我们要感谢阿龙Selya有益的讨论。这项工作是支持由美国国立卫生研究院授予AI29575，CA108835，AI077097的JS，从约翰霍普金斯疟疾研究所的试验赠款和国防部授予的PC 040972部莫

1。 HLA - A2 - IG制作，基于AAPC <ol><li>准备无菌硼酸盐缓冲液<ol><li>硼酸在水中溶解，使0.1M。调整pH至7.0。 </li><li>通过0.22μm的无菌过滤和储存过滤4 ° C。 </li></ol></li><li>准备无菌珠洗涤液<ol><li>以956毫升PBS W / O型的镁或钙。 </li><li>添加30毫升人AB血清。 </li><li>添加EDTA的2MM终浓度。 </li><li> 0.1gsodium叠氮化物。 </li><li>通过0.22μm的无菌过滤和储存过滤4 ° C。 </li></ol></li><li>从Invitrogen公司的M - 450环氧珠1毫升股票，约400万珠（珠可以通过血球计数），并放入无菌，螺杆顶部的玻璃小瓶。 </li><li>把小瓶针对DYNAL磁铁MPC - 1，而珠坚持一侧小瓶，除去吸上清液。一次洗珠，硼酸盐缓冲液1毫升。 </li><li>为1ml硼酸缓冲液HLA - A2免疫球蛋白二聚体的抗人CD28单克隆抗体（克隆9.3）和20微克和20微克的混合物悬浮珠。 </li><li>蛋白质珠共轭：上肩的玻璃小瓶和旋转4 ° 24小时。 </li><li> MPC - 1磁铁放置在管，并删除所有的硼酸盐缓冲。 </li><li>珠用1ml洗涤液洗磁珠两次。 </li><li>孵育1毫升珠洗涤液珠，旋转4 ° 24小时。由于珠洗涤液中含有人AB血清，它会阻止所有剩余的蛋白结合位点上的珠子。 </li><li>从玻璃小瓶中取出上清液，用1ml新鲜珠洗涤液取代。 </li></ol> 2。 AAPC肽加载，存储的质量控制<ol><li> 〜5 × 10 5珠添加100μL流式细胞仪，流式细胞仪管清洗缓冲和染色加入1μl抗鼠IgG1 - PE（识别的HLA - A2免疫球蛋白的Fc部分）和抗鼠IgG2a - FITC加入1μl（承认抗CD28单克隆抗体的Fc部分）。染色洗涤液的流式细胞仪20分钟后，洗一遍，并宣读染色结果立即用流式细胞仪（图4）。 </li><li>肽加载到珠：玻璃小瓶用1ml无菌PBS洗两次珠。用1ml无菌PBS重悬，然后加入加入10μl的CMV肽（1mg/ml的） </li><li>通过血球计数珠，标签的日期和浓度的小瓶。 </li><li> AAPC使用后至少3天肽的潜伏期在4 ° C，准备允许有足够的时间到HLA - A2免疫球蛋白二聚体肽结合。珠可以被保存在4 ° C，并保持至少6个月的功能。 </li></ol> 3。人类的CTL隔离<ol><li>收集来自健康的HLA - A2阳性捐助10 BD肝素钠真空采血管管〜新鲜的外周血百毫升。使用了21号针头或更大，以防止溶血。 </li><li>在室温下离心10分钟，在300xg </li><li>小心地取出吸顶级的等离子层。血浆可以用来作为补充培养基。 </li><li>替换用无菌PBS收集的血浆和血液转移到无菌的T75培养瓶或锥形管50毫升。用PBS的血液充分混合，上下吹打起来。 </li><li>一旦所有的血液收集，准备4个额外的50毫升锥形管和加入聚蔗糖 - 帕确加15毫升。 </li><li>慢慢地覆盖3035毫升上方聚蔗糖各管血细胞。接口之间的聚蔗糖和血细胞明显。 </li><li>在500xg离心机在室温下20分钟。