这项工作得到了一个丹·邓肯的合作研究资助（CMB和EJS），美国国家心脏，肺，和血液研究所（US4HL081007），白血病和淋巴瘤协会临床研究学者奖（CMB），美国国家癌症研究所（RO1 CA06150816 EJS）。

1。单核细胞分离（0天） <ol><li>插入秒杀到出口的20％部分的脐带血阴鲁尔适配器，连接注射器和清除血液中。解冻血液转移到20毫升温热在50ml离心管中的RPMI。用5毫升的RPMI冲洗脐血袋和转移到相同的离心管中。 </li><li>离心细胞以400×g进行10分钟。吸出上清液。 </li><li>在20毫升的温暖的RPMI重悬细胞。到在50ml离心管中的15毫升淋巴制剂层细胞。离心40分钟@ 400×克。 </li><li>含有单核细胞的收获的接口，并在20毫升的RPMI清洗。离心10分钟@ 450×克。 </li><li>吸液上清液，并在20毫升的RPMI清洗。计数细胞和旋转5分钟@ 400×克。 </li><li>吸液上清液和重悬细胞，在5×10 6个细胞/ mL的Cellgenix：直流介质（无血清）。为EBV-LCL代的取出1毫升，放入15毫升CEntrifuge管。 </li></ol> 2。树突状细胞生成（第0天） <ol><li>板2毫升的细胞（已经在5×10 6细胞/ mL的直流媒体）成一个6孔板每孔。发表在1-2个小时的孵化器。 </li><li> 1-2小时后，洗掉非贴壁细胞，用PBS洗涤3-4次。收集非粘附细胞，并冷冻。加入2毫升/ DC含1000 U / mL的IL-4和GM-CSF 800 U / mL的。 </li><li> DC启动后3-4天，补充IL-4，加入100μL/孔含终浓度为1000U/mL IL-4和800 U / mL的GM-CSF和GM-CSF。 </li><li> 5-6天收获DC刮以及用移液管。计数的大细胞，离心5分钟@ 400×克。吸液媒体和重悬DC在2×10 6细胞/ mL的直流媒体。加入0.5 mL /孔24孔培养板。 </li><li>转导DC使用Ad5f35pp65的MOI为每个单元10传染性单位矢量。添加vector到24孔板的各孔中。孵育细胞1.5小时。 1.5小时后，加入1.5毫升/ DC媒体含1000 U / mL的IL-4，800 U / mL的GM-CSF，10 ng / mL的TNF-α，1μg/ mL的PGE-1，100毫微克/毫升IL-6，和10 ng / mL的IL-1β。这些细胞因子成熟的区议会。 </li></ol> 3。 EBV-LCL（从第0天开始）的生成<ol><li>含有5×10 6单核细胞的离心管。添加200μL浓EBV B95.8上清液和完整的媒体的1.8毫升的（RPMI + 10％FBS + 2mM的L-谷氨酰胺）含环孢霉素（1微克/毫升）。 </li><li>等分试样100μL的细胞分为10个孔的96孔板中，分为5口井和200μL。添加100μL完整的媒体+环孢素的孔中只有100μL的细胞。填写剩余的井用无菌水。 </li><li>饲料LCL每周和扩大从96孔板，24孔板后1〜2周。一个星期后，转移到T25烧瓶，并在随后的一星期转移到一个T75烧瓶中。在这个阶段，我们建议您冻结等分LCL。它通常需要1个月，以产生足够数量的LCL。 </li></ol> 4。 CTL启动 - 7天开始后的区议会<ol><li>嘉实区议会和30 Gy的照射。洗4 x和计数。 ：重悬直流在T细胞的介质，含人血清（45％RPMI，45％CLICK，10％人血清，2mM L-谷氨酰胺）@ 1×10 5个区议会/毫升。 </li><li>解冻从步骤2.2的非粘附细胞。洗涤细胞，并计数。重悬在2×10 6在含有人血清的T细胞介质的非贴壁细胞/ mL。加入10纳克/毫升的IL-7和IL-12，和5 ng / mL的IL-15。添加1毫升的细胞在24孔板上每孔。每孔加入1 mL的DC。填充空的24孔板的孔中，用无菌水。 </li><li>后7天，饲料和/或T细胞起始分裂细胞与T细胞的介质，含有人血清。 </li><li> T细胞后9-12天开始，冻结的T细胞，直到LCL准备。 </li><li> ØNCE LCL已经扩大，在T75烧瓶中传代，转导LCL 5分钟@ 400×g下离心。加入到细胞沉淀的载体在100传染性单位每个细胞的MOI。孵育1.5小时。 1.5小时后，重新悬浮在完整的媒体在5×10 5 LCL /毫升，并在24孔板中，每孔加入2毫升12 </li><li>术后第2天，收获LCL在40 Gy的照射。