Name: enrich and expand rare antigen-specific t cells with magnetic nanoparticles
Uploaded: 2018-11-17T14:30:00
Description: Watch this Scientific Journal Video about Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles at JoVE.com

j. w. h. 感谢约翰·霍普金斯生物技术研究所的 nih 癌症纳米技术培训中心、国家科学基金会研究生研究金 (dge-1232825) 和 arcs 研究金支助基金会。这项工作得到了国家卫生研究院 (p01-a072677、r01-ca108835、r21-c185819)、tedco/to01 创新倡议和库尔特基金会 (jps) 的支持。

所有老鼠都按照约翰·霍普金斯大学机构审查委员会批准的指导方针进行维护。
1. 负载二聚类主要组织相容性复合免疫球蛋白融合蛋白 (mhc-ig) 与所需的抗原肽序列。
注: 如果使用 h-2: ig, 则按照步骤1.1 中详细介绍的协议进行操作; 如果使用 h-2 k:ig, 请遵循步骤1.1 中详细介绍的协议。如果使用 h-2db:ig, 则按照步骤1.2 中详细介绍的协议进行操作。
<ol><li>将肽?...

<ol>
	<li>Prasad, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=immunotherapy:+Tisagenlecleucel-the+first+approved+Car-t-cell+therapy:+implications+for+payers+and+policy+makers.">immunotherapy: Tisagenlecleucel-the first approved Car-t-cell therapy: implications for payers and policy makers.</a> Nature Reviews Clinical Oncology. 15 (1), 11 (2018).</li><li>Jenkins, M. K., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+naive+T+cell+precursor+frequency+and+recruitment+in+dictating+immune+response+magnitude.">The role of naive T cell precursor frequency and recruitment in dictating immune response magnitude.</a> The Journal of Immunology. 188 (9), 4135-4140 (2012).</li><li>Rizzuto, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-antigen-specific+CD8++T+cell+precursor+frequency+determines+the+quality+of+the+antitumor+immune+response.">Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response.</a> Journal of Experimental Medicine. 206 (4), 849-866 (2009).</li><li>Newell, E. W., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+model+antigens:+high-dimensional+methods+for+the+analysis+of+antigen-specific+T+cells.">Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells.</a> Nature biotechnology. 32 (2), 149 (2014).</li><li>Perica, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+and+expansion+with+nanoscale+artificial+antigen+presenting+cells+for+adoptive+immunotherapy.">Enrichment and expansion with nanoscale artificial antigen presenting cells for adoptive immunotherapy.</a> ACS nano. 9 (7), 6861-6871 (2015).</li><li>Kosmides, A. K., Necochea, K., Hickey, J. W., Schneck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+T+Cell+Targeting+Components+onto+Magnetically+Clustered+Nanoparticles+Boosts+Activation.">Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation.</a> Nano Letters. , (2018).</li><li>Hickey, J. W., Vicente, F. P., Howard, G. P., Mao, H. Q., Schneck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+Inspired+Design+of+Nanoparticle+Artificial+Antigen-Presenting+Cells+for+Immunomodulation.">Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation.</a> Nano Letters. 17 (11), (2017).</li><li>, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+enrichment+of+antigen-specific+T+cells+by+engineering+particle+properties.">Efficient magnetic enrichment of antigen-specific T cells by engineering particle properties.</a> Biomaterials. , (2018).</li><li>Oelke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+purification+of+CD8++melan-A-specific+cytotoxic+T+lymphocytes+for+adoptive+transfer+in+tumor+immunotherapy.">Generation and purification of CD8+ melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy.</a> Clinical Cancer Research. 6 (5), 1997-2005 (2000).</li><li>Riccione, K., Suryadevara, C. M., Snyder, D., Cui, X., Sampson, J. H., Sanchez-Perez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+CAR+T+cells+for+adoptive+therapy+in+the+context+of+glioblastoma+standard+of+care.">Generation of CAR T cells for adoptive therapy in the context of glioblastoma standard of care.</a> Journal of visualized experiments: JoVE. (96), (2015).</li><li>Ho, W. Y., Nguyen, H. N., Wolfl, M., Kuball, J., Greenberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+generating+CD8++T-cell+clones+for+immunotherapy+from+the+naive+repertoire.">In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire.</a> Journal of immunological methods. 310 (1-2), 40-52 (2006).</li><li>Rudolf, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+costimulation+of+human+CD8+T+cells+by+anti-4-1BB+and+anti-CD28+on+synthetic+artificial+antigen+presenting+cells.">Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells.</a> Cancer immunology, immunotherapy : CII. 57 (2), 175-183 (2008).</li><li>Gulukota, K., Sidney, J., Sette, A., DeLisi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+complementary+methods+for+predicting+peptides+binding+major+histocompatibility+complex+molecules1.">Two complementary methods for predicting peptides binding major histocompatibility complex molecules1.</a> Journal of molecular biology. 267 (5), 1258-1267 (1997).</li><li>Castle, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+mutanome+for+tumor+vaccination.">Exploiting the mutanome for tumor vaccination.</a> Cancer research. 72 (5), 1081-1091 (2012).</li><li>Duan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+bioinformatic+profiling+of+mutational+neoepitopes+reveals+new+rules+to+predict+anticancer+immunogenicity.">Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity.</a> Journal of Experimental Medicine. 211 (11), 2231-2248 (2014).</li><li>Srivastava, P. K., Duan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+antigenic+fingerprint+of+each+individual+cancer+for+immunotherapy+of+human+cancer:+genomics+shows+a+new+way+and+its+challenges.">Harnessing the antigenic fingerprint of each individual cancer for immunotherapy of human cancer: genomics shows a new way and its challenges.</a> Cancer Immunology, Immunotherapy. 62 (5), 967-974 (2013).</li><li>Yadav, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+immunogenic+tumour+mutations+by+combining+mass+spectrometry+and+exome+sequencing.">Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing.</a> Nature. 515 (7528), 572 (2014).</li><li>Gros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+neoantigen-specific+lymphocytes+in+the+peripheral+blood+of+melanoma+patients.">Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.</a> Nature medicine. 22 (4), 433 (2016).</li></ol>

