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

T 细胞和发炎内皮细胞之间的串扰：Crk 家族蛋白的调节

• 批准号: 9118335
• 负责人: Janis K. Burkhardt
• 金额: $ 42万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9118335
• 关键词: Actins; Acute; Adhesions; Affect; Animal Model; Asthma; Binding Proteins; Biochemical; Blood Circulation; CDC42 gene; Cell Shape; Cell physiology; Cell surface; Cells; Complex; Cutaneous; Defect; Deposition; Disease; Dissociation; Elements; Endothelial Cells; Endothelium; Failure; Family; Filopodia; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Immigration; Immune; Immune response; Individual; Inflammation; Inflammatory; Inflammatory Response; Integrins; Intercellular Junctions; Intercellular adhesion molecule 1; Invaded; Knowledge; Lateral; Leukemic Cell; Leukocytes; Ligands; Lymphoid; Lymphoma; MYLK gene; Modeling; Multiple Sclerosis; Myosin Type II; Organ; Pathway interactions; Phosphorylation; Play; Process; Protein Family; Proteins; Reagent; Recycling; Regulation; Rheumatoid Arthritis; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Skin; Stimulus; Surface; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic; Tissues; Transplantation; Vascular Cell Adhesion Molecule-1; cadherin 5; cell motility; cell type; chemokine; graft vs host disease; graft vs leukemia effect; in vivo; intravital imaging; killings; leukemia; migration; monolayer; pathogen; protein function; response; stem; surface coating; trafficking

 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.

 描述（由适用提供）：保护性免疫复杂依赖白细胞对炎症刺激的跨内皮屏障的能力。但是，炎症是一把双刃剑。免疫细胞在哮喘，类风湿关节炎，多发性硬化症和移植等情况下攻击健康的组织。 T细胞迁移到位点F炎症受到局部趋化因子信号的严格调节，该信号激活整联蛋白依赖性粘附和跨内皮屏障所需的细胞骨架变化。跨内皮迁移（TEM）是一个多步骤的过程，涉及绑扎和滚动，坚固的粘合剂，爬行以及最后移民。 TEM代表炎症反应中的一个重要检查点，阻止此过程的试剂具有热有价值。我们发现，CRK家族蛋白在趋化因子途径中起作用，导致整联蛋白活化和细胞骨架重塑，我们发现缺乏这些蛋白质的T细胞具有粘合剂和TEM的缺陷。令人惊讶的是，我们发现这些T细胞正常流动到淋巴器官，但在迁移到炎症部位的缺陷。此外，这些细胞可以使用最小的移植物与宿主疾病（GVHD）进行有效的移植物与白血病（GVL）反应。我们假设CRK蛋白是趋化因子诱导的整联蛋白激活和T细胞中的细胞骨架重塑所必需的，以及允许跨内皮迁移和T细胞运输到发炎组织的植物的相应变化。该假设将通过以下三个目的进行测试：首先，我们将定义T细胞中CRK蛋白信号传导促进TEM的机制。使用剪切流条件下的配体涂层表面和内皮单层，我们将测试CRK/L缺陷T细胞在TEM过程中经历各个步骤的能力，并检验假设CRK蛋白在这种情况下通过RAP1和CDC42的功能。其次，我们将评估内皮细胞对CRK/L缺陷T细胞的反应。我们将测试以下假设：T细胞内CRK依赖性整联蛋白激活的缺陷导致未能适当激活有效TEM所需的一个或多个内皮ELL信号通路。将研究三种功能削弱内皮连接络合物并生成用于T细胞通道的专门导管的途径。第三，我们将分析急性炎症和GVHD/GVL模型中的CRK蛋白依赖性T细胞运输。与它们在控制TEM中的作用一致，我们发现CRK/L缺乏T细胞显示出迁移到发炎组织的缺陷。此外，即使在可以有效消除淋巴瘤的条件下，这些细胞也会引起最小的GVHD。在此目标中，我们将使用动物模型在T细胞运输中探索CRK蛋白功能 体内免疫响应。我们将进行组织学分析和插入式成像，以研究发炎的皮肤中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细胞运输到靶向器官，并测试我们靶向CRK蛋白功能的能力I治疗环境。