Name: detection of polyfunctional t cells in children vaccinated with japanese encephalitis vaccine via the flow cytometry technique
Uploaded: 2022-09-23T15:00:00
Description: Watch this Scientific Journal Video about Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique at JoVE.com

R.W.得到了中国国家自然科学基金（82002130），中国北京自然科学基金（7222059）的支持。ZD.X.获得CAMS医学科学创新基金（2019-I2M-5-026）的支持。

本研究已获得首都医科大学附属北京儿童医院伦理委员会的伦理批准（批准文号：2020-k-85）。志愿者来自首都医科大学附属北京儿童医院。外周静脉血样本取自表面上健康的儿童（2岁），这些儿童以前接种过初免和加强接种乙脑SA14-14-2减毒活疫苗不到半年（接种乙脑疫苗的儿童，n = 5）和未接种疫苗的儿童（6个月大，n = 5）。放弃了人类受试者的知情同意，因为本研究仅使用了经过临床测试后的?...

<ol>
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该协议代表了一种可行的基于流式细胞术的检测方法，用于接种JEV疫苗SA14-14-2的儿童的PBMC中的TPF 谱。本研究使用接种疫苗和未接种疫苗的儿童的静脉血PBMC作为研究材料。随着JEV抗原对PBMC的刺激，那些扩增的抗原特异性TPFs可以通过多色流式细胞术抗体染色进行表征。与传统的酶联免疫点测定方法相比，流式细胞术的突出优势是尽可能多样化地呈现单细胞水平PBMC的功能特征，减少...

