作者感谢James Lee和Christopher Ng对本文稿的评论和编辑。

以下协议符合iQ生物科学人类研究伦理委员会的指导方针。
1. PBMC的分离和NK细胞的富集/纯化
注意:也可以进行外周血单核细胞（PBMC）分离和富集/纯化的其他方法。
<ol><li>在调节条件下将200毫升血液吸入含有肝素的管中。</li>
	<li>将15 mL密度梯度培养基加入到掺入多孔屏障的50 mL管中。以1000 x g 旋转管子30秒。</li>
	<li>将12.5毫升血液加入试管中，然后再加入12.5毫升PBS，并轻轻倒置3倍。在室温（RT）下以 800×g 旋转管子15分钟，没有断裂。</li>
	<li>在细胞旋转时，为每根管准备一个50 mL锥形管，其中40 mL PBS具有多孔屏障。</li>
	<li>小心地取出管子，并在离心后进行检查。检查多孔屏障上方的 20 mL 和 25 mL 分界线之间是否有一层薄而明显的白色 PBMC 层。</li>
	<li>使用移液器小心地从顶部取出尽可能多的液体，而不会干扰薄薄的白色PBMC层。</li>
	<li>使用干净的10 mL血清学移液器，轻轻除去PBMC层以及透明，淡黄色的液体层，直到管的过滤器。</li>
	<li>将PBMC和液体排出到步骤1.4中制备的含有PBS的50 mL锥形中。在室温下旋转管，300 x g 5分钟。</li>
	<li>吸出PBS洗涤液，再加入40 mL PBS。在室温下旋转管，300 x g 5分钟。</li>
	<li>洗涤后，计数细胞并将其重悬在每1×107 个细胞的40μL2%BSA / PBS中。</li>
	<li>使用所选方法进行NK细胞的分离。选择包括手动或自动磁珠基分选9、21。荧光激活的细胞分选也可以进行22。</li>
	<li>通过流式细胞术除去一小块等分试样，以确定NK细胞的纯度。门控策略包括以下内容:基于前向与侧散射曲线对活细胞的门，然后根据特定活体群体中的NK标记物（例如CD3，CD56）评估纯度。典型纯度为>95%。</li>
	<li>分离完成后，在室温下旋转细胞和 300×g5 分钟以沉淀和吸出。</li>
	<li>将细胞沉淀重悬至1 x 107 个细胞/ mL的RPMI 1640中，具有营养物质，10%热灭活FBS，1mM丙酮酸钠，55mM 2-ME和10mM HEPES（pH = 7.2）。</li>
</ol>2. 抗体介导的通过FcγRIIIa激活NK细胞
<ol><li>将冷藏微量离心机设置为4°C。</li>
	<li>将1 x 10 6（100μL）重悬的NK细胞分配到1.5mL管中并置于冰上。 注意:如果样品数量众多，可以将细胞分配到96孔U底板中。</li>
	<li>每个样品制备1μL100μg/ mL利妥昔单抗（或目标抗体），并向细胞中加入1μL，最终浓度为1μg/ mL抗体。 注意:其他分子可以在此时或更早添加到细胞中，以评估组合效应。在这里，利替单抗用于刺激。在先前的研究中，小分子抑制剂已被添加到刺激9中。</li>
	<li>将样品在冰上孵育30分钟。在孵育过程中，在培养基中制备每个样品50μL50μg/ mL抗人κ轻链抗体，并在热块或水浴中加热至37°C。</li>
	<li>在冰上孵育细胞后，加入1mL冰冷培养基，并在4°C下以 135×g 旋转5分钟。 用 1 mL 冰冷培养基再次洗涤。</li>
	<li>在最后一次洗涤后吸出上清液，并加入步骤2.5中制备的50μL抗人κ轻链混合物。</li>
	<li>立即将样品在37°C的加热块或水浴中为最终用户确定的时间点孵育。</li>
	<li>通过加入1mL冰冷培养基停止刺激，并立即在 135×g 的冷藏离心机中旋转样品5分钟。用 1 mL 冰冷培养基再次清洗。</li>
</ol>3. 下游应用和读数
<ol><li>通过蛋白质印迹分析询问FcγRIIIa刺激的NK细胞中的信号分子 注意:可以使用不同的蛋白质分离和膜转移装置。<ol>
