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
1Cancer Division, Burnett School of Biomedical Sciences,University of Central Florida, 2Department of Biochemistry,Hanoi University of Pharmacy, 3Center for Cardiovascular Regeneration, Department of Cardiovascular Sciences,Houston Methodist Research Institute, 4Department of Epidemiology,University of Pittsburgh Graduate School of Public Health, 5Division of Cancer Control and Population Sciences,University of Pittsburgh Medical Center, Hillman Cancer Center
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 第 0 天：准备接收鼠标。 在 BALB/c 背景 （CD45.2+ H2kd+）上使用雌性野生类型 （WT） 小鼠，1…
Representative Results
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?…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.5 M EDTA pH 8.0 100ML | Fisher Scientific | BP2482100 | MACS buffer |
| 10X PBS | Fisher Scientific | BP3994 | MACS buffer |
| A20 B-cell lymphoma | University of Central Florida | In house | GVL experiment |
| ACC1 fl/fl | Jackson Lab | 30954 | GVL experiment |
| ACC1 fl/fl CD4cre | University of Central Florida | GVL experiment | |
| Anti-Biotin MicroBeads | Miltenyi Biotec | 130-090-485 | T-cell enrichment |
| Anti-Human/Mouse CD45R (B220) | Thermo Fisher Scientific | 13-0452-85 | T-cell enrichment |
| Anti-mouse B220 FITC | Thermo Fisher Scientific | 10452-85 | Flow cytometry analysis |
| Anti-mouse CD11c- AF700 | Thermo Fisher Scientific | 117319 | Flow cytometry analysis |
| Anti-Mouse CD25 PE | Thermo Fisher Scientific | 12-0251-82 | Flow staining |
| Anti-Mouse CD4 Biotin | Thermo Fisher Scientific | 13-0041-86 | T-cell enrichment |
| Anti-Mouse CD4 eFluor® 450 (Pacific Blue® replacement) | Thermo Fisher Scientific | 48-0042-82 | Flow staining |
| Anti-mouse CD45.1 PE | Thermo Fisher Scientific | 12-0900-83 | Flow cytometry analysis |
| Anti-Mouse CD8a APC | Thermo Fisher Scientific | 17-0081-83 | Flow cytometry analysis |
| Anti-mouse H-2Kb PerCP-Fluor 710 | Thermo Fisher Scientific | 46-5958-82 | Flow cytometry analysis |
| Anti-mouse MHC Class II-antibody APC | Thermo Fisher Scientific | 17-5320-82 | Flow cytometry analysis |
| Anti-Mouse TER-119 Biotin | Thermo Fisher Scientific | 13-5921-85 | T-cell enrichment |
| Anti-Thy1.2 | Bio Excel | BE0066 | BM generation |
| B6 fB-/- mice | University of Central Florida | In house | Recipients |
| B6.Ly5.1 (CD45.1+) mice | Charles River | 564 | Donors |
| BALB/c mice | Charles River | 028 | Transplant recipients |
| C57BL/6 mice | Charles River | 027 | Donors/Recipients |
| CD11b | Thermo Fisher Scientific | 13-0112-85 | T-cell enrichment |
| CD25-biotin | Thermo Fisher Scientific | 13-0251-82 | T-cell enrichment |
| CD45R | Thermo Fisher Scientific | 13-0452-82 | T-cell enrichment |
| CD49b Monoclonal Antibody (DX5)-biotin | Thermo Fisher Scientific | 13-5971-82 | T-cell enrichment |
| Cell strainer 40 uM | Thermo Fisher Scientific | 22363547 | Cell preparation |
| Cell strainer 70 uM | Thermo Fisher Scientific | 22363548 | Cell preparation |
| D-Luciferin | Goldbio | LUCK-1G | Live animal imaging |
| Fetal Bovine Serum (FBS) | Atlanta Bilogicals R&D system | D17051 | Cell Culture |
| Flow cytometry tubes | Fisher Scientific | 352008 | Flow cytometry analysis |
| FVB/NCrl | Charles River | 207 | Donors |
| Lipopolysacharide (LPS) | Millipore Sigma | L4391-1MG | DC mature |
| LS column | Mitenyi Biotec | 130-042-401 | Cell preparation |
| MidiMACS | Miltenyi Biotec | 130-042-302 | T-cell enrichment |
| New Brunswick Galaxy 170R incubator | Eppendorf | Galaxy 170 R | Cell Culture |
| Penicilin+streptomycinPenicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep) | GIBCO | 15140 | Media |
| RPMI 1640 | Thermo Fisher Scienctific | 11875-093 | Media |
| TER119 | Thermo Fisher Scientific | 13-5921-82 | T-cell enrichment |
| Xenogen IVIS-200 | Perkin Elmer | Xenogen IVIS-200 | Live animal imaging |
| X-RAD 320 Biological Irradiator | Precision X-RAY | X-RAD 320 | Total Body Irradiation |
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Tags
Keywords: Bone Marrow TransplantationDendritic CellsGraft-versus-host DiseaseT-cell DepletionCell IsolationCell CultureImmune Cell PopulationsMacrophagesNeutrophilsCell RegenerationBone Marrow PreparationCell HarvestingCell StrainingCell WashingCell ResuspensionCell IncubationCell CountingCell Pooling
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Nguyen, H. D., Huong, P. T., Hossack, K., Gurshaney, S., Ezhakunnel, K., Huynh, T., Alvarez, A. M., Le, N., Luu, H. N. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. (157), e60083, doi:10.3791/60083 (2020).