Modulation of T cell priming by dendritic cell stiffness

树突状细胞硬度调节 T 细胞启动

• 批准号: 9369929
• 负责人: Janis K. Burkhardt
• 金额: $ 25.2万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-18 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9369929
• 关键词: Abnormal Cell; Actin-Binding Protein; Actins; Affect; Antigens; Atomic Force Microscopy; Autoimmune Diseases; Biochemical; Biological; Biophysics; CD28 gene; CD4 Positive T Lymphocytes; Cell Communication; Cell Maturation; Cell surface; Cells; Complex; Cues; Cytoskeletal Proteins; Cytoskeleton; Data; Dendritic Cells; Development; ERM protein; Effector Cell; Event; Exhibits; Foundations; Future; Genetic screening method; Growth; Hydrogels; Immune response; Immunologic Deficiency Syndromes; Immunologics; Interleukin-2; Investigation; Knockout Mice; Knowledge; Ligands; Light; Measures; Methods; Microfilaments; Molecular; Molecular Conformation; Molecular Genetics; Mutation; Outcome; Pathway interactions; Patients; Peptides; Pharmacology; Phosphorylation; Physiologic pulse; Physiological; Play; Process; Production; Proteins; Research; Role; Side; Signal Transduction; Signaling Molecule; Site; Source; Stimulus; Structure; Surface; Synapses; System; T cell response; T-Cell Activation; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Therapeutic; Thinking; Up-Regulation; Vaccines; Wasps; Work; adaptive immune response; adaptive immunity; base; biophysical properties; biophysical techniques; cell cortex; cell type; cofilin; cytokine; depolymerization; design; experimental study; ezrin; genetic approach; genetic regulatory protein; imaging study; immunological synapse; inhibitor/antagonist; insight; live cell imaging; mechanical force; mechanotransduction; migration; moesin; mutant; pathogen; protein expression; receptor; response; small hairpin RNA; uptake

PROJECT SUMMARY Priming of T-cell responses by dendritic cells (DCs) is an essential initiation step for the adaptive immune response. This process requires intimate cell-cell interactions at a site termed the immunological synapse (IS). Mounting evidence from a variety of sources indicates that this process involves mechanotransduction. Although events on the T cell side of the IS have been extensively studied, little is known about the DC side of the interface, apart from the fact that an intact DC actin cytoskeleton is required. We have found that DC maturation involves changes in cytoskeletal protein expression associated with altered biophysical properties of the cell cortex. Using atomic force microscopy (AFM), we found that cortical stiffness increases from 2kPa to 3.5kPa upon LPS-induced maturation, a large change in biophysical terms. Our preliminary data suggest that changes in the expression or activation state of several key actin regulatory molecules are responsible for regulating DC stiffness. Remarkably, when T cells are stimulated on substrates of different compliance, they exhibit a sharp threshold for activation over the range observed during DC maturation. Thus, we hypothesize that cytoskeletal changes associated with DC maturation alter the stiffness of the DC cortex, and that these events represent a previously unappreciated mechanism through which T cell priming is regulated. To test this hypothesis, we will carry out two specific aims: First, we will characterize the cytoskeletal changes that modulate DC stiffness during maturation. On the basis of preliminary studies using pharmacological inhibitors, we will focus on WASp, formins, cofilin and ERM proteins. Using DCs from knockout mice or WT DCs expressing shRNA or dominant mutants, we will block the expression or activity of candidate proteins and test the effect on cortical stiffness and T cell priming (using peptide loading to focus analysis on events at the IS). In addition, we will manipulate DC cortical stiffness in ways that do not occur naturally, and ask whether this affects T cell priming. Second, we will test T cell priming on substrates that vary in stiffness over the physiological range we have defined in DCs. Using hydrogels coated with T cell ligands, we will determine which T cell subsets are stiffness sensitive, and how stiffness affects proliferation and effector lineage development. We will determine the contribution of TCR, CD28, and LFA-1 to stiffness sensing, and ask how actin dynamics at the IS respond to changes in stiffness. Finally, we will characterize stiffness effects on Ca2+ signaling and key phosphorylation events, with particular emphasis on molecules known to participate in mechanotransduction. If successful, this exploratory project will show that regulated changes in the biophysical properties of the DC cortex function as a previously undiscovered mechanism through which DCs tune the T cell response - a basic feature of DC maturation to be considered along with upregulation of costimulatory molecules and cytokines. Moreover, it will provide a molecular foundation for understanding how T cells sense DC stiffness, guiding future investigation of the underlying mechanobiology. !

项目摘要 树突状细胞（DC）引发T细胞应答是获得性免疫的重要起始步骤， 反应这一过程需要在称为免疫突触（IS）的部位进行密切的细胞-细胞相互作用。 来自各种来源的越来越多的证据表明，这一过程涉及机械转导。 虽然IS的T细胞侧的事件已被广泛研究，但关于IS的DC侧的事件知之甚少。 接口，除了一个完整的DC肌动蛋白细胞骨架是必需的。我们发现DC 成熟涉及与改变的生物物理特性相关的细胞骨架蛋白表达的变化 细胞皮层的一部分。使用原子力显微镜（AFM），我们发现皮质硬度从2kPa增加到2kPa。 3.5 kPa的LPS诱导的成熟，一个大的变化，在生物物理方面。我们的初步数据显示， 几种关键肌动蛋白调节分子的表达或激活状态的变化是导致 调节DC刚度。值得注意的是，当T细胞在不同顺应性的基质上受到刺激时，它们 在DC成熟过程中观察到的范围内，表现出明显的激活阈值。因此，我们假设 与DC成熟相关的细胞骨架变化改变了DC皮质的硬度， 这些事件代表了一种以前未被认识到的机制， 监管.为了验证这一假设，我们将实现两个具体目标：首先，我们将描述 细胞骨架的变化，在成熟过程中调节DC刚度。根据初步研究， 药理学抑制剂，我们将集中在WASp，formins，cofilin和ERM蛋白。使用DC从 敲除小鼠或表达shRNA或显性突变体的WT DC，我们将阻断 并测试对皮质硬度和T细胞引发的影响（使用肽加载以聚焦于 事件的分析）。此外，我们将以不会发生的方式操纵DC皮质刚度 自然地，并询问这是否影响T细胞引发。其次，我们将测试T细胞在不同底物上的引发， 在我们已经定义的DC生理范围内，刚度。使用涂有T细胞配体的水凝胶， 我们将确定哪些T细胞亚群对硬度敏感，以及硬度如何影响增殖， 效应子谱系发育。我们将确定TCR、CD28和LFA-1对僵硬的贡献。 感应，并询问肌动蛋白动力学在IS如何响应刚度的变化。最后，我们将描述 刚度对Ca2+信号传导和关键磷酸化事件的影响，特别强调分子 已知参与机械传导。如果成功的话，这个探索性的项目将表明， DC皮质生物物理特性的变化是一种以前未发现的机制 DC通过其调节T细胞应答-DC成熟的基本特征被沿着考虑， 共刺激分子和细胞因子的上调。此外，它将为以下方面提供分子基础： 了解T细胞如何感知DC刚度，指导未来对潜在机械生物学的研究。 !