在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量
Summary
在这里,我们提出了一个协议,以诱导和评分疾病在异种移植-宿主疾病(xenoGVHD)模型。xenoGVHD为研究人类T细胞的免疫抑制提供了一个体内模型。此外,我们介绍如何使用数字PCR检测组织中的人类T细胞,作为量化免疫抑制的工具。
Abstract
急性移植物对宿主疾病(GVHD)是接受造血干细胞移植治疗血液学缺陷和恶性肿瘤的患者的重大限制。当供体T细胞识别宿主组织为外来抗原并对宿主进行免疫反应时,就会发生急性GVHD。目前的治疗涉及毒性免疫抑制药物,使患者容易感染和复发。因此,有正在进行的研究,以提供急性GVHD治疗,可以有效地瞄准供体T细胞和减少副作用。临床前的大部分工作都使用异种性GVHD(xenoGVHD)鼠模式,该模型允许在人体细胞上测试免疫抑制疗法,而不是体内体内的鼠细胞。该协议概述了如何诱导xenoGVHD以及如何使临床评分失明和标准化,以确保结果一致。此外,该协议还描述了如何使用数字PCR检测小鼠组织中的人类T细胞,随后可用于量化经测试疗法的疗效。xenoGVHD模型不仅提供了一个测试GVHD疗法的模型,而且提供了任何可以抑制人类T细胞的疗法,然后可以应用于许多炎症性疾病。
Introduction
异体造血干细胞移植(HSCT)已成为血液恶性肿瘤患者(如白血病,预后不良)的常规治疗。HSCT 的一个重要并发症是急性移植物与宿主疾病 (GVHD)。2012年的一项研究报告说,39%接受兄弟姐妹移植的HSCT患者和接受不相关捐赠者移植的患者中有59%出现急性GVHD。急性GVHD发生在供体衍生的T细胞攻击受体的器官时。GVHD唯一成功的治疗方法是使用高免疫抑制药物2进行治疗,这种药物毒性极强,增加了感染和肿瘤复发的风险。因此,尽管近年来急性GVHD存活率有所改善,但仍然迫切需要改进GVHD疗法,其毒性最小,能够促进长期缓解。
以下方法的总体目标是诱导和评分异种性GVHD(xenoGVHD)。xenoGVHD模型被开发为一种工具,诱导急性GVHD与人体细胞,而不是鼠细胞允许更直接地翻译临床前GVHD研究临床试验6。该模型涉及静脉注射人外周血单核细胞(PBMC)到NOD-SCID IL-2Rnull(NSG)小鼠,亚致命性辐照。注入的人类T细胞被人类抗原呈现细胞(ApCS)激活,呈现鼠抗原,被激活的T细胞迁移到遥远的组织,导致全身炎症,最终死亡6,7,8,9,10.疾病病理学和进展在xenoGVHD模型密切模仿人类急性GVHD。具体来说,致病性人T细胞对鼠主要组织相容性复合物(MHC)蛋白有反应性,类似于人类GVHD6、9中的T细胞均能活性。xenoGVHD模型比小鼠MHC不匹配模型,另一个广泛使用的GVHD模型的主要优点是,它允许测试在人类细胞,而不是小鼠细胞的治疗。这允许测试产品,可以直接翻译到诊所,没有任何修改,因为它们是针对人类细胞。最近,该模型已用于测试人类抗IL-2抗体11、人类胸腺调控T细胞(Tregs)12和人类中位体干细胞13作为急性GVHD的潜在治疗方法。在更广泛的环境中,该模型可用作任何药物或细胞类型,可以抑制人类T细胞活性的体内抑制测定。例如,Stockis等人14使用xenoGVHD模型来研究阻断整数αV+8对体内Treg抑制活性的影响。因此,xenoGVHD模型可以提供对在体内环境中针对T细胞的任何治疗机制的洞察。
该协议中描述的另一种方法是如何使用数字聚合酶链反应(dPCR)检测小鼠组织中的人类T细胞。该方法的目的是提供一个工具,以量化目标组织中T细胞的迁移和增殖,从而测量在此模型中测试的免疫抑制疗法的疗效。dPCR是核酸15定量的一种相对较新的方法。简单地说,PCR反应混合物被划分为包含少量目标序列或根本没有目标的分区。然后,使用DNA间插染料或荧光靶特异性探针放大和检测目标序列。dPCR根据正分区和泊森的统计数据15、16的百分数对目标序列的拷贝数进行量化。与其他替代方法(包括流式细胞学和组织学)相比,使用 dPCR 检测 T 细胞所需的组织要少得多,并且可以在冷冻或固定组织上执行。dPCR 不需要标准曲线来确定拷贝号,也不需要技术复制。与传统定量PCR(qPCR)16相比,这减少了dPCR所需的试剂和模板DNA的数量。将PCR反应划分为dPCR中的亚反应,有效地集中了目标17。因此,dPCR主要是检测大量非目标DNA中罕见靶点的工具。例如,dPCR用于检测牛奶18中的细菌污染,识别雌激素受体基因19中的罕见突变,并检测患者血液中的循环肿瘤DNA20。在此协议中,dPCR作为检测和量化具有xenoGVHD的小鼠组织中人类T细胞的有效工具。
Protocol
所有小鼠实验均符合堪萨斯大学医学中心机构动物护理和使用委员会的批准。所有健康的人类血液样本都是在知情同意下取得的,并经堪萨斯大学医学中心机构审查委员会批准。 1. NSG小鼠的辐照 在PBMC注射前一天,照射8-12周大的NSG小鼠(可使用任何性别)。在无菌生物安全柜中,将小鼠放入消毒的馅饼笼或微隔离器中。在Cs137源或小型动物辐照器(例如,RS 2000)中照射小…
Representative Results
