Name: 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
Uploaded: 2019-05-23T12:30:00
Description: Watch this Scientific Journal Video about 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 at JoVE

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。

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
<ol><li>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...

<ol>
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J., 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=Quantitation+of+targets+for+PCR+by+use+of+limiting+dilution.">Quantitation of targets for PCR by use of limiting dilution.</a> Biotechniques. 13 (3), 444-449 (1992).</li><li>Ma, H., 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=Evaluation+of+Bacterial+Contamination+in+Goat+Milk+Powder+Using+PacBio+Single+Molecule+Real-Time+Sequencing+and+Droplet+Digital+PCR.">Evaluation of Bacterial Contamination in Goat Milk Powder Using PacBio Single Molecule Real-Time Sequencing and Droplet Digital PCR.</a> Journal of Food Protection. , 1791-1799 (2018).</li><li>Vitale, S. R., 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=An+optimized+workflow+to+evaluate+estrogen+receptor+gene+mutations+in+small+amounts+of+cell-free+DNA.">An optimized workflow to evaluate estrogen receptor gene mutations in small amounts of cell-free DNA.</a> Journal of Molecular Diagnostics. , (2018).</li><li>Gorgannezhad, L., Umer, M., Islam, M. N., Nguyen, N. T., Shiddiky, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+and+liquid+biopsy:+opportunities,+challenges,+and+recent+advances+in+detection+technologies.">Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies.</a> Lab Chip. 18 (8), 1174-1196 (2018).</li><li>Yardeni, T., Eckhaus, M., Morris, H. 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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?...

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。

<table>
	<tbody>
		<tr>
			<td>1.5 mL eppendorf tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>VWR International</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL BD Vacutainers - Green capped with Sodium Heparin</td>
			<td>Becton Dickinson</td>
			<td>366480</td>
			<td></td>
		</tr>
		<tr>
			<td>250 &#181;L Ranin pipette tips</td>
			<td>Rainin</td>
			<td>17001118</td>
			<td>Do not use other pipettes or pipet tips for droplet generation</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR International</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well ddPCR plate</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK (Ammonium-Chloride-Potassium) Lysing Buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Alcohol Wipes</td>
			<td>Fisher Scientific</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Chamber</td>
			<td>World Precision Instruments</td>
			<td>EZ-178</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>Anesthesia Machine</td>
			<td>Parkland Scientific</td>
			<td>PM1002</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>BD Vacutainer Safety-Lok Blood Collection Set</td>
			<td>Becton Dickinson</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridges and Gaskets for QX100/QX200 Droplet Generator</td>
			<td>Bio-Rad</td>
			<td>1864007</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse and RNAse free Molecular Grade H2O</td>
			<td>Life Technologies</td>
			<td>1811318</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure,200 proof, for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Fisher Scientific</td>
			<td>45001750</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>Fisher Scientific</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>CDS019936</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Irradiator Pie Cage</td>
			<td>Braintree Scientific, Inc.</td>
			<td>MPC 1</td>
			<td>Holds up to 11 mice</td>
		</tr>
		<tr>
			<td>Nexcare Gentle Paper Tape (a.k.a. 3M Micropore Surgical Tape / 3/4&#34;)</td>
			<td>Fisher Scientific</td>
			<td>19-027-761</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipetman</td>
			<td>MidSci</td>
			<td>A-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipetman</td>
			<td>MidSci</td>
			<td>A-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seal</td>
			<td>Bio-Rad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetaid Gilson Macroman</td>
			<td>Fisher Scientific</td>
			<td>F110756</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td>Do not use other pipettes or pipet tips for droplet generation</td>
		</tr>
		<tr>
			<td>Qiagen DNeasy Blood and Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>VWR International</td>
			<td>89218-292</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Digital PCR System</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td>Includes droplet generator, droplet reader, laptop computer, software, associated component consumables, for EvaGreen or probe-based digital PCR applications</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generation Oil for EvaGreen</td>
			<td>Bio-Rad</td>
			<td>1864006</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>Bio-Rad</td>
			<td>1864033</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase and DNase-free plate seal</td>
			<td>Thermo Scientific</td>
			<td>12565491</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Advanced 1640</td>
			<td>Life Technologies</td>
			<td>12633012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Pads (2&#34; x 2&#34;, 12-Ply)</td>
			<td>Fisher Scientific</td>
			<td>67522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile reservoir</td>
			<td>VWR International</td>
			<td>89094-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgial Scissors</td>
			<td>Kent Scientific</td>
			<td>INS600393-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650914-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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。

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...

