支持这项工作是由一个细胞疗法（合同NIH - NHLBI（HB - 10 - 03）HHSN26820100000C）（CMR），一个专门的细胞治疗中心的生产援助赠款美国国立卫生研究院NHLBI 1 U54 HL081007（CMR），ASBMT青年研究者奖（UG和合资企业），一个白血病和淋巴瘤协会的临床研究奖（UG）的特别研究员，和艾米Strelzer Manasevit学者奖（AML）的。

1。直流nucleofection <ol><li>收获单核细胞衍生的区议会，已使用塑料的坚持，培养5天使用的细胞Genix媒体与IL - 4（1000U/ml）补充，GMCSF（800IU/ml），并进一步为使用DC的成熟细胞因子IL - 4（1000U 24小时成熟丰富/毫升），GMCSF（800IU/ml），IL6100ng/ml，TNF -α在10ng/ml，IL1 -β在10ng/ml和PGE2（1μg/ml）1，温柔的再悬浮与3毫升移液管。 </li><li>计数可行的区议会，使用，转移到3倍的15毫升管不下 0.5x10 6台盼蓝和不超过2 × 10 6细胞/管。 </li><li>离心10分钟@200克区议会。在这段时间内预温细胞Genix媒体辅以DC成熟的细胞因子（DC成熟媒体） - 2ml/well 12井井组织处理板在37 ° C / 5％CO 2培养箱培养。 </li><li>一旦细胞完成纺纱，吸出上清液，并添加相关的DNA质粒在终浓度为5μgDNA /管到每个管。在这种情况下，添加质粒IE1 - PP65管＃1，六邻体聚氯管＃2，EBNA1 - LMP2 - BZLF1管＃3。 </li><li>悬浮区议会及与DNA的Amaxa nucloefection溶液100μL，拌匀，并转移到nucleofection试管。 </li><li> 4D nucleofector试管，选择方案CB150（Amaxa /龙沙），并按下启动。 </li><li> nucleofection后立即添加2毫升预热细胞Genix DC成熟媒体试管500μL移液器向上和向下的2-3倍，并转移nucleofected区议会到准备好的12孔板包含剩余的1.5毫升的预热，轻轻混匀DC成熟的媒体。转移到37 ° C / 5％的CO 2培养箱中培养12 - 18小时进一步。 </li></ol> 2。 T细胞的刺激<ol><li> nucleofected区议会的收获和计数，并照射在30Gy。洗一次的CTL培养基用10ml（45％，45％的RPMI点击EHAA，10％FBS，2MM Glutamax）和重悬@ 3 × 10 5区议会每毫升的CTL媒体。 </li><li>池最低7.5x10 5（2.5毫升）和15x10 5（5毫升）含有质粒的每个区议会的最大汇集区议会和传输设备将被放置在孵化器的G - REX。 </li><li>对于响应者细胞的制备，使用或者原先被冻结的外周血单核细胞或单核细胞非贴壁后的DC选择（坚持或CD14的选择）保持。解冻的细胞，转移到预热的培养基，洗一次与CTL的媒体。重悬在CTL媒体细胞，细胞计数，并把他们带到浓度每毫升2 × 10 6细胞。以15x10 6细胞或7.5毫升和补充30000U IL - 4（1000U/ml -终浓度）和300ng IL7（在10ng/ml -终浓度）。 </li><li>转移的PBMC7.5毫升（15x10 6个细胞）的G - REX和顶起来的生物反应器的30毫升总量的CTL媒体。 </li><li>文化的G - REX为6-7天，37 ° C / 5％ 二氧化碳培养箱。 </li></ol> 3。 T细胞扩张<ol><li> 6-7天，吸10毫升媒体，再拌入其余20毫升在媒体的细胞与一个10毫升吸管和使用台盼蓝活细胞计数。如果有&lt;50x10 6补充新鲜媒体+因子。如果有&gt; 50x10 6个细胞，取出细胞悬液10毫升，转移到一个新的G - REX，然后与新鲜的CTL媒体+细胞因子的两个G - Rexs饲料。 </li><li>文化为4-6天。一旦足够的细胞已被扩大，执行的表型和功能特性以供将来使用CTL和冻存过剩。 </li></ol> 4。代表性的成果： 美国FDA批准的multivirus特异性CTL生成过程的示意图如图1所示 。相比之下公约multivirus CTL协议，该协议使用adenovectors和EBV - LCL刺激病毒的反应性T 细胞 2我们已经取代传染性病毒DNA质粒材料编码由每个病毒3产生的多种抗原。为了刺激trivirus的CTL，我们设计了三个multicistronic质粒编码的腺病毒六邻体和聚氯，IE1和PP65 CMV和EBNA1，LMP2，和BZLF1 EB病毒。这些抗原被选择的基础上，鼓励我们自己和其他群体显示，T细胞针对ADV -六邻体和聚氯 2,4-6和CMV - 1E1和CMV - PP65 保护 体内 7临床效果。