CONTROL OF ADAPTIVE IMMUNITY BY ACTIN-REGULATORY PROTEINS

描述(由申请人提供)：生产长寿命、高亲和力的抗体对于抵抗感染至关重要。这一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了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。生发中心形成过程中激活的B细胞的复杂迁移是由趋化和黏附信号引导的，但对B细胞运动的分子调控仍然知之甚少。本申请旨在确定肌动蛋白捆绑蛋白L-血浆蛋白在整合趋化和黏附信号方面的作用，这些信号对正常的B细胞运动是必要的，进而形成生发中心。我已经证明，肌动蛋白捆绑蛋白L-血浆蛋白在B细胞的运动和特化的边缘带B细胞的发育中是必不可少的。L-纤溶酶缺乏扰乱了蛋白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产生的类别转换抗体也需要L-纤溶酶。在目标1中，我们将利用已建立的慢病毒表达系统，确定L-纤溶酶原在B细胞运动和边缘带B细胞成熟中所必需的结构域。首先要测试的候选结构域是N-末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。这一目标将决定L-纤溶酶的肌动蛋白结合功能是否对其在B细胞运动中的作用是必要的，或者L-纤溶酶是否独立于肌动蛋白捆绑而作为趋化因子/整合素信号转导的适配蛋白，并将确定上游介质调节L-纤溶酶功能的机制。在目标2中，我们将分析趋化因子和整合素信号在PYK2激活的上下游，以揭示L-纤溶酶在趋化和黏附信号级联中的作用。我们还将确定B细胞中维持Pyk2蛋白水平的机制。在目标3中，新验证的表达条件L-纤溶酶等位基因的小鼠将被用来产生B细胞特异性缺失L-纤溶酶的小鼠。对仅在B细胞中缺乏L-纤溶酶的小鼠体液免疫发育的分析将确定生发中心B细胞形成最依赖于趋化信号的步骤。我有进行这些实验所需的专业知识，以及完成手头的提案所需的所有试剂。这些目标将确立L-纤溶酶作为体液免疫的关键调节因子，并为深入了解趋化和黏附信号级联的整合对抗体产生至关重要。定义B细胞运动的分子调控对于了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.