<li>在最后一次用冰冷培养基洗涤（步骤2.8）后，用20μL含有磷酸酶和蛋白酶抑制剂的RIPA缓冲液裂解细胞在冰上混合30分钟。</li>
		<li>在4°C下以 2100×g 离心管15分钟。 将裂解物转移到干净的1.5 mL管中，并加入蛋白质分离所需的试剂。</li>
		<li>将蛋白质分离到SDS-PAGE凝胶上，并按照标准方案转移到硝酸纤维素或PVDF膜上。</li>
		<li>根据制造商的一级检测抗体数据表（此处使用pAKT，pPRAS 40和pERK1 / 2）阻断和探针膜。</li>
		<li>根据制造商对 PVDF 膜的建议，添加 HRP 偶联二抗和化学发光试剂。使用X射线胶片或检测仪器进行可视化（此处，pAKT在60 kDa下检测到，pPRAS40在40 kDa时检测到，pERK1/2在42 kDa和44 kDa下检测到）。</li>
	</ol></li>
	<li>mRNA的分离和FcγRIIIa刺激的NK细胞基因表达分析的制备（材料表）<ol>
<li>达到刺激的时间点（步骤2.7）后，将管子放在冰上，并立即在135×g的冷藏离心机中旋转5分钟（类似于步骤2.8）。 </li>
		<li>取出上清液，用1 mL冰冷的PBS洗涤2次。</li>
		<li>使用异硫氰酸胍RNA提取裂解细胞（材料表）。使用1μgmRNA使用带有商业试剂盒的随机六聚体进行逆转录（材料表）。 注:任何RNA提取或cDNA合成方法均可采用9、23、24。</li>
		<li>将cDNA在-20°C下冷冻，直到基因表达分析。</li>
	</ol></li>
	<li>FcγRIIIa刺激的NK细胞的细胞骨架重排评估<ol>
<li>在最后一次用冰冷培养基洗涤（步骤2.8）后，用50μL3.7%多聚甲醛在室温下重悬细胞沉淀10分钟。</li>
		<li>加入1 mL PBS，在室温下以135 x g 离心5分钟。</li>
		<li>加入100μL0.1%Triton X-100 / PBS5分钟以透化细胞，并在室温下以 135×g 离心细胞5分钟以沉淀。</li>
		<li>加入100μL5单位/ mL AF488标记的鬼臼素稀释在1%BSA / PBS中，并在室温下孵育20分钟。</li>
		<li>加入1 mL PBS，在室温下以135 x g 离心5分钟以洗涤。将细胞沉淀重悬于所需体积，以进行流式细胞术分析。</li>
	</ol></li>
	<li>使用CD107a表面染色评估FcγRIIIa刺激的NK细胞的脱颗粒<ol>
<li>如步骤2.1-2.4所述，用感兴趣的抗体在冰上孵育细胞。在孵育过程中，每个样品制备50μL抗体混合物。在终浓度为50μg/ mL抗人κ轻链抗体和1μg/ mL荧光染料标记CD107a的细胞培养基中制备混合物。</li>
		<li>在冰上孵育细胞后，加入1mL冰冷培养基。在4°C下以 135×g 旋转5分钟，然后用1mL冰冷介质再次洗涤。</li>
		<li>在最后一次洗涤后吸出上清液，然后加入50μL抗人κ轻链混合物，并在37°C下孵育所需的时间点。</li>
		<li>在所需的时间点，加入1mLPBS，在室温下以 135×g 离心5分钟，吸出上清液并加入100μL4%多聚甲醛。在室温下孵育10分钟。</li>
		<li>加入1 mL PBS，在室温下以135 x g 离心5分钟以洗涤。将细胞沉淀重悬于所需体积，以进行流式细胞术分析。</li>
	</ol></li>
	<li>评估FcγRIIIa刺激的NK细胞的趋化因子/细胞因子产生 注意:可以使用不同的方法和/或试剂盒来评估趋化因子和细胞因子的产生。<ol>
<li>达到刺激的时间点（步骤2.7）后，立即将管子放在冰上，并在4°C下以135×g的冷藏离心机旋转5分钟。</li>
		<li>将上清液转移到干净的容器中。冷冻上清液，直到使用所需的测定评估趋化因子或细胞因子。 注意:细胞沉淀现在可以用冰冷的PBS洗涤并进行处理以进行信号传导，基因表达和细胞骨架重排研究。</li>
	</ol></li>
</ol>

