Chemoattractant-specific T cell navigation of complex environments

项目总结/摘要 T细胞运输对于发育、免疫监视和效应器功能至关重要。移徙是一个复杂 过程，由一系列化学引诱物编排，这些化学引诱物通过GPCR发出信号，指导T细胞细胞骨架 应答体内研究表明趋化因子如CCL 19和CCL 21（CCR 7配体）介导幼稚T细胞 在淋巴组织内的迁移，而脂质化学引诱物S1 P调节出口。发生类似过程 在外周组织中，这些信号控制迁移效应T细胞从发炎组织中迁移出来 进入传入神经系统。我们的实验室最近克服了一个长期存在的技术问题， 研究离体T细胞中的S1 P应答。利用这一进展，我们发现这两种趋化信号 诱导不同T细胞运动模式。CCL 19诱导长时间板状伪足迁移，而S1 P诱导 一种基于水泡的运动性的短暂爆发。这项工作提出了几个问题：这些化学引诱剂如何引起 如此不同的迁徙反应T细胞在面对竞争性信号时会做什么？为什么T细胞 需要多个运动机制吗我们假设CCR 7配体和S1 P激活不同的细胞骨架， 信号通路，指导不同的运动模式，单独或组合工作，使T细胞 通过复杂的环境障碍，比如它们在体内遇到的障碍。为了验证这一假设，我们将 两组研究。在目标1中，我们将继续我们的初步数据，显示CCL 19优先激活 Rac 1依赖性途径导致板状伪足突出，而S1 P优先激活途径 涉及RhoA和磷脂酶活性，其指导肌球蛋白依赖性收缩和水泡形成。到 为了验证这些信号事件与我们观察到的迁移反应有因果关系，我们将治疗 细胞与药理学抑制剂，并评估运动反应和细胞骨架重塑使用transwell 分析和活细胞成像。为了了解细胞如何整合来自多种化学引诱物的信号，细胞将 同时和顺序暴露于S1 P和CCL 19，信号应答和细胞迁移将被抑制。 分析了在目标2中，我们将测试CCL 19诱导的板状伪足运动对于长距离运动是优化的这一想法。 在相对开放的环境中迁移，而S1 P诱导的基于水泡的运动允许细胞通过 狭小的空间为了实现这一点，我们将测试3D胶原凝胶内的趋化性， 通过模拟体内挑战的可变几何形状的微流体通道。作为分析的一部分，我们 将询问肌动蛋白和肌球蛋白如何在细胞中重新分布作为化学引诱物刺激的函数， 禁闭。最后，我们将分析T细胞通过淋巴管内皮屏障使用transwell检测 和来自小鼠耳朵的组织外植体。如果成功，该项目将补充现有的体内研究 通过提供对潜在分子和细胞的急需的机制见解， 生物机制。从长远来看，我们的研究结果将为合理设计 基于调节T细胞运输的治疗方法。

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.