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

描述（由申请人提供）：尽管开发和广泛使用抗菌药物，但感染仍占全球死亡人数的25%以上。病毒、细菌、寄生虫和真菌感染的共同主题是它们需要进入细胞以建立感染。多种病原体包括HIV-1、E.大肠杆菌、恶性疟原虫和白色念珠菌都操纵哺乳动物四跨膜蛋白进行细胞侵入或细胞内运输。顾名思义，四跨膜蛋白跨越脂质双层四次，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区。由于分子冗余和缺乏内在催化活性，四跨膜蛋白的精确功能的表征是非常困难的-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。增加复杂性的是，在不同细胞中表达的相同的四跨膜蛋白通过与不同的伴侣相关联而提供独特的位置依赖性功能。四跨膜蛋白CD 82在癌症的背景下得到了最好的描述，其中表面表达水平与肿瘤转移呈负相关。在树突状细胞中，CD 82与II类MHC和内吞途径的其他组分相关。为了更好地了解CD 82在免疫系统中的作用，我们进行了以下关键观察，这些观察是我们提出的工作的基本原理：1）蛋白质组学数据表明CD 82可能与许多TLR相关2）CpG DNA未能诱导TLR 9依赖性CD 82-/-DC分泌TNF，但其他TLR信号通路保持完整3α）CD 82被募集到含有真菌病原体（包括烟曲霉）的吞噬体中，如通过使用转盘共聚焦显微镜对在原代DC中表达的荧光标记形式的CD 82进行延时成像所确定的。4）在酸化之前，CD 82被募集到含有病原体的吞噬体中，其募集不受溶酶体酸化抑制剂的影响。5）生物化学证据表明II类MHC和CD 82是相关的6）负载有卵清蛋白的CD 82-/-DC不能刺激抗原特异性T细胞以及它们的野生型对应物。 我们假设CD 82直接参与形成TLR 9信号复合物，并直接在DC表面组织负载肽的II类MHC。我们建议：1）确定CD 82在DC中TLR 9介导的信号传导中的作用2）使用CD 82-/-小鼠确定CD 82在对真菌病原体的免疫应答中的作用3）研究CD 82对抗原加工和呈递的贡献。关于CD 82在DC中作用机制的知识对于进一步理解TLR 9信号传导和抗原加工和呈递将是重要的，并且可能导致由A.烟熏。

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.