Crosstalk between T cells and inflamed endothelium: regulation by Crk family proteins

 描述（由申请方提供）：保护性免疫应答依赖于白细胞响应炎症刺激而穿过内皮屏障的能力。然而，炎症是一把双刃剑;在哮喘、类风湿性关节炎、多发性硬化症和移植等情况下，免疫细胞会攻击健康组织。T细胞向炎症部位的迁移受到局部化趋化因子信号的严格调节，其激活跨内皮屏障迁移所需的整合素依赖性粘附和细胞骨架变化。经内皮迁移（TEM）是一个多步骤的过程，涉及束缚和滚动，牢固的粘附，爬行，最后是迁移。TEM代表炎症反应中的重要检查点，阻断该过程的试剂具有治疗价值。我们发现Crk家族蛋白在趋化因子途径中起作用，导致整合素活化和细胞骨架重塑，并且我们发现缺乏这些蛋白的T细胞在粘附和TEM方面存在缺陷。引人注目的是，我们发现这些T细胞正常地运输到淋巴器官，但在迁移到炎症部位时显示出缺陷。此外，这些细胞可以进行有效的移植物抗白血病（GvL）应答，具有最小的移植物抗宿主病（GvHD）。我们假设Crk蛋白是趋化因子诱导的整合素活化和T细胞骨架重塑所必需的，并且是内皮细胞中允许跨内皮迁移和T细胞运输到炎症组织中的相应变化所必需的。这一假设将通过以下三个目的进行检验：首先，我们将确定T细胞中Crk蛋白信号传导促进TEM的机制。使用配体包被的表面和内皮细胞单层在剪切流条件下，我们将测试Crk/L缺陷的T细胞进行TEM过程中的各个步骤的能力，并测试假设Crk蛋白的功能，通过Rap1和Cdc42在这种情况下。其次，我们将评估内皮细胞对Crk/L缺陷型T细胞的反应。我们将测试这样的假设：T细胞内Crk依赖性整合素激活的缺陷导致无法适当激活有效TEM所需的一种或多种内皮细胞信号传导途径。三个途径，共同发挥作用，削弱内皮连接复合物，并产生一个专门的管道T细胞通过将进行研究。第三，我们将分析Crk蛋白依赖性T细胞在急性炎症和GvHD/GvL模型中的运输。与它们在控制TEM中的作用一致，我们发现Crk/L缺陷型T细胞在迁移到发炎组织中时显示缺陷。此外，这些细胞引起最小的GvHD，即使在它们可以有效消除淋巴瘤的条件下。在这个目标中，我们将使用动物模型来探索Crk蛋白在T细胞运输过程中的功能。 体内免疫应答。我们将进行组织学分析和活体成像，以研究炎症皮肤中的T细胞-血管相互作用。此外，我们将使用GvHD和GvHD/GvL模型来研究T细胞向靶器官的运输，并测试我们在治疗环境中靶向Crk蛋白功能的能力。

 DESCRIPTION (provided by applicant): Protective immune responses rely on the ability of leukocytes to cross endothelial barriers in response to inflammatory stimuli. However, inflammation is a double-edged sword; immune cells attack healthy tissues in situations such as asthma, rheumatoid arthritis, multiple sclerosis, and transplantation. T cell migration into sites f inflammation is tightly regulated by localized chemokine signals, which activate integrin dependent adhesion and cytoskeletal changes needed for migration across endothelial barriers. Transendothelial migration (TEM) is a multi-step process involving tethering and rolling, firm adhesion, crawling, and finally transmigration. TEM represents an important checkpoint in the inflammatory response, and reagents that block this process are therapeutically valuable. We have found that Crk family proteins function in the chemokine pathway leading to integrin activation and cytoskeletal remodeling, and we find that T cells lacking these proteins have defects in adhesion and TEM. Strikingly, we find that these T cells traffic normally to lymphoid organs, but show defects in migration to sites of inflammation. Moreover, these cells can carry out an efficient graft vs leukemia (GvL) response, with minimal graft vs host disease (GvHD). We hypothesize that Crk proteins are required for chemokine-induced integrin activation and cytoskeletal remodeling in T cells, and for corresponding changes in the endothelium that allow transendothelial migration and T cell trafficking into inflamed tissue. This hypothesis will be tested by carrying out following three Aims: First, we will define the mechanisms through which Crk protein signaling in T cells promotes TEM. Using ligand-coated surfaces and endothelial monolayers under shear flow conditions, we will test the ability of Crk/L deficient T cells to undergo individual steps in the TEM process, and test hypothesis that Crk proteins function through both Rap1 and Cdc42 in this context. Second, we will assess endothelial cell responses to Crk/L-deficient T cells. We will test the hypothesis that defects in Crk-dependent integrin activation within the T cell result in a failure to appropriately activate one or more endothelial ell signaling pathways that are required for efficient TEM. Three pathways that function together to weaken endothelial junctional complexes and generate a specialized conduit for T cell passage will be investigated. Third, we will analyze Crk protein dependent T cell trafficking in acute inflammation and GvHD/GvL models. Consistent with their role in controlling TEM, we find that Crk/L deficient T cells show defects in migration into inflamed tissues. Moreover, these cells cause minimal GvHD, even under conditions where they can efficiently eliminate lymphoma. In this aim, we will use animal models to explore Crk protein function in T cell trafficking during an in vivo immune response. We will conduct histological analysis and intravital imaging to study T cell-vessel interactions in inflamed skin. In addition, we will use GvHD and GvHD/GvL models to study T cell trafficking to target organs, and to test our ability to target Crk protein function i a therapeutic setting.