日本脑炎病毒（JEV）是一种重要的蚊媒病毒，属于黄病毒科1中的黄病毒属。由于乙型脑炎（JE）造成的巨大疾病负担，许多亚太国家长期以来一直面临巨大的公共卫生挑战，但随着各类疫苗接种的增加，这种情况已得到显著改善2。自然感染或疫苗接种引起的适应性保护性免疫应答有助于预防和抗病毒调节。体液免疫和细胞介导免疫被归类为适应性免疫，前者的诱导一直被视为疫苗设计中的关键策略，尽管过去3的理解相对有限。然而，T细胞介导的免疫在限制黄病毒传播和病毒清除方面的作用越来越受到关注和广泛研究4。此外，T细胞免疫不仅在JEV特异性抗病毒反应中不可或缺，而且在交叉保护异源黄病毒继发感染方面也起着重要作用，这已在先前的研究中得到证实5。据推测，这种效应可能会绕过感染中潜在的抗体介导的增强效应5。值得注意的是，这种交叉反应性T细胞免疫很重要，特别是在没有针对黄病毒的疫苗和抗病毒药物的情况下。尽管已经进行了许多研究来确定T细胞在JEV感染中相对于CD4+和CD8 + T细胞的贡献6，7，但分泌细胞因子的各自谱系及其功能多样化仍未确定，这意味着辅助和杀伤性T细胞的确切功能的阐明受到阻碍。
它们的抗病毒防御规模决定了T细胞反应的质量。CD4+或CD8 + T细胞可以相容地赋予两种或多种功能，包括细胞因子分泌和脱颗粒，在单细胞水平8的特异性刺激下被表征为多功能T细胞（TPFs）。产生单个或多种细胞因子的CD4 + T细胞可能具有各种作用和免疫记忆。例如，IL-2+ IFN-γ+ CD4+ T细胞比IL-2+ CD4+ T细胞更容易形成长期有效的保护性反应9，这可以作为评估疫苗接种效果的重要参数。IL-2+ IFN-γ+ CD4 + T细胞的频率在长期不进展的获得性免疫缺陷综合征（AIDS）患者中增加，而由于IL-2对T细胞增殖的促进作用，艾滋病进展患者中CD4 + T细胞更倾向于单独产生IFN-γ10。此外，IL-2+ IFN-γ+ TNF-α +的一个子集被证明可以在体内长期存活并协同促进杀伤功能11。虽然CD8 + T细胞更有可能表现出细胞毒性活性，但一些CD4 + T细胞还具有细胞毒性活性，作为间接检测到表面CD107a分子的表达12。此外，某些T细胞亚群表达趋化因子MIP-1α，其通常由单核细胞分泌以参与T细胞介导的中性粒细胞募集13。同样，CD8+ TPFs也可用于表征上述标志物的多功能性。研究表明，初免-加强策略可以有效诱导长时间的TPF保护作用13，从而增强疫苗接种引起的保护作用。检查免疫系统的一个核心特征是记忆T细胞能够促进比幼稚T细胞更强，更快，更有效地应对继发性病毒攻击。效应记忆T细胞（TEM）和中央记忆T细胞（TCM）是重要的T细胞亚群，通常通过CD27 / CD45RO或CCR7 / CD45RA14的复合表达进行分化。TCM（CD27+ CD45RO+ 或 CCR7+ CD45RA-）倾向于定位在继发性淋巴组织中，而 TEM（CD27- CD45RO+ 或 CCR7- CD45RA-）定位于淋巴和外周组织15，16。TEM提供即时但不是持续的防御，而TCM通过在次级淋巴器官中增殖并产生新的效应物来维持反应17。因此，鉴于记忆细胞可以介导对病毒的特定和有效的回忆反应，关于这一多功能子集的贡献出现了问题。
随着流式细胞术技术的发展，同时检测10多个簇、表型和分化抗原的标志物已变得普遍，有利于更丰富地注释单个T细胞的功能免疫特征，以减少对T细胞表型的误解和理解困难。这项研究使用 离体 刺激和流式细胞术来分析接种JEV疫苗的儿童外周血单核细胞（PBMC）中的TPF 谱。应用这种方法，将扩大对疫苗接种诱导的短期和长期JEV特异性甚至交叉反应性T细胞免疫的理解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-human CD28</td>
			<td>Biolegend</td>
			<td>302934</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>anti-human CD49d</td>
			<td>Biolegend</td>
			<td>304339</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>APC anti-human MIP-1&#945;</td>
			<td>BD</td>
			<td>551533</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BIO RAD</td>
			<td>TC20</td>
			<td>Cell count</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5</td>
			<td>BD</td>
			<td>A5</td>
			<td>flow Cytometry</td>
		</tr>
		<tr>
			<td>BUV395 anti-human CD4</td>
			<td>BD</td>
			<td>563550</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BUV737 anti-human CCR7</td>
			<td>BD</td>
			<td>741786</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BUV737 anti-human CD27</td>
			<td>BD</td>
			<td>612829</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV421 anti-human CD8</td>
			<td>Biolegend</td>
			<td>344748</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV480 anti-human CD45RA</td>
			<td>BD</td>
			<td>566114</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV480 anti-human CD45RO</td>
			<td>BD</td>
			<td>566143</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV605 anti-human CD107a</td>
			<td>Biolegend</td>
			<td>328634</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV650 anti-human CD3</td>
			<td>BD</td>
			<td>563999</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV785 anti-human IL-2</td>
			<td>Biolegend</td>
			<td>500348</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge Tube</td>
			<td>BD Falcon</td>
			<td>BD-35209715</td>
			<td>15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td>BD</td>
			<td>554714</td>
			<td>Cell fixation and permeabilization</td>
		</tr>
		<tr>
			<td>Density gradient medium</td>
			<td>Dakewe</td>
			<td>DKW-KLSH-0100</td>
			<td>Ficoll-Paque, human lymphocyte separation medium</td>
		</tr>
		<tr>
			<td>FITC anti-human IFN-&#947;</td>
			<td>Biolegend</td>
			<td>502506</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Gibco RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>22400089</td>
			<td>cell culture medium</td>
		</tr>
		<tr>
			<td>High-speed&#160;centrifuge</td>
			<td>Sigma</td>
			<td>&#160;3K15</td>
			<td>Cell centrifugation for 15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>High-speed&#160;centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>Cell centrifugation for 1.5 mL Eppendorf (EP) tube</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL microcentrifuge tube</td>
		</tr>
		<tr>
			<td>PE anti-human TNF-&#945;</td>
			<td>Biolegend</td>
			<td>502909</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>BI</td>
			<td>02-024-1ACS</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (Containing Brefeldin A, GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td>blocks intracellular protein transport processes</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (Containing Monensin)</td>
			<td>BD</td>
			<td>554724</td>
			<td>blocks intracellular protein transport processes</td>
		</tr>
		<tr>
			<td>Round-bottom test tube</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>5 mL test tube</td>
		</tr>
		<tr>
			<td>Trypan Blue Staining Cell Viability Assay Kit</td>
			<td>Beyotime</td>
			<td>C0011</td>
			<td>Trypan Blue Staining</td>
		</tr>
		<tr>
			<td>Zombie NIR Fixable Viability Dye</td>
			<td>Biolegend</td>
			<td>423106</td>
			<td>Dead cell stain</td>
		</tr>
	</tbody>
</table>