对于EB病毒，EBNA1是一个免疫优势的CD4 + T细胞在所有EBV相关的恶性肿瘤，在正常的EB病毒感染的B细胞表达的靶抗原8,9，LMP2是跨多个HLA类型在大多数EB病毒恶性肿瘤的免疫原性和表示，10,11，而BZLF1编码免疫优势，即早裂解周期的抗原刺激CD4 +和CD8 + T细胞和大多数个人的合作是可能的重要ntrol细胞复制病毒12。为了进一步优化我们的制造方法，我们生成的CTL刺激13,14 minimalized，无抗生素（FDA标准）质粒与自然科技合作。使用这一战略，我们坚持实现nucleofection效率&gt; 35％，同时保持较高的细胞活力（数据未显示 ）3，图2显示了病毒特异性T细胞的频率响应优化IFNγELISPOT测定DNA质粒，更大的比pShuttle基于传统的表达质粒，表达相同抗原（N = 8腺病毒，N = 4 CMV和N = 2 EB病毒）。强有力的T细胞的刺激， 如在图3中，其中一个1:50的比例制作的次优激活到一个1:20 s相比所示：R比（N = 2的捐助者）的DC的最佳比例：外周血单个核细胞是重要的。生产足够广泛的抗原特异性CTL的数字是对所有三种病毒的临床疗效的先决条件。这是通过在G - REX，它支持出众的T细胞扩张相比与传统的24孔板（ 图 4A）15的CTL文化，而​​IL - 4和IL7除了增加文化的剧目和特异性如图4B所示反应性T细胞对巨细胞病毒- PP65衍生的HLA - A2的频率只供NLV肽进行了评估，存在或IL - 4和/或IL7 16,17的情况下产生的文化。为了扩大我们进行流式细胞仪分析细胞，细胞内细胞因子染色/γ干扰素ELISPOT，和最终产品的冷冻保存/输液铬51释放试验评估的表型和功能的能力。通常情况下，生成的细胞混合人口与CD4 +和CD8 + T细胞与抗原特异性两个T细胞车厢的检测多克隆。的CTL能够杀死病毒抗原表达的靶细胞，但不是病毒呈阴性反应的部分- HLA匹配的目标，这表明他们不应该诱导体内移植物抗宿主病（GVHD）（图5）。 <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="图1"/> 图1。rCTL代协议。首先，区议会nucleofected与病毒抗原编码质粒，然后与自体外周血单个核细胞在一个R混合：小号10或20:1。在G - REX 10-14天的IL - 4和IL7存在，然后收获，计算，测试功能，身份和不育，然后供临床使用冻存的细胞扩大。 <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="图2"/> 图2。优化的DNA 质粒在体外诱导优越的T细胞活化。区议会是nucleofected与优化，符合美国FDA的质粒编码六邻体和聚氯（ADV），IE1和PP65（CMV）和EBNA1，LMP2，和BZLF1（EB）或常规pShuttle质粒编码相同的抗原。这些都是用来刺激T细胞和特异性是由γ干扰素ELISPOT后10天刺激分析。 <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="图3"/> 图3优化直流：CTL的激活T细胞的比率。 2捐助者的区议会nucleofected与所有三个优化质粒，然后用刺激自体外周血PBMC中的比例在1:20或1:50直流。激活的T细胞的频率γ干扰素ELISPOT 10天评估。 <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="图4"/> 图4。使用增强因子的G - REX的T细胞扩增。 A组的G - REX设备以及外观透氧膜显微镜评估CTL。一个处理板VS的G - REX公约组织文化取得的细胞输出之间的比较也同时显示。 B组显示CMV的五聚体阳性的CTL在无细胞因子，IL - 4单独IL7单独和IL - 4 + IL7存在扩大文化方面取得的频率。 <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="图5"/> 图5。扩大CTL的表型和功能 。 小组一显示了一个在扩大multivirus的CTL，CD4 +（45％ -辅助）的混合多克隆表型的代表性的例子和CD8 +（42％ -细胞毒性）T细胞，其中的大部分（95％ ）表示的内存标记CD45RO + / CD62L +。 小组乙表明，这些细胞是针对所有的刺激抗原，并是多官能为细胞内细胞因子染色评估检测生产的IFNγ和抗原刺激后肿瘤坏死因子。C组显示的扩大的CTL作为衡量功能CR 51检测。自体拼箱，单独或​​与空载体或腺病毒载体表达CMV - PP65转用于焦油获取。 Alloreactivity评估使用异体PHA的爆炸目标。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

