血管内皮细胞过表达PD-L配体上调CTL细胞PD-1抑制信号对脑型疟血脑屏障损伤的保护性作用研究

CM (cerebral malaria) is one of the most serious complications of plasmodium falciparum infection. The BBB（blood-brain barrier）damage caused by cerebral microcirculation disorder and immunopathological injury cooperatively are the key pathological changes of CM. Study shows that CTL (cytotoxic lymphocyte) cells adhering to the brain blood vessels are responsible for vascular endothelial cells injury, which led to BBB damage and CM. In recent years, blockade of PD-1/PD-L negative regulation pathway through antibodies in the anti-tumor research field brings new enlightenment to the immunotherapy of CM. Previous studies of our group have shown that the knock-out of PD-1 gene makes the ECM (experimental cerebral malaria) disease of the mice more seriously, and the survival time is obviously shortened, which demonstrated that the PD-1/PD-L pathway has an obvious effect on the outcome of ECM disease. We speculate that the over-expression of PD-1 ligand, PD-L, led to the reduced activation of CTL through more PD-1 receptor being stimulated, and the injury of brain vascular endothelial cells will also be reduced. Therefore, the recombinant adenovirus expressing a fusion protein contains the extracellular fragment of PD-L and IgG Fc fragment would be constructed, then to study whether more exogenous PD-L expressed by brain endothelial cells have the protective effect on BBB damage through intensify the CTL suppression signal via PD-1 receptor , and provide a new theoretical basis and research ideas for prevention of CM.

脑型疟是恶性疟原虫感染最严重的并发症之一。脑微循环障碍和免疫病理损伤协同造成血脑屏障破坏是脑型疟最主要的病理变化。研究表明：脑血管黏附的CTL细胞靶向杀伤血管内皮细胞是血脑屏障损伤的重要原因。近年来，抗体阻断PD-1/PD-L免疫负调控通路在抗肿瘤研究领域取得的突破性进展给脑型疟免疫治疗带来新的启示。课题组预实验发现，PD-1基因敲除小鼠脑型疟症状明显加重，生存时间明显缩短，说明PD-1/PD-L通路调控对脑型疟转归有明显影响。如果让脑血管内皮细胞过表达PD-L配体，则CTL细胞的活化程度会因为更多PD-1受体被激活而降低，其对内皮细胞的靶向杀伤随之减轻。因此，本研究拟制备PD-L配体胞外段与IgGFc段融合蛋白的重组腺病毒，研究血管内皮细胞特异性过表达外源PD-L配体进而强化CTL细胞PD-1抑制信号对脑型疟血脑屏障损伤的保护性作用，为人类脑型疟防治研究提供新的理论基础和研究思路。

脑型疟是恶性疟原虫感染最严重的并发症之一。脑微循环障碍和免疫病理损伤协同造成血脑屏障破坏是脑型疟最主要的病理变化。研究表明：脑血管黏附的CTL细胞靶向杀伤血管内皮细胞是血脑屏障损伤的重要原因。如果让脑血管内皮细胞过表达PD-L配体，则CTL细胞的活化程度会因为更多PD-1受体被激活而降低，其对内皮细胞的靶向杀伤随之减轻。因此，本研究拟制备PD-L配体胞外段与IgGFc段融合蛋白的重组腺病毒，研究血管内皮细胞特异性过表达外源PD-L配体进而强化CTL细胞PD-1抑制信号对脑型疟血脑屏障损伤的保护性作用。项目组除了构建可以表达PDL1的重组腺病毒外，还制备了两种可溶性融合蛋白，PDL1-IgG1Fc和PDL2-IgG1Fc，以适度增强CD8+ T细胞PD1受体介导的抑制性信号。我们在ECM小鼠脑型疟模型研究中得出以下结论：①PD1基因敲除小鼠感染伯氏疟原虫安卡株PbA后，其CD8+T细胞活化水平明显高于野生型对照组小鼠，生存时间缩短；②与野生型对照组小鼠相比，PDL1-IgG1Fc融合蛋白治疗可延长脑型疟小鼠生存时间；③PDL1-IgG1Fc融合蛋白可有效抑制CD8+T细胞过度活化及其对脑血管内皮细胞的靶向杀伤，减轻血脑屏障损伤，进而缓解小鼠脑型疟症状。综上所述，本研究证实，在疟原虫感染早期给予PDL1-IgG1Fc融合蛋白治疗，对维持脑部免疫微环境平衡具有积极的治疗作用，是一种前景良好的脑型疟免疫治疗新方法。