LMP1/HIF-1α调控PD-L1+外泌体介导鼻咽癌免疫逃逸的作用机制研究

Currently, radiotherapy is the mainstay of treatment for nasopharyngeal carcinoma (NPC). However, NPC patients with upregulated Programmed Death-Ligand 1 (PD-L1) exhibit a poor prognosis after radiotherapy. Therefore, novel integrating treatment is urgently needed for these patients. PD-L1 has been reported to be carried by exosomes and transported to T cells, leading to immune suppression effects. Previous studies indicate a significant correlation between PD-L1 and prognosis of NPC patients. Furthermore, PD-L1 is co-located with HIF-1α in NPC tissues and expression of PD-L1 is regulated by Latent Membrane Protein 1 (LMP1), an important oncogene encoded by Epstein-Barr virus, and HIF-1α. Moreover, exosomes with PD-L1 positive (PD-L1+) were significantly induced in plasma of NPC patients than the control group. Thus, we hypothesize that PD-L1+ exosomes could mediate potent immune suppression and function of PD-L1 may be activated by LMP1/HIF-1α in NPC cells and tumor-derived exosomes. The aim of the study is to isolate and identify plasma exosomes to investigate the correlation between PD-L1+ exosomes in plasma and the prognosis of NPC patients after radiotherapy; to analyze the immune suppression effects of PD-L1+ exosomes to T cell immune response; to clarify the mechanism of LMP-1/HIF-1α in regulating function of PD-L1 in NPC cells and in tumor-derived exosomes in vitro and in vivo. The accomplishment of this study could provide novel insights for NPC immunotherapy.

放射治疗是目前鼻咽癌的主要治疗手段，但PD-L1高表达的患者放疗后预后不佳，此类患者仍需新的综合治疗方法。PD-L1可被外泌体包裹并传递至T细胞，从而发挥免疫抑制效应。前期研究发现，PD-L1与患者不良预后呈正相关并与HIF-1α共定位，其表达受EB病毒编码的潜伏膜蛋白1（LMP1）及HIF-1α调控；且鼻咽癌患者血浆中PD-L1+外泌体数量显著高于正常人。我们因此推断，PD-L1+外泌体可介导鼻咽癌的免疫逃逸，而LMP1/HIF-1α参与鼻咽癌细胞及外泌体中PD-L1功能的活化。本研究拟分析鼻咽癌循环血浆PD-L1+外泌体的免疫抑制效应及其与放疗后患者预后的关系，进行体内外实验阐明LMP1/HIF-1α调控鼻咽癌细胞及外泌体中PD-L1的作用机制，为鼻咽癌免疫治疗提供新的思路。

PD-L1+外泌体可介导鼻咽癌的免疫逃逸，前期研究结果提示而EB病毒编码的潜伏膜蛋白1(LMP1)及乏氧诱导因子-1α(HIF-1α)可能参与鼻咽癌细胞及外泌体中PD-L1功能的活化。本项目研究LMP1及HIF-1α上调鼻咽癌细胞PD-L1表达促进鼻咽癌恶性发展的机制，探索了鼻咽癌细胞顺铂耐药及顺铂促进鼻咽癌细胞表达PD-L1影响免疫效应的机制；通过应用CD63抗体修饰并加入PD-L1适配体，研发磁性纳米颗粒（MNPs）免疫-适配体传感器检测鼻咽癌来源外泌体，并在实际临床样品中验证可行性和实际应用潜力；研究PD-L1+外泌体在EBV阳性/阴性鼻咽癌细胞系间的表达差异并探索LMP1对鼻咽癌细胞外泌体生成及外泌体中PD-L1表达的调控机制及其通过调控PD-L1+外泌体影响CD8+T细胞免疫效应的机制。研究结果提示，顺铂可上调鼻咽癌细胞表达PD-L1；乏氧通过HIF-1α上调鼻咽癌细胞表达PD-L1，这一过程与STAT3/HIF-1α/VEGF通路密切相关；LMP1显著延长了PD-L1的半衰期且增强PD-L1稳定性，从而促进鼻咽癌细胞表达PD-L1；项目成功研发荧光法检测鼻咽癌来源外泌体，通过形成“MNPs-外泌体-适配体”实现荧光信号放大，结果显示检测荧光水平与外泌体浓度高度线性相关，并有较高特异性；研究还发现PD-L1+外泌体在EBV阳性细胞C666-1中表达更为丰富；过表达LMP1后鼻咽癌细胞外泌体中具有更高水平的PD-L1表达, 在此过程中ESCRT III型蛋白CHMP4B与LMP1存在直接相互作用; 过表达LMP1后鼻咽癌细胞外泌体对CD8+细胞产生了更强的免疫抑制作用，具体表现为细胞增殖能力下降，IFN-γ、Granzyme-B分泌水平降低；用PD-L1封闭抗体阻断实验证实LMP-1对于鼻咽癌外泌体的CD8+细胞免疫抑制作用依赖于外泌体中PD-L1的表达。通过上述研究，项目研发成功更为高效的鼻咽癌外泌体定量检测平台，为鼻咽癌外泌体的基础及临床研究提供基础； 系统阐述了LMP1及HIF-1α上调鼻咽癌细胞PD-L1促进鼻咽癌恶性发展的机制，明确了LMP1对鼻咽癌外泌体生成及外泌体中PD-L1表达的调控机制，证实了LMP1调控鼻咽癌细胞PD-L1+外泌体对CD8+T细胞的免疫抑制效应及其机制，为阐明鼻咽癌免疫逃逸机制及免疫治疗提供了新的思路。