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所有作者都是或曾经是iQ Biosciences的员工或顾问。

该协议描述了研究由抗体介导的NK细胞中FcγRIIIa驱动事件的方法。这些技术允许评估治疗性抗体的潜在作用机制，建议为ADCC1，2。具体而言，这些方法为研究负责ADCC的潜在分子信号通路和细胞过程提供了灵活性。它们还允许观察其他效应器功能，如趋化因子和细胞因子的产生。此外，这些方法允许鉴定可能靶向调节ADCC的潜在生物标志物和分子。
该协议的基础是在没有靶细胞的情况下通过FcγRIIIa用抗体对NK细胞进行人工刺激。抗体结合的靶细胞通常用于促进Fc受体的交联，以形成驱动信号传导和下游效应的平台。相反，在该测定中使用抗人κ轻链抗体完成交联，绕过对靶细胞刺激NK细胞的需求。如果没有靶细胞，结果和观察结果可以直接归因于NK细胞，假设纯化过程是成功的。
重要的是，使用抗人κ轻链抗体交联抗体不会干扰Fc部分对FcγRIIIa的结合亲和力，这种相互作用决定了反应的强度10，11，12，13。事实上，研究表明，阿拉伯冰山苷化抗体增加ADCC，因为它们对FcγRIIIa10，11，12，13的亲和力增加。随后的研究表明，这种增加的亲和力不受抗人κ轻链二抗替代品的影响，可用于研究ADCC8增加的基础。为确保NK细胞通过抗体的交联受到刺激，应包括两个阴性对照:1）仅治疗性抗体，没有二级抗人κ轻链抗体，以及2）仅二级抗人κ轻链抗体。在这两种情况下，都不应生成任何信号或效应器功能。
该方法还为研究治疗性抗体对小分子抑制剂的影响提供了灵活性。可以在与二抗交联之前加入抑制剂，以便抑制剂有时间接触其靶标。然而，应进行研究以确定抑制剂预处理的最佳时间;因此，抑制剂对刺激具有最大的作用。话虽如此，研究人员也可能选择研究刺激后抑制剂的作用。在这种情况下，可以在交联后添加抑制剂，以研究它如何影响已经产生的信号和过程。总之，这里描述的方法为研究不同小分子抑制剂与治疗性抗体的组合效应提供了最大的灵活性。
如上所述，刺激后可以执行各种读数。可以使用来自不同供应商的SDS-PAGE和膜转移系统进行蛋白质印迹以研究信号传导。同样，也可以使用各种RNA提取方法，逆转录试剂和基因表达仪器来评估基因表达。最后，还可以进行细胞内或细胞外蛋白的染色，其中可以使用不同的流式细胞仪分析样品。对于细胞内细胞因子和CD107a染色（步骤3.4，可同时评估），应添加莫宁酶和/或brefeldin A以最大化信号。我们对每个实验目标使用了不同的平台，并且仍然观察到类似的结果。因此，根据研究情况，该方法可以与各种试剂，平台和仪器相辅相成。
