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

 描述(由申请人提供)：保护性免疫反应依赖于白细胞对炎症刺激的反应跨越内皮屏障的能力。然而，炎症是一把双刃剑；免疫细胞在哮喘、类风湿性关节炎、多发性硬化症和移植等情况下攻击健康组织。T细胞向炎症部位的迁移受到局部趋化因子信号的严格调控，趋化因子信号激活整合素依赖的黏附和细胞骨架变化，这些变化是跨越内皮屏障迁移所需的。跨内皮细胞迁移是一个多步骤的过程，包括系绳和滚动、牢固粘连、爬行和最后的移行。TM代表了炎症反应中的一个重要检查点，而阻断这一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发挥作用，导致整合素活化和细胞骨架重塑，我们发现缺乏这些蛋白的T细胞在黏附和透射电子显微镜下存在缺陷。令人惊讶的是，我们发现这些T细胞正常地向淋巴器官运输，但在向炎症部位迁移方面显示出缺陷。此外，这些细胞可以进行有效的移植物抗白血病(GVL)反应，移植物抗宿主病(GvHD)最少。我们假设，Crk蛋白是趋化因子诱导的T细胞整合素激活和细胞骨架重塑所必需的，也是内皮细胞发生相应变化所必需的，从而允许跨内皮细胞迁移和T细胞向炎症组织的运输。这一假说将通过实现以下三个目标来验证：第一，我们将确定T细胞中Crk蛋白信号促进透射电子显微镜的机制。利用剪切流条件下的配体涂层表面和内皮单分子层，我们将测试Crk/L缺陷T细胞在透射电子显微镜过程中经历各个步骤的能力，并验证Crk蛋白在这一背景下通过Rap1和CDc42发挥作用的假设。其次，我们将评估内皮细胞对Crk/L缺陷T细胞的反应。我们将测试这一假设，即T细胞内依赖于Crk的整合素激活缺陷导致未能适当地激活有效的TEM所需的一个或多个内皮细胞信号通路。将研究三种共同作用的途径，它们可以削弱内皮细胞连接复合体，并为T细胞传递产生特殊的管道。第三，我们将分析急性炎症和GvHD/GVL模型中依赖Crk蛋白的T细胞转运。与它们在控制透射电子显微镜中的作用一致，我们发现Crk/L缺陷的T细胞在向炎症组织迁移方面存在缺陷。此外，即使在可以有效消除淋巴瘤的条件下，这些细胞也能最大限度地减少移植物抗宿主病。为此，我们将使用动物模型来探索Crk蛋白在T细胞运输中的作用。 体内免疫反应。我们将进行组织学分析和活体成像，以研究炎症皮肤中T细胞与血管的相互作用。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.