NK Cell Activation and Function in HIV-1 Exposed Uninfected IV Drug Users

描述（由申请方提供）：HIV血清阴性的多次暴露未感染IV阿片类药物使用者（EU-IVDU）是极少数可以与研究这些受试者感染风险的流行病学团队联合研究活HIV暴露的临床队列之一。所描述的已知通过与未知状态的人共用针头行为而处于感染风险的受试者的血清阴性状态提高了对鉴定免疫控制机制的兴趣，所述免疫控制机制可提供对与HIV暴露和药物使用相关的感染的抗性。在EU-IVDU中，NK细胞活化、组成性脱粒增加和溶解活性增加被认为是与保护相关的关键特征，但仍不清楚什么样的特定NK膜蛋白谱和功能反应最能定义或识别EU-IVDU中的NK或DC细胞活化程序。药物使用对NK或PDC激活程序的影响，即使在没有重复HIV暴露的情况下，也是未知的。我们建议分析三组缺乏艾滋病毒感染（serongative）与不同的历史，静脉阿片类药物的使用。本研究将招募三组，每组50名受试者，如下：第1组：EU-IVDU>3年，第2组：清洁使用者（不共用针头）-IVU使用者，第3组：未感染-非IVU使用者。对于每个吸毒者群体，将在纯类阿片吸毒者与类阿片和非注射毒品使用者之间进行1：1的招募，但对后者无依赖性。对于所有组，我们将采集血液并通过流式细胞术表征循环免疫细胞亚群，同时制备PBMC和分离的NK细胞亚群，用于在有和无干扰素-α急性刺激的情况下进行分析。如初步数据所示，基于干扰素-α在激活HIV感染靶的NK裂解中的作用，选择干扰素-α作为参考激活刺激物。对于目标1，我们将通过以下方式完成定义EU-IVDU中NK细胞的分子表型的分析：（1）测量活化（即，CD 69）、调节（即，Np 44等），IFN-γ/趋化因子分泌和STAT-1信号传导潜力;（2）使用基于蛋白质组学的方法比较NK细胞上的整体和细胞表面蛋白质谱，以鉴定与EU-IVDU选择性相关的关键细胞途径和细胞表面受体（KIR 3DL 1，KIR 3DS 1）。我们预计在第1组中发现与第2组和第3组相比的独特差异。目的2将分析NK和DC依赖性NK活化潜力，以及TLR-7刺激后与疾病控制或浆细胞样DC分泌体相关的KIR蛋白表达（与HIV依赖性PDC介导的NK活化相关）。当与自体HIV-1感染的靶标（有和没有IFN-1刺激）共培养时，将进行组成型循环NK CD 107 a和PDC的体外活性以及NK细胞的细胞毒性功能，预期与2或3相比，第1组中显示出更大的NK活化和裂解。综上所述，拟议的工作将测试的假设，一个定义的NK和pDC激活表型介导的可溶性和直接的抗病毒功能在EU-IVDU，从而降低HIV-1感染效率后，IV暴露。 公共卫生相关性：对HIV-1感染和吸毒成瘾之间的相互关系知之甚少。通过使用全球无偏见的蛋白质组学和基因组发现方法，我们希望发现新的生物标志物和分子机制，通过帮助指导临床研究和基于HIV-1和吸毒者状态的患者干预措施来改善患者护理。

DESCRIPTION (provided by applicant): Multiply-exposed uninfected IV opioid drug users (EU-IVDU) remaining HIV sero-negative are one of very few clinical cohorts where live HIV exposure can be studied in conjunction with epidemiological teams that are studying risk of infection in these subjects. The described seronegative state of subjects known to be at risk of infection via shared needle behavior with persons of unknown status has heightened interest in identifying the mechanism(s) of immune control that may provide resistance to infection in association with HIV exposure and drug use. NK cell activation, increased constitutive degranulation and increased lytic activity, have been proposed in EU-IVDU as a critical feature associated with protection, yet it remains unknown what specific NK membrane protein profiles and functional responses best defines or identifies an NK or DC cell activation program in EU-IVDU. The effects of drug use on NK or PDC activation programs, even in the absence of repeated HIV exposure, are also unknown. We propose to analyze three groups identified by lack of HIV infection (serongative) with different histories of IV opioid drug use. Three groups of 50 subjects each will be recruited for this study as follows: Group 1: EU-IVDU for >3 yrs, Group 2: Clean users (not needle sharing)-IVU users and Group 3: uninfected-non IVU users. For each of the drug user groups a 1:1 recruitment will take place between exclusive opioid users and opioid and non-injectable drug use in absence of dependence to the latter. For all groups, we will collect blood and characterize circulating immune cell subsets by flow cytometry while preparing PBMC and isolated NK cell subsets for analysis with and without an acute stimulation with Interferon-alpha. Interferon-alpha is chosen as a reference activation stimuli based for its role in activating NK lysis of HIV-infected targets as shown in preliminary data. For Aim 1, we will complete an analysis defining the molecular phenotype of NK cells in the EU-IVDU by (1) measuring activation (i.e., CD69), regulatory (i.e., Np44, etc.), IFN-gamma/chemokine secretion, and STAT-1 signaling potential; (2) using proteomics-based methods to compare global and cell surface protein profiles on NK cells to identify key cellular pathways and cell surface receptors selectively associated with EU-IVDU (KIR3DL1, KIR3DS1). We anticipate to find unique differences in group 1 as compared to groups 2 and 3. Aim 2 will analyze NK and DC-dependent NK activation potential in conjunction with protein expression of KIR associated with disease control or the Plasmacytoid DC secretome after TLR-7 stimulation (associated with HIV-dependent PDC-mediated NK activation). Constitutive circulating NK CD107a and in vitro activity of PDC and cytotoxic function of NK cells when co-cultured with autologous HIV-1 infected targets (with and without IFN-1 stimulation) will be done anticipating to show greater NK activation and lysis in Group 1 as compared to 2 or 3. Taken together, the proposed work will test the hypothesis that a defined NK and pDC activation phenotype mediates both soluble and direct antiviral function in EU-IVDU, thereby decreasing HIV-1 infection efficiency upon IV exposure. PUBLIC HEALTH RELEVANCE: The inter-relationship between HIV-1 infection and drug addiction is poorly understood. By using global unbiased proteomic and genomic discovery approaches, we expect to discover novel biomarkers and molecular mechanisms that will improve patient care by helping to guide clinical research and interventions of patients based on their HIV-1 and drug user status.