刺激的交联时间将取决于研究的目标。如果需要信号传导研究，典型的交联刺激时间在2分钟至10分钟之间，pAKT，pPRAS40和pERK1 / 2在2分钟时达到积累峰值，并在10分钟后消失8，9。对于功能研究（即涉及趋化因子/细胞因子产生的研究），必须刺激细胞至少30分钟，具体取决于分析物9。基因表达分析通常还需要30分钟的刺激9。使用RANTES基因表达作为读数时应谨慎行事，因为RANTES mRNA的产生与转录激活无关，因为它已经储存在细胞中，以便在刺激时及时翻译和释放蛋白质25。相反，脱颗粒需要至少3小时的刺激。尽管有这些一般观察结果，研究人员仍应进行动力学研究，以确定特定目标分子的最佳刺激时间。
同样，研究人员应该滴定感兴趣的抗体以确定最佳浓度，因为具有不同特异性的抗体与具有不同亲和力的FcγRIIIa结合，即使它们具有相同的同种型。例如，利妥昔单抗和曲妥珠单抗都是IgG1同种型，但曲妥珠单抗与FcγRIIIa的缬氨酸多态性结合比利妥昔单抗26，27更强。这种亲和力的差异可能导致功能差异，例如脱颗粒，如已发表的研究8中观察到的那样。
确定最佳浓度也很重要，因为抗体的Fc部分对FcγRIIIa具有低亲和力。这可能导致洗掉抗体，因为该协议包括抗体与Fc受体结合后的洗涤步骤。这可能导致测定中缺乏敏感性，正如刺激后CD107a阳性细胞的低百分比所表明的那样（图5）。然而，确定最佳浓度应提供信心，即结果不是由于缺乏敏感性。此外，细胞在生化和功能测定中被明显激活，这些测定使用体积细胞而不是单细胞读数（图2，图3，图6）。
该协议也是有限的，因为它不完全模仿生理上发生的事情。使用的二级抗人K抗体是模仿细胞上表达的靶抗原产生的交联。在这里，添加饱和量的二抗以产生最大反应。然而，不同的靶细胞会表达不同水平的抗原，这将影响交联和反应。目前，该平台尚未优化以模仿不同抗原表达水平的影响。
进行这些实验时要考虑的另一个因素是由于个体之间不同的遗传背景和免疫学历史，供体与供体之间的变异性。因此，在比较来自不同供体的NK细胞反应时，必须注意相同的测定。同样，在使用不同的捐助方时，只应作出一般性结论。
总而言之，所描述的方法是一种简单而灵活的刺激平台，用于研究NK细胞中抗体驱动的FcγRIIIa介导的事件。它已被用于更好地了解使用阿福球菌基化抗体8观察到的ADCC增加和功效的基础。该方法还被用于结合治疗性抗体和PI3K小分子抑制剂9的研究中。此外，还鉴定出由pS6调节的先前未知的趋化因子和细胞因子产生机制9。因此，使用这种人工信号平台的未来研究可以进一步阐明由FcγRIIIa驱动的效应器功能的调节机制。它还可能潜在地识别对这些机制重要的新分子，以及已知分子的新作用。