洗4 x和计数。重悬LCL在T细胞介质中含有2.5×10 5细胞/ mL的人血清。加入1 mL的LCL的24孔培养板每孔。此外，添加到的Grex40的培养装置，以及5 ng / mL的IL-15的5×10 6个 LCL 13 </li><li>收获的T细胞。离心机@ 400×g离心5分钟，吸出上清液，并重新悬浮在含有1×10 6个 T细胞/ mL的人血清的T细胞介质。加入5 ng / mL的IL-15，并加入1毫升/孔的T细胞（总数为1×10 6个 T细胞），已经在24孔板上的LCL。在的Grex40，带来介质的总体积达30毫升。 </li><li>每天3-4刺激后，如果细胞是汇合，然后将它们分割（板）1:1，并添加新鲜培养基中含50 U / mL的IL-2。如果他们不汇合，然后简单地吸½媒体和更换含50 U / mL的IL-2。在的Grex40，吸一半的媒体，更换新鲜的培养基，并添加50 U / mL的IL-2。 </li><li>在第7天，重复在步骤4.6，但使用的IL-2的刺激的天，IL-15的。 </li></ol> 5。代表性的成果按照GMP标准FDA批准的制造协议的示意图， 如图1所示。这个过程需要50天左右。典型的产生病毒特异性T细胞扩大预先存在的记忆T细胞，然而，脐血缺乏病毒经验丰富的T细胞，因此，我们需要素幼稚T细胞的体外方法。要做到这一点，我们使用的树突状细胞，以及细胞因子IL-7，IL-12和IL-15，必要产生病毒特异性。 经过3刺激，收率 ​​应该超过100×10 6个细胞。如果有足够的细胞是不可用时，可以进行额外的刺激，直到所需的号码的CTLs可用。这些细胞的大部分应该是CD3 +，CD4 +和CD8 + T细胞的混合物。应该有小于15％的NK细胞（CD3-/CD56 +）且小于1％CD19 + B细胞（ 图2）。 扩大的CTL识别的抗原病毒pp65从巨细胞病毒，六邻体和聚氯从腺病毒，以及众多的EBV抗原表达于EBV-LCL。测试时，酶联免疫斑点法，CTL分泌IFN-？响应于比不相干抗原（ 图3）的这些抗原。 ，CTL还应该裂解抗原肽脉冲目标这样的PHA爆炸。在51 Cr释放试验，CTL裂解LCL，CMVpp65脉冲和腺病毒六邻体和/或聚氯脉冲的目标，但不是脉冲与目标不相关肽（ 图4）。 <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="图1。"/> 图1。生成多病毒特异性CTL从脐带血 。示意图显示CTL制造的全过程，从脐带血。首先，脐带血单核细胞分离出20％的馏分的分馏脐血单元。散货代保存中的5×10 6个细胞的异常，然后，所有的细胞铺板在树突状细胞的介质为1-2小时，在该点的非粘附细胞的收获和冻结。的DC，然后输入DC媒体中IL-4和GM-CSF。经过5天的文化，是成熟的DC和巨细胞病毒抗原的免疫细胞病毒pp65的腺病毒载体转导的。发起，这些DC的非贴壁细胞，以及细胞因子IL-7，IL-12的结合，和IL-15。在随后的刺激，使用相同的腺病毒载体转导EBV-LCL，被用作抗原呈递细胞。 IL-15是用于在第二次刺激和其后的IL-2。 <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="图2。"/> 图2。产生的T细胞表型 。所示出的是活细胞的百分比，在淋巴细胞门控。 CTL是CD3 +，CD4 +或CD8 +，但主要CD3-/CD56-和CD19-。 <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="图3。"/> 图3的T细胞的功能 。由IFN-γELISPOT的T细胞特异性进行了测试。脉冲跨越整个六邻体蛋白，聚氯，病毒pp65的重叠肽，T细胞和不相关的抗原IE-1。 CTL单独表示单独的媒体。自体LCL照射，并添加1×10 5 /孔。显示的是的平均斑点形成细胞C指望。2-15的三个孔。 <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="图4。"/> 图4的CTL的细胞溶解活性 。所得的CTL裂解表达病毒抗原的目标的能力进行了测试中的51 Cr的检测。，跨越整个抗原或51 Cr-标记的自体LCL与CTL共培养的重叠肽脉冲的51 Cr-标记的自体的PHA爆炸。 4小时后，γ释放γ计数器上计数。

