小鼠淋巴细胞通过标签<sup> 64</sup>的Cu-抗体受体靶向用于<em>体内</em>由PET / CT细胞运输
Summary
以下64的Cu改性的单克隆抗体结合到转基因小鼠的T细胞受体的制备中，T细胞在体内的放射性标记的，用于存活力，功能，标记稳定性和细胞凋亡分析，并过继转移到小鼠的气道迟发型超敏反应用于非侵入性成像通过正电子发射断层成像/计算机断层扫描（PET / CT）反应。
Abstract
该协议说明了生产64的Cu和随后的鼠淋巴细胞的细胞培养和64的Cu-抗体受体的细胞的靶向缀合的螯合剂/单克隆抗体（mAb）的放射性标记。 在的在气道迟发型超敏反应（DTHR）由PET / CT的动物模型的放射性标记和体内细胞跟踪非侵入性地描述体外评价。
详细地说，与螯合剂1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）mAb的共轭被示出。以下生产放射性64的Cu，所述DOTA缀合的mAb的放射性标记的描述。接着，鸡卵清蛋白（科瓦） -特异性CD4 +干扰素的膨胀（IFN）-γ的T辅助细胞（科瓦-TH1）和科瓦-TH1细胞的放射性标记随后被描绘。各种体外技术被提出来评价EF对细胞64的Cu-放射性标记的fects，如细胞活力的测定通过台盼蓝排除，染色的细胞凋亡与膜联蛋白V用于流式细胞术，和功能通过IFN-γ酶联免疫吸附测定法评估（ELISA） 。此外，放射性摄取进入细胞和标记稳定性的确定被详细描述。该协议还描述了如何在动物模型中进行小区跟踪研究为气道DTHR和，因此，在BALB / c小鼠科瓦引起的急性呼吸道DHTR的诱导是包括在内。最后，一个强大的PET / CT工作流程，包括图像采集，重建和分析被呈现。
相比普通PET-示踪剂细胞标记与随后受体内在64的Cu-抗体受体靶向方法提供高特异性和稳定性，降低的细胞毒性，和低流出速率， 例如 64铜-pyruvaldehyde双（N4-甲基氨基硫脲）（64的Cu-PTSM）。最后，我们的方法使得能够在由PET / CT 体内细胞跟踪非侵入性具有最佳信号-背景比为48小时。此实验方法可以被转移到不同的动物模型和细胞类型与被内化的膜结合受体。
Introduction
非侵入性细胞跟踪是监视在体内细胞的功能，迁移和归巢的多功能工具。最近细胞跟踪研究在再生医学中，自体外周血白细胞中在对抗癌症3,4过继细胞疗法的炎症或T淋巴细胞的上下文中都集中于间充质1,2或骨髓来源的干细胞3。作用部位和细胞疗法的基本生物学原理的阐明是极其重要的。 CD8 +细胞毒性T淋巴细胞，遗传工程嵌合抗原受体（CAR）的T细胞或肿瘤浸润淋巴细胞（TIL的）被广泛认为是金标准。然而，肿瘤相关抗原特异性T H 1细胞已被证明是一种有效的替代治疗方案4，</ SUP> 5，6，7。
如在炎症的主要参与者，器官特异性自身免疫性疾病（ 例如类风湿性关节炎或支气管哮喘），并在癌症免疫疗法高度感兴趣的细胞，它表征TH1细胞的时间分布和归巢模式是重要的。非侵入性体内通过PET成像提出了一种定量的，高度敏感的方法8检测细胞迁移模式， 在体内归巢，和的T细胞作用和响应过程中的炎症，过敏，感染或肿瘤排斥9，10，11中的位点。
在临床上，在111-喔星用于白细胞显像对炎症和感染12的辨别，而2-脱氧-2-（18F）氟-D-葡萄糖（18 F-FDG）由PET 3,13通常用于细胞跟踪研究。此PET示踪剂的一个主要缺点，但是，是的放射性核素（18）F在109.7分钟，阻碍在稍后的时间点成像后过继细胞转移的低细胞内稳定性的半衰期短。用于在由PET 体内细胞跟踪研究长期来看，虽然在不稳定的细胞中，64的Cu-PTSM经常用于非特异性地标记细胞14，15与T细胞活力最小化有害作用和功能16。
这个协议描述了一种方法，以进一步减少使用T细胞受体（TCR）特异性的放射性标记的mAb对细胞存活力和功能的不利影响。首先，生产放射性同位素64的Cu，所述的mAb KJ1-26与第共轭的ë螯合剂DOTA，和随后的64的Cu-放射性标记被示出。在第二步骤，分离和DO11.10供体小鼠的科瓦-TH1细胞的扩增，并用64的Cu-加载DOTA-缀合的mAb KJ1-26（64的Cu-DOTA-KJ1-26）放射性标记进行详细说明。通过γ计数，分别摄取值，并用一个剂量校准器的放射性流出的和评估，以及对64的Cu-放射性标记对细胞活力通过台盼蓝排除和功能用IFN-γELISA呈现效果的评价。对于体内细胞跟踪非侵入性的，由PET / CT过继细胞转移后科瓦引起的急性呼吸道DTHR和图像采集的小鼠模型的启发进行说明。
此外，这种标记方法可以被转移到不同的疾病模型，小鼠T细胞与不同的TCR或与膜结合受体或表达擦伤兴趣一般细胞KERS底层连续膜穿梭17。
Protocol
安全注意事项：当处理放射性，存储后面的2英寸厚的铅砖64 Cu和使用各自的屏蔽，用于携带活性的所有容器。使用适当的工具来间接处理非屏蔽来源，以避免直接用手接触，并尽量减少暴露在放射性物质。始终佩戴辐射剂量监测徽章和个人防护装备，并检查自己和污染的工作区域要立即解决这一问题。留在使用放射性物质的区域之前丢弃可能被污染的个人保护装备。直到放射性64?…
Representative Results
图1总结了科瓦-TH1细胞与64的Cu-DOTA-KJ1-26单抗和实验设计用于在体外和在包括在本协议体内研究的标记。 图1：64的Cu-DOTA-KJ1-26单抗贴标过程＆实验设计。放射性细胞标记的（A）示意性表示具有64</sup…
Discussion
该协议提出了一种可靠和简单的方法来通过PET稳定地放射性标记用于体内跟踪细胞。利用这种方法，科瓦-TH1细胞，分离的和在从供体小鼠体外扩增，可能是放射性标记的具有64的Cu-DOTA-KJ1-26单抗和它们的归巢被跟踪到肺和淋巴结perithymic如在科瓦呈现的网站COVA引起的急性呼吸道DTHR。
与螯合剂的mAb的修改需要快速高效的工作和无胺的痕迹使用超纯解?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢朱医生曼海姆，瓦尔特·埃利奇曼，拉莫纳·斯特姆，丰达礁，丹尼尔·布卡拉，马伦·哈兰特以及纳塔利·阿尔特迈耶的实验和数据分析过程中的支持。这项工作是由沃纳西门子基础上，通过SFB685东风集团（项目B6）和财富（2309-0-0）的支持。
