We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

1.准备在无菌条件下材料（见材料和​​设备表） <ol><li>准备3升的PBS / EDTA缓冲液补充有人血清白蛋白（HSA），以0.5％的终浓度（重量/体积）。 </li><li>制备1升的临床级0.9％氯化钠（NaCl）中溶液袋和2升GMP级细胞培养基。 </li><li>由重构CMV pp65抗原的一小瓶用8ml无菌水中制备60纳摩尔的CMV特异性抗原肽的鸡尾酒。 </li><li>转移CMV病毒pp65肽混合物成使用鲁尔/穗互连50毫升的容量冷冻袋，并用钳钳锁，以避免后续的分配鸡尾酒到管道组。打开细胞富集管道组（TS 500）在无菌条件下。 </li><li>使用无菌管道焊接，连接肽混合物冷冻袋成管路的阀门2 TS 500，不要在这个时候打开肽混合物袋夹管连接。 </li><li>删除1×10 9吨数控从开始细胞产物和暂停在含有2.5％HSA至50毫升的总体积的PBS / EDTA缓冲液。细胞产品注入150ml的输液袋。 </li></ol> 2.准备和使用自动化的细胞富集系统（见材料和​​设备表） <ol><li>切换细胞富集系统 （图1B），选择节目“CCS_IFN-γ富集 ”。观察显示具有说明书和图片通过程序引导操作员屏幕的用户界面。 </li><li>输入参数“ 操作员 ”和“ 管路P / N号 ”。接下来，安装管500设置到自动细胞富集设备按交互式监视器屏幕上显示的指令。 </li><li>遵循由显示在屏幕上以连接介质和缓冲液至设备步骤的指令的步骤。记录产品编号和connectin前试剂的批号克至仪器。 </li><li>管道组的最后检查后，打开肽混合物袋的夹子。打开介质包和发起管路装置的自动启动。 </li><li>后引发步骤完成后，补充的HSA（2.5％）为NaCl缓冲液中贮存袋（200毫升）与无菌管道焊机的帮助。起始细胞产品转移到“应用程序包”使用无菌管道焊工。 </li><li>通过适配器连接CCS（IFNγ）试剂进入各自的管道。输入首选时间浓缩过程之前收集细胞物质的一小部分。审查和核实所有数据的准确度/参数输入。开始处理。 </li><li>自动细胞富集过程开始之前，除去质量控制袋（QCB，原始分数（ORI）约含1.3毫升出用PBS / EDTA缓冲液稀释百毫升室的内容）。密封的QCB，称重，并在4℃下保存。 </li><li>启动enrichmeNT进程。在该过程结束时，靶细胞将被洗脱的洗脱缓冲液从贮存袋中的近似体积。 </li><li>密封的非目标单元格袋（NTCB，负分数=负）和靶细胞袋（TCB，阳性率= POS）和权衡每个袋子。的权重将被用于以后的细胞数的计算。 </li><li>立即富集过程后收集每级分两等份用于流式细胞分析，样品的其余部分存储在4℃。用细胞计数测定和富集性能分析（表1），另取试样一个样本等份。 </li><li>拆下油管从细胞富集仪器设置。日志文件传送到USB驱动器以备将来使用。 注：所有的试剂应该在无菌条件下制备。使用生物安全II型吸油烟机的强烈建议。使用稳态单采蜂窝产品（非动员），从健康的隔离巨细胞病毒阳性捐助，充实巨细胞病毒抗原特异性T细胞。唯一获得FDA许可的HSA应该被使用。缓冲对细胞制剂应保持在19℃至25℃的较低或较高的环境温度会导致降低纯度和靶细胞的产率降低。 </li></ol> 3.