流式细胞仪为基础的检测方法的NK细胞功能的监控
Summary
一种简单和可靠的方法是此处描述的分析的一组NK细胞的功能，如不同的NK细胞亚群内脱粒，细胞因子和趋化因子生产。
Abstract
Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.
Introduction
作为先天性免疫系统天然杀伤的一部分（NK）细胞有助于针对病毒感染或恶性转化的细胞防御的第一道防线。抑制和激活受体的一种系统使他们没有相反的T细胞之前，抗原引发健康和转化的细胞之间进行区分。当靶细胞相遇NK细胞释放它们的细胞毒性颗粒（ 如穿孔素，颗粒酶）的含量为免疫突触杀死他们的目标。此外，NK细胞产生和分泌不同种类的细胞因子（ 例如，干扰素γ：干扰素γ;肿瘤坏死因子α：TNF-α）和趋化因子（ 例如，巨噬细胞炎性蛋白1β：MIP-1β）在靶细胞相互作用或细胞因子刺激1。
足够的NK细胞功能，如细胞毒性，趋化因子和细胞因子的产生有不同的存款保险计划的命运产生重要影响简化。白血病患者显示增加的复发率，如果他们在诊断显示出有缺陷的NK细胞轮廓由减少的IFN-γ生产和活化NK细胞受体2的表达减少。 NK细胞数量和功能，包括细胞因子的产生对靶细胞相互作用的早期恢复与接收异基因造血干细胞移植3例患者减少复发和改善存活率有关。此外，当在C型肝炎病毒感染的病人的干扰素治疗开始外周血NK细胞的脱粒能力是在早期应答比在非应答者4更强。 NK细胞数（> 80 /微升）在第15天在从淋巴瘤或多发性骨髓瘤的患者的自体干细胞移植（autoSCT）之后是预测性的改进的无进展生存率和总生存率5。在黑素瘤患者的T细胞中的表达immunoglobulin-和粘蛋白结构域containin克分子-3（TIM-3），NK细胞上的免疫调节蛋白，与疾病分期和预后6相关。
科学家们在整个过去几十年监测NK细胞的功能。抗肿瘤细胞NK细胞细胞毒性的不预先起动最初的分析是使用51 Cr-释放测定法7解决。最近，科学家开发了一种非放射性方法来评估膨胀NK细胞8的细胞毒性。细胞因子和趋化因子生产已使用酶联免疫吸附测定（ELISA）技术9,10-频繁评估。在过去几十年来，这些方法已通过流式细胞术基于测定补充。与常规的表面染色方案结合使用蛋白转运抑制剂（ 如，布雷菲德菌素A和莫能菌素）和细胞透方法已使科学家研究在不同的特定淋巴趋化因子和细胞因子产生ocyte子集（ 例如 ，T，B或NK细胞）11。此外，不同的流量基于流式细胞术的测定法已被开发并监控T和NK细胞的细胞毒性。在2004年阿尔特等人在目标小区遭遇所述NK细胞的溶酶体相关蛋白CD107a（灯1）的表面上表达作为细胞毒性颗粒12的脱粒标记。自宽范围的不同的荧光染料和多通道的流式细胞仪的是在我们的日子可用，它已成为可能同时监测在不同NK细胞亚群多样NK细胞功能（细胞毒性，细胞因子和趋化因子生产）。这成为在的情况下的样本大小是有限的， 例如 ，特别重要的是，在活检或从白细胞减少患者的血液样本。
为了测试全局NK细胞的功能，不同的流基于流式细胞仪的测定法可以有效地结合起来。 Theorell 等从HEA刺激NK细胞lthy捐助者与肿瘤细胞株K562和分析通过流式细胞仪13 NK细胞脱颗粒，由内向外信号和趋化因子的生产。最近NK细胞亚群，表型和肿瘤患者的功能autoSCT期间使用流式细胞仪为基础的试验进行了分析。据表明，NK细胞能够脱粒和autoSCT 11后在非常早的时间点产生在肿瘤细胞识别细胞因子/趋化因子。
这里一个协议描述用一个流式细胞术为基础的测定法，使得它能够同时监视在不同子集的NK细胞功能的肿瘤细胞，包括脱粒能力，趋化因子和细胞因子的产生来评估相互作用后NK细胞的功能。
Protocol
Representative Results
Discussion
所描述的方法是一种简单，快速和可靠的方法来研究健康人或病人全血标本NK细胞功能。这种方法提供了很大的优势，以直接净化从全血NK细胞，避免了费时密度梯度离心，这是强制性的许多其他纯化方法15。此外，它需要比较“经典的”NK细胞分离/富集方法较小的样本大小，这使得它对于儿科和/或免疫缺陷患者的样品的合适的替代。该协议可以被用来获得NK细胞功能的离体的基础?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the “Alfred- und Angelika Gutermuth-Stiftung”, Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.
