CONTROL OF ADAPTIVE IMMUNITY BY ACTIN-REGULATORY PROTEINS

描述（由申请方提供）：长寿命、高亲和力抗体的产生对于抗感染至关重要。这个过程需要形成生发中心。生发中心形成的失调导致免疫缺陷和自身免疫。因此，阐明控制生发中心形成的调节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线索引导的，但对B细胞运动的分子调控仍然知之甚少。本申请提出定义肌动蛋白捆绑蛋白L-纤维蛋白酶原在整合正常B细胞运动性所必需的趋化性和粘附性线索中的作用，并通过扩展来定义生发中心的形成。我已经表明，肌动蛋白捆绑蛋白L-纤维蛋白是必不可少的B细胞的运动和专门的边缘区B细胞的发展。L-纤维蛋白原缺乏破坏蛋白质水平的维持和整合素相关激酶Pyk 2的活化。在抗原刺激后，Germinant中心的形成和类别转换抗体的产生也需要L- plastin。在目的1中，我们将使用已建立的慢病毒表达系统来定义L-纤维蛋白酶的结构域，这些结构域对于B细胞运动和边缘区B细胞成熟中的功能是必不可少的。首先要测试的候选域是N-末端丝氨酸磷酸化位点、钙结合域和肌动蛋白捆绑域。该目的将确定L-纤溶蛋白的肌动蛋白捆绑功能是否对其在B细胞运动中的功能是必不可少的，或者L-纤溶蛋白是否在独立于肌动蛋白捆绑的趋化因子/整合素信号传导中充当衔接蛋白，并且将定义上游介质可调节L-纤溶蛋白功能的机制。在目标2中，我们将分析Pyk 2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-纤溶酶原激活蛋白在趋化和粘附信号传导级联中的作用。我们还将确定Pyk 2蛋白水平在B细胞中维持的机制。在目标3中，表达条件性L-塑性蛋白等位基因的新验证小鼠将用于产生具有L-塑性蛋白的B细胞特异性缺失的小鼠。分析仅在B细胞中缺乏L-类活蛋白的小鼠的体液免疫的发展，将确定最依赖于趋化性线索的生发中心B细胞形成的步骤。我有进行这些实验所需的专业知识和完成手头提案所需的所有试剂。这些目标将确立L-纤维蛋白酶作为体液免疫的关键调节因子，并提供对抗体生成所必需的趋化性和粘附性信号级联的整合的见解。B细胞运动的分子调控的定义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是必不可少的。

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.