Lymphatic Microenvironment: Altering Cell Traffic by Targeting Glycans

描述（由申请方提供）：在炎症反应和瘤形成期间，淋巴管细胞从外周流向淋巴结。在这两种情况下，由淋巴管内皮产生的独特趋化因子（趋化性细胞因子）在驱动先天免疫细胞（如树突细胞）或肿瘤细胞的转运中发挥核心作用。该提案研究了独特的复合碳水化合物（聚糖）在淋巴微环境中介导趋化因子依赖性细胞运输的遗传重要性。我们专注于硫酸乙酰肝素（HS），由硫酸多糖栓系到独特的细胞表面结合以及分泌的蛋白多糖核心蛋白。HS与几种趋化因子的富含碱性氨基酸的结构域相互作用。本文的重点是由淋巴内皮产生的HS，因为初步工作表明淋巴HS可能介导两种关键的趋化因子功能：（1）主要淋巴趋化因子的支架（如CCL21和CXCL12）在淋巴周围空间梯度上的表达，和（2）趋化因子对运输细胞上的同源受体的独特成簇呈递，其中分泌的淋巴HS蛋白聚糖可以作为趋化因子共受体。该提议解决了以下假设：靶向淋巴聚糖生物合成将改变树突状细胞或肿瘤细胞以趋化因子依赖性方式向淋巴管迁移并运输至淋巴结的能力。目的是：（1）检查遗传改变淋巴HS对体内淋巴微环境中细胞运输的影响。树突状细胞和肿瘤细胞从外周组织向局部淋巴结的迁移将在淋巴HS生物合成中携带基因缺陷的小鼠中进行检查。还将检查抗原驱动运输研究中的T细胞应答。突变体将表征淋巴HS结构和蛋白聚糖表达。(2)确定HS在建立淋巴趋化因子梯度中的作用。HS链将被纯化并测试其结合主要淋巴趋化因子的能力。在基于淋巴内皮基质的测定中，将检查在静态和流动条件下遗传改变HS生物合成对趋化因子梯度形成的影响。还将在体内检查突变状态下的趋化因子分布。(3)描述淋巴HS作为趋化因子依赖性细胞迁移和信号传导的共受体的重要性。将确定树突细胞向HS生物合成中携带突变的淋巴内皮迁移的能力，并测试来自突变淋巴内皮的条件培养基寡聚化（聚集）淋巴趋化因子和激活树突细胞中趋化因子依赖性迁移信号传导的能力。最后，将在体内检查淋巴HS突变对淋巴趋化因子和运输细胞上的同源受体之间的关联的影响。总的来说，这项工作可能会揭示淋巴微环境中的聚糖如何控制多种趋化因子的作用的新机制。它也可能为新的治疗发现奠定基础。

DESCRIPTION (provided by applicant): Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the transit of either innate immune cells, such as dendritic cells, or tumor cells. This proposal examines the genetic importance of unique complex carbohydrates (glycans) in mediating chemokine-dependent cell traffic in the lymphatic microenvironment. We focus on heparan sulfate (HS), composed of sulfated glycans tethered to unique cell-surface bound as well as secreted proteoglycan core proteins. HS interacts with basic amino acid-rich domains of several chemokines. The focus herein is on HS produced by the lymphatic endothelium, as preliminary work suggests that lymphatic HS may mediate two critical chemokine functions: (1) scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) onto peri-lymphatic spatial gradients, and (2) unique clustered presentation of chemokines to cognate receptors on trafficking cells, wherein secreted lymphatic HS proteoglycans may serve as chemokine co-receptors. This proposal addresses the hypothesis that targeting lymphatic glycan biosynthesis will alter the ability of dendritic cells or tumor cells to migrate toward lymphatic vessels and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Examine the effects of genetically altering lymphatic HS on cell trafficking in the lymphatic microenvironment in vivo. Migration of dendritic cells and tumor cells from peripheral tissue to regional lymph nodes will be examined in mice bearing gene defects in lymphatic HS biosynthesis. T cell responses in antigen-driven trafficking studies will also be examined. Mutants will be characterized for lymphatic HS structure and proteoglycan expression. (2) Determine the role of HS in establishing lymphatic chemokine gradients. HS chains will be purified and tested for their ability to bind major lymphatic chemokines. In lymphatic endothelial matrix-based assays, the effects of genetically altering HS biosynthesis on chemokine gradient formation in static and flow conditions will be examined. Chemokine distribution in the mutant state will also be examined in vivo. (3) Characterize the importance of lymphatic HS as a co-receptor for chemokinedependent cell migration and signaling. The ability of dendritic cells to migrate toward lymphatic endothelium bearing mutations in HS biosynthesis will be determined, and conditioned medium from mutant lymphatic endothelia will be tested for its ability to oligomerize (cluster) lymphatic chemokines and activate chemokine-dependent migration signaling in dendritic cells. Finally, the effect of lymphatic HS mutation on the association between lymphatic chemokines and cognate receptors on trafficking cells will be examined in vivo. Collectively, this work may uncover novel mechanisms for how the actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a basis for novel therapeutic discovery.