打开刹车“关闭”和加速度尽可能低，以保持层与层之间的界面清晰。 </li><li>使用seriological吸管，小心吸外周血单个核细胞层，并收集到一个新的50ml的锥形管的PBMC中。当所有PBMC的收获添加400xg PBS和自旋30毫升10分钟。丢弃的上清，用PBS30毫升更多的时间来删除所有剩余的聚蔗糖。 </li><li>通过美天旎人力，CD8 + T细胞分离试剂盒，根据制造商的协议进行的CD8 + T细胞的分离。该试剂盒的高度丰富CD8 +细胞（通常&gt; 95％）消耗CD8 -细胞。 </li><li> CD8 + T细胞计数。预期的纯度应大于95％。为了证实这一点使用2 × 10 5细胞和执行CD4/3/8流式细胞仪分析。其余的CTL可以马上使用抗原特异性AAPC刺激，也可以冻结供日后使用。 </li></ol> 在体外 AAPC为基础的文化系统4。 <ol><li>准备培养基<ol><li>为TF（T细胞生长因子，在实验室4）2X的培养基：完全RPMI中加5％自体血浆和8％的T细胞生长因子的捐助。 </li><li&gt;捐助者的自体血浆可以代替人AB血清热灭活。 </li></ol></li><li>悬浮1万TF 2X培养液中加完全RPMI培养基8毫升8毫升的CTL，加1 × 10 6 AAPC，拌匀。 </li><li>到96 ü底部组织培养板，使用多渠道pipetter板细胞。 （每孔160μL） </li><li>培养细胞在37℃，5％的CO 2 incubater 7天。第4天与80μL/ TF 2X中等饲料细胞。 </li><li>细胞是准备7天收获。收获后，将细胞对磁铁，并删除旧的AAPC。 </li><li>抗原特异性，可根据制造商的协议，由四聚体染色。根据我们以前的研究3表型染色和细胞内细胞因子染色。 </li><li> replated提取的干细胞可以在相同条件下与AAPC再次。反复刺激后预计将增加细胞数量和抗原特异性。 </li></ol> 5。代表性的成果： AAPC后HLA A2 - IG和抗CD28的共轭的例子如图4所示。成功的蛋白共轭是显而易见的，由相应的抗体染色的明显转变。虽然外周血中的巨细胞病毒特异性CTL的频率通常是0.5-1％的AAPC介导的刺激单周后，特异性可以达到55 - 93％（图2和3）。抗原特异性CTL的扩张可以是非常不同的捐助者之间的变量，但结果是在同一捐助重现。扩大巨细胞病毒特异性细胞可以推断，数千倍，易制毒化学水平直接体外（数据未显示） 。细胞内细胞因子染色（图3）表明，这些扩大的CTL官能，而不是消耗殆尽，经过长时间的细胞培养和显着增生。 <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="图1"/> 图1代表AAPC基于前人类CTL的体外扩增流在异体造血干细胞移植的过继免疫治疗的图表<img src="/files/ftp_upload/2801/2801fig2.jpg" alt="图2"/> 图2代表四聚体染色结果AAPC产生一个文化周后巨细胞病毒特异性的CTL <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="图3"/> 图3。代表AAPC产生的巨细胞病毒特异性的CTL细胞内细胞因子染色结果细胞病毒（CMV特异性为61％ ） <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="图4"/> 蛋白共轭抗鼠IgG1 - PE和抗鼠IgG2的- FITC染色后的M - 450环氧珠代表染色结果图4。