细胞计数的测定<ol><li>取QCB，NTCB和TCB的用于细胞计数的等分试样如表 1所示。加入CD45-VoBlue至每个等分试样（效价1点11分）和孵化在黑暗中进行10分钟，在4℃。 </li><li>加1.5毫升新制备的红细胞溶解溶液（1倍）的原始分数和阴性部分，450微升新鲜制备的红细胞溶解溶液的阳性分数，孵化的所有级分进行15分钟，在室温。 </li><li>只是在分析之前，碘化丙锭添加于1μg/ ml的终浓度（1：100稀释的100微克/毫升）。使用自动细胞计数，以确定细胞计数和vi能力。用细胞计数设备推荐的软件进行流式细胞分析。确定白细胞绝对计数的原创，消极和积极的部分。 注：每毫升采取细胞计数分析样品的可行的白细胞的细胞数，使用的细胞分析软件建议确定的。 </li><li>设置区域，如图2（区域5，可行的白细胞）。使用以下的门控策略，以确定细胞计数。在原级分A可行白细胞见图2。 </li><li>所指示的区域（图2，1-6）是分层如下： 1：时间门→2：单细胞→3：CD45 +细胞 →4：白细胞（碎片除外）→5：活白细胞→6：活的淋巴细胞 </li><li>重复相同的步骤，以确定细胞计数的消极和积极的部分。计算整个部分的细胞计数通过考虑在样品和分数（表2）的总体积的稀释剂因子。 </li></ol> 4.考试的分离性能<ol><li>预冷的PBS / EDTA缓冲液/ 0.5％AB血清洗QCB，NTCB和TCB级分的细胞的等分试样。离心细胞，在300×g离心5分钟，在4℃，吸出上清液。 </li><li>悬浮细胞在含有100微升抗体 - 荧光染料染色混合物：CD3-FITC，CD4-APC，CD8-APC-Vio770，CD14-PerCP，CD20-PerCP，CD45-VioBlue和抗IFNγ-PE（滴度一时11）和孵育在黑暗中在4℃下10分钟。 </li><li>加入1毫升新鲜制备的红细胞溶解溶液（1x）和孵育15分钟，在室温。离心机在300×g离心5分钟，在4℃，吸出上清液。重悬在PBS / EDTA缓冲/ 0.5％AB血清的货量充足的细胞。 </li><li>加入碘化丙啶至1微克/毫升之前，为了分析（1最终浓度：100 diluti上的100微克/毫升）。进行流式细胞分析来评价样品的纯度。 </li><li>使用以下的门控策略来计算的CD3 + T细胞在活白细胞门控策略用于确定CD3 + T细胞示出在CCS浓缩过程后阳性分数。所指示的区域（ 图3A和3B，1-6）分层联如下： 1：时间门→2：单细胞→3：CD45 +细胞 →4：电池（碎片除外）→5：活白细胞→6：活CD3 +细胞群 </li><li>确定CD4 +，CD8 +，CD4 + IFN-γ+和CD8 + IFN-γ+的CCS富集处理后的T细胞（表2）的频率。 </li><li>使用门控策略来确定CD4 +，CD8 +，CD4 + IFN-γ+和CD8 + IFN-γ+ T细胞示出所有低于f的频率还是原始和丰富（捕获）阳性CCS处理后部分。所指示的区域被分层链接并命名如下： 1：时间门→2：单细胞→3：CD45 +细胞 →4：电池（碎片除外）→5：活白细胞→6：活CD3 +细胞→7：CD4 +细胞→7A：CD4 + IFN-γ+细胞（盒）→8：CD8 +细胞→8A：CD8 + IFN-γ+细胞（盒） </li></ol>注：层级链接的前6所示区域是相同的。图3中，（1-6）和最后2个区域示于图4（6-8a）。

