Name: bone marrow transplantation platform to investigate the role of dendritic cells in graft-versus-host disease
Uploaded: 2020-03-17T12:00:00
Description: Watch this Scientific Journal Video about Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease at JoVE.com

这项研究由中佛罗里达大学医学院启动补助金（对HN）、匹兹堡大学医学中心希尔曼癌症中心启动补助金（至HL）、美国NIH赠款#1P20CA210300-01和越南卫生部赠款#4694/QD-BYT（至PTH）支持。我们感谢南卡罗来纳医科大学的余学忠博士为这项研究提供了材料。

实验程序得到了中佛罗里达大学机构动物护理和使用委员会的批准。
1. GVHD 感应
注：异体骨髓（BM）细胞移植（步骤1.2）在辐照后24小时内进行。下面描述的所有程序都在无菌环境中执行。在组织培养罩中执行该过程，并使用过滤试剂。
<ol><li>第 0 天：准备接收鼠标。<ol>
<li>在 BALB/c 背景 （CD45.2+ H2kd+）上使用雌性野生类型 （WT） 小鼠，1...

<ol>
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The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Nguyen, 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=Complement+C3a+and+C5a+receptors+promote+GVHD+by+suppressing+mitophagy+in+recipient+dendritic+cells.">Complement C3a and C5a receptors promote GVHD by suppressing mitophagy in recipient dendritic cells.</a> Journal of Clinical Investigation Insight. 3, (2018).</li><li>McNutt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy.">Cancer immunotherapy.</a> Science. 342, 1417 (2013).</li><li>Negrin, R. S., Contag, C. 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使用干细胞来适应特定个体是治疗晚期和抗药性癌症的有效方法。然而，小分子药物长期以来一直是个性化癌症治疗的主要焦点。另一方面，在细胞治疗中，供体和宿主之间的多种相互作用可以决定性地影响治疗结果，如BMT1后GVHD的发展。
BMT的主要MHC错配小鼠模型是了解GVHD生物学和测试药物治疗效果的宝贵工具。其中，C57BL/6（H2b）</sup...

