NK/DC Cross-Talk and Antiviral Response

描述（由申请方提供）：IFN-？/利巴韦林治疗HCV/HIV-1合并感染或HCV单一感染仍不清楚。本提案的目标是通过研究自然杀伤（NK）细胞和树突状细胞（DC）功能与适应性T细胞水平的关系，确定先天免疫效应器在HCV和HCV/HIV-1共感染受试者治疗反应中的作用。反应和治疗诱导的HCV抑制。我们将检验先天性效应细胞（即自然杀伤细胞和树突状细胞功能）对IFN-γ的持续功能反应。利巴韦林治疗是先天性和适应性（HCV特异性）应答水平以及最终早期和持续HCV病毒学抑制的决定因素。作为一个推论，我们假设，先天表型和细胞介导的反应与HCV控制将选择性地丰富与记录的SVS（独立于HIV-1感染）的受试者相比，健康或HIV-1感染的捐助者没有HCV感染。我们将通过两个目的对接受聚乙二醇-IFN-α/利巴韦林治疗的KIR/HLA-C型HCV感染和HCV/HIV共感染受试者测试该假设：（1）分析随访至SVR达到12周EVR的受试者的前瞻性纵向队列，与未能达到EVR但仍在治疗的受试者相比，收集以下数据：（a）NK和DC亚群分布水平、DC成熟状态和通过流式细胞术测定的与HCV病毒载量相关的总体细胞免疫活化;（B）PDC亚群内IRF-7增加的程度，作为IFN-α/STAT 1/IRF-7反馈环和保留的IFN-1功能的反映，如通过流式细胞术检测PBMC亚群中IFN-α诱导的STAT 1磷酸化所测量的;（c）NK组成性和IFN-α诱导的针对HLA无效或病毒感染的靶（包括HCV感染的肝细胞）的裂解功能;和（d）先天功能性和HCV T细胞记忆应答水平之间的关系，如通过IFN-？EVR和治疗完成时的ELISPOT和四聚体测定。第二个具体目的是分析与未感染对照相比，已经实现HCV抑制和SVR状态的先前单HCV或双重HCV/HIV感染受试者的横断面队列，测量（a）NK/DC亚群频率的流式细胞术分析;（B）NK细胞功能应答（细胞毒性和脱粒、STAT 1磷酸化、活化标志物的表达）对IFN-α、HLA耗尽的靶细胞和HCV感染的肝细胞的作用;（c）树突细胞对IFN-α和TLR配体（CpG，瑞喹莫特）的功能性应答（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免疫疾病治疗中心的假设驱动的合作努力（费城战斗），宾夕法尼亚大学传染病部，德雷克塞尔大学艾滋病诊所，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 Bioscience（San Diego CA）和University of Massachusetts-Amherst的生物统计学系。公共卫生相关性声明 公共卫生相关性：在HCV/HIV-1共感染或HCV单一感染的受试者中，IFN-α/利巴韦林治疗后HCV根除的机制仍不清楚。本提案的目标是通过研究与适应性T细胞应答水平和治疗诱导的HCV病毒抑制相关的自然杀伤（NK）细胞和树突细胞（DC）功能性，确定先天免疫效应物在HCV和HCV/HIV-1共感染受试者中IFN-α/利巴韦林治疗应答中的作用。由于对治疗的反应率从单一感染的约60%到HIV/HCV合并感染个体的<25%不等，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.