接受人类PBMC的8-12周大NSG小鼠在注射后第10天左右开始出现GVHD的临床症状,而只接受PBS的阴性对照小鼠则开始出现GVHD的临床症状(图1A)。XenoGVHD小鼠的中位存活期为23.5天(图1B)。使用数字PCR,CD3 epsilon阳性人类T细胞可以在接受人类PBMC的小鼠的肺和肝脏样本中检测到。注射PBS的小鼠的组织样本被用作对照组(图2)。</strong…
Discussion
在 xenoGVHD 模型中,疾病进展通常是一致的,即使从不同的捐赠者注射 PBMC,因此可以组合多个实验。保持这种一致性所需的关键步骤是适当的 i.v. 注射技术、致盲和一致的评分。Nervi等人25日的研究表明,与静脉注射尾静脉注射相比,PBMC的逆轨注射可产生更一致的移植和更严重的GVHD。Leon-Rico等人26日还表明,与尾静脉注射相比,逆转轨道注射在小鼠体内的造血干细胞移植?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Lane Christenson 实验室提供这些实验中使用的数字 PCR 机器以及提供的技术支持。我们还要感谢托马斯·扬基博士的指导和指导。这些研究得到了特里普家庭基金会的支持。
Materials
| 1.5 mL eppendorf tubes | Fisher | 05-408-129 | |
| 10 mL serological pipet | VWR International | 89130-898 | |
| 10mL BD Vacutainers – Green capped with Sodium Heparin | Becton Dickinson | 366480 | |
| 250 µL Ranin pipette tips | Rainin | 17001118 | Do not use other pipettes or pipet tips for droplet generation |
| 50 mL conical tube | VWR International | 89039-656 | |
| 96-Well ddPCR plate | Bio-Rad | 12001925 | |
| ACK (Ammonium-Chloride-Potassium) Lysing Buffer | Lonza | 10-548E | Optional |
| Alcohol Wipes | Fisher Scientific | 6818 | |
| Anesthesia Chamber | World Precision Instruments | EZ-178 | Provided by animal facility |
| Anesthesia Machine | Parkland Scientific | PM1002 | Provided by animal facility |
| BD Vacutainer Safety-Lok Blood Collection Set | Becton Dickinson | 367281 | |
| DG8 Cartridges and Gaskets for QX100/QX200 Droplet Generator | Bio-Rad | 1864007 | |
| DNAse and RNAse free Molecular Grade H2O | Life Technologies | 1811318 | |
| Ethyl alcohol, Pure,200 proof, for molecular biology | Sigma-Aldrich | E7023-500ML | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
| Ficoll | Fisher Scientific | 45001750 | |
| Insulin Syringe | Fisher Scientific | 329424 | |
| Isoflurane | Sigma-Aldrich | CDS019936 | Provided by animal facility |
| Liquid nitrogen | N/A | N/A | |
| Mouse Irradiator Pie Cage | Braintree Scientific, Inc. | MPC 1 | Holds up to 11 mice |
| Nexcare Gentle Paper Tape (a.k.a. 3M Micropore Surgical Tape / 3/4") | Fisher Scientific | 19-027-761 | |
| P1000 pipetman | MidSci | A-1000 | |
| P200 pipetman | MidSci | A-200 | |
| Pierceable Foil Heat Seal | Bio-Rad | 1814040 | |
| Pipetaid Gilson Macroman | Fisher Scientific | F110756 | |
| Pipet-Lite Multi Pipette L8-200XLS+ | Rainin | 17013805 | Do not use other pipettes or pipet tips for droplet generation |
