Functional Role of the Tetraspanin CD82/Kai1 in Innate and Adaptive Immunity

描述（由申请人提供）：尽管抗菌素的发展和广泛使用，感染仍然占全球死亡人数的25%以上。病毒、细菌、寄生虫和真菌感染的一个共同主题是它们需要进入细胞以建立感染。包括HIV-1、大肠杆菌、恶性疟原虫和白色念珠菌在内的多种病原体都能操纵哺乳动物的四联蛋白进行细胞入侵或细胞内运输。顾名思义，四跨蛋白跨越脂质双分子层四次，被认为发挥结构作用，与其他蛋白质横向相互作用并形成膜微域。众所周知，由于分子冗余和缺乏内在催化活性，对四跨蛋白的精确功能进行表征是非常困难的——它们的功能与其他蛋白质作为膜蛋白的横向组织者的作用有关。增加复杂性的事实是，在不同细胞中表达的相同的四联蛋白通过与不同的伴侣相关联而提供独特的位置依赖功能。四联蛋白CD82在肿瘤表面表达水平与肿瘤转移呈负相关的情况下得到了最好的描述。在树突状细胞中，CD82与II类MHC和内吞途径的其他成分相关。为了更好地理解CD82在免疫系统中的作用，我们做了以下关键观察，这是我们提出工作的基本原理：1)蛋白质组学数据表明CD82可能与许多TLRs相关2)CpG DNA未能诱导tlr9依赖性；CD82-/- dc分泌TNF-，但其他TLR信号通路保持完整3)CD82被招募到含有真菌病原体的吞噬体，包括烟曲霉（Aspergillus fumigatus），这是通过使用旋转盘共聚焦显微镜对原发性dc中表达的CD82荧光标记版本的时移成像确定的4)CD82在酸化前被招募到含病原体的吞噬体，其招募不受溶酶体酸化抑制剂的影响。CD82的募集与II类MHC的到来同时发生，并且发生在CD63之前。生化证据表明II类MHC和CD82是相关的。6)携带卵清蛋白的CD82-/- dc不能像野生型dc一样刺激抗原特异性T细胞。我们假设CD82直接参与TLR9信号复合物的形成，并直接在dc表面组织装载肽的II类MHC。我们建议：1)确定CD82在dc中TLR9介导的信号传导中的作用；2)利用CD82-/-小鼠确定CD82在真菌病原体免疫应答中的作用；3)研究CD82在抗原加工和递呈中的作用。关于CD82在dc中的作用机制的知识将有助于进一步了解TLR9信号和抗原加工和呈递，并可能导致烟曲霉引起的侵袭性真菌感染的新治疗方法。

DESCRIPTION (provided by applicant): Infections still account for over 25% of deaths worldwide despite the development and widespread use of antimicrobials. A common theme for viral, bacterial, parasitic and fungal infections is their need to gain entry into cells to establish infection. Diverse pathogens including HIV-1, E. coli, Plasmodium falciparum and Candida albicans all manipulate mammalian tetraspanins for cell invasion or intracellular trafficking. As the name suggests, tetraspanins span lipid bilayers four times and are thought to play a structural role, interacting laterally with other proteins and forming membrane microdomains. Characterization of precise function of tetraspanins has been notoriously difficult as a result of molecular redundancy and the lack of intrinsic catalytic activity-- their function is linked to other proteins in their role as lateral organizers of membrane proteins. Adding to the complexity is the fact that the same tetraspanin expressed in different cells provide unique location-dependent functions by associating with distinct partners. The tetraspanin CD82 has been best described in the context of cancer where levels of surface expression are inversely correlated with tumor metastasis. In dendritic cells, CD82 associates with Class II MHC and other components of the endocytic pathway. In order to understand better the role of CD82 in the immune system, we have made the following key observations that are the rationale for our proposed work: 1) Proteomic data indicates that CD82 may be associated with a number of TLRs 2) CpG DNA failed to induce TLR9-dependent, TNF-α secretion from CD82-/- DCs but other TLR signaling pathways remained intact 3) CD82 is recruited to phagosomes containing fungal pathogens including Aspergillus fumigatus as determined by time-lapse imaging of a fluorescentlytagged version of CD82 expressed in primary DCs using a spinning-disk confocal microscope 4) CD82 is recruited to pathogen-containing phagosomes prior to acidification with its recruitment unaffected by inhibitors of lysosomal acidification. CD82 recruitment is coincident with the arrival of Class II MHC and occurs before CD63 5) Biochemical evidence indicates that class II MHC and CD82 are associated 6) CD82-/- DCs loaded with ovalbumin failed to stimulate antigen-specific T cells as well as their wild-type counterparts. We hypothesize that CD82 directly participates in forming the TLR9 signaling complex and directly organizes peptide-loaded class II MHC on the surface of DCs. We propose to: 1) Determine the role of CD82 in TLR9- mediated signaling in DCs 2) Define the role of CD82 in the immune response to fungal pathogens using CD82-/- mice 3) Investigate the contribution of CD82 to antigen processing and presentation. Knowledge gained regarding the mechanism of action of CD82 in DCs will be important in furthering our understanding of TLR9 signaling and of antigen processing and presentation, and could lead to novel treatments of invasive fungal infections caused by A. fumigatus.