HPV AND ANTIGEN PRESENTING CELLS

项目摘要 人乳头瘤病毒(HPV)是常见的性传播病毒，其感染是一种重要的 人口的健康负担。持续感染高危HPV基因导致 一种人类癌症，最突出的是宫颈癌、肛门癌和头颈癌的亚型。人类乳头瘤病毒 通过逃避免疫系统在宿主内建立持续感染。HPV的一种方法是 这是通过操纵朗格汉斯细胞(LC)来实现的，朗格汉斯细胞是该部位的抗原提呈细胞 负责启动对上皮细胞入侵病毒的免疫反应的感染。这个 HPV通过膜联蛋白A2异源四聚体(A2t)与LC相互作用及其抑制信号转导 事件充当刹车机制，停止LC激活，导致LC不完全成熟和 预防有效的抗病毒T细胞免疫反应。我们的长期目标是了解HPV是如何 操纵LC激活和抗原处理以逃避免疫系统并测试新的治疗方法 人乳头瘤病毒相关疾病。目标1将重点放在使用“刹车，油门踏板”的效果上。 联合方法激活暴露于HPV的LC。这将通过细胞表面标记进行调查 细胞因子分析和功能免疫学检测。目标2将研究信号通路和 与A2T介导的LC免疫功能抑制相关的A2T相互作用伙伴被 使用生化和细胞生物学方法的人乳头瘤病毒。这将通过广泛的询问来分析 磷蛋白激活和细胞信号网络，抗原提呈细胞特异性图谱基因阵列， 和一种新开发的单分子下拉试验(SiMPull)。目标3将评估A2T的贡献 目的：探讨LC Birbeck颗粒的结构、功能及其在HPV抗原加工中的作用。这将通过以下途径进行调查 LC中Birbeck颗粒成分的免疫电子显微镜分析及A2t蛋白的鉴定 基因敲除对Birbeck颗粒形成的影响，以及蛋白质基因敲除对细胞周期的影响 LC能够正确处理HPV衍生的多肽并将其呈递给人类白细胞抗原相合的T细胞克隆。另外， 这些目标将利用创新的皮肤外植体和3D培养系统来验证所提出的HPV16-A2t 在活体内，相互作用对LC生物学很重要。总的来说，拟议的研究将提供对 基本的LC生物学和功能。此外，我们将研究一种新的双管齐下的方法的效果 在LC中预防负性信号事件的同时提供正向刺激激活LC 作为一种治疗HPV感染的新策略，从而预防HPV相关癌症。

Project Summary Human papillomaviruses (HPV) are common sexually transmitted viruses and their infections are a significant health burden for the population. Persistent infection with high-risk HPV genotypes result in the development of a variety of human cancers, most prominently cervical, anal, and subtypes of head and neck cancers. HPV establishes persistent infection within the host through evasion of the immune system. One of the ways HPV accomplishes this is by manipulating Langerhans cells (LC), which are the antigen presenting cells at the site of infection that are responsible for initiating immune responses against epithelium invading viruses. The interaction of HPV with LC through the annexin A2 heterotetramer (A2t) and subsequent suppressive signaling events act as a braking mechanism that stops LC activation, resulting in incomplete LC maturation and prevention of an effective antiviral T cell immune response. Our long-term goals are to understand how HPV manipulates LC activation and antigen processing to evade the immune system and test new ways to treat HPV-associated diseases. AIM 1 will focus on the effects of utilizing a “brakes off, gas pedal down” combination approach for activating LC exposed to HPV. This will be investigated through cell surface marker and cytokine analysis and functional immunological assays. AIM 2 will investigate the signaling pathways and A2t interaction partners associated with A2t-mediated suppression of LC immune function that are hijacked by HPV using biochemical and cell biological approaches. This will be analyzed through broad interrogation of phosphoprotein activation and cell signaling networks, antigen presenting cell specific profiling gene arrays, and a newly developed Single Molecule Pulldown (SiMPull) assay. AIM 3 will evaluate the contribution of A2t to LC Birbeck granule structure, function, and role in HPV antigen processing. This will be investigated through immuno-electron microscopic analysis of Birbeck granule components in LC, assessment of A2t protein knockdown effects on Birbeck granule formation, and elucidation of the effects of protein knockdown on the ability of LC to properly process and present HPV-derived peptides to HLA-matched T cell clones. Additionally, these aims will utilize innovative skin explant and 3-D culture systems to validate that the proposed HPV16-A2t interactions are important for LC biology in vivo. Collectively the proposed studies will provide deep insight into basic LC biology and function. In addition we will examine the effects of a novel two-pronged approach focused on preventing negative signaling events in LC while simultaneously providing a positive stimulus to activate LC as a novel strategy to treat HPV infections, thereby preventing HPV-associated cancers.