在过去的几十年里，人们一直非常关注使用抗体开发靶向癌症疗法。治疗性抗体，如曲妥珠单抗和利妥昔单抗，通过多种机制运作，包括防止信号分子的二聚化和免疫系统的动员1，2。后者是通过抗体依赖性细胞毒性（ADCC）完成的，其中称为自然杀伤（NK）细胞的淋巴细胞通过抗体1，2带入靶细胞。通过将细胞彼此靠近放置，NK细胞被激活，并且可以通过分泌效应分子3来裂解肿瘤/靶细胞。
在分子水平上，抗体的Fab部分结合其在肿瘤细胞上表达的同源抗原，而其Fc部分与NK细胞上表达的FcγRIIIa结合，使两个细胞结合在一起1，2。在FcγRIIIa参与后，信号通路（即MAPK和PI3K途径）驱动细胞骨架重排，细胞因子产生和细胞毒性4，5，6，7，8，9。因此，ADCC是由NK细胞和抗体介导的FcγRIIIa驱动的事件。
由于ADCC被认为是这些治疗性抗体的作用机制，研究人员寻找通过修饰抗体来增加ADCC的方法。一项修改是去除附着在天冬酰胺297上的寡糖链上的岩藻糖，这增加了抗体Fc部分与FcγRIIIa10，11，12的结合亲和力。在动物研究中，与用岩藻糖基化对应物13处理的小鼠相比，接受酸根基化抗体的小鼠表现出较慢的肿瘤生长。更重要的是，在诊断为慢性淋巴细胞白血病或滤泡性淋巴瘤的患者中，obinutuzumab（例如，Gazyva，一种批准的阿福球菌基化抗体）相对于利妥昔单抗（例如，Rituxan，其岩藻糖基化对应物）显示出更好的疗效14，15。
直到最近，通过芳香糖基化抗体增加ADCC的机制尚不清楚。结合有许多研究计划开发治疗性抗体以利用FcγRIIIa驱动的机制靶向癌细胞的事实，必须开发体外测定来检查这些抗体促进的分子和细胞方面。这提供了对作用机制以及发现生物标志物的潜力的基本理解。因此，开发了一种人工活化测定法，以研究抗体依赖性FcγRIIIa介导的功能以及信号传导和细胞特征8。通过这些研究，已经阐明了硫代葡萄糖基化抗体功效增加的机制，其中增强的结合亲和力增加信号传导以促进细胞特性和细胞毒性特征8。
目前临床试验的趋势是使用治疗分子16的组合。最常见的突变途径之一是PI3K途径，这促使人们努力开发靶向该途径17，18，19，20的小分子抑制剂。然而，这些分子如何与治疗性抗体联合作用是相对未知的，特别是在抑制剂可能影响需要PI3K途径才能发挥作用的分子的组合中，例如由治疗性抗体驱动的分子。
为此，用于化武菌丝化抗体研究的体外测定也用于研究PI3K抑制剂和治疗性抗体的组合。这些研究定义了PI3K抑制对治疗性抗体PI3K驱动事件的分子特征，并描述了酸洗酚糖基化抗体如何抵消这种信号传导的损失9。这些发现是相关的，因为它们为设计临床试验提供了潜在的指导。此外，这一系列实验还导致了首次描述的PI3K信号通路动力学调控的观察结果，以调节趋化因子/细胞因子的转录和产生，这可能作为潜在的生物标志物9。
用于定义上述信号传导和细胞特征的人工体外活化测定已被设计用于研究在没有靶细胞的情况下由抗体介导的NK细胞中的FcγRIIIa驱动事件。系统中没有靶细胞，观察到的所有信号传导事件和功能都可以直接归因于NK细胞。在所提出的测定中，将抗体加入到纯化的NK细胞中，此时Fc部分结合FcγRIIIa。随后是使用抗人κ轻链抗体对抗体进行交联，以人为地刺激细胞。抗体的交联模拟靶抗原的结合，以产生引发下游事件的信号平台。根据刺激的长度，研究人员可以评估信号传导，细胞过程，细胞毒性特征和效应器功能8，9。同样，当抗体与其他分子结合时，该测定也为研究这些事件提供了灵活性9。
总之，这是一种理想的体外测定，用于研究通过其FcγRIIIa引发NK细胞反应的治疗性抗体，作为作用机制的一部分。该协议描述了这种体外活化测定的性能，并提供了对可以执行的各种读数的见解。