Materials
| HCl, Suprapur | Merck, Darmstadt, Germany | 1.00318 | 64Cu production |
| Methanol, Suprapur | Merck, Darmstadt, Germany | 1.06007 | 64Cu production |
| Isopropanol, Suprapur | Merck, Darmstadt, Germany | 1.0104 | 64Cu production |
| Pt/Ir (90/10) plate | Ögussa | Custom made | 64Cu production |
| PEEK chamber | Ögussa | Custom made | 64Cu production |
| 64Ni | Chemotrade | 64Cu production | |
| Polygram SIL G/UV 254 plate | Macherey-Nagel | 805021 | 64Cu production |
| Ion exchange column | BioRad | AG1-X8 | 64Cu production |
| Solid state target system for PETtrace | WKL | costum made | 64Cu production |
| 64Cu work-up module | WKL | costum made | 64Cu production |
| Dose calibrator | Capintec | CRC-25R | |
| PETtrace cyclotron | General Electric Medical Systems | ||
| DOTA-NHS | Macrocyclics | B-280 | DOTA-conjugation |
| Anti-cOVA-TCR antibody (KJ1-26) | Isolated from hybridoma cell culture | DOTA-conjugation | |
| Na2HPO4 | Sigma-Aldrich | 71633 | DOTA-conjugation |
| H+ Chelex 100 | Sigma-Aldrich | C7901 | DOTA-conjugation |
| Amicon Ultra-15 filter unit | Merck Millipore | UFC910008 | DOTA-conjugation |
| Rotipuran ultrapure water | Carl Roth | HN68.3 | DOTA-conjugation |
| Ammonium acetate | Sigma-Aldrich | 32301 | DOTA-conjugation |
| PBS | University Tuebingen | DOTA-conjugation | |
| Micro Bio-spin P-6 column | Bio-Rad Laboratories | 7326221 | DOTA-conjugation |
| Sodium citrate | Sigma-Aldrich | 71497 | DOTA-conjugation |
| Cyclone Plus PhosphorImager | Perkin-Elmer | L2250116 | DOTA-conjugation |
| DMEM | Merck Millipore | 102568 | ingredient for T cell medium |
| FCS | Merck Millipore | S0115/1004B | ingredient for T cell medium |
| Sodium pyruvate | Merck Millipore | L0473 | ingredient for T cell medium |
| MEM-amino acids | Merck Millipore | K0293 | ingredient for T cell medium |
| HEPES | Merck Millipore | L 1613 | ingredient for T cell medium |
| Penicillin/Streptomycin | Merck Millipore | A2212 | ingredient for T cell medium |
| 0.05 mM 2-β-mercaptoethanol | Sigma-Aldrich | M3148 | ingredient for T cell medium |
| DO11.10 mice | in-house breeding | TH1 cell culture | |
| DPBS | Gibco | 14190144 | TH1 cell culture |
| Cell strainer 40 µm | Corning | 352340 | TH1 cell culture |
| ACK Lysing Buffer | Lonza | 10-548E | TH1 cell culture |
| CD4 MicroBeads, mouse | Miltenyi Biotech | 130-097-145 | TH1 cell culture |
| QuadroMACS separator | Miltenyi Biotech | 130-090-976 | TH1 cell culture |
| LS column | Miltenyi Biotech | 130-042-401 | TH1 cell culture |
| anti-CD4 antibody (Gk1.5) | Isolated from hybridoma cell culture | TH1 cell culture | |
