【作者】 邹丽云；

【导师】 吴玉章；

【作者基本信息】 第三军医大学 ， 免疫学， 2008， 博士

【摘要】 研究表明:抗原呈递细胞(antigen presentation cell,APC)在呈递和处理外源性抗原和内源性抗原时存在不同的途径即MHC-Ⅰ类分子(Major Histocompatibility Complex class ?)途径和MHC-II类分子(Major Histocompatibility Complex classИ)途径。经典的MHC-I类途径认为内源性合成蛋白在胞浆降解后,转运到内质网与MHC-I类分子结合形成复合物后转运的细胞膜上,活化CD8+ T细胞;而外源性抗原被APC细胞内化后,与MHC-II类分子结合形成MHC-II类分子肽复合物,转运到细胞膜上,活化CD4+ T细胞。然而这两条途径并非完全的分离,事实上,外源性抗原同样也可以进入MHC-Ⅰ类分子处理途径,这种现象被称为交叉呈递(cross presentation)。最初Bevan等人在1976年发现抗移植物的免疫应答过程中存在此抗原递呈途径,到90年代Sigal等人在Science报道:在生理情况下,此抗原途径在机体抗病毒免疫应答中起着重要作用。随后越来越多的研究显示,抗原交叉递呈特异性启动细胞毒T细胞应答,构成防御病毒、肿瘤和胞外病原微生物的重要一环,而且此抗原递呈途径参与维持机体免疫耐受的形成,与I型糖尿病、哮喘等自身免疫性疾病的发病机制有关。因此进一步研究抗原交叉递呈的机制将有助于自身免疫性疾病治疗策略的发展以及新型疫苗的制备。树突状细胞(dendritic cell,DC)是机体进行外源抗原交叉递呈的主要抗原递呈细胞。外源抗原经DC内化形成吞噬体,经过一系列胞内囊泡融合、转运,最终将形成的MHC-I类肽复合物转运到细胞膜上才完成整个抗原递呈过程。近年来为了解此递呈途径的胞内机制开展了大量研究,但其相关机制尚不明确。Rodriguez A在Nature报道吞噬可溶性抗原转运到胞浆,进入MHC-I类分子递呈途径,却一直未能鉴定外源抗原从内吞囊泡到胞浆的转运机制。Houde M和Guermonprez P在Nature提出内质网与吞噬体融合模型来解释抗原交叉递呈的途径,而在2005年Touret N在Nature发表实验数据对此模型提出异议。因此,需进一步开展树突状细胞抗原递呈相关的胞内机制研究,有助于我们深入了解此抗原递呈途径及其在免疫系统的功能。在真核细胞中,膜转运和融合主要由Rab蛋白和SNARE(soluble NSF attachment protein receptor,SNARE)蛋白控制。Rab蛋白是一类属于Ras蛋白家族的小GTP酶,其家族的不同成员高度局限性地定位在各种囊泡的膜表面。通过GDP结合的无活性形式和GTP结合的活性形式之间循环,Rab蛋白在胞浆和膜质结构之间进行转位,进而在时间和空间上调控囊泡的运输。另一方面Rab蛋白通过脂酰链锚着于胞内各种膜结构,包括质膜的胞浆面,通过与大量的下游效应分子相互作用,并以严格受调节的方式与细胞骨架蛋白相联系,实现胞内膜结构(及其负载的蛋白)的定向转运并介导转运膜结构与靶向囊泡之间的锚着和膜融合过程,在细胞内膜性结构移动中执行着中心调动功能。鉴于吞噬体的蛋白质组研究显示多种Rab蛋白招募到内吞囊泡,参与吞噬体的形成和转运。因此,我们拟通过研究Rab蛋白对树突状细胞抗原交叉递呈的影响,进而探讨树突状细胞抗原递呈中胞内转运机制和调控机制。近年大规模siRNA干扰技术的进展,为系统性研究与树突状细胞抗原交叉递呈相关的Rab蛋白提供了可能。首先,我们采用基于慢病毒的siRNA库筛选到12个与抗原交叉递呈相关的Rab分子,包括:Rab3b,3c,4a,5b,6,8b,10,27a,32,33a,34,35。进而,我们合成针对12个Rab分子siRNA进一步验证了筛选结果,避免siRNA库筛选中的“off-target”效应。其次,构建EYFP-Rab融合蛋白,采用激光共聚焦观察阳性Rab分子的分布以及吞噬细菌抗原后招募到吞噬体的情况,发现一些Rab分子如Rab8b,10,27a,33a,34,35以不同的动力学招募到吞噬体,而其他一些与外吐相关的Rab分子如Rab3b,3c,32与吞噬体不共聚,但位于吞噬体的周围,并且部分形成管状结构与细胞膜融合,提示该结构可能参与吞噬体内物质往细胞膜转运;分析Rab3b,3c,32阳性外吐囊泡的性质,发现该囊泡为LAMP+ Tfn+的非酸性囊泡,提示该囊泡为溶酶体性质的回运囊泡;进一步分析Rab3b,3c囊泡与抗原交叉递呈中的重要装配装置MHC-I类分子的共聚情况,发现MHC-I类分子聚集在Rab3b,3c阳性囊泡中;采用β2微球蛋白标识回运的MHC-I类分子,发现储存在Rab3b,3c阳性囊泡中的MHC-I类分子为回运性质的MHC-I类分子;而干扰Rab32的DC2.4细胞吞噬细菌抗原后,Rab3b,3c与细胞膜回运MHC-I类分子的共聚明显减少,提示干扰Rab32阻断了MHC-I类向Rab3b,3c囊泡的转运。由于吞噬体由部分细胞膜形成,细胞膜上的MHC-I类分子可能与吞噬颗粒一起形成吞噬体结构,因此该结果提示干扰Rab32阻断了吞噬体内MHC-I类分子(很有可能是MHC-I类分子抗原肽复合物)向Rab3b,3c囊泡的转运,进而向细胞膜上转运,从而影响抗原交叉递呈(见示意图);鉴于此,我们采用慢病毒介导的转基因制备了树突状细胞特异性的Rab32基因沉默转基因小鼠(简称CD11c-GFP-Rab32mir),通过定量PCR检测CD11c-GFP-Rab32mir转基因小鼠骨髓来源树突状细胞中Rab32表达,发现Rab32转基因鼠中树突状细胞Rab32表达明显低于对照组,提示转基因小鼠的制备成功,进一步采用该细胞进行抗原递呈分析,结果CD11c-GFP-Rab32mir转基因小鼠来源树突状细胞对细菌抗原交叉递呈能力显著低于对照组,但是MHC-II类分子限制性的抗原递呈没有影响,提示Rab32特异性的调控树突状细胞的交叉递呈。由于树突状细胞功能缺失导致T细胞功能异常,为此我们还分析了CD11c-GFP-Rab32mir转基因小鼠T细胞功能状态,结果发现T细胞的活化、记忆均没有明显变化,CD4+ T细胞,CD8+T细胞的比例以及调节性T细胞的比例也正常。说明CD11c-GFP-Rab32mir转基因小鼠T细胞功能状态比较正常,但是在负载抗原后,其T细胞状态和亚群是否有改变需要进一步的实验来证实。总之,本研究通过RNA干扰筛选获得一些与树突状细胞抗原交叉递呈相关的Rab分子,进一步分析发现树突状细胞中存在回运性质的Rab3b,3c,32阳性溶酶体相关细胞器参与抗原交叉递呈,并且干扰Rab32阻断了吞噬体内回运MHC-I类向Rab3b,3c囊泡的转运,进而影响抗原交叉递呈。提示回运相关的囊泡在联系吞噬体和细胞膜之间的物质转运中发挥作用,该结果为进一步探讨树突状细胞抗原交叉递呈胞内机制的理论模型提供依据,为基于树突状细胞抗原递呈的治疗策略奠定基础。更多还原

