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）。

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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><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%&amp;nbsp;</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 &amp;mu;m&amp;nbsp;</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>&lt;strong&gt;Flow Cytometry Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</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-&amp;gamma;-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 &amp;micro;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>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>CliniMACS Prodigy Device&amp;nbsp;</td><td>Miltenyi Biotec GmbH</td><td>200-075-301</td><td></td></tr><tr><td>Software V1.0.0.RC</td><td></td><td></td><td></td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec GmbH</td><td>130-096-343</td><td></td></tr><tr><td>Software 2.4</td><td></td><td></td><td></td></tr><tr><td>Centrifuge 5415R&amp;nbsp;</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

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

科研

癌症研究

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

免疫与感染

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

用于分离多功能循环肿瘤细胞的临床微流控芯片平台

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...