Determining the role of oxysterols in lymphocyte homing to lymph nodes in homeostasis and inflammation

项目摘要/摘要 淋巴细胞从血液进入淋巴结节(LNS)是有效启动AFP的关键动态平衡过程 对病原体的适应性免疫反应。幼稚的B和T细胞通过LNS来扫描外来抗原 从潜在感染的不同部位传递到这些器官并集中在这些器官中。如果没有抗原 遇到时，淋巴细胞在返回血液并开始淋巴循环之前退出淋巴循环 再骑一次。这种持续的淋巴细胞再循环需要大量渗入淋巴组织 非炎症性条件下，在LNS中支持这一过程的特殊血管称为高 内皮小静脉(HEV)。HEV表达和发光呈现几种血管“地址蛋白”和趋化因子 这些共同支持了淋巴细胞进入LNS的多步骤黏附级联反应。信号促进了 HEV壁上的淋巴细胞停滞已被很好地阐明，但驱动跨膜转运的化学诱导剂 对血管内皮细胞的了解还不完全。阐明介导这一基本步骤的配体和受体 对于理解和操纵免疫细胞在癌症和癌症等疾病中的贩运是必不可少的 自身免疫力。从胆固醇中产生25-羟基胆固醇(25HC)的酶CH25H具有很高的 与LNS中的毛细血管内皮细胞相比，HEV中有选择性表达。此外，CYP7B1，它 羟化25HC产生7𝛼𝛼，25-二羟基胆固醇(7，25HC)在周围的基质细胞中表达 在混合动力车里。7，25HC是EBI2的有效配体，EBI2是一种GPCR，有助于引导激活的B细胞在LNS内的运动 在幼稚的B细胞和T细胞中也高度表达。HEV合成氧固醇的能力表明 EBI2和7，25HC可能在介导淋巴细胞进入LNS中发挥作用。我们假设一个梯度为 跨HEV的7，25HC支持天然B细胞和T细胞的黏附后跨内皮细胞迁移。在预赛中 研究表明，EBI2KO B细胞和CD4T细胞在过继转移中对LNS有募集缺陷，并且 在缺乏CH25H或CYP7B1的小鼠中，类似的归巢缺陷也很明显。此外，对EBI2的依赖 在病毒感染的情况下，进入LNS的淋巴细胞增加。我们推测这是由于725HC升高所致 炎症的LNS中趋化因子水平和降低。根据这些结果，我们的目标是确定LN的步骤 EBI2通过体内归巢试验、活体成像和体外迁移相结合的方式引导的条目 化验(目标1)。此外，我们还将研究EBI2的S在维持炎症的LNS淋巴细胞归巢中的作用。 表征LN氧化类固醇和趋化因子水平的变化，以及这些变化的中介信号 感染(目标2)。总体而言，这项工作将定义一种新的化学诱导剂，推动淋巴细胞迁移到LNS 并加深了我们对轮回过程的理解。除了它们在LNS中的作用外，HEV还在肿瘤中形成 相关的第三级淋巴组织和慢性炎症部位。我们的发现可以为发展提供信息 在这些疾病环境中调节淋巴细胞渗透的新疗法。

PROJECT SUMMARY/ABSTRACT Lymphocyte entry into lymph nodes (LNs) from blood is a key homeostatic process for efficient initiation of an adaptive immune response to pathogens. Naïve B and T cells traffic through LNs to scan for foreign antigens delivered to and concentrated in these organs from diverse sites of potential infection. If no antigens are encountered, lymphocytes exit LNs into lymphatic circulation before returning to the blood and beginning the cycle again. This constant lymphocyte recirculation requires large scale extravasation into lymphoid tissue under non-inflammatory conditions, and the specialized vessels supporting this process in LNs are called high endothelial venules (HEVs). HEVs express and luminally present several vascular ‘addressins’ and chemokines that together support a multi-step adhesion cascade for lymphocyte entry into LNs. The signals promoting lymphocyte arrest on HEV walls are well-elucidated, but the chemoattractants driving transmigration across the endothelium are not fully understood. Elucidating the ligands and receptors that mediate this fundamental step of entry is essential for understanding and manipulating immune cell trafficking in diseases such as cancer and autoimmunity. The enzyme Ch25h, which produces 25-hydroxycholesterol (25HC) from cholesterol, is highly and selectively expressed in HEVs compared to capillary endothelium in LNs. Furthermore, Cyp7b1, which hydroxylates 25HC to generate 7𝛼𝛼,25-dihydroxycholesterol (7,25HC), is expressed in stromal cells surrounding LN HEVs. 7,25HC is a potent ligand for EBI2, a GPCR that helps guide activated B cell movement within LNs and is also highly expressed in naïve B and T cells. The capability for oxysterol synthesis by HEVs suggests EBI2 and 7,25HC may play a role in mediating lymphocyte entry into LNs. We hypothesize that a gradient of 7,25HC across HEVs supports post-adhesion transendothelial migration of naïve B and T cells. In preliminary studies, EBI2 KO B cells and CD4 T cells displayed a recruitment defect to LNs in adoptive transfers, and a similar homing defect was evident in mice lacking Ch25h or Cyp7b1. Furthermore, dependency on EBI2 for lymphocyte entry into LNs increased in a viral infection setting. We hypothesize this is due to elevated 7,25HC levels and reduced chemokine levels in inflamed LNs. From these results, we aim to determine the step of LN entry that EBI2 directs via a combination of in vivo homing assays, intravital imaging, and in vitro migration assays (Aim 1). In addition, we will examine EBI2’s role in maintaining lymphocyte homing in inflamed LNs by characterizing the changes, and signals mediating these changes, in LN oxysterol and chemokine levels during infection (Aim 2). Overall, this work will define a novel chemoattractant driving lymphocyte migration into LNs and advance our understanding of the transmigration process. As well as their role in LNs, HEVs form in tumor associated tertiary lymphoid tissues and at sites of chronic inflammation. Our findings could inform development of novel therapeutics that modulate lymphocyte infiltration in these disease settings.