detection of polyfunctional t cells in children vaccinated with japanese encephalitis vaccine via the flow cytometry technique

T细胞介导的免疫在控制黄病毒感染方面起着重要作用，无论是在接种疫苗后还是在自然感染后。T细胞的“质量”需要通过功能来评估，而更高的功能与更强大的免疫保护有关。在单细胞水平上可以同时产生两种或两种以上细胞因子或趋化因子的T细胞称为多功能T细胞（TPFs），其通过多种分子机制介导免疫应答，表达脱颗粒标志物（CD107a）并分泌干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-2或巨噬细胞炎症蛋白（MIP）-1α。越来越多的证据表明，TPFs与维持长期免疫记忆和保护密切相关，其增加的比例是保护性免疫的重要标志物，对有效控制病毒感染和再激活具有重要意义。该评估不仅适用于特异性免疫反应，也适用于交叉反应性免疫反应的评估。本文以乙型脑炎病毒（JEV）为例，对接种乙型脑炎接种患儿外周血单核细胞产生的JEV特异性TPFS的检测方法和流式细胞术配色方案进行了检测，为类似研究提供参考。

图1显示了用于从接种JE疫苗的儿童的代表性JEV刺激组中分离CD8 +或CD4 + T细胞的TCM或TEM的门控策略。FSC-A/SSC-A 点图用于识别淋巴细胞，FSC-A/FSC-W 点图用于识别单个细胞。在活/死/SSC-A点图上选择活细胞。CD3 / SSC-A点图用于识别CD3 + T细胞。CD4 / CD8点图分别用于识别CD3 + CD4 +和CD3 + CD8 + T细胞。CD8+或CD4...

Watch this Scientific Journal Video about Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique at JoVE.com

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

本协议结合了 离体 刺激和流式细胞术，以分析日本脑炎病毒（JEV）疫苗接种的儿童中外周血单核细胞（PBMC）中的多功能T细胞（TPF）谱。对JEV特异性TPF的检测方法和流式细胞术配色方案进行了测试，为类似研究提供参考。

流式细胞术 检测 接种乙型脑炎疫苗的儿童多功能T细胞

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...

