小鼠致耐受性树突状细胞的发育及功能表征
Summary
在这里, 我们提出了一个协议, 开发和表征致耐受性树突状细胞 (TolDCs), 并评估其免疫治疗效用。
Abstract
免疫系统的运作方式是在协调对外来抗原的反应和保持对自抗原的无反应状态以及从共生生物体中提取的抗原之间保持紧密的平衡。这种免疫稳态的破坏会导致慢性炎症和自身免疫的发展。树突状细胞 (DCs) 是一种专业的抗原呈现细胞的先天免疫系统参与激活幼稚 T 细胞启动免疫应答外来抗原。然而, DCs 也可以被区分为 TolDCs, 以维持和促进 T 细胞耐受性和抑制效应细胞, 促进发展的任何一种自身免疫或慢性炎症条件。最近在我们理解 TolDCs 的进展表明, 直流耐受可以通过调节它们的分化条件来实现。这种现象已经导致了巨大的增长, 发展 TolDC 疗法的许多免疫障碍造成的免疫耐受性的突破。对前体免疫性小鼠模型的成功研究进一步证实了 TolDCs 治疗自身免疫性疾病的免疫治疗效用。今天, TolDCs 已成为临床上一个有希望的免疫治疗工具, 以恢复免疫耐受的各种免疫障碍的目标致病性自身免疫反应, 同时保持保护免疫完好。虽然多个实验室提出了一系列的策略来诱导 TolDCs, 但在表征细胞和功能表型方面没有一致性。该协议为骨髓源 DCs 的大量开发提供了一步一步的指导, 一种独特的方法, 用于将其与合成萜 2-氰基-312-dioxooleana-19-奠 28-伊斯兰会议组织区分为 TolDCs。酸-difluoro-丙酰胺 (CDDO-DFPA), 以及用于确认其表型的技术, 包括分析 TolDCs 的基本分子特征。最后, 通过在多发性硬化的临床前模型中测试其免疫抑制反应体外和体内, 我们展示了一种评估 TolDC 功能的方法。
Introduction
树突状细胞 (DCs) 是先天免疫系统不可分割的一部分, 并首次发现和特征的拉尔夫斯坦曼和 Zanvil 科恩在1973年作为主要的专业抗原呈现细胞1。DCs 在免疫活化方面发挥了重要作用, 通过在次级淋巴器官中通过主要的组织相容性复合物 (MHC) 向 T 细胞和 B 细胞呈现经过处理的抗原, 将先天免疫系统和自适应的免疫力结合在一起2。在哺乳动物免疫系统中, 至少有两类 dcs 被描述为髓系 dcs 和浆 dcs (pDCs)3。髓细胞 dcs, 也称为常规 dcs (招揽), 其特点是 CD11c 的表达, 并可区分为未成熟的 dcs (iDCs)体外从骨髓前体细胞或外周血单核单位使用粒细胞-巨噬细胞集落刺激因子 (GM-CSF) 和 IL-4 的小鼠或人类物种, 分别为4。
激活 ‘ 危险 ‘ 信号, 如病原体相关的分子模式 (玉兰) 或损伤相关的分子模式 (潮湿), 将推动 iDCs 成熟免疫 dcs 作为成熟的 dcs (mDCs) 通过捆绑各种模式识别受体对DC 曲面5。免疫 DCs 进一步通过上调 MHCII2、刺激配体 (CD80、CD86 和 CD40)6、细胞因子和其他可溶性调解人7, 进一步促进天真的 T 细胞增殖和分化。免疫 DCs 对细胞因子介导的 T 细胞分化至关重要。例如, 干扰素和 IL-12 都是 Th1 分化所必需的8和 IL-1、IL-6 和 IL-23 对于天真的 T 细胞极化对 Th17 细胞9是至关重要的。虽然成熟的 DCs 对外来抗原有反应, 但自抗原控制的 dc 活化可能导致耐受性消融, 并通过生成自身反应性 T 细胞来促进自身免疫性疾病的发展, 其活化导致组织破坏10.