Watch this Scientific Journal Video about 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 at JoVE

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

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

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。

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

该协议概述了如何诱导异种移植与宿主疾病，或异种GVHD，以及如何盲目和标准化临床评分，以确保一致的结果。XenoGVHD模型提供了一种体内方法，用于测试针对人类而非穆林T细胞的免疫抑制疗法，从而改进了这些研究的转化过程。演示这个程序的将是阿马拉·森，一个研究生，从我的实验室。注射前一天，将8至12周大、非肥胖的糖尿病scid伽玛（NSG）小鼠放在灭菌馅饼笼中，并在一个分量为150百分源的铀-137源中照射小鼠，并缓慢旋转以确保均匀的辐照。然后，将老鼠放入无菌生物安全柜中过夜的清洁笼中。第二天早上，收集足够的健康人类血液，每只小鼠分离1.1倍至7个外周血单核细胞（PBMC），并在PBS中以2%的相同体积稀释肝素化血液，即FBS。在 50 毫升锥形管中小心地将高达 25 毫升稀释的血液覆盖到 15 毫升淋巴细胞分离密度梯度介质上，用于密度梯度离心。在分离结束时，在接口上收获PBMC，并在新的50毫升锥形管中清洗10毫升PBS中的细胞。吸上一液，轻拂管子以松开颗粒。将 PBMC 重新用 10 毫升新鲜 PBS 重新离心，然后重新用 5 毫升新鲜 PBS 进行计数。然后，用最终离心收集细胞，并在每毫升PBS浓度的1倍10至8PBMC下重新暂停颗粒。为了将分离的人类PBMC的逆轨传递到小鼠眼中，将一个装有28米针的100微升细胞装入一个1毫升注射器。确认对脚趾捏没有反应，用拇指和中指轻轻约束鼠标。将针斜面向下插入，横向插入中侧手根，通过结膜，直到到达眼部后面。稍微缩回针头，慢慢注射整个细胞体积。然后，完全缩回针头，以正确处置注射器和针头。关闭眼睑，用纱布海绵对注射部位施加轻度压力。然后，检查注射部位有无肿胀或其他可见的创伤，让小鼠在无菌笼内恢复知觉，并监测，然后再将动物移到其家庭笼子。要测量 GVHD 分数，请将笼子放在层流罩中，然后从笼子中取出食物和水。要对活动进行评分，请观察小鼠的行为五分钟。要获得减肥分数，请将每只鼠标放在玻璃杯中。当鼠标仍在烧杯中时，检查动物的姿势、毛皮质地和皮肤完整性。收获感兴趣的组织后，从器官中切出一块约30.5毫米的组织，并在平衡上称量样品。将加权组织转移到无菌的1.5毫升管中，将样品冷冻在液氮中，在零下80摄氏度下隔夜储存。第二天早上，根据基因组DNA分离试剂盒的指示，在解解前解冻样品。对于通过数字聚合酶链反应（PCR）进行人类T细胞定量，根据用于数字PCR机的DNA结合染料协议准备数字PCR反应，并使用适当的底塑剂用于人类CD3 epsilon基因组DNA。然后，在适当的反应条件下进行数字PCR反应。与仅接受PBS治疗的负对照小鼠相比，接受人类PBMC的8至12周大NSG小鼠在注射后第10天左右开始显示GVHD的临床症状。异种GVHD小鼠的中位存活期为23.5天。使用数字PCR，CD3 epsilon阳性的人类T细胞可以在接受人类PBMC的小鼠的肺和肝脏样本中检测。关键是，评分必须始终如一地进行，并且给小鼠打分的研究人员对小鼠的治疗视而不见。按照这个程序，可以从安乐死小鼠身上采集血清进行外周细胞因子表达分析，也可以从脾脏中采集免疫细胞，通过流式细胞学进行分析。

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...