The mechanochemical control of T-cell directional migration under flow

流动条件下T细胞定向迁移的机械力化学控制 丹尼尔·A·哈默(PI)和贾尼斯·K·伯克哈特(共同调查者) 项目摘要 T淋巴细胞是获得性免疫反应中的关键分子，其运动性对其功能至关重要。T- 细胞配备了多种不同的黏附分子，这些黏附分子与表达的配体相互作用 在整个免疫系统中是不同的。此外，T细胞通常必须在外加的流场下活动。 当它们通过血管系统和淋巴系统流动时。我们的目标是了解T细胞如何应对 不同的黏附配体和剪切率，它们有效地迁移到炎症和 免疫沟通。在分子水平上了解这一过程对于发展 治疗炎症性和感染性疾病以及癌症的治疗策略 最近，我们发现T细胞的定向迁移与它们的配体类型有关 以及它们所暴露的剪切率。当放置在带有血管细胞的表面时 与1整合素受体VLA-4结合的黏附分子-1，T细胞向下游爬行 向下流动(沿着流动方向)。然而，当放置在具有细胞间粘连的表面时 与2受体LFA-1结合的分子-1(ICAM-1)，T细胞沿着流动的方向爬行，就像一个 鲑鱼逆流而上。上游迁移的幅度取决于切变率，其中T细胞更多 致力于随着剪切率的增加而向上游迁移。在黏附分子所在的表面上 混合，任何数量的ICAM-1都支持上游迁移。当流被移除时，T细胞表现出 迁移记忆，但仅当它们同时暴露于ICAM-1和VCAM-1时。这个观察点 涉及两个不同整合素受体之间的串扰的新机制。 我们建议研究在流动状态下驱动T细胞上游迁移的机制 ICAM-1和迁移记忆的起源。我们假设上游迁移是由2引起的 整合素形成捕获键，当整合素连接产生的信号时，它将细胞固定在适当的位置 加强黏附相互作用，推动前沿肌动蛋白聚合，推动 迁移。为了测试这一点，我们将使用分子工程、流动室、微图案表面和 微型制造的柱阵列探测器(MPAD)用于测量迁移细胞所施加的力。我们有 初步证据表明，其他可移动的阿米巴细胞，如永生化的KG1a细胞系，显示出 同样的现象，有助于我们使用分子工程工具和成像方法来识别 相关分子。通过剖析这种引人入胜的现象背后的机制，我们希望 阐明整合素依赖的T细胞运输的关键特征。我们这项工作的目的是：1.测量 T细胞和KG1a定向运动和迁移记忆的动力学2.识别信号和离合器 负责1和2整合素配体在流动下的差异迁移的分子；以及3. 测量当T细胞在整合素配体上定向扩散和爬行时的力产生机制。

The mechanochemical control of T-cell directional migration under flow Daniel A. Hammer (PI) and Janis K. Burkhardt (co-Investigator) Project Summary T-lymphocytes are key players in the adaptive immune response, and motility is critical to their function. T- cells are equipped with multiple different adhesion molecules that interact with ligands that are expressed differentially throughout the immune system. Furthermore, T-cells often must act under an imposed flow field as they traffic through the vasculature and lymphic system. Our goal is to understand how T-cells respond to the different adhesion ligands and shear rates they encounter to effectively migrate to sites of inflammation and immune communication. Understanding this process at the molecular level is important for development of therapeutic strategies to treat inflammatory and infectious diseases, and cancer Recently, we have discovered that directional T-cell migration varies as a function of the type of ligand they encounter and the shear rate to which they are exposed. When placed on a surface bearing vascular cell adhesion molecule-1 (VCAM-1), which engages the 1-integrin receptor VLA-4, T-cells crawl downstream under flow (in the direction of flow). However, when placed on a surface bearing intercellular adhesion molecule-1 (ICAM-1), which engages the 2-receptor LFA-1, T-cells crawl against the direction of flow, like a salmon swims upstream. The magnitude of upstream migration depends on shear rate, with T-cells more committed to upstream migration as the shear rate increases. On surfaces in which adhesion molecules are mixed, any amount of ICAM-1 supports upstream migration. When the flow is removed, T-cells exhibit migrational memory, but only if they have been exposed to both ICAM-1 and VCAM-1. This observation points to a novel mechanism of crosstalk between two distinct integrin receptors. We propose to investigate the mechanisms that drive the upstream migration of T-cells under flow on ICAM-1, and the origins of migrational memory. We hypothesize that upstream migration is caused by 2 integrin forming a catch bond, which holds the cell in place while signals generated by integrin ligation strengthen adhesive interactions and spur the polymerization of actin at the leading edge, driving forward migration. To test this, we will use molecular engineering, flow chambers, micropatterned surfaces, and microfabricated post array detectors (mPADs) to measure forces exerted by the migrating cell. We have preliminary evidence that other motile amoeboid cells such as the immortalized KG1a cell line display the same phenomenon, facilitating our use of molecular engineering tools and imaging methods to identify the relevant molecules. By dissecting the mechanisms that underlie this fascinating phenomenon, we expect to elucidate key features of integrin-dependent T cell trafficking. Our aims in this work are to: 1. Measure the dynamics of T-cell and KG1a directional motion and migrational memory; 2. Identify the signals and clutch molecules responsible for the differential migration under flow in response to 1 and 2 integrin ligands; and 3. Measure the mechanisms of force generation when T-cells spread and crawl directionally on integrin ligands.