对乙型脑炎病毒特异性TPFS的检测方法和流式细胞术配色方案进行了测试，为类似研究提供参考。演示该程序将由我们实验室的两名学生张琳琳和张萌进行。首先，在单克隆抗体CD28和CD49d，CD107a，GolgiPlug和monensin存在下，用浓缩的灭活JEV颗粒在37摄氏度下刺激JEV刺激组的PBMC16小时。然后，在单克隆抗体CD28和CD49d，CD 107a，GolgiPlug和monensin存在下，在37摄氏度下刺激没有浓缩病毒颗粒的对照组的PBMC16小时。将每组的细胞悬液收集在1.5毫升微量离心管中。在室温下以500g离心5分钟，然后用移液管除去上清液。将细胞重悬于1毫升PBS中。将可固定的活性染料添加到细胞悬液中，并在室温下在黑暗中孵育10分钟。在室温下以500g离心五分钟，并除去上清液。将细胞重悬于1毫升PBS中后，再次在室温下以500g离心5分钟，并如前所述弃去上清液。为了进行细胞表面标志物染色，将细胞重悬于100微升PBS中，并在每个管中的细胞悬液中加入两微升每种表面标志物抗体。将试管在室温下孵育 30 分钟，同时保护它们免受光照。以500g离心五分钟，并如前所述除去上清液。再次，将细胞重悬于一毫升PBS中。在室温下以500g离心五分钟，然后小心地弃去上清液。为了进行固定和膜破裂，用500微升破膜固定溶液重悬细胞，并在室温下在黑暗中固定细胞20分钟。然后以500g离心五分钟，除去上清液。将细胞重悬于一毫升PBS中后，在室温下以500g离心五分钟，并小心除去上清液，如前所述。然后，将细胞重悬于100微升PBS中，并在每管中的细胞悬液中加入2微升每种细胞因子抗体以进行细胞内细胞因子染色。将试管在室温下在黑暗中孵育30分钟。再次，以500g离心五分钟，并如前所述除去上清液。再次将细胞重悬于一毫升PBS中。在室温下以500g离心5分钟，并如前所述小心除去上清液。然后，加入500微升PBS以重悬细胞。如前所述，单独分离PBMC样品后，将细胞悬液在手稿中描述的1.5毫升微量离心管中分成12等份。类似地，如前所述，在加入细胞表面标志物和细胞内细胞因子抗体后，加入500微升PBS以重悬细胞并以低速涡旋。使用未染色的样品，调整前向散射、侧向散射和不同的荧光染料电压，同时调整流式细胞术补偿以消除不同荧光团之间的污染信号，使用单个染色样品。通过前向散射区绘制多边形门，侧向散射区点图选择完整的淋巴细胞群，同时排除碎片，并通过前向散射区绘制矩形门，前向散射宽度点图选择单个细胞。接下来，通过活的矩形门绘制一个矩形门，死边散射区点图以选择活细胞，并通过CD3侧散射区点图绘制一个矩形门以识别CD中的三个阳性T细胞。然后，通过CD4，CD8点图绘制四栅门以识别CD4阳性或CD8阳性T细胞，并通过CD45RO，CD27点图将CD4阳性或CD8阳性T细胞细分为TCM和TEM。在抽取CD107a门后，干扰素γ、肿瘤坏死因子α、白细胞介素2和小噬细胞炎症蛋白一α分别从中医或透射电镜中取出CD8阳性或CD4阳性T细胞，分别确定不同反应模式的频率。将样品依次加载到细胞仪上。采用门控策略，先用前向散射面积宽度，然后侧向散射区域点图鉴定CD8阳性或CD4阳性T细胞及其亚群的TCM或TEM。CD4阳性和CD8阳性T细胞对JEV应答的多功能表征表明，与未接种疫苗的儿童相比，接种疫苗的儿童在JEV刺激后CD8阳性中医细胞中检测到CD107a，干扰素γ，肿瘤坏死因子α和白细胞介素2水平升高。然而，两组巨噬细胞炎症蛋白一α的水平没有差异。与未接种疫苗组相比，在JEV刺激下，接种疫苗组的CD8阳性TEM细胞中检测到更高水平的CD107a和干扰素γ。而肿瘤坏死因子α，白细胞介素2和巨噬细胞炎症蛋白α在这些阳性细胞中没有显着升高。与未接种组相比，JEV抗原成功诱导接种组CD4阳性中药细胞CD107a、干扰素γ、肿瘤坏死因子α和巨噬细胞炎症蛋白1α水平较高。然而，在JEV刺激下，两组白细胞介素2阳性细胞的比例没有差异。在JEV存在下，接种组CD107a阳性、干扰素γ阳性和肿瘤坏死因子α阳性亚群、亚CD4阳性TEM细胞的比例高于未接种组。刺激的T细胞免疫原性和兼容的颜色匹配策略是该协议中的关键步骤。

Research

JoVE Journal

Immunology and Infection

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

流式细胞术 检测 接种乙型脑炎疫苗的儿童多功能T细胞

流式细胞术检测接种乙型脑炎疫苗的儿童多功能T细胞