascades essential for antibody generation. Definition of the molecular regulation of B cell motility is essential to understanding the pathogenesis of immunodeficiency and autoimmunity, as well as to designing and improving vaccines and immunomodulatory drugs.