人γδT细胞对A型流感病毒血凝素蛋白及假病毒的免疫应答研究

发布时间：2018-04-23 12:05

本文选题：γδT细胞 + HA蛋白　；参考：《北京协和医学院》2011年博士论文

【摘要】：血凝素蛋白(hemagglutinin,HA)是流感病毒表面最主要也是最重要的蛋白,负责与宿主受体结合,主要引起体液免疫应答,也能引起固有免疫应答和细胞免疫应答。γδT细胞是一种表面表达TCRγδ分子的T细胞亚群,能被病毒分子快速活化,释放细胞因子,杀伤感染细胞,而产生抗感染的功能。但γδT细胞对流感病毒的免疫应答人们所知甚少,对γδT细胞与流感病毒最重要的成分HA蛋白相互作用的研究更是空白。因此,我们拟通过研究HA蛋白与γδT细胞的相互作用及机制,来加深人们对流感病毒HA引起的免疫反应的了解,为了更好的预防和治疗流感感染提供新的策略。这一研究,也为进一步探寻γδT细胞识别的配体的特异性和多样性、了解γδT细胞的生物学功能尤其是抗病毒感染方面的功能奠定基础。在本研究中,我们主要关注以下四个问题。第一,流感病毒入侵人体时,其表面的HA蛋白能否刺激外周血中的γδT细胞产生免疫应答? 第二,如果能产生免疫应答,那么免疫应答的机制是怎样的?HA蛋白能否被γδT细胞直接识别,受体分子是什么? 第三,如果HA蛋白不能被γδT细胞直接识别,是否还有其他的作用机制介导了γδT细胞对HA蛋白的免疫应答作用? 第四,有报道,活化的γδT细胞能杀伤病毒感染的宿主细胞,那么这种杀伤是由什么分子经由什么通路介导的? 针对上述四个科学问题,我们进行了以下研究,得到了下述结果。 (1)利用昆虫真核表达系统表达多株重组A型流感病毒HA蛋白,并包装了外被上述HA蛋白的假病毒颗粒。 (2)利用重组表达的流感HA蛋白刺激人外周血单个核细胞(PBMC),检测发现,能促进其中的γδT细胞表达早期活化分子CD69等,也能促进其活化性细胞因子IFN-γ等表达。同样在假病毒水平,也取得与此一致的结果。 (3)利用ELISA、流式细胞术、激光共聚焦扫描显微镜(Confocal)等多种方法我们发现HA蛋白能与γδT细胞结合。而短肽、蛋白、细胞水平的多重检验证明了γδT细胞上与HA结合的特异性受体不是TCRγδ。抗体封闭实验同样排除了NKG2D、TLRs、NCR等可能的特异性受体分子的结合作用。最终发现γδT细胞上结合HA蛋白的受体是非特异性的α-2,3和α-2,6唾液酸糖苷受体分子。 (4)进一步检测分析发现,HA蛋白和假病毒不能直接活化单一的γδT细胞,需要其他免疫细胞的辅佐作用,这种辅佐作用可能是通过HA蛋白刺激PBMC中其他细胞分泌产生的可溶性分子如细胞因子等进一步诱导了γδT细胞产生免疫应答。 (5)利用假病毒感染巨噬细胞,发现活化的γδT细胞对其有杀伤活性,且这种杀伤作用可能是通过NKG2D分子识别感染的巨噬细胞表面表达上调的应激分子ULBP等介导的。综上,A型流感病毒血凝素蛋白能刺激外周血中的γδT细胞活化,分泌细胞因子。但是这种活化是一种非直接的的相互作用,需要其他免疫细胞的辅佐,可能是由其他细胞产生的可溶性分子如细胞因子引起的。活化的γδT细胞能通过NKG2D分子识别应激分子对假病毒感染的靶细胞产生杀伤作用。上述结果证明,在流感病毒感染机体时,γδT细胞快速参与了机体的抗感染免疫过程。HA蛋白是这一过程的重要一环,它刺激了γδT细胞快速活化,产生免疫应答。本研究还初步阐明了在外周血中HA蛋白活化γδT细胞的作用机制和在感染部位γδT细胞发挥杀伤作用的作用机制。对γδT细胞抗病毒感染免疫的研究做了补充,也启示我们结合γδT细胞对HA蛋白的快速免疫应答开发抗流感病毒尤其是高致病性禽流感病毒治疗和预防的新手段。
[Abstract]:Hemagglutinin (HA) is the most important and most important protein on the surface of the influenza virus. It is responsible for combining with the host receptor, mainly causing the humoral immune response, and also causing the inherent immune response and cellular immune response. The gamma delta T cell is a T cell subgroup of the surface expressing TCR gamma delta, which can be quickly activated by the virus and release. Cytokines, which kill infected cells, produce anti infection functions. But the immune response of T cells to influenza viruses is poorly understood. The study of the interaction between the T cells and the most important component of the influenza virus, HA protein, is more blank. Therefore, we intend to deepen people by studying the interaction and mechanism of HA egg white and gamma delta T cells. Our understanding of the immune response to influenza virus HA provides a new strategy for the prevention and treatment of influenza infection. This study also provides a basis for further exploration of the specificity and diversity of the ligand for the recognition of gamma delta T cells and the understanding of the biological functions of the gamma delta T cells, especially the function of the disease resistant infection.
In this study, we focus on the following four questions.
First, can influenza virus invade the human body, can its surface HA protein stimulate the gamma delta T cells in peripheral blood to produce immune response?
Second, if an immune response is generated, then what is the mechanism of the immune response? Can HA protein be directly recognized by the gamma delta T cell and what is the receptor molecule?
Third, if HA protein can not be directly identified by gamma delta T cells, is there any other mechanism that mediates the immune response of T cells to HA protein?