【Abstract】 It is supposed that exogenous and endogenous antigens are presented in two different pathways classified as MHC class I pathway and MHC class II pathway. Classical MHC class I pathway indicates that intracellular synthesized antigens, for example of viral or tumor origin, are presented by MHC I molecules and activate CD8+ cytotoxic T cells. Classical MHC class II pathway indicates extracellular antigens are presented by professional antigen-presenting cells (APCs) with MHC II molecules to CD4+ T helper cells. However, two pathways are not completely separated. In fact, exogenous antigens can also be presented in MHC class I restricted pathway. This process now called cross presentation, is first discovered by Bevan`s research in CD8+ T cell response to an exogenous cellular antigen (minor histocompatibility antigens from transplanted cells) in 1976. In 1992, Sigal group reported that this antigen presentation pathway play an important role in antiviral immune response at physiological situation. More and More evidences demonstrate that cross presentation is an important route for priming CD8+ T cells response and effectively clearing tumor, virus-infected cells and intracellular microorganism. On the other hand, this antigen presentation pathway is involved in maintaining immune tolerance in vivo and participate the process of autoimmune diseases such as type I diabetes and asthma. Further investigations on the mechanisms of cross presentation may provide opportunities to develop new immunotherapy strategies for autoimmune disease and vaccine.Dendritic cells are major antigen cross presentation cells in vivo as dendritic cell deleting transgenic mouse lack the ability to response to virus and cellular antigens. Cross presentation of dendritic cells is a complex process which includes antigens uptake, phagosome formation and finally MHC I-peptide complexes translocation onto plasma membrane. The mechanisms of this intracellular transport process were explored by many researches. Rodriguez A reported that soluble antigens are transport to cytosol and enter the classic MHC I restricted pathway, while the mechanisms of exogenous antigen transport from phagosome to cytosol remains uncovered. Houde M. and Guermonprez P. proposed that ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. However this model recently has been refuted by the Tourset`s data. It is a reasonable consideration that detailed understandings of antigen cross presentation would benefit from a comprehensive analysis of membrane transport related proteins in participating in the process.In eukaryotes, membrane trafficking and fusion is under control of Rab GTPase and SNAREs. Rabs are small G proteins that are characterized by their specific localization on intracellular organelles. In the extensive studies on Rab functions, it have been found that specific Rab GTPases transposition between membrane