最近的报告提供了直流可塑性的明确证据, 其例证是它们能够在组织微环境中与不同的线索相互作用, 并区分为不同的效应/抑制 dc 子集。抗炎介质 (如 IL-1011、TGF β12和 HO-113 ) 在诱导致耐受性 DCs (TolDCs) 的免疫抑制中发挥了重要作用。这些 TolDCs 获取调节功能并抑制 T 细胞增殖14。此外, DCs 缺乏协同刺激和 TolDCs 的抗炎介质的产生, 都有助于诱导调节 T 细胞 (Tregs), 同时也有效抑制 Th1 和 Th17 分化和扩张15。在过去的两年中, TolDCs 的治疗潜力已经被几个调查员报告。在这些研究中, 体外产生的 TolDCs 不仅改善了自身免疫性疾病的不同临床前模型的病理症状 16 , 而且还导致了患者免疫耐受的发展17 ,18。有趣的是, 今天 TolDCs 疗法已被认为是一种替代或辅助方法的自身免疫性疾病的几个临床试验, 包括1型糖尿病19, 类风湿关节炎20, 21, 多发性硬化症 (MS)22,23,24, 克罗恩病25。
有各种各样的协议被使用了开发 TolDCs 和几个实验室报告了方法为 TolDCs 的世代和表型特征。这些方法可用于从造血祖重现性生成 TolDCs体外, 并稳定地维护它们在致耐受性状态在体内26,27,28,29。iDCs 可以通过接触各种免疫调节药理剂或抗炎性细胞因子转化为 TolDCs。例如, 维生素 D3 是已知的一种已知的药理剂, 可以增加 IL-10 的生产和抑制 IL-12 分泌物, 从而增强其免疫抑制功能30。此外, 当 DCs 暴露于强烈的炎症刺激, 如脂多糖 (LPS), 一些药理剂, 如地塞米松 31, 雷帕霉素 32, 皮质类固醇33已显示, 以诱导TolDC 表型通过减少 CD40、CD80、CD86 和 MHCII34的 DC 表面表达式。IL-10 和 TGF β是最受研究的抗炎性细胞因子, 以诱导直流耐受35和伴随暴露于这两种细胞因子已被证明诱导致耐受性表型在dc36。
由于致耐受性 DC 是由功能特征而不是表型标记来定义的, 所以需要对 TolDCs 的细胞和功能特性进行一致的方法。此外, 必须建立一个严格和一致的协议, 以便对致耐受性 DC 表型进行一致的评价和表征, 如果我们要有效和重现性比较新的药物诱导 TolDC 表型的能力实验室。在这里, 我们提供了一个详细的协议, 分步方法分离 iDCs 与造血祖细胞的小鼠, 并随后分析的功效, 评估新的药物的能力, 以转换 iDCs 为 TolDCs, 提供了一个强大的TolDCs 的功能和表型特性体外和体内。这个描述包括一个精心的方法来表征 TolDCs 的表面配体, 细胞因子剖面和免疫抑制功能在体内。我们还提供了一个方法的例子, 探讨这些 TolDCs 的潜在治疗应用在 MS, 实验性自身免疫脑脊髓炎 (EAE) 的临床前模型。这项既定的议定书将帮助调查人员评估新的药物促进诱导 TolDCs 的能力, 并将促进扩大 TolDC 治疗发展范围的努力。
Protocol
所有的研究都是按照西方储备大学医学院的机构动物护理和使用委员会批准的程序进行的。 1. 制备骨髓源性树突状细胞 (BMDCs) 通过热处理消毒所有手术器械, 并在第二类生物安全柜中进行实验, 并进行安全程序。 弄死 C57BL/6 小鼠 8-10 周的年龄通过使用 CO2室。将鼠标放在解剖板上, 用70% 乙醇冲洗。使用外科剪刀将胫骨-腓骨和股骨骨放在10厘米的培养皿中,…
Representative Results
BMDCs 的分化与选择: 骨髓祖细胞在全 RPMI 培养基中培养, 在 GM-CSF 和 IL-4 的存在下, 可分化为 iDCs 7 天 (图 1A)。1天, 细胞体积小, 呈球形形态。在3天更换新鲜培养基之前, 使用 PBS 进行洗涤有助于细胞形成簇群, 也增加了 CD11c+细胞的数量。在4天, BMDCs 扩大了规模, 并开始了集群的形成。黏?…
Discussion
本文描述了一种有效的协议, 可用于重现性生成 iDCs, 并随后将其分化为 TolDCs, 我们建议, 这可以用于评估新分子靶剂的能力, 以诱导 TolDC表。如本报告所述, 我们遵循了一个序列, 我们首先分析了流式细胞术对表面配体的 TolDC 表达, 然后通过 qRT PCR 和 ELISA 测定对 DC 细胞因子剖面的评价。最后, 利用 MS 的临床前小鼠 EAE 模型, 证明了 TolDCs在体外抑制 T 细胞增殖的能力, 以及在体内抑癌的功效, 证…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Reata 制药公司提供 CDDO DFPA。我们也承认, 简和李塞德曼主席在儿科癌症创新 (约翰 Letterio) 的支持。这项工作得到了国防部的支持 [W81XWH-12-1-0452];安吉福勒青少年和青年癌症研究倡议在案例综合癌症中心;琼斯卡拉汉基金会的卡拉汉研究生奖学金。
Materials
| CDDO-DFPA (RTA-408) | Reata Pharmaceuticals | in house synthesis | Cell culture |
| Mouse GM-CSF | Peprotech Inc. | 315-03 | BMDC differentiation |
| Mouse IL-4 | Peprotech Inc. | 214-14 | BMDC differentiation |
| Lipopolysaccharides (LPS) | Sigma Aldrich Inc. | L2880 | Cell culture |
| β-mercaptoethanol | Sigma Aldrich Inc. | 516732 | Cell culture |
| Pertussis toxin (PTX) | R&D systems | 3097 | EAE induction |
| MOG (35–55) peptide | 21stCentury Biochemicals | in house synthesis | EAE induction |
| Trypan blue | Gibco, Life Technologies | 15250-061 | Cell culture |
| RPMI-1640 plus L-glutamine | ThermoFisher Scientific | 11875-093 | Cell culture |
| Non-essential amino acid (100X) | ThermoFisher Scientific | 11140050 | Cell culture |
| HEPES | ThermoFisher Scientific | 15630080 | Cell culture |
| penicillin/streptomycin | ThermoFisher Scientific | 15140122 | Cell culture |
| 40 μm cell strainer | Corning | 352340 | Cell isolation |
| PE-conjugated CD80 | BD Biosciences | 557227 | Flow cytometry |
| PE-conjugated CD86 | BD Biosciences | 555665 | Flow cytometry |
| PE-conjugated PD-L1 | BioLegend | 124307 | Flow cytometry |
| APC-conjugated MHCII | Miltenyi Biotec Inc. | 130-112-388 | Flow cytometry |