Fourth, it has been reported that activated gamma delta T cells can kill host cells infected by virus. What pathway is the molecular pathway mediated by this killing molecule?
In view of the above four scientific problems, we carried out the following research and got the following results.
(1) using the insect eukaryotic expression system to express multiple recombinant influenza A virus HA protein and package the fake virus particles which are coated with the above HA protein.
(2) the recombinant expression of influenza HA protein was used to stimulate human peripheral blood mononuclear cells (PBMC), and it was found that it could promote the expression of the early activation molecule CD69 in the T cells, and also promote the expression of the activated cytokine IFN- gamma. The same results were obtained at the level of the pseudo virus.
(3) we found that using ELISA, flow cytometry, laser confocal scanning microscope (Confocal) and other methods, we found that HA protein can combine with gamma delta T cells. And the multiple tests of short peptide, protein, and cell level prove that the specific receptor on the T cell with HA is not TCR - Delta. The antibody blocking experiment also excludes the possible NKG2D, TLRs, NCR and so on. The binding of specific receptor molecules revealed that the receptor binding to HA protein on gamma delta T cells was nonspecific alpha -2,3 and alpha -2,6 sialoglycoside receptor molecules.
(4) further detection and analysis found that HA protein and pseudo virus can not directly activate a single gamma delta T cell, which requires the adjuvant effect of other immune cells, which may be further induced by HA protein to stimulate the other cells secreted by other cells in PBMC, such as cytokines, such as cytokine, to induce the immune response of the gamma delta T cells.
(5) using a pseudo virus to infect macrophages, it is found that the activated gamma delta T cells have a killing activity, which may be mediated by the NKG2D molecule to identify the up-regulated stress molecule ULBP on the surface of infected macrophages.
To sum up, the A influenza virus hemagglutinin protein can stimulate the activation of the delta T cells in the peripheral blood and secrete cytokine. But this activation is a non direct interaction that requires the adjuvant of other immune cells and may be caused by the soluble molecules produced by other cells, such as cytokine. The activated gamma delta T cells can pass through the NKG2D fraction. Sub identified stress molecules produce lethal effects on target cells infected by false virus.
These results show that when the influenza virus infected the body, the.HA protein of the gamma delta T cells is an important part of this process. It stimulates the rapid activation of the gamma delta T cells and produces the immune response. This study also preliminarily clarified the mechanism of the activation of the HA protein in the peripheral blood and the mechanism of the activation of the T cells in the peripheral blood and the infection part. The mechanism of the killing effect of the bits of gamma delta T cells, supplementing the study of the immune response to the anti-virus infection of the gamma delta T cells, also enlightens us that we combine the rapid immune response of the T cells to the HA protein to develop a new method for the treatment and prevention of the influenza virus, especially the highly pathogenic avian influenza virus.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

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