Materials
| RPMI 1640 + glutamine | Invitrogen | 6187-044 | |
| penicilin/streptomycin | Invitrogen | 15140-122 | |
| BD Falcon Round Bottom Tube | BD | 352008 | |
| fetal calf serum | Invitrogen | 10270-106 | heat inactivated before use |
| T-flask | Greiner Bio-One | 690195 | |
| K562 tumor cell line | DSMZ GmbH | ACC 10 | |
| ammonium-chloride-potassium (ACK) lysis buffer | made in house | / | components are listed in the text |
| Distilled water: Ampuwa Spüllösung 1000ml Plastipur | Fresenius Kabi | 1088813 | |
| Megafuge 40R Centrifuge | Heraeus | / | |
| EDTA blood collector tubes | Sarstedt | 386453 | S-Monovette 7,5 ml, K3 EDTA |
| UltraPure 0.5M EDTA, pH 8.0 | Life Technologies | 15575-020 | |
| Hematopoietic media (XVIVO) | Lonza Group Ltd | BE04-743Q | |
| human serum | DRK Blutspendedienst, Frankfurt/M | / | healthy donors with blood type AB; heat inactivated before use |
| Neubauer-improved counting chamber, bright line | Marienfeld superior/ LO-Laboroptik Ltd. | 0640030/ 1110000 | |
| trypan blue solution (0,4%) | Invitrogen | 15250-061 | |
| 3% acetic acid with methylene blue | Stemcell Technologies | 07060 | |
| Corning 96 Well Clear V-Bottom TC-treated Microplates | Corning | 3894 | |
| Falcon 96 Well Round Bottom Not Treated Microplates | Corning | 351177 | |
| DPBS (Ca2+– and Mg2+-free) | Gibco Invitrogen | 14190-169 | |
| BSA | Sigma Aldrich | A2153-100G | |
| NaN3 | Sigma Aldrich | 08591-1ML-F | |
| phorbol 12-myristate 13-acetate (PMA) | Merck | 524400-1MG | |
| ionomycin | PromoKine | PK-CA577-1566-5 | |
| interleukin 15 (IL-15) | PeproTech | 200-15 | |
| Proleukin S (IL-2) | Novartis Pharma | 730523 | |
| Golgi Stop, Protein Transport Inhibitor (containing Monensin) | BD Biosciences | 554724 | This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested. |
| Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A) | BD Biosciences | 555029 | |
| paraformaldehyde | AppliChem | UN2209 | |
| saponin | Sigma Aldrich | 47036 | |
| flow cytometer: Canto10C | BD Biosciences | / | |
| FlowJo | TreeStar Inc. | / | |
| Graph Pad | Graph Pad Inc. | / | |
| MACSxpress Separator | Miltenyi Biotec | 130-098-308 | |
| MACSxpress NK isolation kit | Miltenyi Biotec | 130-098-185 | |
| MACSxpress Erythrocyte Depletion Kit, human | Miltenyi Biotec | 130-098-196 | |
| MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 | |
| anti-human CD3 APC | Biolegend | 300412 | |
| anti-human CD3 V450 | BD Biosciences | 560366 | |
| anti-human CD14 PerCP | Miltenyi Biotec | 130-094-969 | |
| anti-human CD14 V450 | BD Biosciences | 560349 | |
| anti-human CD16 PE | Biolegend | 302008 | |
| anti-human CD16 PerCP | Biolegend | 302029 | |
| anti-human CD19 PE-Cy7 | Biolegend | 302216 | |
| anti-human CD19 V450 | BD Biosciences | 560353 | |
| anti-human CD45 BV510 | BD Biosciences | 563204 | |
| anti-human CD56 FITC | Biolegend | 345811 | |
| anti-human CD107a PE | Biolegend | 328608 | |
| anti-human IFN-γ AF-647 | BD Biosciences | 557729 | |
| anti-human MIP-1β APC-H7 | BD Biosciences | 561280 | |
| DAPI | Biolegend | 422801 | |
| Zombie Violet Fixable Viability Kit | Biolegend | 423113 | fixable dead cell marker |
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