| Qiagen DNeasy Blood and Tissue Kit | Qiagen | 69506 | |
| qPCR plates | VWR International | 89218-292 | |
| QX200 Droplet Digital PCR System | Bio-Rad | 12001925 | Includes droplet generator, droplet reader, laptop computer, software, associated component consumables, for EvaGreen or probe-based digital PCR applications |
| QX200 Droplet Generation Oil for EvaGreen | Bio-Rad | 1864006 | |
| QX200 ddPCR EvaGreen Supermix | Bio-Rad | 1864033 | |
| RNase and DNase-free plate seal | Thermo Scientific | 12565491 | |
| RPMI Advanced 1640 | Life Technologies | 12633012 | |
| Sterile Gauze Pads (2" x 2", 12-Ply) | Fisher Scientific | 67522 | |
| Sterile Phosphate Buffered Saline | Fisher Scientific | 21040CV | |
| Sterile reservoir | VWR International | 89094-662 | |
| Surgial Scissors | Kent Scientific | INS600393-4 | |
| Surgical Forceps | Kent Scientific | INS650914-4 |
References
- Jagasia, M., et al. Risk factors for acute GVHD and survival after hematopoietic cell transplantation. Blood. 119 (1), 296-307 (2012).
- Bolanos-Meade, J., et al. Phase 3 clinical trial of steroids/mycophenolate mofetil vs steroids/placebo as therapy for acute GVHD: BMT CTN 0802. Blood. 124 (22), 3221-3227 (2014).
- Gooley, T. A., et al. Reduced mortality after allogeneic hematopoietic-cell transplantation. New England Journal of Medicine. 363 (22), 2091-2101 (2010).
- Hahn, T., et al. Significant improvement in survival after allogeneic hematopoietic cell transplantation during a period of significantly increased use, older recipient age, and use of unrelated donors. Journal of Clinical Oncology. 31 (19), 2437-2449 (2013).
- Khoury, H. J., et al. Improved survival after acute graft-versus-host disease diagnosis in the modern era. Haematologica. 102 (5), 958-966 (2017).
- King, M. A., et al. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex. Clinical & Experimental Immunology. 157 (1), 104-118 (2009).
- Lucas, P. J., Shearer, G. M., Neudorf, S., Gress, R. E. The human antimurine xenogeneic cytotoxic response. I. Dependence on responder antigen-presenting cells. Journal of Immunology. 144 (12), 4548-4554 (1990).
- Ito, R., et al. Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice. Transplantation. 87 (11), 1654-1658 (2009).
