NK/DC Cross-Talk and Antiviral Response

描述（由申请人提供）：IFN-？/利巴韦林治疗HCV/HIV-1合并感染或HCV单一感染尚不清楚。本提案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HCV抑制的关系，确定先天免疫效应物在HCV和HCV/HIV-1共感染受试者的治疗反应中的作用。我们将测试先天效应细胞（即自然杀伤细胞和树突状细胞功能）对IFN-?？/利巴韦林治疗是先天和适应性（HCV特异性）反应水平以及最终早期和持续HCV病毒学抑制的决定因素。作为推论，我们假设与没有HCV感染的健康或HIV-1感染的供者相比，与HCV控制相关的先天表型和细胞介导反应在有记录的SVS受试者（独立于HIV-1感染）中选择性地富集。我们将通过两个目标对接受peg-IFN- a/利巴韦林治疗的KIR/HLA-C型HCV感染和HCV/ hiv合并感染的受试者进行验证这一假设：(1)对EVR达到12周并随访SVR的受试者进行前瞻性纵向队列分析，与未达到EVR但仍在治疗的受试者进行比较，数据收集如下：(a) NK和DC亚群分布、DC成熟状态和整体细胞免疫激活水平与HCV病毒载量的关系；(b)通过流式细胞术检测PBMC亚群中IFN-a诱导的STAT1磷酸化，IRF-7在PDC亚群中增加的程度反映了IFN-a/STAT1/IRF-7反馈回路和保留的ifr -i功能；(c) NK组成型和ifn -a诱导的对hla无效或病毒感染目标（包括hcv感染的肝细胞）的裂解功能；(d)先天功能与HCV T细胞记忆反应水平之间的关系，通过IFN-？ELISPOT和四聚体检测EVR时间和治疗结束。第二个特定目标将分析以前单一HCV或双重HCV/ hiv感染受试者的横断面队列，与未感染的对照组相比，已达到HCV抑制和SVR状态，测量(a) NK/DC亚群频率的流式细胞分析；(b) NK细胞对IFN-a、hla缺失靶细胞和hcv感染肝细胞的功能反应（细胞毒性和脱颗粒、STAT1磷酸化、激活标志物的表达）；(c)树突状细胞对IFN-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是Wistar研究所、乔纳森·拉克斯免疫疾病治疗中心（Philadelphia FIGHT）、宾夕法尼亚大学传染病部、德雷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生物科学（San Diego CA）、以及马萨诸塞大学阿姆赫斯特分校生物统计学系。公共卫生相关性声明

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.