Measurement of CCR5 and CCL3L1 on Single Cell by Fluorescent Metal Nanoparticle

描述(由申请人提供)：在CD4+T淋巴细胞的细胞表面存在CC-趋化因子共受体CCR5是成功感染HIV-1所必需的。然而，CCR5与其配体CCL3L1的相互作用对HIV-1感染具有很强的抵抗力。越来越多的证据表明，CCL3L1基因拷贝数在不同个体和种族群体中存在遗传变异。CCL3L1的高基因组拷贝数及其与CCR5的遗传相互作用与降低HIV-1的易感性和减缓艾滋病的进展有关。这一发现提出了一种可能的策略，即简单、灵敏和准确地定量检测CD4+T淋巴细胞表面的CCR5和CCL3L1分子，这可能为评估个体感染HIV-1的风险或易感性提供一个诊断标志。此外，它还可能提供一个预测HIV感染患者疾病进展的预后工具，以及一个可能的替代标记物，用于测试未来基于CCR5的抗HIV疗法的疗效。本研究的目的是建立一种利用金属荧光纳米颗粒作为探针的单细胞成像新策略，以研究CD4+T淋巴细胞表面CCR5和CCL3L1分子的数量、分布和相互作用。将针对CCR5或CCL3L1的特异性单抗(MAb)偶联到荧光金属纳米颗粒上，或先荧光标记后再结合到没有荧光团的金属纳米颗粒上。单抗结合的金属颗粒具有较强的发射、与细胞自身荧光不同的寿命、较好的光稳定性和较少的光闪烁，将作为荧光显像剂与CD4+T淋巴细胞表面的靶CCR5和/或CCL3L1进行免疫偶联。用共聚焦显微镜记录细胞荧光图像，从单细胞水平上的发射强度或寿命估计CCR5或CCL3L1在细胞表面的数量和分布。将开发一种合理的统计方法来分析经验数据。结果将通过基因组拷贝数法、流式细胞术和免疫金属电子显微镜图像以及与表面已知量的CCR5和CCL3L1结合的合成珠子的荧光图像进行校准。将研究CCR5和CCL3L1的相互作用。建立的荧光图像方法将用于研究从疾病进展快和慢的HIV感染患者中分离的CD+T淋巴细胞表面CCR5和CCL3L1分子，并进一步使我们能够评估个体对HIV-1感染的易感性和HIV感染患者的相关疾病进展。公共卫生相关性：在CD4+T淋巴细胞的细胞表面存在CC-趋化因子共受体CCR5是成功感染HIV-1所必需的。CCR5通过其配体CCL3L1的保护性相互作用抵消病毒的入侵。越来越多的证据表明，CCR5和CCL3L1的拷贝数与个体对HIV-1感染的易感性和HIV感染者的疾病进展有关。本研究的目的是建立一种新的方法，利用金属等离子体偶联探针(PCPs)通过单细胞的荧光图像来定量和定位CCR5和CCL3L1在细胞表面的靶分子。抗CCR5和/或CCL3L1的特异性单抗将结合在标记的金属颗粒上或用荧光团标记，然后与金属颗粒结合。单抗-金属颗粒将与CD4+T淋巴细胞表面的CCR5或/和CCL3L1结合。共聚焦显微镜将被用来记录标记细胞的荧光强度和寿命图像。细胞表面CCR5或CCL3L1分子的数量将从单个细胞水平的细胞图像中检测出来。CCR5和CCL3L1的定量将通过使用分析模拟模型进行校准，以确保准确性，并使用已建立的具有已知CCR5拷贝数的CCR5阳性细胞系进行进一步验证。经过验证的方法将与其他常用的方法进行比较，如流式细胞仪分析，并应用于从确诊的疾病进展快和慢的患者分离的HIV感染的CD4+T淋巴细胞上测量CCR5的分子水平及其与CCL3L1的相互作用。

DESCRIPTION (provided by applicant): Presence of the CC-chemokine co-receptors CCR5 on the cell surface of CD4+ T-lymphocytes is necessary for successful HIV-1 infection. However, the interaction of CCR5 by its ligand CCL3L1 provides strong counteraction against HIV-1 infection. Increasing evidence suggests that there is a genetic variability of the CCL3L1 gene copy number among different individuals and racial populations. High genomic copy number of CCL3L1 and its genetic interaction with CCR5 are associated with a reduced susceptibility to HIV-1 and slow progression to AIDS. This finding raises a possible strategy with easy, sensitive, and accurate quantification of CCR5 and CCL3L1 molecules on the surface of CD4+ T-lymphocytes that may provide a diagnostic marker for assessing individual risk or susceptibility to HIV-1 infection. In addition, it may also provide a prognostic tool to predict disease progression of HIV-infected patients and a possible surrogate marker for testing efficacy of future CCR5-based anti-HIV therapies. Objective of this proposal is to develop a new strategy about the single cell imaging using fluorescent metal nanoparticles as probes to investigate the number, distribution, and interaction of CCR5 and CCL3L1 molecules on the surfaces of CD4+ T-lymphocytes. Specific monoclonal antibodies (mAb) to CCR5 or CCL3L1 will be conjugated on the fluorescent metal nanoparticles or first fluorescently labeled and then bound on the metal nanoparticles without fluorophores. The