在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞
Summary
In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.
Abstract
浆细胞样树突细胞（pDC细胞）是强有力的I型干扰素（IFN-I）的生产被响应于感染或在炎症反应的活化细胞。不幸的是，的pDC功能的研究是由它们的低频在淋巴器官受阻，并在体外直流代现有的方法主要有利于生产cDC上的过度的pDC。在这里，我们提出了一个独特的方法来有效地生成常见淋巴祖（的CLP） 在体外 pDC的。具体地，该协议描述细节如何从骨髓纯化的CLPs和通过用γ照射的AC-6饲养细胞在Flt3配体的存在下共培养产生的pDC。该培养系统的一个独特的特征是所述的CLPs迁移对AC-6细胞的下面，并成为鹅卵石区形成细胞，用于扩展的pDC的关键步骤。形态不同的DC，即的pDC和cDC上，产生后约2周为7的组合物最佳条件下0-90％的pDC。通常情况下，用这种方法产生的pDC的数目大约是100倍的CLPs播种的数目。因此，这是一种新的系统，用以有力产生大量以促进进一步的研究进入这些细胞的发育和功能所需的pDC的。
Introduction
树突细胞（DC）是专业抗原呈递细胞，在控制免疫反应1起着重要的作用。而DC是异质的，它们大致可分为两个群体，常规的DC（cDC上）和浆树突（pDC细胞）2,3-。除了 淋巴器官，和cDC上的pDCs还发现在组织中，包括肺，肠，皮肤和2。 cDC上和的pDC的形态不同，与cDC上呈现枝蔓状凸起和的pDCs更加浆细胞样的形状。此外，常见的小鼠DC标记，CD11c的，更高度表达于cDC上比上的pDC。此外，cDC上可进一步分成的CD11b + CD24 – cDC上和CD11b – CD24 + cDC上，这两者都表达MHC II类的更高水平和共刺激分子比的pDC 2。成熟的pDCs，另一方面，已经显示出选择性表达的Siglec-H和BST2 4。 Functionally，cDC上有更好的抗原呈递细胞比是pDC的;然而，pDC细胞可以产生类型的大量的I型干扰素后，病毒感染或炎症刺激5。
既cDC上和的pDCs是短暂的，因此，必须不断从骨髓（BM）的6,7-内祖细胞补充。继转移的常见髓系祖细胞（中医）和公共淋巴祖细胞（的CLPs）到致死照射的小鼠表明cDC上和pDC细胞可以从两个谱系8-10产生。然而，有表达RAG1 / 2和具有重新排列的DJ片段在IgH基因基因座11,12的pDC的子集。这些细胞也与B淋巴细胞，包括B220标志物的表达，核酸感应受体（TLR7 / TLR9），和转录因子（SPIB和BCL11A）13共享分子的相似性，提供所有认为是淋巴谱系的签名。因此，电S可以是用于体外代因为谱系相似的pDC的很好的选择。
同时兼具cDC上和的pDC在人类和小鼠的频率很低6，DCS，尤其cDC上， 可以在细胞因子，如GM-CSF或11,14 Flt3配体的存在下，从BM或祖细胞体外生成（佛罗里达州）采用无饲养系统11,15,16。然而不幸的是，它是不可能产生大量使用FL 11,15,16 体外的pDC的。先前我们表明pDC细胞可以有效地在体外从使用AC-6馈线系统17的CLPs生成。使用AC-6基质细胞系在培养系统的优点是，它提供了细胞 – 细胞接触和支持大量的pDC的产生从体CLPs细胞因子的分泌。虽然生产该系统是相当强劲，手续如下所述小心复制需要我n阶确保有效产生的pDC的。
Protocol
Representative Results
Discussion
这里，我们描述的体外培养体系为鲁棒生成的DC，和pDC细胞特别是从少数的CLPs的。该培养体系的独特性是由于使用AC-6细胞，基质细胞系，如送料器。这种方法已被证明提供的不仅是细胞因子，如IL-7，SCF，M-CSF和FL 20，而且在细胞-细胞接触21必要支持直流发展。 AC-6细胞已被先前用于促进体外直流发展的研究从多个祖细胞8,16,22。我们发现，这是至关重要的种子?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢博士。马库斯·曼茨和欧文·韦斯曼提供试剂。我们也承认在医学和台大医院台大医学院提供的流式细胞仪分析，并第一次细胞分选核心设施和第二核实验室的服务，分别为。这项工作得到了科技部，台湾的支持部（MOST 102-2320-B-002-030-MY3）和国家卫生研究院，台湾地区（国家人权机构，EX102-10256SI和国家人权机构 – EX103-10256SI）。
Materials
| Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5 | Biolegend | 108408 | linage marker |
| Anti-mouse NK1.1 (PE), clone PK136 | Biolegend | 108708 | linage marker |
| Anti-mouse CD11b (PE), cloneM1/70 | Biolegend | 101208 | linage marker |
| Anti-mouse CD19 (PE), clone eBio1D3 | Biolegend | 115508 | linage marker |
| Anti-mouse B220 (PE), clone RA3-6B2 | Biolegend | 103208 | linage marker/FACS |
| Anti-mouse CD3 (PE), clone 17A2 | Biolegend | 100308 | linage marker |
| Anti-mouse CD8a (PE), clone 53-6.7 | Biolegend | 100707 | linage marker |
| Anti-mouse MHC-II (PE), clone NIMR-4 | Biolegend | 107608 | linage marker |
| Anti-mouse Ter119 (PE), clone TER-119 | Biolegend | 116208 | linage marker |
| Anti-mouse Thy1.1 (PE), clone HIS51 | eBioscience | 12-0900-83 | linage marker |
| Anti-mouse M-CSFR (APC), clone AFS98 | Biolegend | 135510 | FACS |
| Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8 | Biolegend | 105824 | FACS |
| Anti-mouse Sca-1 (FITC), clone D7 | Biolegend | 108106 | FACS |
| Anti-mouse IL-7Ra (PE/Cy7), clone A7R34 | Biolegend | 135014 | FACS |
| Anti-mouse CD11c (PerCP/Cy5.5), clone N418 | Biolegend | 117328 | FACS |
| Anti-mouse CD11b (FITC), clone M1/70 | Biolegend | 101206 | FACS |
| FACSAria III | BD Biosciences | Cell sorter | |
| FACS sorting tube | BD Biosciences | 352054 | |
| FlowJo | FlowJo LLC | Flow analysis sofrware |
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