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

描述(申请人提供)：尽管抗菌药的开发和广泛使用，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一个共同主题是它们需要进入细胞以确定感染。包括HIV-1、大肠杆菌、恶性疟原虫和白色念珠菌在内的各种病原体都能操纵哺乳动物的四肽类物质进行细胞入侵或细胞内转运。顾名思义，Tetraspanins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，形成膜微域。众所周知，由于分子冗余和缺乏内在催化活性，很难确定Tetraspanins的确切功能--它们的功能与其他蛋白质联系在一起，因为它们的作用是膜蛋白的侧向组织者。增加复杂性的事实是，在不同细胞中表达的相同的Tetraspanin通过与不同的伙伴结合来提供独特的位置依赖功能。Tetraspanin CD82在癌症的背景下得到了最好的描述，在癌症中，表面表达水平与肿瘤转移呈负相关。在树突状细胞中，CD82与第二类MHC和内吞途径的其他成分相关。为了更好地了解CD82在免疫系统中的作用，我们做了以下主要观察：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、肿瘤坏死因子-&#945；CD82被招募到含有真菌病原体的吞噬小体，包括烟曲霉，通过旋转圆盘共聚焦显微镜对原代DC表达的CD82荧光标记版本的时间推移成像确定。4)CD82在酸化之前被招募到含有病原体的吞噬小体中，它的招募不受溶酶体酸化抑制剂的影响。CD82的募集与II类MHC的到来是一致的，并且发生在CD63之前。5)生化证据表明，II类MHC和CD82是相关的。6)负载卵白蛋白的CD82-/-DC不能刺激抗原特异性T细胞和它们的野生型DC。我们推测CD82直接参与TLR9信号复合体的形成，并直接在树突状细胞表面组织载肽的II类MHC。我们建议：1)确定CD82在TLR9介导的DC信号转导中的作用2)利用CD82-/-小鼠确定CD82在对真菌病原体的免疫应答中的作用3)研究CD82在抗原处理和提呈中的作用。了解CD82在DC中的作用机制将有助于我们进一步了解TLR9信号和抗原的处理和提呈，并可能导致由烟曲霉菌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.