<ol>
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这两个MD安德森癌症中心和Cooper博士拥有财务利益ZIOPHARM肿瘤，公司和Intrexon公司。 5月7日，2015年，Cooper博士被任命为ZIOPHARM肿瘤首席执行官。 Cooper博士现在是一个访问学者在MD安德森。 Cooper博士创立，拥有InCellerate，Inc.的他拥有的专利与Sangamo生物科学人工核酸酶。他担任过Targazyme公司（前身为美国干细胞公司），GE医疗集团，辉凌制药，命运治疗，扬森制药和百时美施贵宝。他是Cellectis公司的科学顾问委员会。他收到来自美天旎酬金。

继T细胞疗法已成为一个可行的办法来治疗B细胞恶性肿瘤4。其治疗潜力是依赖于注入缺乏复制衰老2靶抗原特异性T细胞的期望数量。这可以通过用当前的良好生产规范整理抗原特异性T细胞的纯人口从扩增的T细胞的合规性来实现。两个分拣程序被广泛应用，即，荧光激活细胞分选（FACS）和磁性活化细胞分选（MACS）来产生的CMV抗原特异性T细胞因为我们最近5审查。使用一个?...

造血干细胞移植（HSCT）1可以与过继性T细胞治疗相结合，以提高移植物抗肿瘤效果，并提供免疫机会感染2。产生抗原特异性供体来源的T细胞输注历来所需的技能型人才和使用专门的设施，是按照GMP标准。这种T细胞的递送导致的机会性感染3的分辨率以及治疗潜在恶性肿瘤4。最近，研究人员已经证明，只有几千病毒特异性T细胞的过继转移（〜1×10 4 - 2.5×10 5细胞/千克接受者体重）可以成功地治疗同种异体HSCT后5-9机会性巨细胞病毒感染。数量有限的GMP设施，相关精湛的制造要求和电池生产相关的成本高了，但是，restricted患者获得希望的T细胞疗法10。的一种方法分离抗原特异性T细胞是基于使用一个双特异性试剂来识别CD45和IFN-γ在CCS。如图所示，这种方法可以用于产生临床级的CMV特异性T细胞采用一种自动化细胞富集的CCS装置（ 图1B）。 通过将重叠肽从CMV病毒pp65抗原用感染CMV血清阳性供体白细胞分离总有核细胞（TNC）产生CMV特异性T细胞。这些肽在人类白细胞抗原（HLA）的环境中显示的，激活该TNC内CMV启动病毒pp65特异性T细胞分泌的IFN-γ。这些T细胞可以被“捕获”和磁性分离。第一代细胞富集装置（图1A）的操作所需的在细胞培养人员熟练GMP条件下，和协调工作人员承担的多个STEPS必须产生一个“俘获”的产品。 该过程通常需要10至12小时的连续运行，因此，人员可能需要工作在两班在GMP的设施。这些约束现在由第二代装置（ 图 1B所示）的执行避免。该装置进行磁性富集，与第一代设备，但在自动化的漂白的方法的CCS的其他方面。这显著降低作为大多数步骤可以完成无人参与由工作人员在GMP的球队的负担。另外，由于装置作为一个封闭系统中，抗原特异性T细胞可以被捕获并在除参与白细胞分离的分离和制备的材料启动仪器之前的步骤台式处理。完整的仪器和第二代细胞富集设备的功能性的细节已经酒馆lished 11。 这里，我们描述的步骤，从使用自动细胞富集的CCS系统稳态单采产品丰富的CMV pp65抗原特异性T细胞。一旦分离，这些CMV特异性T细胞可以被立即注入到病人体内。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Tubing Set TS 500</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Cytokine Capture System (IFN-gamma)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>279-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (Human) 25%&#160;</td>
			<td>Grifols</td>
			<td>58516-5216-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer/Spike Interconnector</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-701</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 % NaCl Solution (1 L)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP PepTivator HCMV pp65</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLEX GV Filter Unit 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS Freezing Bag 50</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Syringes, sterile</td>
			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Materials</td>
			<td>Manufacturer</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Serum, GemCell</td>
			<td>Gemini Bio-Products, West Sacramento, USA</td>
			<td>100-512</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-FITC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-080-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-APC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-Vio770</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-VioBlue</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
			<td></td>
		</tr>
		<tr>
			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-940</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Solution (100 &#181;g/mL)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">200-075-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Software V1.0.0.RC</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Software 2.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R&#160;</td>
			<td>Eppendorf AG</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2</td>
			<td>Nexelom Bioscience, Lawrence, MA</td>
			<td>LB-001-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubing welder SCDIIB</td>
			<td>Terumo Medical Corp., Elkton, MA</td>
			<td>7811</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

病原体特异性T细胞的过继转移可用于预防和治疗机会性感染如巨细胞病毒（CMV）感染异基因造血干细胞移植后发生。从供者，包括第三方捐助者病毒特异性T细胞，可以传播体外 ，符合现行良好生产规范（cGMP），采用多轮抗原驱动的刺激有选择性地繁殖所需的T细胞。抗原特异性T细胞的鉴定和分离，也可基于T细胞的细胞因子的捕获系统，该系统已经被激活以分泌γ-干扰素（IFN-γ）进行。然而，广泛的人类应用的细胞因子捕获系统（CCS），以帮助恢复免疫的已受到了限制生产过程是耗时，并且需要熟练的操作人员。第二代细胞富集设备，如CliniMACS神童发展到现在使研究者以产生使用自动化，更少劳动密集的系统的病毒特异性T细胞。该装置中分离磁使用磁激活细胞分选技术，产生临床级产品未标记细胞标记的细胞，被设计为封闭系统，并且可以进行访问和操作上的台式。我们证明这个新的自动化细胞富集装置的操作来制造从CMV启动血清阳性供体获得一个稳态单采产物获得的CMV pp65抗原特异性T细胞。这些分离的T细胞可以被直接注入机构和联邦监管监督下的患者。所有的生物处理步骤包括去除红血细胞，T细胞，抗原特异性T细胞，纯化和洗涤分离的刺激完全自动化。设备，如这提高的可能性，T细胞对人类应用能奉献良好生产规范GMP外（制造）设施，而是在血库设施的工作人员可以监控自动化协议，以生产多种产品的生产。

在这项研究中，一个自动细胞富集的CCS系统被用于自动化生产的CMV pp65抗原特异性T细胞。 CMV特异性T细胞从三个血液分离细胞产物富集。稳态单采产品收获了2小时从CMV血清阳性供体和生成的10 10总有核细胞（TNC）。 10 9 TNC然后用CMV病毒pp65衍生肽（60纳摩尔）4小时，所述IFN-γ的分泌使用自动化细胞富集设备上的CCS的T细胞中分离激活。操作者需要在实验开始时以装载所有的试剂和管?...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

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

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 ...

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  U.T. MD Anderson Cancer Center. 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.

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

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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 ...

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

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 antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

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 ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.