| anti-CD8 antibody (5367.2) | Isolated from hybridoma cell culture | TH1 cell culture | |
| Anti-rat antibody (MAR18.5) | Isolated from hybridoma cell culture | TH1 cell culture | |
| Rabbit complement MA | tebu-Bio | CL3221 | TH1 cell culture |
| Anti-IL-4 antibody (11B11) | Isolated from hybridoma cell culture | TH1 cell culture | |
| cOVA 323-339-peptide | EMC-micro-collections | Custom order | TH1 cell culture |
| CPG1668-oligonucleotides | Eurofins MWG Operon | Custom order | TH1 cell culture |
| IL-2 | Novartis | 65483-116-07 | TH1 cell culture |
| 96-well plates | Greiner | 655180 | TH1 cell culture |
| 24-well plates | Greiner | 662160 | TH1 cell culture |
| cell culture flask | Greiner | 660175 | TH1 cell culture |
| 48-well plates | Greiner | 677 180 | cell labeling |
| Gammacell 1000 | Best Theratronics | via inquiry | |
| Gulmay RT225 | Gulmay | via inquiry | |
| Trypan blue | Merck Millipore | L6323 | in vitro evaluation |
| Mouse IFN-γ ELISA | BD Biosciences | 558258 | in vitro evaluation |
| PE Annexin V Apoptosis Detection Kit | BD Biosciences | 559763 | in vitro evaluation |
| Tube 5 ml | Sarstedt | 55.476 | in vitro evaluation |
| Round-bottom tubes | BD Biosciences | 352008 | in vitro evaluation |
| Wizard γ-counter | Perkin-Elmer | 2480-0010 | in vitro evaluation |
| ELISA Reader MultiscanEX | Thermo Fisher Scientific | 51118177 | in vitro evaluation |
| Microscope | Leica | via inquiry | in vitro evaluation |
| BD LSRII | BD Biosciences | via inquiry | in vitro evaluation |
| BALB/c mice | Charles River | 028 | in vivo cell trafficking |
| Aluminum gel | Serva Electrophoresis | 12261.01 | in vivo cell trafficking |
| Xylazine | Bayer HealthCare | Ordered via University hospital | in vivo cell trafficking |
| Ketamine | Ratiopharm | Ordered via University hospital | in vivo cell trafficking |
| Isoflurane | CP-Pharma | Ordered via University hospital | in vivo cell trafficking |
| 30G needle | BD Biosciences | 304000 | in vivo cell trafficking |
| Syringe | BD Biosciences | 11612491 | in vivo cell trafficking |
| Capillaries 10 µl | VWR | 612-2439 | |
| Inveon PET scanner | Siemens Healthineers | no longer available | in vivo cell trafficking, alternative companies: Bruker, Mediso |
| Inveon SPECT/CT scanner | Siemens Healthineers | no longer available | in vivo cell trafficking |
| Inveon Research Workplace | Siemens Healthineers | image analysis, alternative software: Pmod |
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Cite This Article
Hoffmann, S. H. L., Maurer, A., Reck, D. I., Reischl, G., Pichler, B. J., Kneilling, M., Griessinger, C. M. Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT. J. Vis. Exp. (122), e55270, doi:10.3791/55270 (2017).