H7N9感染时机体的细胞免疫应答及其保护作用研究

Since March of 2013, H7N9 has caused 355 cases with 112 deaths (31.5%). In human flu T cell immunity, although it is extensively studied in mice model, it is still not clear about the kinetics of CD8 or CD4 response against flu and its role in recovery of flu infection. Albeit that previous studies showed the preexisting CD4 or CD8 could be associated with recovery from experimentally mild or naturally mild flu infection, very little is known about whether virus specific CD4 or CD8 is still associated with the recovery of H7N9 severe infection and how the host immune response look like, In 2013, we have collected the longitunal PBMC samples from 18 hospitalized H7N9 patients and are still collecting samples from new cases in our hospital. In the meantime, we developed one new method containing 12 color of fluorescence labeled antibodies panel based on flow cytometer to detect the innate cell immunity (including monocytes, mucaso-associated invariant T cell (MAIT), γδT and NK cells) and adaptive T cell immunity (including CD4 and CD8). In this study, we will investigate: 1) whether virus-specific T cell immunity (CD4 and/or CD8) and innate immunity are associated with the recovery of H7N9 severe infection. 2) the kinetics of CD8 or CD4 response in human severe infection, to see whether it is similar to the known CD8 kinetics in mice model; 3) the role of preexisting CD8 (memory CD8) and the new developed effector CD8 T cell in the progress of H7N9 disease course, to see whether it will make difference in recovery; 4) whether there is frequency difference in NK，monocytes， γδT and MAIT between young people and the elderly, which may explain why the elderly are more venerable to H7N9 than young people; 5) the cooperative network between the CD8, CD4 and neutralizing antibodies in H7N9 severe infection. This study will help us understand the human immune response against severe flu infection such as H7N9 and H5N1, and make up the shortcoming of mice model in flu T cell immunity research, providing information for possible immune intervention in H7N9 treatment and for the design of proper flu vaccine in the future.

H7N9已引起355例感染，其中112例死亡。在流感T细胞领域，虽用小鼠模型做了大量CD4、CD8研究，但对人感染流感后，病毒特异CD4或CD8的动力学及其在患者康复中的作用尚有争议。据报道，感染前预存CD4或CD8 T 细胞数量与实验性或自然感染轻微流感的康复相关，但与H7N9类似的严重感染关系尚不清楚。我们已采集H7N9感染的18位病人不同时间点的样本，且已建立一种基于12色流式细胞术的新方法，可同时检测天然免疫细胞（MAIT、γδT 和 NK）和T细胞（CD4和CD8）的免疫应答。本课题将研究：1）天然免疫细胞及T细胞与H7N9患者康复的关系；2）CD4和CD8 T细胞的动力学特征及记忆性CD8二次应答与新产生的效应性CD8 在患者康复中的作用；3）老年人易感H7N9的原因：与青年人天然免疫细胞频率的比较；4）CD4、CD8和抗体的协同作用。本研究将为临床治疗和疫苗研究提供新思路。

目前对H7N9为什么引起严重感染的宿主因素尚不清楚。我们在国家自然基金面上项目（81471556）的支持下，利用上海16例H7N9病人的外周血细胞，首次阐述了中国人基因型（IFITM3-C/C）感染H7N9后易发重症，并且肺部感染的细胞因子水平是血液中的几百倍（Proc Natl Acad Sci U S A. 2014 Jan 14;111(2):769-74.），并进一步发现H7N9感染患者的细胞免疫应答需要在不同时期招募CD8, CD4，NK 细胞或者抗体进行抗病毒免疫，而且早期的CD8+反应至关重要（Nat Commun. 2015 May 13;6:6833.）。为了阐明H7N9死亡病人为什么没有有效的CD8 应答，我们更深一步发现H7N9感染，可以导致病人体内 “细胞免疫疲劳”，该疲劳现象在病人早期尤其严重，康复期恢复，并发现“细胞免疫疲劳”与细胞线粒体的呼吸链代谢紊乱和抗原呈递功能减弱有关 （Nat Commun. 2018 Feb 26;9(1):824.）。针对H7N9病人T细胞免疫为什么产生免疫疲劳，我们通过RNA array分析，找到了数个相关基因，发现与T细胞的脂类代谢相关，正在CRISP基因敲除鼠上分析是否与死亡组病人的免疫功能低下相关。同时，我们对免疫疲劳的CD8 T 细胞进行了单细胞转录组学测定，并发现几个潜在的可以调控细胞免疫疲劳的基因，相关研究目前在验证中。上述系统性研究，将人高致病性禽流感细胞免疫应答研究推到了一个新高度，也使本项目资助的研究在流感病毒临床免疫研究领域内处于世界最领先的地位。了解上述免疫应答因素在H7N9感染者疾病康复过程中的作用，不仅为H7N9感染，而且为其他流感病毒导致的严重感染，提供预后或者临床干预指标，从而对H7N9等引起的重症流感的临床治疗及感染防控具有重要意义。