| APC-conjugated CD11c | BD Biosciences | 340544 | Flow cytometry |
| Isotype matched PE | Miltenyi Biotec Inc. | 130-091-835 | Flow cytometry |
| Isotype matched APC | Miltenyi Biotec Inc. | 130-091-836 | Flow cytometry |
| CFSE | BioLegend | 423801 | T cell proliferation assay |
| Pan dendritic cell isolation kit | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| FcR Blocking Reagent | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| Pan Dendritic Cell Biotin-Antibody Cocktail | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| Anti-Biotin MicroBeads | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| CD4+ T cell isolation kit | Miltenyi Biotec Inc. | 130-104-454 | T cell proliferation assay |
| CD4+ T cell Biotin-Antibody Cocktail | Miltenyi Biotec Inc. | 130-104-454 | T cell proliferation assay |
| Anti-Biotin MicroBeads | Miltenyi Biotec Inc. | 130-104-454 | T cell proliferation assay |
| ACK lysing buffer | ThermoFisher Scientific | A1049201 | BMDC differentiation |
| 1 ml syringe | BD Biosciences | 309626 | T cell proliferation assay |
| 3 ml syringe | BD Biosciences | 309588 | BMDC differentiation |
| 25G needle | BD Biosciences | 309626 | T cell proliferation assay |
| 23G needle | BD Biosciences | 309588 | BMDC differentiation |
| BSA | Sigma Aldrich Inc. | A2058 | T cell proliferation assay |
| EDTA | ThermoFisher Scientific | 15575020 | T cell proliferation assay |
| LS Column | Miltenyi Biotec Inc. | 130-042-401 | T cell proliferation assay |
| Pre-Separation Filter | Miltenyi Biotec Inc. | 130-095-823 | T cell proliferation assay |
| collagenase D | Sigma Aldrich Inc. | 11088858001 | T cell proliferation assay |
| HBSS | ThermoFisher Scientific | 14025076 | T cell proliferation assay |
| ovalbumin (OVA) peptide 323–329 | Sigma Aldrich Inc. | O1641 | T cell proliferation assay |
| Mouse IFN-γ TaqMan probe | ThermoFisher Scientific | Mm01168134_m1 | qRT-PCR |
| Mouse IL-12a TaqMan probe | ThermoFisher Scientific | Mm00434165 | qRT-PCR |
| Mouse IL-12 p70 DuoSet ELISA | R&D systems | DY419-05 | ELISA |
| Mouse EDN-1 ELISA | RayBiotech | ELM-EDN1-1 | ELISA |
| TNF-α TaqMan probe | ThermoFisher Scientific | Mm00443258 | qRT-PCR |
| Mouse TNF-α Quantikine ELISA Kit | R&D systems | MTA00B | ELISA |
| IL-6 TaqMan probe | ThermoFisher Scientific | Mm00446190 | qRT-PCR |
| Mouse IL-6 Quantikine ELISA Kit | R&D systems | M6000B | ELISA |
| IL-23a TaqMan probe | ThermoFisher Scientific | Mm01160011 | qRT-PCR |
| Mouse IL-23 DuoSet ELISA | R&D systems | DY1887-05 | ELISA |
| IL-4 TaqMan probe | ThermoFisher Scientific | Mm99999154_m1 | qRT-PCR |
| IL-10 TaqMan probe | ThermoFisher Scientific | Mm01288386_m1 | qRT-PCR |
| TGF-β TaqMan probe | ThermoFisher Scientific | Mm01178820_m1 | qRT-PCR |
| Anti-Heme Oxygenase 1 antibody | Abcam | ab13248 | Western blotting |
| Anti-β-actin antibody | Abcam | ab8226 | Western blotting |
| CFX96 Touch Real-Time PCR Detection System | Bio-Rad Inc. | qRT-PCR | |
| BD FACSCalibur Cell Analyzer | BD Biosciences | Flow cytometry |
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Cite This Article
Wei, H., Letterio, J. J., Pareek, T. K. Development and Functional Characterization of Murine Tolerogenic Dendritic Cells. J. Vis. Exp. (135), e57637, doi:10.3791/57637 (2018).