作者想感谢安德烈·汉和劳伦斯日元的技术援助，成员的詹金斯实验室的帮助，在此协议的发展。

1。淋巴组织细胞分离<ol><li>加入1毫升:冰:冷cEHAA（EHAA + 10％FBS，钢笔/链球菌，庆大霉素，2mM的L-谷氨酰胺，55 mM的2 - 巯基乙醇）或其它等效的T细胞的介质，以60毫米的培养皿中含有一个小正方形100μm的尼龙网和冰。 </li><li>安乐死鼠标。 </li><li>取出脾和淋巴结尽可能方便。这些措施应包括至少在腹股沟，腋窝，上臂，子宫颈癌，肠系膜淋巴结肿大。将它们放置在顶部的尼龙网眼的培养皿中。 </li><li>使用平顶一个封闭的1.5 ml离心管中， 轻轻捣烂的尼龙网，解放淋巴细胞的淋巴组织。的cEHAA和移液管向上和向下添加另一1毫升的工作到悬浮液中的细胞。通过另一块尼龙筛放在15ml的聚丙烯离心管中的顶部，将细胞转移。洗净的菜和另1毫升冰冷的cEHAA，宝网oling卷到同一管内。重复实现了最终的细胞悬浮液体积为4ml。 </li><li>加入冷的分拣机缓冲液（PBS + 2％FBS，0.05％叠氮化物），至终体积为15毫升，离心管在300×g离心5分钟，4℃下</li><li>小心地吸出上清液，确保没有在管的侧面上留下的液体微滴。中:Fc的嵌段（分拣机缓冲液+ 2.4G2抗体）中重悬细胞沉淀，至终体积等于大约两倍的颗粒本身。例如，一个天真的小鼠的脾和淋巴结肿大，通常会产生一个约100微升细胞沉淀。在这种情况下，加入100μl的Fc块，使体积至200μl。如果已经发生了很大程度的细胞结块，小心地取出细胞聚集在这一点上与枪头。 </li></ol> 2。四聚体染色<ol><li>添加PE-或APC-标记的住房抵押贷款公司四聚体，使最终浓度为10nM（或凭经验优化的浓度）。 </li><li> Mix和温浴特1小时，在室温下（或凭经验优化时间和温度）。 </li><li>加入冷分拣机缓冲液至体积为15毫升，离心管，4℃，300×g离心5分钟将样品在冰上或4°C从现在开始。 </li><li>小心地吸出上清液，确保没有在管的侧面上留下的液体微滴。分拣机，至终体积为200μl的缓冲液中重悬细胞沉淀。对于双四聚体富集，重悬，至终体积为150μl。 </li></ol> 3。磁富集<ol><li>美天旎抗-PE或抗-APC微球中加入50微升。对于双四聚体浓缩，加入50微升两个。 </li><li>混合孵育20分钟，在4°C。 </li><li>加入冷分拣机缓冲液至体积为15毫升，离心管，4℃，300×g离心5分钟</li><li>在等待过程中，成立了美天旎LS在MidiMACS或QuadroMACS磁铁的磁性列。上一个开放的15毫升的聚丙烯离心管中放置机架的正下方列。 </li><li> 3毫升分拣机缓冲液加入到柱顶部，允许它排放到15毫升的试管。 </li><li>放置为100μm的尼龙网正方形柱顶部。 </li><li>当细胞已完成纺纱，小心地吸出上清液，将沉淀重悬于3毫升分拣机缓冲液。 </li><li>细胞悬浮液转移到柱顶部通过尼龙筛。 </li><li>当细胞悬浮液已完全耗尽成列，与另外3毫升分拣机缓冲液冲洗的原管和传输缓冲器通过尼龙筛入列，漂洗的过程中啮合。丢弃的尼龙网。 </li><li>当缓冲区已完全耗尽入列，另外3毫升分拣机的缓冲列。 </li><li>共3×3毫升洗涤，重复步骤10。 </li><li>列磁铁和一个新的15毫升的聚丙烯离心管中取出。 </li><li>加入5毫升分拣机的缓冲吨他列。 </li><li>立即将柱塞压入列的顶部，在一个连续的动作，推动柱塞一路下来，强迫将缓冲区列管。 </li><li>含有洗脱的结合的部分和流过的未结合的部分在300×g离心5分钟，4℃下离心管</li><li>小心地吸出上清液从绑定的馏分，确保没有在管的侧面上留下的液体微滴。细胞沉淀重悬在分拣机的缓冲液中，至终体积正好95微升。吸液和未结合的级分重新悬浮至终体积为2ml。 </li></ol> 4。流式细胞仪<ol><li>对于每个馏分，取出5微升，并加至200μl的计数珠（调整到200,000 /毫升在分拣机的缓冲液中的浓度）在5毫升的FACS管。抛开在4°C后进行分析。为了节省时间，珠可以预先等分标记的试管中，在实验开始之前。 </li><li>准备是翠菊混合的抗体染色的细胞表面标志物（ 表1）。为了节省时间，这可以做到在实验开始前的。 </li><li>对于绑定的馏分，直接添加一个剂量的抗体鸡尾酒〜90微升的细胞在管内。如果未结合的部分的分析，需要的话，90微升的细胞转移到一个5毫升的FACS管中，并添加一个剂量的抗体鸡尾酒。 </li><li>设置为流式细胞仪单色补偿控制面板。以使用对于每个荧光染料，混合5毫升FACS管中的剩余的未结合的部分细胞加入50μl用1μl的共轭到相应的荧光染料的抗CD4抗体。记住设立染色的控制，以及。 </li><li>涡街流量计和培育所有样品为30分钟，在4°C </li><li>在300×g离心5分钟，4℃，加入5毫升分拣机的缓冲液中，以每管和离心机</li><li>绑定馏分，小心吸上清液和细胞悬浮在200μl分拣机buf中FER。转移到了1.2毫升的FACS微管细胞。冲洗管到相同的微管200微升分拣机的缓冲区和游泳池。如果细胞团块是显而易见的，通过细胞通过一个50微米的过滤器。 </li><li>对于未结合的部分补偿控制，倒出或吸出上清液，重悬在2毫升分拣机的缓冲区。 </li><li>设置的流式细胞仪使用单色的补偿控制。作为一个额外的参数被记录，选择侧向散射宽度（SSC-W）。 </li><li>分析连续列入门使用的序列，以确定CD4 +或CD8 + T细胞，如在图1和图2所示的染色样品。结合态，收集尽可能多的细胞尽可能最大的200事件总数。对于未绑定的部分，收集1,000,000总额事件。保持或低于3000事件每秒采集速率。 </li><li>使用同一台机器上设置，计数珠样品进行分析。收集10,000个事件。&lt;/ P&gt; <li> FCS文件保存所有数据。 </li></ol> 5。数据分析<ol><li> FCS数据文件的计数珠样本，使用FlowJo软件分析。绘制前方散射由FITC和设置栅极aroung的计数珠（请参阅图1和图2）。确定计数每个样品中检测到的小珠的总数。从收集的事件总数中减去的数量来确定的细胞收集的事件。 </li><li>计算在每个样品中的所有细胞，使用框1中列出的方程的总数。 </li><li>分析FCS数据文件的彩色绑定和未绑定的部分样品。连续列入门设定一个序列，以确定淋巴+侧分散宽度卤味 ，转储，CD3 +，CD4 +或CD8 +，四聚体+ T细胞在每个样品中，如在图1和图2中示出。 </li><li>总量乘以频率每个列入门用于定义CD4 +四聚体的样品中的细胞数，由+或CD8 +四聚体+细胞的表位特异性T细胞（专栏1）计算的总数量。 </li></ol>

