CONTROL OF ADAPTIVE IMMUNITY BY ACTIN-REGULATORY PROTEINS

肌动蛋白调节蛋白对适应性免疫的控制

基本信息

批准号：
9035349
负责人：
Sharon Celeste Morley
金额：
$ 38.13万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9035349
关键词：
Actins Adaptor Signaling Protein Adhesions Adhesives Affinity Alleles Anti-Inflammatory Agents Anti-inflammatory Antibodies Antibody Affinity Antibody Formation Antibody Response Antigens Autoimmune Diseases Autoimmunity B cell differentiation B-Cell Development B-Lymphocytes Binding Biological Assay Bone Marrow Bundling CD19 gene CD4 Positive T Lymphocytes Calcium-Binding Domain Cell Maturation Chimera organism Clinical Complex Cues Cytoskeleton Data Defect Development Diagnosis Follicular Dendritic Cells Generations Hand Health Helper-Inducer T-Lymphocyte Humoral Immunities Immunoglobulin Class Switching Immunologic Deficiency Syndromes In Vitro Infection Integrins Knowledge L-Plastin Lentivirus Vector Life Link Location Lymphoid Maintenance Measures Mediating Mediator of activation protein Memory Modeling Molecular Mus Mutate N-terminal Organ Pathogenesis Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Plasma Plasma Cells Process Production Proteins Publishing Reagent Recurrence Regulation Reporting Resistance to infection Role Serine Serine Phosphorylation Site Signal Transduction Signaling Molecule Site Staging Structure of germinal center of lymph node System T-Cell Development T-Lymphocyte Testing Therapeutic Vaccines adaptive immunity cell motility chemokine design extracellular genetic regulatory protein improved in vitro Assay in vivo insight migration mutant neutralizing antibody novel reconstitution research study vaccine development

项目摘要

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.

描述（由申请人提供）：长寿高亲和力抗体的产生对于抵抗感染至关重要。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性。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细胞卵泡中迁移，它们首先与同源T细胞相互作用，然后与卵泡树突状细胞相互作用。在生殖中心形成过程中活化B细胞的复杂迁移由趋化性和粘合性提示引导，但B细胞运动的分子调节仍然尚不清楚。该应用建议定义肌动蛋白捆绑蛋白L-普拉斯汀在整合正常B细胞运动至关重要的趋化性和粘合性提示中的作用，并通过扩展为生发中心形成。我已经表明，肌动蛋白捆绑蛋白L-普拉斯汀在B细胞运动性和专门边缘区B细胞的发展至关重要。 L-倍蛋白的缺乏破坏了蛋白质水平的维持和与整合素相关激酶PYK2的激活。抗原刺激后的生发中心形成和类切换的抗体的产生也需要l-帕斯汀。在AIM 1中，我们将使用已建立的慢病毒表达系统来定义L-普拉斯汀的结构结构域，这对于B细胞运动性和边缘区B细胞成熟至关重要。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结构域。该目标将确定L-普拉斯汀的肌动蛋白捆绑功能是否对于其在B cel运动中的功能至关重要，或者L-普拉斯汀在趋化因子/整合素信号中用作肌动蛋白捆绑的衔接蛋白，并且将通过肌动蛋白捆绑，并定义上游介体可以调节L-Plastin的功能的机制。在AIM 2中，我们将分析PYK2激活上游和下游的趋化因子和整合素信号传导，以描绘L-普拉斯汀在趋化性和粘合信号级联反应中的作用。我们还将确定B细胞中PYK2蛋白水平保持的机制。在AIM 3中，新近验证的表达有条件L-普拉斯汀等位基因的小鼠将用于生成具有L- plastin的B细胞特异性缺失的小鼠。仅在B细胞中缺乏L-普拉斯汀的小鼠中体液免疫发展的分析将定义生发中心B细胞形成的步骤最依赖于趋化性提示。我有进行这些实验以及手头完成提案所需的所有试剂所需的专业知识。这些目的将建立L-普拉斯汀作为体液免疫的关键调节剂，并洞悉趋化性和粘合剂信号传导级联对于抗体产生必不可少的。 B细胞运动的分子调节的定义对于理解免疫缺陷和自身免疫的发病机理以及设计和改善疫苗和免疫调节药物至关重要。