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（DCS）对T细胞反应的启动是适应性免疫的重要启动步骤 回复。此过程需要在称为免疫突触（IS）的部位上进行亲密的细胞 - 细胞相互作用。 来自各种来源的越来越多的证据表明，此过程涉及机械转移。 尽管已经对IS的T细胞侧的事件进行了广泛的研究，但对DC的DC侧知之甚少 除了需要完整的直流肌动蛋白细胞骨架的事实外，该界面还需要。我们发现DC 成熟涉及与生物物理特性改变相关的细胞骨架蛋白表达的变化 细胞皮质的。使用原子力显微镜（AFM），我们发现皮质刚度从2KPA增加到 3.5KPA在LPS诱导的成熟后，生物物理术语发生了巨大变化。我们的初步数据表明 几种关键肌动蛋白调节分子的表达或激活状态的变化是负责的 调节直流刚度。值得注意的是，当T细胞在不同依从性的底物上刺激时，它们 在直流成熟期间观察到的范围内，表现出急剧的激活阈值。因此，我们假设 与直流成熟相关的细胞骨架变化改变了直流皮质的刚度，并且 这些事件代表了一种先前未欣赏的机制，T细胞启动是 受监管。为了检验这一假设，我们将执行两个具体的目标：首先，我们将表征 细胞骨架变化，可在成熟过程中调节直流刚度。根据初步研究 药理学抑制剂，我们将重点放在黄蜂，formins，cofilin和ERM蛋白上。使用DC 敲除小鼠或表达shRNA或显性突变体的WT DC，我们将阻止 候选蛋白质并测试对皮质刚度和T细胞启动的影响（使用肽载荷进行聚焦 对IS的事件的分析）。此外，我们将以不发生的方式操纵直流皮质刚度 自然，询问这是否会影响T细胞启动。其次，我们将测试T细胞启动的底物 在DC中定义的生理范围内的刚度。使用涂有T细胞配体的水凝胶， 我们将确定哪些T细胞子集对刚度敏感，以及刚度如何影响增殖和 效应子谱系的发展。我们将确定TCR，CD28和LFA-1对刚度的贡献 感测，并询问IS肌动蛋白动力学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 已知参与机械转导。如果成功，这个探索性项目将表明受监管 DC皮层的生物物理特性的变化作为先前未发现的机制 DC通过其中调整T细胞响应 - 与DC成熟的基本特征一起考虑 共刺激分子和细胞因子的上调。此外，它将为 了解T细胞如何感知DC刚度，并指导对潜在机械生物学的未来研究。 呢