<ol>
	<li>Walter, E. A. <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, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

AAPC系统，我们在这里描述的是一个有效率的制度，为人类CTL体外对多种抗原扩张。应特别注意在96孔板文化方面所采取的蛋白共轭的质量和均匀分布AAPC和CTL。使用这种方法我们已经能够扩大的CTL，为8周以上，在此期间，我们扩大抗原特异性CTL百万倍 4 。有各种人造的APC系统，利用细胞株或其他无细胞平台 5，但是，根据公布的数据，每个系统都有其独特的外形扩张和特异性方面支持不同的应用。更重要的是，自CTL的质量和数量的重要，我们的系统所产生的巨细胞病毒特异性CTL polyfunctionality预计赋予优异的抗病毒的效率。

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th></tr><tr><td>真空采血管管（含肝素） </td><td>碧迪</td><td> 367874 </td></tr><tr><td>人类CD8 + T细胞分离试剂盒</td><td>美天旎</td><td> 130-094-156 </td></tr><tr><td>磁珠M - 450环氧</td><td> Invitrogen公司</td><td> 140.11 </td></tr><tr><td> DYNAL MPC - 1磁铁</td><td> Invitrogen公司</td><td> 120 - 01D </td></tr><tr><td>聚蔗糖 - 帕确加</td><td> GE医疗集团</td><td> 17-1440-03 </td></tr><tr><td>的RPMI培养基1640 </td><td> Gibco公司</td><td> 11875 </td></tr><tr><td> HLA - A2免疫球蛋白二聚体X </td><td>碧迪</td><td> 551263 </td></tr><tr><td> iTAgMHC四聚体（HLA - A2，CMV）PE </td><td> Beckman Coulter公司</td><td> T20099 </td></tr><tr><td>猎鹰明确的96孔微孔板</td><td>碧迪</td><td> 353077 </td></tr><tr><td>鼠抗鼠IgG2a - FITC </td><td>碧迪</td><td> 553390 </td></tr><tr><td>羊抗鼠IgG1 - PE </td><td> Invitrogen公司</td><td> P21129 </td></tr><tr><td>人血清AB型</td><td>亚特兰大生物制品</td><td> S40110 </td></tr><tr><td>鼠抗人CD8a - FITC </td><td> Sigma - Aldrich公司</td><td> F0772 </td></tr><tr><td>鼠抗人CD8a - APC </td><td>碧迪</td><td> 340684 </td></tr><tr><td>鼠抗人IFNγ- FITC </td><td>碧迪</td><td> 340449 </td></tr><tr><td>小鼠抗人肿瘤坏死因子- PE </td><td>碧迪</td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL的最佳效应功能发挥的关键作用，在调解对各种细胞内感染和癌症的保护。然而，个人可能会出现抑制免疫微环境，在激活的CTL，其自体的抗原提呈细胞可能倾向于tolerize或anergize抗原特异性CTL。因此，虽然仍处于实验阶段，CTL为基础的过继免疫治疗已经发展成为一个很有前途的治疗各种疾病，如癌症和病毒感染。在最初的实验中，前体外扩增巨细胞病毒（巨细胞病毒）特异性CTL已用于免疫功能低下的异基因骨髓移植患者巨细胞病毒感染的治疗。虽然是很常见的危及生命的巨细胞病毒血症，在这些患者中没有接收病人扩大的CTL发展巨细胞病毒有关的疾病，这意味着抗CMV免疫过继转移的 CTL 1。可喜的成果也被观察到黑色素瘤，并有可能延长向其他类型的癌症2。 虽然有很多方法体外刺激和扩大人类的CTL，目前的办法是限制的成本和技术的限制。例如，目前的黄金标准是基于使用的自体DC。这就要求每一个病人捐出大量的白细胞，也很昂贵，费力。此外，在体外直流扩大CTL的特性详细透露，这些有唯一不理想的效应功能3。 这里我们提出一个高效的AAPC体外扩增人类巨细胞病毒特异性CTL过继免疫治疗（图1）为基础的系统。 AAPC作了耦合细胞大小的磁珠与人类HLA - A2免疫球蛋白二聚体和抗CD28mAb 4。一旦AAPC是，他们可以满载着各种感兴趣的肽，并保持个月的功能。在这份报告中，AAPC加载与巨细胞病毒，PP65（NLVPMVATV）占主导地位的肽。经过培养纯化与AAPC为期一周的健康捐赠者的人类CD8 + CTL，巨细胞病毒特异性CTL可显着增加的特异性高达98％（图2）和放大10,000倍多。如果需要更多的巨细胞病毒特异性CTL，进一步扩大可轻松实现与AAPC重复刺激。表型和功能分析显示，这些扩大的细胞效应记忆体的表型和TNFα和γ干扰素（图3）大量。

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

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

描述一个新的DC抗原特异性T细胞的诱导和扩张的独立方法。基于HLA A2 - Ig的人工抗原提呈细胞（AAPC）加载与HLA - A2限制性肽能够有效地扩大多样化的抗原特异性CTL的。该技术拥有CTL为基础的过继免疫治疗的巨大潜力。

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 ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

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

15.3K 次观看.  Johns Hopkins University. 描述一个新的DC抗原特异性T细胞的诱导和扩张的独立方法。基于HLA A2 - Ig的人工抗原提呈细胞（AAPC）加载与HLA - A2限制性肽能够有效地扩大多样化的抗原特异性CTL的。该技术拥有CTL为基础的过继免疫治疗的巨大潜力。

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

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

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

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

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

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

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...