and cytosol through its GDP/GTP binding switch and regulate the vesicles transport in time and spatial fashion. On the other hand, Rab protein anchor at membrane and recruit different effectors and mediate intracellular membrane trafficking and organelle-targeted membrane fusion. Recently, phagosome proteome revealed that some Rabs are recruiting to phagosome and participating phagosome formation.In this study, using ovalbumin expressing bacteria as a particle antigen, we screened an RNAi library against 57 mouse Rab GTPases, and identified twelve Rab proteins that associated with cross-presentation in dendritic cells. Further studies with fluorescent protein tagged Rabs and Rab27a found Rab3b, 3c, 32 positive compartments extend from the juxtaposition of phagosome to the plasma membrane, suggesting that that these vesicles participating the transport from phagosome to plasma. Analysis the characteristics of Rab3b, 3c, 32 compartments revealed that these vesicles are LAMP+, Tfn+. These data suggest that a previously unrecognized lysosome related organelles (LRO) participate in exogenous antigen cross-presentation. To explore the function of these LROs, the co-localization of fluorescent protein tagged MHC class I molecules with Rab3b, 3c was analyzed by confocal microscope and found that a fraction of MHC class I molecules were stored at the Rabs (3b, 3c) positive compartment, which was resistant to BFA treatment and labeled as recycled MHC class I molecules byβ2 microglobulin. However, the concentration of MHC class I in Rab3b, 3c LROs was inhibited by silencing Rab32. The results suggested that silencing Rab32 block the transport of recycled MHC class I to Rab3b,3c LROs, which in return, affect antigen cross presentation. To investigate the function of Rab32 in vivo, dendritic cell specific Rab32 silencing transgenic mouse was established by using lentiviral vector expressing shRNA under CD11c promoter. The 50% decreased expression of Rab32 in dendritc cells derived from Rab32 silencing transgenic mouse was analyzed by realtime PCR. The efficency of bacteria antigen cross presentation by dendritic cells derived from Rab32 silencing transgenic mouse was also analyzed.A threefold decrease in the effiency of bacteria antigen cross presentation by Rab32 silencing dendritic cells was obtained, whereas the MHC class II restricted antigen presentation was unaffected. These data suggested that Rab32 expression in dendritic cells is critical for the efficient cross presentation of at least bacteria antigens, but not required for MHC class II restricted antigen presentation. Some reported that impaired function of dendritic cells may induce the abnormal phenotype of T cells. Then the phenotype of T cells was analyzed in Rab32 silencing transgenic mouse. Both T cell subsets and function was normal. But whether the function of T cells remains unaffected after antigen challenged need further investigation.更多还原