NK/DC Cross-Talk and Antiviral Response

描述(申请人提供)：在丙型肝炎病毒/HIV-1混合感染或单一丙型肝炎病毒感染中，干扰素/利巴韦林治疗后丙型肝炎病毒根除的机制尚不清楚。这项建议的目的是通过研究自然杀伤细胞(NK)和树突状细胞(DC)功能与适应性T细胞反应水平和治疗诱导的丙型肝炎病毒抑制水平的关系，确定先天免疫效应器在丙型肝炎病毒和丙型肝炎病毒/HIV-1混合感染者治疗反应中的作用。我们将验证这样一种假设，即天然效应细胞(即自然杀伤细胞和树突状细胞功能)对干扰素-β/利巴韦林治疗的持续功能反应是先天性和适应性(丙型肝炎病毒特异性)反应以及最终早期和持续的丙型肝炎病毒抑制的决定因素。作为推论，我们假设与丙型肝炎病毒控制相关的先天表型和细胞介导性反应在记录在案的SVS(独立于HIV-1感染)的受试者中将选择性地丰富，与健康或未感染丙型肝炎病毒的HIV-1感染的捐赠者相比。我们将在接受聚乙二醇化干扰素/利巴韦林治疗的KIR/HLAC型丙型肝炎病毒感染者和丙型肝炎病毒/艾滋病病毒混合感染者上通过两个目的来验证这一假设：(1)分析接受SVR随访后达到12wk EVR的受试者的前瞻性纵向队列，与未能达到EVR但仍在接受治疗的受试者进行比较，收集数据以收集以下数据：(A)通过流式细胞仪分析与丙型肝炎病毒载量相关的NK和DC亚群分布、DC成熟状态和总体细胞免疫激活水平；(B)PDC亚群中IRF-7的升高程度，反映了干扰素-a/STAT1/IRF-7反馈环和保留的IFNR-I功能，通过流式细胞仪检测干扰素-a诱导的PBMC亚群中STAT1的磷酸化来衡量；(C)NK构成和干扰素-a诱导的针对HLA阴性或病毒感染靶细胞的溶解功能，包括感染丙型肝炎病毒的肝细胞；以及(D)天然功能与丙型肝炎病毒T细胞记忆反应水平之间的关系，如用干扰素-？？在EVR和治疗结束时检测ELISPOT和四聚体。第二个特定目标将分析与未感染对照相比，既往单一丙型肝炎病毒感染者或双重丙型肝炎病毒感染者的横断面队列，与未感染对照相比，获得了丙型肝炎病毒抑制和SVR状态，测量(A)流式细胞术分析NK/DC亚群频率；(B)NK细胞功能反应(细胞毒性和脱颗粒，STAT1磷酸化，激活标记物的表达)；(C)树突状细胞对干扰素a和TLR配体(CpG，Resiquimod)的功能反应(细胞因子产生，IRF-7表达)。这项基础和临床研究代表了Wistar研究所、Jonathan Lax免疫疾病治疗中心(Philadelphia Fight)、宾夕法尼亚大学传染病分部、Drexel大学艾滋病诊所、国家癌症研究所基因组多样性实验室和实验免疫学实验室(Frederick，MD)、BD生物科学(圣地亚哥CA)和马萨诸塞大学阿默斯特分校生物统计系在假设驱动下的合作努力。公共卫生相关声明 公共卫生相关性：在丙型肝炎病毒/艾滋病毒-1混合感染者或单一丙型肝炎病毒感染者中，干扰素/利巴韦林治疗后根除丙型肝炎病毒的机制尚不清楚。这项建议的目的是通过研究自然杀伤细胞(NK)和树突状细胞(DC)功能与适应性T细胞反应水平和治疗诱导的丙型肝炎病毒抑制水平的关系，确定先天免疫效应器在丙型肝炎病毒和丙型肝炎病毒/HIV-1混合感染者的干扰素-a/利巴韦林治疗反应中的作用。由于对治疗的应答率在单一感染者中约为60%，在艾滋病毒/丙型肝炎病毒混合感染者中为25%，因此必须进行更多的研究，以了解清除机制。

DESCRIPTION (provided by applicant): The mechanisms of HCV eradication following IFN-?/ribavirin therapy in HCV/HIV-1 co-infection or HCV mono-infection remain unclear. The goal of this proposal is to determine the role of innate immunity effectors in therapy response in HCV and HCV/HIV-1 co-infected subjects by investigating Natural Killer (NK) cell and Dendritic Cell (DC) functionality in relation to level of adaptive T cell responses and therapy-induced HCV suppression. We will test the hypothesis that the sustained functional response of innate effector cells (i.e. Natural Killer cell and Dendritic cell function) to IFN-??/ribavirin therapy is a determinant of both innate and level of adaptive (HCV-specific) responses and ultimately early and sustained HCV virologic suppression. As a corollary, we hypothesize that innate phenotypes and cell-mediated responses associated with HCV control would be selectively enriched in subjects with documented SVS (independent of HIV-1 infection) as compared to healthy or HIV-1-infected donors without HCV infection. We will test this hypothesis by two aims on KIR/HLA-C typed HCV-infected and HCV/HIV-co-infected subjects undergoing treatment with peg-IFN- a/ribavirin by: (1) Analyzing a prospective