Identification and eliminationof HIV reservoirs in oral lymphoid tissues by engineered NK cells.

 描述（由申请人提供）：实现HIV的功能性治愈，允许停止抗逆转录病毒治疗（ART），将需要开发特定的策略来识别和耗尽受感染的细胞库。HIV-1在初次感染期间建立了潜伏感染的静息记忆CD 4 + T细胞库，这些细胞在ART治疗期间持续存在，并可在治疗中断后数周内恢复活跃的病毒复制。潜伏库在ART存在下非常稳定，半衰期为44个月，可在间歇性病毒血症期间或通过潜伏感染细胞的稳态增殖得到补充，并且不会因ART强化而显著减少。重要的是，活跃和潜伏的HIV储库的建立主要发生在身体的次级淋巴组织中，因此这些组织是HIV免疫治疗的中心战场。口腔中的粘膜免疫系统是广泛的和区室化的粘膜相关淋巴组织（MALT），其具有适于防御口腔病原体的专门的显微解剖结构和组成。虽然HIV不常通过口腔途径传播，但口腔MALT的丰富淋巴环境，特别是扁桃体，使这些组织对感染和HIV储库的建立高度敏感。根除患者中的HIV感染的努力无疑需要确定口腔中携带潜伏病毒的特定细胞群，并制定有效破坏这些细胞的策略。迄今为止，大多数对HIV体内潜伏期的研究都来自于对循环静止T细胞的分析，这些T细胞与支持感染个体中大部分病毒复制的淋巴组织显著不同，并作为潜伏创始者病毒群体的初始播种地。本申请的首要目标是确定口腔MALT中易受HI感染的细胞-因此是潜在的潜伏感染库-并探索一种新的自然杀伤（NK）细胞方法来消除潜伏感染的细胞。为了实现这一目标，我们建立了一个高度合作的HIV病毒学和潜伏期（Karn，McDonald和Tilton博士）和口腔粘膜免疫学（Pandayan博士）专家团队，并利用新颖而强大的流式细胞术和成像技术，结合使用离体培养的扁桃体组织的强大组织外植体模型。我们已经建立了创新的方案来模拟扁桃体组织中的潜伏感染，以及扩增和工程化自体NK细胞群以破坏扁桃体中的感染细胞的方法。在这项应用中，Tilton博士的团队将采用他的团队开发的新型流式细胞术检测方法，以确定口腔中感染和潜伏期的目标（目标1）。McDonald博士将利用他在活细胞荧光显微镜方面的专业知识，直接观察活淋巴组织内的感染事件（目标2）。 Karn博士是该团队的成员，他在使用染色质免疫沉淀（ChIP）和深度测序技术确定负责进入艾滋病毒潜伏期和从艾滋病毒潜伏期重新激活的信号机制方面发挥了核心作用。Karn小组将调整他们开发的方案，以扩增和遗传修饰能够特异性杀死重新激活的HIV感染细胞的活化NK细胞（目标3和4）。这项工作的成功完成将首次确定口腔中易受生产性和潜伏性HIV-1感染的细胞群体，建立这些组织中潜伏感染的生物学相关模型，并探索一种有前途的新策略，使用活化和扩增的自体或工程NK细胞根除感染细胞。我们相信，我们的系统方法，表征HIV感染的细胞，监测NK细胞杀伤离体和工程NK细胞与特异性嵌合抗原受体将提供前所未有的洞察口腔粘膜MALT细胞群体易受潜伏感染，并允许开发新的和有效的技术，以清除潜伏的HIV水库在口腔。

 DESCRIPTION (provided by applicant): Achieving a functional cure for HIV that allows cessation of anti-retroviral therapy (ART) will require development of specific strategies to recognize and deplete infected cell reservoirs. HIV-1 establishes a pool of latently infected, resting memory CD4+ T cells during primary infection that persist during ART treatment with and can resume active viral replication within weeks following treatment interruption. The latent reservoir is very stable with a half-life of 44 months in the presence of ART, may be replenished during intermittent viremia or by homeostatic proliferation of latently infected cells, and is not significantly diminished by ART intensification. Importantly, establishment of active and latent HIV reservoirs occurs primarily in the secondary lymphoid tissues of the body, and therefore these tissues are the central battleground for HIV immunotherapy. The mucosal immune system in the oral cavity is an extensive and compartmentalized mucosaassociated lymphoid tissue (MALT) with a specialized microanatomy and composition adapted for defense against oral pathogens. While HIV is uncommonly transmitted via the oral route, the rich lymphoid environment of the oral MALT, particularly the tonsils, makes these tissues highly susceptible to infection and establishment of HIV reservoirs. Efforts to eradicate HIV infection in patients will undoubtedly need to identify the specific populations of cells harboring latent virus in the oral cavity and devise strategies to effectively destroy these cells. To date, the majority of research into HIV latency in vivo has been derived from analysis of circulating quiescent T cells, which differ significantly from lymphoid tissues that support the majority of viral replication in infectd individuals and serve as the initial seeding ground for latent founder virus populations. The overarching goal of this application is to define cells in the oral MALT that are susceptible to HI infection-and therefore potential reservoirs of latent infection-and to explore a novel natural killer (NK) cell approach to eradicating latently infected cells. To achieve this goal, we have established a highly collaborative team of experts in HIV virology and latency (Drs. Karn, McDonald, and Tilton) and oral mucosal immunology (Dr. Pandayan) and leveraged novel and powerful flow cytometry and imaging technologies coupled to a robust tissue explant model using ex vivo cultured tonsillar tissues. We have established innovative protocols to model latent infection in tonsillar tissues as well as methods to expand and engineer autologous NK cell populations for destruction of infected cells in the tonsils. In this application, Dr. Tilton's grop will adapt novel flow cytometry assays developed by his group to identify targets of infection and latency in the oral cavity (Aim 1). Dr. McDonald will utilize his expertise in live cell fluorescene microscopy to directly visualize those infectious events within the living lymphoid tissue (Aim 2). Anchoring the team is Dr. Karn, who has played a central role in identifying the signaling machinery responsible for entry into and reactivation from HIV latency using chromatin immunoprecipitation (ChIP) and deep sequencing technologies. The Karn group will adapt the protocols they have developed to expand and genetically modify activated NK cells that are capable of specifically killing reactivated HIV infected cells (Aims 3 & 4). The successful completion of this work will for the first time identify cellular populations in the oral cavity susceptible to productive and latent HIV-1 infection, establish biologically relevant models of latent infection in these tissues, and explore a promising and novel strategy to eradicate infected cells using activated and expanded autologous or engineered NK cells. We believe our systematic approach of characterizing the HIV infected cells, monitoring NK cell killing ex vivo and engineering NK cells with specific chimeric antigen receptors will provide unprecedented insight into oral mucosal MALT cell populations susceptible to latent infection and permit development of new and effective technologies to clear latent HIV reservoirs in the oral cavity.