Chemoattractant-specific T cell navigation of complex environments

复杂环境中化学引诱剂特异性 T 细胞导航

• 批准号: 10741224
• 负责人: Janis K. Burkhardt
• 金额: $ 22.25万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741224
• 关键词: 3-Dimensional; Actins; Actomyosin; Autoimmune Diseases; Back; Biological; Biological Assay; Bulla; CCL19 gene; CCL21 gene; Cell physiology; Cells; Chemotactic Factors; Chemotaxis; Clinical; Collagen; Complement; Complex; Confined Spaces; Cues; Cytoskeleton; Data; Defect; Development; Disease; Ear; Environment; Event; Exhibits; Exposure to; G-Protein-Coupled Receptors; Gel; Geometry; Host Defense; Image; Immune; Immunologic Surveillance; Inflammatory; Knowledge; Ligands; Light; Link; Lipids; Lymphatic; Lymphatic Endothelium; Lymphoid Tissue; Malignant Neoplasms; Mediating; Microfilaments; Microfluidics; Modeling; Molecular; Movement; Mus; Myosin ATPase; Myosin Light Chains; Nonmuscle Myosin Type IIA; Pathway interactions; Peripheral; Phospholipase; Phosphorylation; Polymers; Process; Proteins; Regulation; Role; Signal Induction; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Speed; Stimulus; Surface; T cell response; T-Lymphocyte; Testing; Therapeutic; Tissues; Transplantation; Work; cell motility; cell type; chemokine; constriction; effector T cell; in vivo; inhibitor; insight; interest; interstitial; live cell imaging; lymphatic vessel; migration; novel therapeutic intervention; pharmacologic; polymerization; pressure; rational design; receptor; response; rho; sphingosine 1-phosphate; therapeutic target; trafficking; 3-Dimensional; Actins; Actomyosin; Autoimmune Diseases; Back; Biological; Biological Assay; Bulla; CCL19 gene; CCL21 gene; Cell physiology; Cells; Chemotactic Factors; Chemotaxis; Clinical; Collagen; Complement; Complex; Confined Spaces; Cues; Cytoskeleton; Data; Defect; Development; Disease; Ear; Environment; Event; Exhibits; Exposure to; G-Protein-Coupled Receptors; Gel; Geometry; Host Defense; Image; Immune; Immunologic Surveillance; Inflammatory; Knowledge; Ligands; Light; Link; Lipids; Lymphatic; Lymphatic Endothelium; Lymphoid Tissue; Malignant Neoplasms; Mediating; Microfilaments; Microfluidics; Modeling; Molecular; Movement; Mus; Myosin ATPase; Myosin Light Chains; Nonmuscle Myosin Type IIA; Pathway interactions; Peripheral; Phospholipase; Phosphorylation; Polymers; Process; Proteins; Regulation; Role; Signal Induction; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Speed; Stimulus; Surface; T cell response; T-Lymphocyte; Testing; Therapeutic; Tissues; Transplantation; Work; cell motility; cell type; chemokine; constriction; effector T cell; in vivo; inhibitor; insight; interest; interstitial; live cell imaging; lymphatic vessel; migration; novel therapeutic intervention; pharmacologic; polymerization; pressure; rational design; receptor; response; rho; sphingosine 1-phosphate; therapeutic target; trafficking