<table><tbody><tr><td>16% paraformaldehyde</td><td>Thermo Fisher Scientific</td><td>50-980-487</td><td></td></tr><tr><td>Alexa Fluor 488 phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td></td></tr><tr><td>anti-mouse HRP antibody</td><td>Cell Signaling Technologies</td><td>7076</td><td></td></tr><tr><td>AutoMACS instrument</td><td>Miltenyi</td><td></td><td>NK cell isolation method; another isolation instrument may be used</td></tr><tr><td>B-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>21985023</td><td></td></tr><tr><td>Bovine Serum Albumin Fraction V, fatty acid free</td><td>Millipore Sigma</td><td>10775835001</td><td></td></tr><tr><td>CD107a APC antibody</td><td>Biolegend</td><td>328620</td><td></td></tr><tr><td>Cytokine 30-Plex Human Panel</td><td>Thermo Fisher Scientific</td><td>LHC6003M</td><td>chemokine/cytokine method; another chemokine/cytokine analysis method may be used</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.01</td><td></td></tr><tr><td>Goat anti-human &amp;kappa; light chain antibody</td><td>Millipore Sigma</td><td>AP502</td><td></td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail</td><td>Thermo Fisher Scientific</td><td>78440</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Thermo Fisher Scientific</td><td>4368814</td><td>cDNA transcription method; used according to manufacturer&#039;s protocol</td></tr><tr><td>IFN-? primers</td><td>Thermo Fisher Scientific</td><td>Hs00989291_m1</td><td></td></tr><tr><td>Leukosep tube (50 ml conical with porous barrier)</td><td>Greiner</td><td>227290</td><td></td></tr><tr><td>Lymphoprep (density gradient medium)</td><td>Stemcell</td><td>7851</td><td></td></tr><tr><td>MIP-1? primers</td><td>Thermo Fisher Scientific</td><td>Hs00234142_m1</td><td></td></tr><tr><td>MIP-1&amp;beta; primers</td><td>Thermo Fisher Scientific</td><td>Hs99999148_m1</td><td></td></tr><tr><td>NK cell isolation kit</td><td>Miltenyi</td><td>130-092-657</td><td>NK cell isolation kit; another isolation kit may be used</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td></td><td>another protein separation system may be used</td></tr><tr><td>pAKT (S473) antibody</td><td>Cell Signaling Technologies</td><td>4060</td><td></td></tr><tr><td>pERK1/2 antibody</td><td>Cell Signaling Technologies</td><td>4370</td><td></td></tr><tr><td>pPRAS40 antibody</td><td>Cell Signaling Technologies</td><td>13175</td><td></td></tr><tr><td>PVDF membrane</td><td>Thermo Fisher Scientific</td><td></td><td>nitrocellulose may be used</td></tr><tr><td>RANTES primers</td><td>Thermo Fisher Scientific</td><td>Hs00982282_m1</td><td></td></tr><tr><td>RIPA buffer</td><td>Millipore Sigma</td><td>R0278</td><td></td></tr><tr><td>RPMI w/glutamax</td><td>Thermo Fisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher Scientific</td><td>11360070</td><td></td></tr><tr><td>TNF-? primers</td><td>Thermo Fisher Scientific</td><td>Hs00174128_m1</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>85111</td><td></td></tr><tr><td>TRIzol</td><td>Thermo Fisher Scientific</td><td>15596018</td><td>RNA isolation method; used according to manufacturer&#039;s protocol</td></tr><tr><td>Xcell Blot II Transfer Module</td><td>Thermo Fisher Scientific</td><td></td><td>another protein separation system may be used</td></tr><tr><td>Xcell SureLock Protein Gel Electrophoresis Chamber System</td><td>Thermo Fisher Scientific</td><td></td><td>another protein separation system may be used</td></tr><tr><td>&amp;beta;-actin HRP antibody</td><td>Abcam</td><td>ab6721</td><td></td></tr><tr><td>&amp;beta;-actin primers</td><td>Thermo Fisher Scientific</td><td>Hs00982282_m1</td><td></td></tr></tbody></table>

assessment of human natural killer cell events driven by fc&#947;riiia engagement in the presence of therapeutic antibodies