longitudinal cohort of subjects reaching 12 wk EVR with follow-up to SVR, as compared to subjects failing to achieve EVR but remaining on therapy, with data collection for: (a) Levels of NK and DC subset distribution, DC maturation status and overall cellular immune activation by flow cytometry in relation to HCV viral load; (b) Degree of IRF-7 increase within PDC subsets as a reflection of IFN-a/STAT1/IRF-7 feedback loop and retained IFNR-I function, as measured by flow cytometry detection of IFN-a-induced STAT1 phosphorylation in PBMC subsets; (c) NK constitutive and IFN-a-induced lytic function against HLA-null or viral infected targets, including HCV-infected hepatocytes; and (d) Relation between innate functionality and levels of HCV T cell memory responses, as measured by IFN-? ELISPOT and tetramer assay at time of EVR and therapy completion. The second specific aim will analyze a cross-sectional cohort of previous mono-HCV or dual HCV/HIV-infected subjects having achieved HCV suppression and SVR status, as compared to uninfected controls, measuring (a) Flow-cytometric analysis of NK/DC subset frequency; (b) NK cell functional response (cytotoxicity and degranulation, STAT1 phosphorylation, expression of activation markers) to IFN-a, HLA-depleted target cells and HCV-infected hepatocyte; (c) Dendritic cell functional response (cytokine production, IRF-7 expression) to IFN-a and TLR ligands (CpG, Resiquimod). This basic and clinical research study represents a hypothesis-driven collaborative effort by the Wistar Institute, The Jonathan Lax Center for the Treatment of Immune Disorders (Philadelphia FIGHT), The Infectious Disease Division for the University of Pennsylvania, The AIDS clinic of Drexel University, The National Cancer Institute's Laboratory of Genomic Diversity and Laboratory of Experimental Immunology (Frederick, MD), BD Bioscience (San Diego CA), and the Department of Biostatistics of the University of Massachusetts-Amherst. Public Health Relevance Statement PUBLIC HEALTH RELEVANCE: The mechanisms of HCV eradication following IFN-a/ribavirin therapy in HCV/HIV-1 co-infected or HCV mono-infected subjects remain unclear. The goal of this proposal is to determine the role of innate immunity effectors in IFN-a/ribavirin therapy response in HCV and HCV/HIV-1 co- infected subjects by investigating Natural Killer (NK) cell and Dendritic Cell (DC) functionality in relation to level of adaptive T cell responses and therapy-induced HCV viral suppression. As the rate of response to therapy varies from about 60% in mono-infection to <25% in HIV/HCV co-infected individuals, it is imperative that added research be conducted in understanding clearance mechanisms.