我们创造了一种基于纳米粒子人工抗原呈现细胞 (aAPCs) 的抗原特异性 t 细胞分离技术。纳米粒子 aAPCs 表面含有含肽的 mhc, 可在共同刺激激活的同时, 实现抗原特异性 t 细胞的结合和激活。aAPCs 也是顺磁性的, 因此可以利用磁场来丰富罕见的抗原特异性 t 细胞。我们对纳米颗粒的尺寸、配体密度、配体选择及其对结合、富集、活化和细胞富集的影响等关键特性进行了优化和研究 (补充文件 1-box ...

随着许多免疫疗法的兴起, 有必要能够描述和控制免疫反应。特别是, 由于细胞的特异性和耐久性, 适应性免疫反应是有意义的。最近, 嵌合体抗原受体 t 细胞疗法已被批准用于癌症治疗;然而, 抗原受体根据共同的细胞表面抗原 cd19, 而不是抗原特异性的癌症1。除了特异性之外, 免疫疗法还可能缺乏控制, 对癌症或自身免疫功能内的动态免疫反应了解有限。
研究抗原特异性反应的挑战之一是其极低的频率,例如, 抗原特异性 t 细胞是每 10 4 至 106 t 细胞 2,3中的1个。因此, 为了研究哪些 t 细胞存在或响应, 细胞要么需要丰富和扩大, 要么需要放大其信号。使用目前专注于抗原特异性细胞扩张的技术来维护馈线单元是昂贵和困难的。目前的技术, 重点是放大抗原特异性 t 细胞的信号, 如酶联免疫罐 (elispot) 检测, 限制了这些 t 细胞4的再利用。最后, 由于灵敏度较低, 通常需要将这两种技术结合起来才能进行抗原特定的枚举。
为了解决这些问题, 我们开发了基于磁性纳米粒子的人工抗原呈现细胞 (aAPC)5,6,7,8。aAPC 可以功能化与抗原特异性信号肽加载主要组织相容性复合体 (pmhc) 和共同刺激分子-例如, 抗 cd28抗体既丰富抗原特异性 t 细胞, 然后随后刺激它们的扩张 (图 1)。因此, 这些颗粒可以是一种经济高效的现成产品, 既可以定制, 以满足抗原特定的刺激, 但在实验和患者之间实现标准化。执行富集和膨胀过程可使抗原特异性 cd8 + t 细胞膨胀数到千倍, 并可在短短一周内将频率提高到 60%, 从而实现大型 cd8 + t 细胞的表征或治疗使用。细胞的数量。本文介绍了纳米粒子 aAPCs 的制备方法, 在选择纳米粒子性能时的一些关键设计考虑因素, 并展示了利用这些粒子分离和扩展稀有抗原特异性 cd8 + t 细胞的一些典型结果。