mAb- bound metal particles, which are known to have stronger emission, different lifetime from the cellular autofluorescence, better photostability, and less photoblinking, will be used as fluorescence imaging reagents to immuno-conjugate with the target CCR5 and/or CCL3L1 on the surfaces of CD4+ T- lymphocytes. The cell fluorescence images will be recorded by confocal microscopy, and the number and distribution of CCR5 or CCL3L1 on the cell surfaces will be estimated from the emission intensity or lifetime over the cell images at single cell level. A reasonable statistic approach will be developed to analyze the empirical data. The results will be calibrated with the genomic copy number method, flow cytometry, and immunometal electron microscopy images, as well as the fluorescent images of synthetic beads bound with known amounts of CCR5 and CCL3L1 on surfaces. The interaction of CCR5 and CCL3L1 will be investigated. The established fluorescence images approach will be used to study the CCR5 and CCL3L1 molecules on the surfaces of CD+ T-lymphocytes isolated from the fast and slow disease progressing HIV-infected patients, and furthermore allow us to evaluate individual susceptibility to HIV-1 infection and correlate disease progression of HIV-infected patients. PUBLIC HEALTH RELEVANCE: Presence of the CC-chemokine co-receptors CCR5 on the cell surface of CD4+ T-lymphocytes is necessary for successful HIV-1 infection. The protective interaction of CCR5 by its ligand CCL3L1 counteracts viral invasion. Increasing evidence suggests that copy numbers of CCR5 and CCL3L1 are associated with individual susceptibility to HIV-1 infection and disease progression of HIV- infected patients. Objective of this proposal is to develop a novel approach to quantify and map the target molecules of CCR5 and CCL3L1 on the cell surface by the fluorescence image of single cell using metal plasmon-coupled probes (PCPs). Specific monoclonal antibodies (mAb) to CCR5 and/or CCL3L1 will be conjugated on the labeled metal particles or labeled with the fluorophores before binding to the metal particles. The mAb-metal particles will be bound with the CCR5 or/and CCL3L1 on the surfaces of CD4+ T-lymphocytes. Confocal microscopy will be utilized to record fluorescence intensity and lifetime images of the labeled cells. The amount of the CCR5 or CCL3L1 molecules on the cell surfaces will be detected from the cell images at single cell level. Quantification of CCR5 and CCL3L1 will be calibrated by using an analytic simulation model to ensure accuracy and further validated using established CCR5-positive cell lines with known copy numbers of CCR5. The validated method will be compared with other commonly used methods such as flow cytometric analysis and applied to measure the molecular level of CCR5 and its interaction with CCL3L1 on HIV-infected CD4+ T-lymphocytes isolated from the established fast and slow disease progressing patients.