<ol>
	<li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Kearney, E. R., Pape, K. A., Loh, D. Y., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+peptide-specific+T+cell+immunity+and+peripheral+tolerance+induction+in+vivo.">Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.</a> Immunity. 1, 327-339 (1994).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+epitope-specific+T+cells.">Tracking epitope-specific T cells.</a> Nat Protoc. 4, 565-581 (2009).</li><li>Hataye, J., Moon, J. J., Khoruts, A., Reilly, C., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+and+memory+CD4++T+cell+survival+controlled+by+clonal+abundance.">Naive and memory CD4+ T cell survival controlled by clonal abundance.</a> Science. 312, 114-116 (2006).</li><li>Marzo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+T+cell+frequency+dictates+memory+CD8++T+cell+lineage+commitment.">Initial T cell frequency dictates memory CD8+ T cell lineage commitment.</a> Nat Immunol. 6, 793-799 (2005).</li><li>Davis, M. M., Altman, J. D., Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+the+repertoire:+broadening+the+scope+of+peptide-MHC+multimer+analysis.">Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis.</a> Nature reviews. Immunology. 11, 551-558 (2011).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4(+)+T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27, 203-213 (2007).</li><li>Seah, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linear+range+for+accurately+quantifying+antigen-specific+T-cell+frequencies+by+tetramer+staining+during+natural+immune+responses.">The linear range for accurately quantifying antigen-specific T-cell frequencies by tetramer staining during natural immune responses.</a> European Journal of Immunology. 41, 1499-1500 (2011).</li><li>Obar, J. J., Khanna, K. M., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+naive+CD8++T+cell+precursor+frequency+regulates+primary+and+memory+responses+to+infection.">Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection.</a> Immunity. 28, 859-869 (2008).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+3+domain+mutants+of+peptide/MHC+class+I+multimers+allow+the+selective+isolation+of+high+avidity+tumor-reactive+CD8+T+cells.">Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.</a> Journal of Immunology. 171, 1844-1849 (2003).</li><li>Choi, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+avidity+antigen-specific+CTL+identified+by+CD8-independent+tetramer+staining.">High avidity antigen-specific CTL identified by CD8-independent tetramer staining.</a> Journal of Immunology. 171, 5116-5123 (2003).</li><li>Chu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+optimizes+the+number+and+function+of+MHCII-restricted+CD4++T+cell+clones+in+the+naive+polyclonal+repertoire.">Positive selection optimizes the number and function of MHCII-restricted CD4+ T cell clones in the naive polyclonal repertoire.</a> Proc Natl Acad Sci U S A. 106, 11241-11245 (2009).</li><li>Chu, H. H., Moon, J. J., Kruse, A. C., Pepper, M., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Selection+and+Peptide+Chemistry+Determine+the+Size+of+Naive+Foreign+Peptide-MHC+Class+II-Specific+CD4++T+Cell+Populations.">Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide-MHC Class II-Specific CD4+ T Cell Populations.</a> J Immunol. 185, 4705-4713 (2010).</li><li>Legoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+TCR+reactivity+and+HLA+phenotype+on+naive+CD8+T+cell+frequency+in+humans.">Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.</a> J Immunol. 184, 6731-6738 (2010).</li><li>Alanio, C., Lemaitre, F., Law, H. K., Hasan, M., Albert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+human+antigen-specific+naive+CD8++T+cells+reveals+conserved+precursor+frequencies.">Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies.</a> Blood. 115, 3718-3725 (2010).</li><li>Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+Epitope-Specific+Naive+CD4++T+Cells+Correlates+with+Immunodominance+in+the+Human+Memory+Repertoire.">Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire.</a> Journal of Immunology. 188, 2537-2544 (2012).</li><li>Jenkins, M. K., Chu, H. H., McLachlan, J. B., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+composition+of+the+preimmune+repertoire+of+T+cells+specific+for+Peptide-major+histocompatibility+complex+ligands.">On the composition of the preimmune repertoire of T cells specific for Peptide-major histocompatibility complex ligands.</a> Annu Rev Immunol. 28, 275-294 (2010).</li><li>Matechak, E. O., Killeen, N., Hedrick, S. M., Fowlkes, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-specific+T+cells+can+develop+in+the+CD8+lineage+when+CD4+is+absent.">MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent.</a> Immunity. 4, 337-347 (1996).</li><li>Burchill, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+T+cell+receptor+and+cytokine+signaling+govern+the+development+of+the+regulatory+T+cell+repertoire.">Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire.</a> Immunity. 28, 112-121 (2008).</li><li>Pepper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+routes+of+bacterial+infection+induce+long-lived+TH1+memory+cells+and+short-lived+TH17+cells.">Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells.</a> Nature Immunology. 11, 83-89 (2010).</li></ol>