治疗性抗体临床疗效的一种作用机制是促进免疫相关功能，如细胞因子分泌和细胞毒性，由自然杀伤（NK）细胞上表达的FcγRIIIa（CD16）驱动。这些观察结果导致研究重点是增加Fc受体介导事件的方法，其中包括去除在抗体的Fc部分发现的岩藻糖部分。进一步的研究已经阐明了信号传导，细胞过程和细胞毒性特征的机制变化，这些变化增加了酸锌酰化抗体的ADCC活性。此外，其他研究表明，这些抗体与小分子抑制剂联合使用具有潜在的益处。这些实验证明了在组合环境中使用酸根基化抗体的益处背后的分子和细胞机制。其中许多观察结果基于人工体外活化测定，其中人NK细胞上的FcγRIIIa被治疗性抗体激活。该测定为研究下游效应NK细胞功能（如细胞因子产生和脱颗粒）提供了灵活性。此外，该测定已被用于询问信号通路并鉴定可调节或用作生物标志物的分子。最后，其他治疗分子（即小分子抑制剂）已被添加到系统中，以提供对这些治疗药物与治疗性抗体组合的见解，这在当前的临床空间中至关重要。本手稿旨在为进行这种人工NK细胞活化测定提供技术基础。该协议展示了细胞活化的关键步骤以及潜在的下游应用，从功能读数到更机械的观察。

NK细胞纯度高至关重要，因为抗体的Fc部分可以结合在其他细胞类型（如单核细胞）上表达的FcγRIIIa。由于纯度高，所进行的观察可以直接归因于NK细胞中的FcγRIIIa驱动事件。在这里，基于CD56和CD3染色剂，NK细胞具有大于90%的纯度（图1）。此外，存活率为>95%。使用存活率较低的隔离物时应小心谨慎。为了确保事件由FcγRIIIa驱动，进行了磷酸化AKT（pAKT），磷酸化PRAS40（pPRAS40）和磷酸化-ERK1 / 2（pERK1 / 2）的蛋白质印迹，其中NK细胞被激活1-5分钟。如图所示，观察到这些分子的积累（图2）。类似地，活化的NK细胞表达MIP-1α，MIP-1β，IFN-γ和TNF-α，如基因表达分析所示（图3）。
此外，在活化细胞中观察到细胞骨架重排（图4）。刺激4小时的NK细胞的百分比在细胞表面表达CD107a（图5）。此外，在刺激3小时后，在上清液中检测到IFN-γ，TNF-α，MIP-1α，MIP-1β和RANTES（图6）。这些读数预计将基于已发表的研究，因为激活的NK细胞将具有这些磷酸化蛋白，以及上述趋化因子和细胞因子8，9的基因表达和产生。在所有实验中，刺激条件应包括仅抗人κ轻链对照和抗体无抗人κ光交联对照。这些NK细胞不应显示任何磷酸信号传导，脱颗粒，趋化因子/细胞因子产生或趋化因子/细胞因子基因表达。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61144/61144fig1v4.jpg"> 图1:从PBMC中分离出后NK细胞纯度的代表性流动曲线。从健康供体的血液中分离PBMC，然后使用阴性选择方法富集NK细胞进行人NK细胞分离（材料表）。在富集之前和之后用CD56和CD3染色细胞以确定纯度。隔离前后CD56与CD3的代表性点阵图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig1v4large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61144/61144fig02.jpg"> 图2:FcγRIIIa激活的NK细胞表现出磷酸化的信号分子。用利妥昔单抗刺激细胞2分钟，并根据方案制造细胞裂解物。裂解物在4%-12%的凝胶上分离，然后转移到PVDF膜上。用针对pAKT，pPRAS40，pERK1 / 2和肌动蛋白的抗体探针膜。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61144/61144fig03.jpg"> 图3:FcγRIIIa激活的NK细胞表达趋化因子和细胞因子基因。用利妥昔单抗刺激NK细胞0小时，0.5小时，收集1小时mRNA，反向转录并进行qPCR分析，以检测MIP-1α，MIP-1β，RANTES，IFN-γ和TNF-α（材料表）。（A） MIP-1α、MIP-1β 和 RANTES 在每个时间点的相对表达。（B） IFN-γ和TNF-α在每个时间点的相对表达。值归一化为肌动蛋白基因。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61144/61144fig04.jpg"> 图4:FcγRIIIa激活的NK细胞表现出细胞骨架重排。用利妥昔单抗刺激 NK 细胞 0 分钟， 5 分钟， 15 分钟和 30 分钟，然后通过染色和流式细胞术评估细胞骨架重排。实验时间点的MFI与用利妥昔单抗（圆形，实线）或单独二抗（方形，虚线）刺激的NK细胞的时间0处的MFI与MFI的比率。条形表示四个仿行的 SD。星号表示基于双尾不成对学生 t 检验的统计显著性（*p < 0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig04large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61144/61144fig05.jpg"> 图5:FcγRIIIa活化的NK细胞表达CD107a。用利妥昔单抗刺激NK细胞4小时，然后通过CD107a和流式细胞术评估脱颗粒。（A）用利妥昔单抗（白条）或抗人κ轻链抗体（灰条）刺激0小时和4小时后表达CD107a的NK细胞的百分比。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig05large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61144/61144fig06.jpg"> 图6:FcγRIIIa激活的NK细胞分泌趋化因子和细胞因子。用利妥昔单抗刺激NK细胞0，0.5，1，3和6小时。收集上清液以通过基于流动和珠状的细胞因子评估方法测量MIP-1α，MIP-1β，RANTES，IFN-γ和TNF-α的释放（材料表）。（A）每个时间点的MIP-1α，MIP-1β和RANTES的产生。（B） IFN-γ和TNF-α在每个时间点的生产。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61144/61144fig06large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Assessment of Human Natural Killer Cell Events Driven by FcÎ³RIIIa Engagement in the Presence of Therapeutic Antibodies at JoVE.com