胡安楼维拉威尔逊狼制造顾问。其他作者有没有相关的信息披露。

病毒感染占谁是他们的疾病，其治疗免疫功能低下的患者的发病率和死亡率大幅。例如，移植后，如EB病毒和巨细胞病毒，以及由呼吸道病毒如呼吸道合胞体病毒（RSV）的持久性疱疹病毒感染引起的，众所周知的，而ADV，BK病毒引起的感染的重要性，和人类疱疹病毒-6型（HHV）最近受到了赞赏。虽然药理代理商，他们都具有相当的毒性，对一些感染的标准疗法产生抗药性的变种，并经常无效。与此相反，病毒特异性T细胞干细胞捐献者所得已被证明是安全的和有效的预防和治疗病毒感染或疾病的造血干细胞移植（HSCT） 的设置2,5,6,18-21。然而，T细胞免疫的更广泛的执行最终是有限的煤制油生产成本，复杂性和所需的时间。 我们的新的和快速的方法来生成multivirus的CTL，在当前的手稿描述，应该大幅增加细胞毒性T细胞治疗病毒性疾病的可行性，使战略成为护理免疫功能低下的主机的标准。质粒nucleofected区议会作为装甲运兵车的使用，使两个MHC I类和II不受病毒载体或多个单个细胞内表达，因为不同的直流人群利用每个质粒3的病毒抗原的竞争的抗原提呈。使用IL - 4 / 7增加T细胞的生存和增殖，从而相应增加的频率和响应抗原特异性T细胞16,17剧目。最后，在G - REX文化极大地减少了在文化T细胞凋亡。气体交换（O 2和CO 2出）发生跨透氧的硅膜在烧瓶底部，防止缺氧的同时，允许更大的中等深度以上的细胞，提供更多的营养物质和稀释废物的产品。这个平台也可以扩展至额外的病毒保护性抗原，当确定。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005年</td></tr><tr><td> IL - 4 </td><td> R + D系统</td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td>胎牛血清的RPMI 1640 </td><td> Thermo Scientific的</td><td> SH30096.01 </td></tr><tr><td>人AB血清</td><td>谷生物医学公司</td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa /龙沙</td><td> AAF - 1001B AAF - 1001X </td></tr><tr><td> Nucleofection套件</td><td> Amaxa /龙沙</td><td> V4XP - 3012 </td></tr><tr><td>质粒</td><td> NTC </td><td> N / A </td></tr><tr><td> GM - CSF的</td><td>研发</td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td>研发</td><td> 201 - LB - 025 </td></tr><tr><td> IL - 6 </td><td>研发</td><td> 206 - IL - CF </td></tr><tr><td>肿瘤坏死因子</td><td>研发</td><td> 210 - TA - 010 </td></tr><tr><td>前列腺素E2 </td><td>西格玛</td><td> P6532 - 1MG </td></tr><tr><td> G - REX </td><td>威尔逊狼制造</td><td> AY11 - 00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

病毒感染引起的异体造血干细胞移植（HSCT）受助人的发病率和死亡率。我们和其他人已经成功地生成和注入EB病毒（EBV）的，巨细胞病毒（CMV）和腺病毒（ADV）使用单核细胞和T细胞的具体EB病毒转化的淋巴母细胞（EB病毒LCL）基因修饰的腺病毒载体抗原提呈细胞（APCs）。由几个日志数为2 × 10 5 /公斤trivirus，特定的细胞毒性T淋巴细胞（CTL）的增殖和输液后出现甚至是严重的病毒病，耐其他可用的治疗，以预防和治疗。这个令人鼓舞的做法更广泛的执行是有限的，生产成本高，制造复杂和准备时间延长（4-6周EBV拼箱货代4-8周的CTL制造 - 共10-14周）。为了克服这些限制，我们已经开发了一个新的GMP标准CTL的生产协议。首先，在地方adenovectors刺激T细胞，我们使用的树突状细胞（DCs）DNA质粒编码的LMP2，EBNA1和BZLF1（EB），六邻体和聚氯（ADV），和PP65和IE1病毒（CMV）抗原提呈nucleofected细胞。这些装甲运兵车，所有的刺激抗原激活T细胞具体。其次，文化激活T细胞中存在的IL - 4（1000 U /毫升）和IL - 7（在10ng/ml）增加，并维持在我们行的剧目和特异性T细胞的频率。第三，我们已经使用了一个新的，透氧培养装置（G - REX），以促进一个单一的刺激后大细胞数量的扩张和生存，从而消除了EBV - LCLS的要求，减少技术人员的干预。通过实施这些变化，我们现在可以生产multispecific的CTL，EB病毒，CMV和高级定位在每 10 6细胞减少&gt; 90％的成本，并在短短10天，而不是10个星期使用可扩展到其他的方法防护病毒抗原。我们的美国FDA批准的的方法，应为高风险的异基因造血干细胞移植接受者预防和治疗的应用价值。

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

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

一种快速，简便和成本效益的协议为代捐助衍生multivirus特异性CTL（rCTL）输注异体造血干细胞移植（HSCT）在发展巨细胞病毒，高级或EBV感染的风险收件人。这制造过程是按照GMP标准，并应确保实施专门的中心之外的更广泛的T细胞免疫。

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 ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K 次观看.  Baylor College of Medicine. 一种快速，简便和成本效益的协议为代捐助衍生multivirus特异性CTL（rCTL）输注异体造血干细胞移植（HSCT）在发展巨细胞病毒，高级或EBV感染的风险收件人。这制造过程是按照GMP标准，并应确保实施专门的中心之外的更广泛的T细胞免疫。

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

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 ...

科研

免疫与感染

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

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

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

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

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

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

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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

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

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

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

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

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...