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enrich and expand rare antigen-specific t cells with magnetic nanoparticles

我们开发了一种工具, 既能丰富又能扩大抗原特异性 t 细胞。这在 a) 检测抗原特异性 t 细胞的存在等情况下很有帮助, b) 探测抗原特异性反应的动态, c) 了解抗原特异性反应如何影响疾病状态, 如自身免疫性, d) 消除异质性的疾病状态抗原特异性 t 细胞 (e) 的反应利用抗原特异性细胞进行治疗。该工具是基于一个磁性粒子, 我们结合抗原特异性和 t 细胞共刺激信号, 我们将其称为人工抗原呈现细胞 (aAPCs)。因此, 由于该技术生产简单, 可方便地被其他实验室采用;因此, 我们在这里的目的是详细描述的制造和随后的使用的 apc。我们解释了如何将抗原特异性和共刺激信号附加到 aAPCs, 如何利用它们来丰富抗原特异性 t 细胞, 以及如何扩大抗原特异性 t 细胞。此外, 我们将根据我们在抗原特异性 t 细胞特征方面的经验的实验和生物学信息, 重点介绍工程设计方面的考虑。

为了成功地富集和扩展抗原特异性 t 细胞, 应成功地将含肽的 mhc-ig 和共刺激分子附着在 aAPC 粒子上。基于3种粒子连接方法, 我们为成功的共轭过程结果提供了一些有代表性的数据 (图 5a)。事实上, 如果配体密度太低, 那么在我们的经验中, 抗原特异性 cd8 + t 细胞就不会发生在100纳米以上配体之间的线性间距附近 (图 5b)<sup class="xre...

Watch this Scientific Journal Video about Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles at JoVE.com

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

抗原特异性 t 细胞由于其频率极低, 很难在治疗中表征或利用。在此, 我们提供了一个协议, 以开发一个磁性粒子, 它可以结合到抗原特异性 t 细胞, 以丰富这些细胞, 然后扩大他们的几百倍, 用于表征和治疗。

