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

描述(由申请人提供)：多次接触未感染的静脉注射阿片类药物使用者(EU-IVDU)保持艾滋病毒血清阴性是少数几个可以与正在研究这些受试者感染风险的流行病学小组一起研究活体艾滋病毒暴露的临床队列之一。已知通过与未知状态的人共用针头行为而面临感染风险的受试者的上述血清阴性状态，提高了人们对识别免疫控制机制(S)的兴趣，该机制可能提供与艾滋病毒暴露和药物使用相关的感染抵抗力。在EU-IVDU中，NK细胞的激活、更多的结构性脱颗粒和更高的裂解活性被认为是与保护相关的关键特征，但目前尚不清楚什么特定的NK膜蛋白谱和功能反应最好地定义或识别EU-IVDU中的NK或DC细胞激活计划。即使在没有反复接触艾滋病毒的情况下，药物使用对NK或PDC激活程序的影响也是未知的。我们建议分析三组被确认为缺乏艾滋病毒感染(血清学)的不同静脉注射阿片类药物使用史的人群。这项研究将招募三组受试者，每组50人：第一组：EU-IVDU，为期3年；第二组：清洁使用者(不是共用针头)-IVU使用者，以及第三组：未感染-非IVU使用者。对于每个吸毒者群体，将在专属阿片类药物使用者和不依赖阿片类药物和非注射药物的非注射药物使用者之间进行1：1招募。对于所有组，我们将采集血液并通过流式细胞仪鉴定循环免疫细胞亚群，同时准备PBMC和分离的NK细胞亚群，以便在干扰素-α急性刺激和不急性刺激的情况下进行分析。根据初步数据显示，干扰素-α在激活HIV感染靶点的NK裂解方面的作用被选为参考激活刺激。对于目标1，我们将通过(1)检测激活(即CD69)、调节(即Np44等)、干扰素-γ/趋化因子分泌和STAT-1信号潜能来完成定义EU-IVDU中NK细胞的分子表型的分析；(2)使用基于蛋白质组学的方法比较NK细胞的整体和细胞表面蛋白质谱，以确定与EU-IVDU(KIR3DL1，KIR3DS1)选择性相关的关键细胞通路和细胞表面受体。我们预计将发现组1与组2和组3相比的独特差异。目的2将分析依赖NK和DC的NK激活潜力以及与疾病控制相关的KIR蛋白表达或TLR-7刺激后的血浆细胞样树突状细胞分泌体(与HIV依赖的PDC介导的NK激活相关)。当与自体HIV-1感染靶点(有或没有干扰素-1刺激)共同培养时，组1将进行结构性循环NK CD107a、PDC的体外活性和NK细胞的细胞毒功能的研究，预期与2或3组相比，组1显示出更大的NK激活和裂解。综上所述，拟议的工作将检验这样的假设，即明确的NK和PDC激活表型介导了EU-IVDU中可溶性和直接的抗病毒功能，从而降低了IV暴露时HIV-1的感染效率。

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.