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>

<ol>
	<li>Hudis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trastuzumab+-+Mechanism+of+Action+and+Use+in+Clinical+Practice.">Trastuzumab - Mechanism of Action and Use in Clinical Practice.</a> New England Journal of Medicine. 357 (1), 39-51 (2007).</li><li>Weiner, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rituximab:+mechanism+of+action.">Rituximab: mechanism of action.</a> Seminars inHhematology. 47 (2), 115-123 (2010).</li><li>Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+function+of+the+lytic+NK-cell+immunological+synapse.">Formation and function of the lytic NK-cell immunological synapse.</a> Nature Reviews Immunology. 8 (9), 713-725 (2008).</li><li>Bonnema, J. D., Karnitz, L. M., Schoon, R. A., Abraham, R. T., Leibson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptor+stimulation+of+phosphatidylinositol+3-kinase+in+natural+killer+cells+is+associated+with+protein+kinase+C-independent+granule+release+and+cell-mediated+cytotoxicity.">Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity.</a> The Journal of Experimental Medicine. 180 (4), 1427-1435 (1994).</li><li>Jiang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+role+of+phosphoinositide-3+kinase+in+regulation+of+cytotoxicity+in+natural+killer+cells.">Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells.</a> Nature Immunology. 1 (5), 419-425 (2000).</li><li>Kanakaraj, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-3+kinase+activation+induced+upon+Fc+gamma+RIIIA-ligand+interaction.">Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA-ligand interaction.</a> The Journal of Experimental Medicine. 179 (2), 551-558 (1994).</li><li>Orange, J. S., Harris, K. E., Andzelm, M. M., Valter, M. M., Geha, R. S., Strominger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mature+activating+natural+killer+cell+immunologic+synapse+is+formed+in+distinct+stages.">The mature activating natural killer cell immunologic synapse is formed in distinct stages.</a> Proceedings of the National Academy of Sciences. 100 (24), 14151-14156 (2003).</li><li>Liu, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afucosylated+Antibodies+Increase+Activation+of+Fc&#947;RIIIa-Dependent+Signaling+Components+to+Intensify+Processes+Promoting+ADCC.">Afucosylated Antibodies Increase Activation of Fc&#947;RIIIa-Dependent Signaling Components to Intensify Processes Promoting ADCC.</a> Cancer Immunology Research. 3 (2), 173-183 (2015).</li><li>Romeo, V., Gierke, S., Edgar, K. A., Liu, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+PI3K+Inhibition+on+Afucosylated+Antibody-Driven+Fc&#947;RIIIa+Events+and+Phospho-S6+Activity+in+NK+Cells.">Effects of PI3K Inhibition on Afucosylated Antibody-Driven Fc&#947;RIIIa Events and Phospho-S6 Activity in NK Cells.</a> The Journal of Immunology. 203 (1), 137-147 (2019).</li><li>M&#246;ssner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+efficacy+of+CD20+antibody+therapy+through+the+engineering+of+a+new+type+II+anti-CD20+antibody+with+enhanced+direct+and+immune+effector+cell-mediated+B-cell+cytotoxicity.">Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity.</a> Blood. 115 (22), 4393-4402 (2010).</li><li>Shields, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+fucose+on+human+IgG1+N-linked+oligosaccharide+improves+binding+to+human+Fcgamma+RIII+and+antibody-dependent+cellular+toxicity.">Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity.</a> The Journal of Biological Chemistry. 277 (30), 26733-26740 (2002).</li><li>Shinkawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+absence+of+fucose+but+not+the+presence+of+galactose+or+bisecting+N-acetylglucosamine+of+human+IgG1+complex-type+oligosaccharides+shows+the+critical+role+of+enhancing+antibody-dependent+cellular+cytotoxicity.">The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.</a> The Journal of Biological Chemistry. 278 (5), 3466-3473 (2003).</li><li>Junttila, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+In+vivo+Efficacy+of+Afucosylated+Trastuzumab+in+the+Treatment+of+HER2-Amplified+Breast+Cancer.">Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer.</a> Cancer Research. 70 (11), 4481-4489 (2010).</li><li>Goede, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obinutuzumab+(GA101)+plus+chlorambucil+(Clb)+or+rituximab+(R)+plus+Clb+versus+Clb+alone+in+patients+with+chronic+lymphocytic+leukemia+(CLL)+and+preexisting+medical+conditions+(comorbidities):+Final+stage+1+results+of+the+CLL11+(BO21004)+phase+III+trial.">Obinutuzumab (GA101) plus chlorambucil (Clb) or rituximab (R) plus Clb versus Clb alone in patients with chronic lymphocytic leukemia (CLL) and preexisting medical conditions (comorbidities): Final stage 1 results of the CLL11 (BO21004) phase III trial.</a> Journal of Clinical Oncology. 31, 7004 (2013).</li><li>Sehn, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+Phase+II+Trial+Comparing+Obinutuzumab+(GA101)+With+Rituximab+in+Patients+with+Relapsed+CD20++Indolent+B-Cell+Non-Hodgkin+Lymphoma:+Final+Analysis+of+the+GAUSS+Study.">Randomized Phase II Trial Comparing Obinutuzumab (GA101) With Rituximab in Patients with Relapsed CD20+ Indolent B-Cell Non-Hodgkin Lymphoma: Final Analysis of the GAUSS Study.