异体造血干细胞移植（BMT）是治疗血液恶性肿瘤的有效疗法1、22通过移植对白血病（GVL）效应3。然而，供体淋巴细胞总是对受体组织进行不需要的免疫攻击，这个过程称为移植与宿主疾病（GVHD）4。4
GVHD的毛利模型是研究GVHD生物学和GVL反应5的有效工具。老鼠是一种具有成本效益的研究动物模型。它们很小，在发育的早期阶段就有效地与分子和生物制剂结合。老鼠是基因操作研究的理想研究动物，因为它们在基因上定义得很好，非常适合研究生物途径和机制。几种小鼠主要组织相容性复合（MHC）MHC不匹配的GVHD模型已经建立，如C57BL/6（H2b）到BALB/c（H2d）和FVB（H2q）_C57BL/6（H2qbb）5，7。,7这些是确定单个细胞类型、基因和影响GVHD的因素的作用的特别重要的模型。从C57/BL/6（H2b）父母捐赠者移植到MHC I（B6.C-H2bm1）和/或MHC II（B6.C-H2bm12）突变的接受者，表明MHC I类和II类的不匹配是急性GVHD发展的重要要求。这表明CD4+和CD8+T细胞都是疾病发展所需的7，8。8GVHD还参与了被称为"亲炎细胞因子风暴"的炎症级联。在鼠模型中最常见的调理方法是 X 射线或137C 的总身体辐照 （TBI）。这导致接受者的骨髓消融，从而允许供体干细胞移植，并防止移植的排斥。这是通过限制受体T细胞的增殖来响应供体细胞来实现的。此外，遗传差异在疾病诱导中起着重要作用，这也取决于轻微的MHC不匹配10。因此，骨髓照射剂量在不同的小鼠菌株中有所不同（例如，BALB/c=C57BL/6）。
宿主抗原呈现细胞（APC）激活供体T细胞对GVHD发育至关重要。在APC中，树突状细胞（DC）是最有效的。它们具有遗传性诱导GVHD的能力，因为它们具有优越的抗原摄入、T细胞共刺激分子的表达以及产生使T细胞极化成致病子集的亲炎细胞因子。接受性D对促进移植后,T细胞充注和GVHD诱导至关重要。因此，在治疗GVHD12时，DC已成为有趣的目标。
需要TBI来增强供体细胞的移植。由于TBI效应，接受者DC在移植12后被激活并存活一小段时间。尽管在使用生物发光或荧光方面取得了重大进步，但建立一个有效的模型来研究受体D在GVHD中的作用仍然具有挑战性。
由于供体T细胞是GVL活动的驱动力，使用免疫抑制药物如类固醇抑制T细胞过敏反应的治疗策略往往导致肿瘤复发或感染13。因此，针对受体 DC 可以提供治疗 GVHD 的替代方法，同时保持 GVL 效果并避免感染。
简而言之，目前的研究提供了一个平台，以了解接收D中不同类型的信令如何调节GVHD的发展和BMT后GVL效应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA pH 8.0 100ML</td>
			<td>Fisher Scientific</td>
			<td>BP2482100</td>
			<td>MACS buffer</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>MACS buffer</td>
		</tr>
		<tr>
			<td>A20 B-cell lymphoma</td>
			<td>University of Central Florida</td>
			<td>In house</td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>ACC1 fl/fl</td>
			<td>Jackson Lab</td>
			<td>30954</td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>ACC1 fl/fl CD4cre</td>
			<td>University of Central Florida</td>
			<td></td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse CD45R (B220)</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0452-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 FITC</td>
			<td>Thermo Fisher Scientific</td>
			<td>10452-85</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c- AF700</td>
			<td>Thermo Fisher Scientific</td>
			<td>117319</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25 PE</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-0251-82</td>
			<td>Flow staining</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0041-86</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 eFluor&#174; 450 (Pacific Blue&#174; replacement)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-0042-82</td>
			<td>Flow staining</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.1 PE</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-0900-83</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD8a APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-0081-83</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse H-2Kb PerCP-Fluor 710</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-5958-82</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC Class II-antibody APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-5320-82</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse TER-119 Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5921-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Thy1.2</td>
			<td>Bio Excel</td>
			<td>BE0066</td>
			<td>BM generation</td>
		</tr>
		<tr>
			<td>B6 fB-/- mice</td>
			<td>University of Central Florida</td>
			<td>In house</td>
			<td>Recipients</td>
		</tr>
		<tr>
			<td>B6.Ly5.1 (CD45.1+) mice</td>
			<td>Charles River</td>
			<td>564</td>
			<td>Donors</td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Charles River</td>
			<td>028</td>
			<td>Transplant recipients</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Donors/Recipients</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0112-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD25-biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0251-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD45R</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0452-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD49b Monoclonal Antibody (DX5)-biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5971-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Cell strainer 40 uM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>Cell strainer 70 uM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363548</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td>Live animal imaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Bilogicals R&#38;D system</td>
			<td>D17051</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Flow cytometry tubes</td>
			<td>Fisher Scientific</td>
			<td>352008</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>FVB/NCrl</td>
			<td>Charles River</td>
			<td>207</td>
			<td>Donors</td>
		</tr>
		<tr>
			<td>Lipopolysacharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391-1MG</td>
			<td>DC mature</td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Mitenyi Biotec</td>
			<td>130-042-401</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>MidiMACS</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>New Brunswick Galaxy 170R incubator</td>
			<td>Eppendorf</td>
			<td>Galaxy 170 R</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Penicilin+streptomycinPenicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scienctific</td>
			<td>11875-093</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>TER119</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5921-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Xenogen IVIS-200</td>
			<td>Perkin Elmer</td>
			<td>Xenogen IVIS-200</td>
			<td>Live animal imaging</td>
		</tr>
		<tr>
			<td>X-RAD 320 Biological Irradiator</td>
			<td>Precision X-RAY</td>
			<td>X-RAD 320</td>
			<td>Total Body Irradiation</td>
		</tr>
	</tbody>
</table>

bone marrow transplantation platform to investigate the role of dendritic cells in graft-versus-host disease

异体骨髓移植（BMT）是治疗血液恶性肿瘤的有效疗法，由于移植对白血病（GVL）的作用来根除肿瘤。然而，其应用受到移植与宿主疾病（GVHD）的发展的限制，这是BMT的主要并发症。当供体移植物中的T细胞识别受体细胞表达的抗核抗原并对受体健康组织进行不需要的免疫攻击时，GVHD被唤起。因此，传统疗法旨在抑制供体T细胞同位活性。然而，这些方法大大削弱了GVL效应，使接受者的生存得不到改善。因此，了解治疗方法对BMT、GVL和GVHD的影响至关重要。由于抗原呈现和细胞因子分泌能力刺激供体T细胞，受体树突状细胞（DC）在GVHD的诱导中起着重要作用。因此，针对收件人 DC 成为控制 GVHD 的潜在方法。本工作描述了一个新的BMT平台，用于研究宿主DC如何调节移植后的GVH和GVL反应。介绍了一种有效的BMT模型，用于研究移植后GVHD和GVL的生物学。

主要的MHC不匹配的B6（H2k b）-BALB/C（H2kd）模型与移植后GVHD的发展密切相关（图2）。b库克等人16日建立的所有6个GVHD临床体征均发生在使用WT-B6 T细胞移植的接受者中，但不包括单独移植BM的接受者（步骤1.5），后者代表GVHD阴性组。此模型中 GVHD 开发有两个阶段。首先，严重程度的峰值在移植后大约11天，随后临床评?...

Watch this Scientific Journal Video about Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease at JoVE.com

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

移植与宿主疾病是异体骨髓移植后的主要并发症。树突状细胞在移植与宿主疾病的发病机制中起着关键作用。本文介绍了一种新型的骨髓移植平台，用于研究树突状细胞在移植与宿主疾病发展中的作用以及移植对白血病的影响。

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 ...