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

本文描述了通过人类自然杀伤细胞中的治疗性抗体研究FcγRIIIa驱动事件的方案。该人工刺激平台允许询问下游效应器功能，例如脱颗粒，趋化因子/细胞因子产生以及由参与结合的抗体的FcγRIIIa和Fc部分介导的信号通路。

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

assessment-human-natural-killer-cell-events-driven-fcriiia-engagement

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.4K 次观看.  iQ Biosciences. 本文描述了通过人类自然杀伤细胞中的治疗性抗体研究FcγRIIIa驱动事件的方案。该人工刺激平台允许询问下游效应器功能，例如脱颗粒，趋化因子/细胞因子产生以及由参与结合的抗体的FcγRIIIa和Fc部分介导的信号通路。

视频: Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

科研

癌症研究

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

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.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

免疫与感染

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

从人类扩增的潜在干细胞中产生自然杀伤细胞

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

发育生物学

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

肝肿瘤细胞背景下自然杀伤细胞介导细胞毒性和迁移的测量

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

医学

<meta charset="utf8"></head><body>甲状腺癌细胞作为癌症免疫治疗的 ADCC 生物测定中的效应子

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

<meta charset="utf8"></head><body>作者聚焦:通过抗体依赖性细胞介导的细胞毒性测定优化基于抗体的癌症治疗

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

摘要

探索更多视频

此视频中的章节

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

流式细胞仪为基础的检测方法的NK细胞功能的监控

从人类扩增的潜在干细胞中产生自然杀伤细胞

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

肝肿瘤细胞背景下自然杀伤细胞介导细胞毒性和迁移的测量

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性