</a> Journal of Clinical Oncology. 33 (30), 3467-3474 (2015).</li><li>Pons-Tostivint, E., Thibault, B., Guillermet-Guibert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+PI3K+Signaling+in+Combination+Cancer+Therapy.">Targeting PI3K Signaling in Combination Cancer Therapy.</a> Trends in Cancer. 3 (6), 454-469 (2017).</li><li>Engelman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+PI3K+signalling+in+cancer:+opportunities,+challenges+and+limitations.">Targeting PI3K signalling in cancer: opportunities, challenges and limitations.</a> Nature Reviews. Cancer. 9 (8), 550-562 (2009).</li><li>Fruman, D. A., Rommel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+and+cancer:+lessons,+challenges+and+opportunities.">PI3K and cancer: lessons, challenges and opportunities.</a> Nature Reviews. Drug Discovery. 13 (2), 140-156 (2014).</li><li>Janku, F., Yap, T. A., Meric-Bernstam, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+PI3K+pathway+in+cancer:+are+we+making+headway.">Targeting the PI3K pathway in cancer: are we making headway.</a> Nature Reviews. Clinical Oncology. 15 (5), 273-291 (2018).</li><li>Samuels, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+of+mutations+of+the+PIK3CA+gene+in+human+cancers.">High frequency of mutations of the PIK3CA gene in human cancers.</a> Science. 304 (5670), 554 (2004).</li><li>Kanevskiy, L. M., Telford, W. G., Sapozhnikov, A. M., Kovalenko, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+induces+IFN-&#947;+production+in+human+NK+cells.">Lipopolysaccharide induces IFN-&#947; production in human NK cells.</a> Frontiers in Immunology. 4, (2013).</li><li>Romagnani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+human+NK+cells+by+plasmacytoid+dendritic+cells+and+its+modulation+by+CD4++T+helper+cells+and+CD4++CD25hi+T+regulatory+cells.">Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells.</a> European Journal of Immunology. 35 (8), 2452-2458 (2005).</li><li>Sivori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+and+double-stranded+RNA+trigger+human+NK+cells+by+Toll-like+receptors:+Induction+of+cytokine+release+and+cytotoxicity+against+tumors+and+dendritic+cells.">CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: Induction of cytokine release and cytotoxicity against tumors and dendritic cells.</a> Proceedings of the National Academy of Sciences. 101 (27), 10116-10121 (2004).</li><li>Hanna, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Insights+on+Human+NK+Cells'+Immunological+Modalities+Revealed+by+Gene+Expression+Profiling.">Novel Insights on Human NK Cells' Immunological Modalities Revealed by Gene Expression Profiling.</a> The Journal of Immunology. 173 (11), 6547-6563 (2004).</li><li>Swanson, B. J., Murakami, M., Mitchell, T. C., Kappler, J., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANTES+production+by+memory+phenotype+T+cells+is+controlled+by+a+posttranscriptional,+TCR-dependent+process.">RANTES production by memory phenotype T cells is controlled by a posttranscriptional, TCR-dependent process.</a> Immunity. 17 (5), 605-615 (2002).</li><li>Jo, M., Kwon, H. S., Lee, K. H., Lee, J. C., Jung, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+aglycosylated+full-length+IgG+Fc+variants+exhibiting+improved+Fc&#947;RIIIa+binding+and+tumor+cell+clearance.">Engineered aglycosylated full-length IgG Fc variants exhibiting improved Fc&#947;RIIIa binding and tumor cell clearance.</a> mAbs. 10 (2), 278-289 (2018).</li><li>Isoda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+Side+Chain+at+Position+296+of+Antibody+Fc+in+Interactions+with+Fc&#947;RIIIa+and+Other+Fc&#947;+Receptors.">Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with Fc&#947;RIIIa and Other Fc&#947; Receptors.</a> PLoS ONE. 10 (10), 0140120 (2015).</li></ol>

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 &#954; 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&#39;s protocol</td>
		</tr>
		<tr>
			<td>IFN-&#611; 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&#9082; primers</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hs00234142_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>MIP-1&#946; 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-&#9082; 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&#39;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>&#946;-actin HRP antibody</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-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/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/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/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/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/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/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 FcÃŽÂ³RIIIa 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

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...