这种新型骨髓移植方案可用于研究宿主树突状细胞在移植后如何调节移植与宿主和移植与白血病反应。外体细胞再生允许此协议通过修改培养条件来适应其他免疫细胞群体（如巨噬细胞和嗜中性粒细胞）的作用。与我展示这个程序的将是实验室经理克里斯塔尔·霍萨克和本科生桑吉耶夫·古尔沙尼。对于供体T细胞耗尽的C57BL/6骨髓制剂，根据安乐死供体小鼠的标准方案收获股骨和头骨，将骨骼转移到含有1%RPMI培养基的92毫米直径的培养皿中。使用剪刀，从每根骨头上取下两端，用新鲜的 1% RPMI 介质填充一个装有 26 量表针的三毫升注射器。将针头插入一个骨的一端，压压柱塞，将骨髓从腔中冲出并放入收集细胞过滤器中。收集所有骨髓后，通过75微米网状过滤器将骨髓片段应变，将单细胞悬浮液转移到新的50毫升管中。通过离心沉淀细胞，并在PBS中每毫升浓度的20倍10至6个细胞重新悬浮细胞，并辅以0.5%BSA。接下来，在4摄氏度下，在6个细胞中每10次加入0.05微克THI1抗体，进行30分钟的孵育。在孵育结束时，用10毫升冰冷PBS洗两次细胞，将颗粒以每毫升浓度的2倍10重新暂停至0.5%BSA的7个细胞，辅以10%幼兔补充剂和2%DNAAse在无菌水中，在37摄氏度下进行45分钟的孵育。在孵育结束时，每次洗涤用15毫升新鲜PBS清洗细胞两次，并在含有0.5%BSA的20毫升PBS中重新暂停颗粒。将淋巴结和脾脏汇集在40微米孔隙过滤器中，并使用注射器柱塞通过过滤器将组织滤芯吸收，以释放细胞。用RPMI介质用1%FBS进行洗涤，收集所有细胞，通过离心沉淀细胞。向细胞颗粒中加入五毫升的 ACK 解液缓冲液。在室温下五分钟后，停止用5毫升的中量辅以1%FBS的反应，通过离心对白细胞进行颗粒化。将颗粒重新浓缩在五毫升 MACS 缓冲液中，用于计数，并在新鲜 MACS 缓冲液中将细胞稀释至每毫升 8 个细胞的浓度 2 倍至 8 个细胞。将抗红细胞、抗粒细胞、抗B细胞和抗NK细胞耗竭抗体的浓度适当添加到6个细胞中，在4摄氏度下孵育15分钟。在孵育结束时，用10毫升的冰冷MACS缓冲液清洗细胞，并在新鲜 MACS 缓冲液中每毫升浓度的10倍至8个细胞中重新填充颗粒。接下来，在6个细胞中加入0.22微升抗生物素微珠，在4摄氏度下混合15分钟孵育。在孵育结束时，使用10毫升冰冷MACS缓冲液洗涤细胞，然后根据标准珠子分离器使用MACSxpress分离器，通过磁珠分离收集未绑定的富T细胞。在分离结束时，通过离心沉淀T细胞，将颗粒重新悬浮在5毫升新鲜MACS缓冲液中进行计数。为了产生骨髓衍生的树突状细胞，将骨髓从野生型或因子B型敲除小鼠的股骨中分离，并在5毫升的CK解压缓冲液中分离红血球，在5分钟后用10毫升的RPMI和1%FBS补充停止反应。通过离心收集整个血细胞后，将培养基的10毫升中颗粒重新注入每毫升浓度的2倍10至6个细胞，并在每毫升GM-CSF中加入20毫微克，然后将10毫升细胞在37摄氏度的100毫升15毫米培养皿中播种，在6天内将5%的二氧化碳植入体内。第六天，每盘LPS为25微克，激活骨髓衍生的树突状细胞培养物。至少85%的培养骨髓细胞分化成树突状细胞。磁珠富集后T细胞纯度为90%主要MHC不匹配的C57BL/6 BALB/c模型与移植后GVHD的发展密切相关。严重程度的峰值发生在细胞转移后大约11天，随后临床评分和体重恢复减少，一直持续到第16天。接受者在移植后30至40天统一屈服于GVHD。有趣的是，具有因子B的带突挖树突状细胞的移植可提高接受者存活率和GVHD临床得分。路西法酶转导A20 B细胞淋巴瘤允许监测活动物的肿瘤生长。在这个具有代表性的实验中，所有仅接受骨髓的野生BALB/c接受者加上 A20 死于肿瘤复发。野生型BALB/c接受骨髓衍生的ACC1-耗尽的供体T细胞移植后死于GVHD。此外，接受供体骨髓和ACC1耗尽T细胞的动物死于GVHD和肿瘤复发。为了产生足够的骨髓细胞，在骨髓矫正之前，收集的头骨和股骨必须完全没有肌肉组织。使用生物素酶抗CD3和抗生物素微珠对骨髓细胞进行负纯化，也可以根据手术从骨髓中消耗淋巴细胞。

Research

JoVE Journal

Immunology and Infection

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

An In Vivo Mouse Model to Measure NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

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 ...

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

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