没有利益冲突的声明。

本协议提出的住房抵押贷款公司四聚体为基础的细胞富集方法的研究表位特异性T细胞内源性T细胞剧目是一个功能强大的工具。住房抵押贷款公司四聚体的使用，可以直接基于绑定同源住房抵押贷款公司配体的能力，其电阻温度系数的表位特异性的T细胞的检测。的富集提供了一个极为罕见的群体可以检测到抗原特异性T细胞的T细胞内源性剧目没有他们的基因构成任何操作或前体频率的灵敏度水平?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂</strong</td&gt;<td<strong&gt;供应商</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PE或APC标记的住房抵押贷款公司四聚体（多聚体）</td&gt;<td由研究者，得到了美国国立卫生研究院四聚体核心，或从商业渠道购买</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td反PE标记的磁珠</td&gt;<td&gt;美天旎</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt;防APC共轭磁珠</td&gt;<td&gt;美天旎</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; LS磁列</td&gt;<td&gt;美天旎</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS或QuadroMACS磁铁</td&gt;<td&gt;美天旎</td&gt;<td&gt; 130-042-302或130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt;细胞计数珠</td&gt;<td&gt;生命科技</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

研究与体内实验模型的适应性免疫识别能力，根据他们的T细胞抗原受体（TCR）的特异性的T细胞的基本需要。许多的间接的方法是可在其中通过测量确定的官能反应，如增殖，细胞因子的产生，或活化标志物的表达的1与特定的抗原和表位的特异性T细胞在体外刺激T细胞堆积人口。然而，这些方法只能识别特定表位的T细胞表现出的许多可能的功能之一，它们是不够敏感检测表位特异性的T细胞在幼稚前体频率。一种流行的选择是TCR基因转殖继转移模型，其中单克隆T细胞的TCR转基因小鼠接种到组织相容性的主机创建一个大的前体人口的表位特异性的T细胞，可以EASI光年跟踪与一个同类系的标记抗体2,3使用。虽然功能强大，该方法遭受从实验与工件的非生理性T细胞的频率与一个单一的抗原决定簇特异性的4,5。此外，该系统不能被用于研究的功能内的异质性的表位特异性的T细胞克隆的多克隆人口。 理想的方式来学习适应性免疫应包括使用的方法，区分TCR特异性完全由结合到同源肽:MHC（住房抵押贷款公司）的直接检测表位特异性T细胞的内源性T细胞库。住房抵押贷款公司的四聚体和流式细胞仪的使用来完成这6，但被限制在检测高频只符合下列抗原诱导的克隆扩增的特定表位的T细胞群体。在这个协议中，我们描述了一种方法，协调使用的住房抵押贷款公司四和磁学抽动细胞富集技术，使极低频小鼠淋巴组织3,7的特定表位的T细胞的检测。利用这种技术，人们可以全面跟踪整个在小鼠内源性T细胞表位的特定人群的免疫反应的所有阶段。

图1描述了代表性的流式细胞仪地块pMHCII四聚体富集的脾脏和淋巴结从幼稚小鼠的样品，而图2示出了有代表性的数据，先前与相关肽+ CFA免疫小鼠。串行浇注删除的分析CD4 + T细胞群的自体荧光和其他有害事件。的CD8 + T细胞群体作为一个有用的内部pMHCII四聚体染色的CD4 + T细胞的阴性对照。需要注意的是结合态的富集通常包含一个显着较高比例的自体荧光的细胞比未结合的?...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

此协议描述了使用的肽:MHC四聚体和磁性微珠分离低频表位特异性的T细胞群体，并通过流式细胞术分析它们。这种方法能够直接研究内源性T细胞群的兴趣<em在体内</em实验系统。

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

peptidemhc-tetramer-based-enrichment-of-epitope-specific-t-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

17.8K 次观看.  Massachusetts General Hospital and Harvard Medical School. 此协议描述了使用的肽:MHC四聚体和磁性微珠分离低频表位特异性的T细胞群体，并通过流式细胞术分析它们。这种方法能够直接研究内源性T细胞群的兴趣

视频: Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...