PROJECT SUMMARY/ABSTRACT T cell trafficking is crucial for development, immune surveillance, and effector function. Migration is a complex process, choreographed by a host of chemoattractants that signal through GPCRs to direct T cell cytoskeletal responses. In vivo work shows that chemokines like CCL19 and CCL21 (CCR7 ligands) mediate naïve T cell migration within lymphoid tissues, while the lipid chemoattractant S1P regulates egress. A similar process occurs in peripheral tissues, where these signals control migration of migratory effector T cells out of inflamed tissues and into afferent lymphatics. Our lab recently overcame a longstanding technical problem that made it difficult to study S1P responses in ex vivo T cells. Using this advance, we discovered that these two chemotactic signals induce distinct modes of T cell motility. CCL19 induces long-duration lamellipodial migration while S1P induces a shorter burst of bleb-based motility. This work raises several questions: How do these chemoattractants elicit such different migratory responses? What do T cells do when confronted with competing cues? Why do T cells need multiple motile mechanisms? We hypothesize that CCR7 ligands and S1P activate different cytoskeletal signaling pathways that direct distinct modes of motility, which work alone or in combination to allow T cells to navigate complex environmental obstacles like those they encounter in vivo. To test this hypothesis, we will carry out two sets of studies. In Aim 1, we will pursue our preliminary data showing that CCL19 preferentially activates a Rac1-dependent pathway leading to lamellipodial protrusion, while S1P preferentially activates a pathway involving RhoA and phospholipase activity, which directs myosin-dependent contractility and bleb formation. To verify that that these signaling events are causally linked to the migratory responses we observe, we will treat cells with pharmacological inhibitors and assess motile responses and cytoskeletal remodeling using transwell assays and live cell imaging. To ask how cells integrate signals from multiple chemoattractants, cells will be exposed to S1P and CCL19 simultaneously and sequentially, and signaling responses and cell migration will be analyzed. In Aim 2, we will test the idea that CCL19-induced lamellipodial motility is optimized for long-distance migration in relatively unconfined settings, while S1P-induced bleb-based motility permits cells to pass through small, highly confined spaces. To achieve this, we will test chemotaxis within 3D collagen gels and passage through microfluidic channels with variable geometries that mimic in vivo challenges. As part of this analysis, we will ask how actin and myosin are redistributed in the cell as a function of chemoattractant stimulus and confinement. Finally, we will analyze T cell passage across lymphatic endothelial barriers using transwell assays and tissue explants derived from mouse ears. If successful, this project will complement existing in vivo studies of T cell trafficking by providing much needed mechanistic insights into the underlying molecular and cell biological mechanisms. In the long run, our findings will reveal valuable targets for the rational design of therapeutic approaches based on modulating T cell trafficking.

项目摘要/摘要 T细胞运输对于发育，免疫监视和效应子功能至关重要。迁移是一个复杂的 过程，由许多通过GPCR发出信号的趋化因子编排，以引导T细胞骨架 回答。体内工作表明，诸如CCL19和CCL21（CCR7配体）之类的趋化因子介导了幼稚的T细胞 淋巴组织中的迁移，而脂质趋化剂S1P调节出口。发生类似的过程 在外围组织中，这些信号控制着迁移效应T细胞的迁移。 并进入传入淋巴管。我们的实验室最近克服了一个长期存在的技术问题，这使得很难 研究过体T细胞中的S1P反应。使用此进步，我们发现这两个趋化信号 诱导T细胞运动的不同模式。 CCL19诱导长持续的薄膜迁移，而S1P诱导 较短的基于BLEB的运动。这项工作提出了几个问题：这些化学吸引剂如何引起 如此不同的迁徙反应？当面对竞争提示时，T细胞会做什么？为什么T细胞 需要多种图案机制吗？我们假设CCR7配体和S1P激活不同的细胞骨架 引导不同运动模式的信号通路，单独或组合起作用，以使T细胞得以实现 像在体内遇到的那样驾驶复杂的环境障碍。为了检验这一假设，我们将携带 进行两组研究。在AIM 1中，我们将追求我们的初步数据，表明CCL19优先激活 Rac1依赖性途径导致层状蛋白质，而S1P优先激活途径 涉及RhoA和磷脂酶活性，该活性指导肌球蛋白依赖性收缩力和BLEB形成。到 确认这些信号事件有时与我们观察到的迁移反应有关，我们将对待 具有药物抑制剂和评估母亲反应和细胞骨架重塑的细胞使用Transwell 测定和活细胞成像。要询问细胞如何整合来自多种趋化剂的信号，细胞将是 简单，顺序暴露于S1P和CCL19，信号响应和细胞迁移将是 分析。在AIM 2中，我们将测试CCL19诱导的lamellipodial运动的想法 在相对无限制的设置中迁移，而S1P诱导的基于BLEB的运动允许细胞通过 小，高度狭窄的空间。为此，我们将在3D胶原蛋白凝胶和密码中测试趋化性 通过微流体通道具有可变的几何形状，可以模仿体内挑战。作为该分析的一部分，我们 会问如何将肌动蛋白和肌球蛋白作为趋化剂刺激的函数重新分布在细胞中， 结局。最后，我们将使用Transwell测定法分析T细胞通过淋巴内皮屏障的通道 和源自小鼠耳朵的组织外植体。如果成功，该项目将完成现有的体内研究 通过向基础分子和细胞提供急需的机械洞察来进行T细胞运输 生物学机制。从长远来看，我们的发现将为合理设计的宝贵目标揭示 基于调节T细胞运输的治疗方法。