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

项目总结/摘要 淋巴细胞从血液进入淋巴结（LN）是有效启动免疫应答的关键稳态过程。 对病原体的适应性免疫反应幼稚B和T细胞通过淋巴结运输以扫描外来抗原 从不同的潜在感染部位输送到这些器官并集中在这些器官中。如果没有抗原， 淋巴细胞在返回血液之前进入淋巴循环， 再循环一次。这种恒定的淋巴细胞再循环需要在一定条件下大规模外渗到淋巴组织中。 非炎症性疾病，在LN中支持这一过程的专门血管被称为高 内皮小静脉（HEVs）。HEV表达并在管腔内呈递几种血管“地址素”和趋化因子 它们共同支持淋巴细胞进入LN的多步粘附级联。信号促进 HEV壁上的淋巴细胞阻滞已经很好地阐明了，但是驱动穿过细胞壁的化学引诱物 内皮还不完全清楚。阐明介导这一基本步骤的配体和受体 对于理解和操纵癌症等疾病中的免疫细胞运输至关重要， 自身免疫从胆固醇产生25-羟基胆固醇（25 HC）的酶Ch 25 h是高度 并且与LN中的毛细血管内皮相比，选择性地在HEV中表达。此外，Cyp 7 b1 羟基化25 HC以产生7-羟基胆固醇，25-二羟基胆固醇（7，25 HC），在周围的基质细胞中表达。 LN HEV。7，25 HC是EBI 2的有效配体，EBI 2是一种GPCR，有助于引导活化的B细胞在LN内运动 并且在幼稚B和T细胞中也高度表达。HEV合成氧固醇的能力表明 EBI 2和7，25 HC可能在介导淋巴细胞进入淋巴结中起作用。我们假设， 7，25 HC跨HEV支持幼稚B和T细胞的粘附后跨内皮迁移。初步 研究中，EBI 2 KO B细胞和CD 4 T细胞在过继转移中显示出对LN的募集缺陷， 类似的归巢缺陷在缺乏Ch 25 h或Cyp 7 b1的小鼠中是明显的。此外，对EBI 2的依赖性 淋巴细胞进入淋巴结增加病毒感染设置。我们推测这是由于7，25 HC升高所致 水平和降低的趋化因子水平。从这些结果中，我们的目的是确定LN的步骤 EBI 2通过体内归巢测定、活体成像和体外迁移的组合引导进入 测定（目的1）。此外，我们将通过以下方式研究EBI 2在维持炎症淋巴结中淋巴细胞归巢中的作用 表征LN氧化固醇和趋化因子水平的变化，以及介导这些变化的信号， 感染（目标2）。总之，这项工作将确定一种新的趋化因子驱动淋巴细胞迁移到淋巴结 并促进我们对轮回过程的理解。除了它们在淋巴结中的作用外，HEV在肿瘤中形成， 相关的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 在这些疾病中调节淋巴细胞浸润的新疗法。

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.