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

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。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。 公共卫生相关性：人们对艾滋病毒-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和基因组发现方法，我们希望发现新的生物标记物和分子机制，通过帮助指导基于患者的HIV-1和药物使用者状况的临床研究和干预，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.