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

因此，我们新技术的主要优点，我们把它称为E+E，它代表富集加膨胀，是我们可以同时丰富和扩展这些细胞，罕见的抗原特异性细胞。这使我们能够开发足够数量的细胞，我们将能够用它来调查它们在不同的设置和不同的临床环境中的作用，甚至允许我们现在开发大量的细胞，我们可以利用免疫治疗的方式，我们不能之前。我们开发的方法，这个E加E，可以帮助回答基本免疫学中关于新皮素存在的关键问题。什么是新皮托佩斯？这些个性化表位，我们知道现在是非常重要的癌症免疫治疗和抗原特异性动力学，以及这些因素如何影响疾病状态和有效的细胞治疗。这种技术的影响延伸到免疫治疗，因为纳米粒子人工抗原呈现细胞可能是一种现成的产品，很容易为单个患者的免疫反应量身定做。为了产生纳米粒子人工抗原呈现细胞，将冻干颗粒重新悬浮在一毫升的再悬浮缓冲液中，并大力涡旋悬浮液至少15分钟，直到没有聚集物可见。在与蛋白质发生反应之前，未充分涡旋和再悬浮的粒子将导致更大的聚合颗粒无法分离。将磁性颗粒放在磁性支架上，取出上经剂，然后用 0.5 毫升的新鲜再悬浮缓冲液重新悬浮。将颗粒悬浮液转移到玻璃闪烁小瓶中进行涡流，直到没有聚集物可见，每一毫克重新悬浮的颗粒添加0.1毫克的总刺激信号。涡旋后，让悬浮液在室温下反应2.5小时，在旋转器中混合，然后加入0.1毫升淬火缓冲液，在室温下与混合混合，再孵育30分钟。在第二次反应结束时，将闪烁小瓶放在磁性支架上，将颗粒用一毫升的再悬浮缓冲液洗涤四次，每次洗涤后变清时取出缓冲液，并用新鲜的再悬浮缓冲液代替，以便随后对磁铁进行洗涤。上次洗涤后，吸出溶液并去除磁性支架上的颗粒，然后在四摄氏度的新鲜再悬浮缓冲液中储存颗粒长达六个月。为了分离抗原特异性CD8+T细胞，通过一个无菌的70微米细胞过滤器，通过频繁的PBS冲洗，从野生C57黑色6J小鼠中分离出的血红细胞和淋巴结，并按照制造商的指示使用无接触CD8+T细胞隔离套件，以消除非CD8+T细胞。计数后，通过离心收集分离的CD8+T细胞，并在PBS的100微升中重新暂停颗粒，并辅以0.5%牛血清白蛋白和2毫摩尔EDTA， 然后用纳米粒子人工抗原呈现细胞孵育细胞，使11肽加载的MHC-ig有1倍10倍，10倍到第六分离的CD8+T细胞在无菌的，五毫升聚苯乙烯圆底管在四摄氏度下持续混合一小时。在孵化结束时，如演示，在磁支架上三次清洗纳米粒子细胞悬浮液，在上次洗涤后用1%T细胞生长因子在500微升的新鲜、辅以的培养基中重新悬浮人工抗原呈现细胞和富集的CD8+T细胞。为了获得最大的细胞回收，将粒子和细胞留在磁柱上至少两分钟，并在不破坏受磁场影响的粒子和细胞的情况下小心吸气缓冲器至关重要。经过计数，种子2.5倍10至第五富集CD8+T细胞和磁性粒子混合物每160微升的补充介质加上1%TCGF在96井U底板。第三天，用80微升补充培养的细胞每井2%T细胞生长因子喂养细胞，将板回细胞培养箱四天以上。在第七天，将受刺激的细胞收获成一个五毫升的圆底管，用于计数和通过离心收集细胞。将颗粒在0.5毫升PBS中补充0.05%的阿齐德钠和2%的胎儿牛血清进行计数，5倍至4至5倍，第五细胞放入新的5毫升圆底管中，用于抗原特异性染色。用生物基化 MHC-ig 和抗小鼠 CD8a 标记适当的管，在 4 摄氏度下放置一小时， 然后在新鲜PBS中进行离心清洗，以去除任何多余的生物素化免疫球蛋白，然后用适当的链黄素结合二次抗体和适当的活死可修复死细胞菌株在4摄氏度下染色15分钟，并在流动细胞仪上读取细胞，根据标准协议确定抗原特异性CD8+T细胞的特异性和数量。在这里，使用三种不同的蛋白质附着在粒子上的方法实现了成功的蛋白质结合。如果APC配体密度过低，对抗原特异性CD8+T细胞不会进行有效刺激。生物素化二丁车的质量控制可以在转基因CD8+T细胞上完成，以验证染色。例如，这些代表性的结果表明，gp100特异性CD8+T细胞的阳性染色与非抗原特异性b6 CD8+T细胞作为背景对照。此外，了解生物素化二丁胺的背景染色至关重要，因为任何百分比低于同源染色管中的这种染色率都应被视为负面结果。经过七天的浓缩和扩张，从2倍10到第四和第二倍10到第五抗原特异性和5至50%CD8+T细胞可以预期从原来的5倍10到第六个内源CD8+T细胞起始群体。同样的方法可用于分离和刺激人类抗原特异性CD8+T细胞，在浓缩后仅一周后，观察到的抗原特异性CD8+T细胞的百分比和数量也类似增加。在尝试此过程时，必须记住执行相应的试剂质量检查，如前所述。按照这个程序，其他方法，如流细胞学细胞内细胞因子染色或表面蛋白质表达可以执行，以回答有关细胞的功能或表型的其他问题。这项技术为基础免疫学领域的研究人员探索其他生物医学领域（如癌症、传染病或自身免疫研究）抗原特异性反应的广度、深度和动力学铺平了道路。

Research

JoVE Journal

Engineering

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

相移窄粒度分布的纳米乳液的合成声波液滴蒸发和泡增强的超声介导消融

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

科研

工程学

Optical Trapping of Nanoparticles

纳米粒子的光学捕获

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

在固体氧化物燃料电池电极表面的探测和测绘

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ...

Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers

黄金纳米粒子作为软木塞氮掺杂碳纳米管杯的合成与功能构造

Nitrogen-doped carbon  nanotubes consist of many cup-shaped graphitic compartments termed as  nitrogen-doped carbon nanotube cups (NCNCs).  These ...

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties

电活性聚合物纳米粒子参展光热特性

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

电浆捕获和纳米粒子的释放在监控环境

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

采用部分氧化铝阴极增强电子注入和激子约束的纯蓝色量子点发光二极管

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

用畸变校正扫描透射电镜进行单数字纳米电子束光刻

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

大面积地下纳米化的可控、可定制金纳米粒子通过包盖湿法

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

用于寡核苷酸传递的可生多孔硅纳米粒子的合成、功能化和表征

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...