- Kawasaki, Y., et al. Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes, and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease. Biology of Blood and Marrow Transplantation. 24 (8), 1563-1574 (2018).
- Ito, R., et al. A Novel Xenogeneic Graft-Versus-Host Disease Model for Investigating the Pathological Role of Human CD4(+) or CD8. T Cells Using Immunodeficient NOG Mice. American Journal of Transplantation. 17 (5), 1216-1228 (2017).
- Trotta, E., et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism. Nature Medicine. 24 (7), 1005-1014 (2018).
- Dijke, I. E., et al. Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells. American Journal of Transplantation. 16 (1), 58-71 (2016).
- Huang, F., et al. Human Gingiva-Derived Mesenchymal Stem Cells Inhibit Xeno-Graft-versus-Host Disease via CD39-CD73-Adenosine and IDO Signals. Frontiers in Immunology. 8, 68 (2017).
- Stockis, J., et al. Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin alphaVbeta8. Proceedings of the National Academy of Sciences of the United States of America. 114 (47), E10161-E10168 (2017).
- Vogelstein, B., Kinzler, K. W. Digital PCR. Proceedings of the National Academy of Sciences of the United States of America. 96 (16), 9236-9241 (1999).
- Quan, P. L., Sauzade, M., Brouzes, E. dPCR: A Technology Review. Sensors (Basel). 18 (4), (2018).
- Sykes, P. J., et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques. 13 (3), 444-449 (1992).
- Ma, H., et al. Evaluation of Bacterial Contamination in Goat Milk Powder Using PacBio Single Molecule Real-Time Sequencing and Droplet Digital PCR. Journal of Food Protection. , 1791-1799 (2018).
- Vitale, S. R., et al. An optimized workflow to evaluate estrogen receptor gene mutations in small amounts of cell-free DNA. Journal of Molecular Diagnostics. , (2018).
- Gorgannezhad, L., Umer, M., Islam, M. N., Nguyen, N. T., Shiddiky, M. J. A. Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies. Lab Chip. 18 (8), 1174-1196 (2018).
- Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. Retro-orbital injections in mice. LabAnimal. 40 (5), 155-160 (2011).
- Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. Manual restraint and common compound administration routes in mice and rats. Journal of Visualized Experiments. (67), (2012).
- Cooke, K. R., et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood. 88 (8), 3230-3239 (1996).
- Weisdorf, D. J., et al. Prospective grading of graft-versus-host disease after unrelated donor marrow transplantation: a grading algorithm versus blinded expert panel review. Biology of Blood and Marrow Transplantation. 9 (8), 512-518 (2003).
- Nervi, B., et al. Factors affecting human T cell engraftment, trafficking, and associated xenogeneic graft-vs-host disease in NOD/SCID beta2mnull mice. Experimental Hematology. 35 (12), 1823-1838 (2007).
- Leon-Rico, D., et al. Comparison of haematopoietic stem cell engraftment through the retro-orbital venous sinus and the lateral vein: alternative routes for bone marrow transplantation in mice. LabAnimal. 49 (2), 132-141 (2015).
- Ali, N., et al. Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rgammanull mice display a T-effector memory phenotype. PLoS One. 7 (8), e44219 (2012).
- van Rijn, R. S., et al. A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2-/- gammac-/- double-mutant mice. Blood. 102 (7), 2522-2531 (2003).
- Wunderlich, M., et al. OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood. 123 (24), e134-e144 (2014).
- Schroeder, M. A., DiPersio, J. F. Mouse models of graft-versus-host disease: advances and limitations. Disease Models & Mechanisms. 4 (3), 318-333 (2011).
- Zeiser, R., Blazar, B. R. Preclinical models of acute and chronic graft-versus-host disease: how predictive are they for a successful clinical translation?. Blood. 127 (25), 3117-3126 (2016).
Cite This Article
Seng, A., Markiewicz, M. A. Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR. J. Vis. Exp. (147), e59107, doi:10.3791/59107 (2019).