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作者有没有透露。

<html>目的是在控制病毒感染后，CBT目前的战略是有效的，但他们都与显著的毒性，价格昂贵，并且不授予长期保护，对以后的感染。事实上，一些抗病毒药物的使用可能会限制病毒特异性T细胞，否则将保护的膨胀。14另一种选择是来自供体的病毒特异性T细胞的输注。我们和其他人表明，这种T细胞是安全，有效和符合成本效益的。15日至17日在这里，我们表明，这种方法也可以扩展...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen公司</td><td> 21870-076 </td></tr><tr><td> DC媒体</td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA（点击的中等） </td><td> Irvine Scientific公司</td><td> 9195 </td></tr><tr><td>人血清</td><td>双子座生物制品</td><td> 100-110 </td></tr><tr><td>透气性Cultureware 18 </td><td>威尔逊狼</td><td> 80040S </td></tr><tr><td> IL-2的</td><td>凯龙（TCH药剂） </td><td></td></tr><tr><td> IL-12的</td><td> NCI / CTEP的</td><td></td></tr><tr><td> IL-15的</td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7的</td><td> R＆D </td><td> AFL207 </td></tr><tr><td> IL-1β </td><td> R＆D </td><td> AFL201 </td></tr><tr><td> IL-6的</td><td>细胞Genix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH药剂</td><td></td></tr><tr><td> IL-4的</td><td> R＆D </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-α的</td><td> R＆D </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGT载体生产设施</td><td></td></tr><tr><td>等离子传输带阴Luer适配器</td><td> “联合国宪章”医疗</td><td> 89-550-66J </td></tr><tr><td>淋巴制剂</td><td>奈科明</td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

干细胞移植后的病毒感染是最常见的死亡原因，尤其是在脐带血（CB）移植（CBT），CB不包含相当数量的病毒有经验的T细胞可以保护收件人感染。1 -我们和其他人表明，病毒特异性CTL产生的血清学阳性的捐助者，并注入到收件人的安全和保护。5-8然而，直到最近，病毒特异性T细胞不能产生脐带血，可能是由于病毒特异性记忆T细胞的缺乏。 为了更好地模仿体内吸幼稚T细胞的条件，我们建立了一个方法，该方法使用CB来源的树突状细胞（DC）的腺病毒载体（Ad5f35pp65）含有的免疫CMV抗原病毒pp65转，因此驱动T细胞特异性对巨细胞病毒和腺病毒。在开始，我们利用这些马捕获区议会，以及CB来源的T细胞的细胞因子的存在下，IL-7，IL-12和IL-15。[10]在所述第二刺激我们使用EBV-转化的B细胞，或EBV-LCL，同时表达潜在的和裂解EB病毒抗原。 ad5f35pp65转导的EBV-LCL是用于刺激的T细胞中的IL-15的存在，在第二次刺激。后续的刺激使用Ad5f35pp65转导的EBV-LCL和IL-2。 从50×10 6 CB单核细胞中，我们能够产生向上的150×10 6病毒特异性T细胞，裂解抗原脉冲的目标和释放细胞因子响应于抗原刺激11将这些细胞在GMP兼容的方式制造，使用仅分馏脐带血单元分数的20％，并已被翻译供临床使用。

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

在这里，我们描述的第一个好生产质量管理规范（GMP）标准的方法从脐带血幼稚T细胞的来源主要是病毒特异性细胞毒性T淋巴细胞（CTL）。

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 ...

expanding-cytotoxic-t-lymphocytes-from-umbilical-cord-blood-that

Research

JoVE Journal

Immunology and Infection

12.7K 次观看.  Baylor College of Medicine. 在这里，我们描述的第一个好生产质量管理规范（GMP）标准的方法从脐带血幼稚T细胞的来源主要是病毒特异性细胞毒性T淋巴细胞（CTL）。

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

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

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

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

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

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

科研

免疫与感染

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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 ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

径向移动性和逆转录病毒复制的载体转导，非粘附Alloresponsive T细胞的杀伤功能

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...