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

描述（由申请人提供）：多次暴露的未感染的静脉注射阿片类药物使用者（EU-IVDU）剩余的艾滋病毒血清阴性是少数临床队列之一，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的艾滋病毒暴露。已知与身份不明的人共用针头行为有感染风险的受试者血清呈阴性状态，这提高了人们对确定与艾滋病毒暴露和药物使用相关的免疫控制机制的兴趣，这种机制可能提供对感染的抵抗力。在EU-IVDU中，NK细胞激活，增加的组成性脱颗粒和增加的裂解活性被认为是与保护相关的关键特征，但目前尚不清楚哪些特定的NK膜蛋白谱和功能反应最好地定义或识别了EU-IVDU中的NK或DC细胞激活程序。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。我们建议分析三组通过缺乏艾滋病毒感染（血清）确定与不同的静脉阿片类药物使用历史。本研究将招募三组50名受试者，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，排他性阿片类药物使用者与阿片类药物和非注射药物使用者之间将进行1:1的招募，而不依赖后者。对于所有组，我们将收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NK细胞亚群，以便在有和没有干扰素- α急性刺激的情况下进行分析。根据初步数据显示的干扰素- α在激活NK裂解hiv感染靶标中的作用，我们选择干扰素- α作为参考激活刺激。在Aim 1中，我们将通过(1)测量活化（即CD69）、调节（即Np44等）、ifn - γ /趋化因子分泌和STAT-1信号传导电位来完成对EU-IVDU中NK细胞分子表型的分析；(2)利用基于蛋白质组学的方法比较NK细胞的全局和细胞表面蛋白谱，以鉴定与EU-IVDU选择性相关的关键细胞通路和细胞表面受体（KIR3DL1, KIR3DS1）。我们期望在第1组中发现与第2组和第3组相比的独特差异。Aim 2将分析NK和DC依赖性NK激活电位与TLR-7刺激后与疾病控制或浆细胞样DC分泌组相关的KIR蛋白表达（与hiv依赖性pdc介导的NK激活相关）。当与自体HIV-1感染靶标（有或没有IFN-1刺激）共培养时，将进行组成性循环NK CD107a和PDC的体外活性以及NK细胞的细胞毒性功能的研究，预计与2或3组相比，第1组的NK活化和裂解程度更高。综上所述，本研究将验证一种假设，即明确的NK和pDC激活表型介导EU-IVDU的可溶性和直接抗病毒功能，从而降低IV暴露后HIV-1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.