从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法
Summary
Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.
Abstract
刺激后迅速分泌细胞因子的能力是不变的天然杀伤T（的iNKT）细胞谱系的功能特性。因此iNKT细胞的特征是一种天生的T细胞群体能够激活和转向适应性免疫应答。的改进的技术用于小鼠的iNKT细胞的培养和扩大发展能促进的iNKT细胞生物学中的体外和体内模型系统研究。这里，我们描述了分离和扩增的鼠脾iNKT细胞的优化过程。
从C57BL / 6小鼠脾脏被除去，解剖和紧张，将所得细胞悬浮液被层叠在密度梯度介质。离心后，脾单核细胞（MNCs）被收集和CD5阳性（CD5 +）淋巴细胞富集使用磁珠。是在CD5 +分数在iNKT细胞随后用^5;神经酰胺加载的CD1d四聚体和通过荧光激活细胞分选（FACS）纯化。 FACS分选iNKT细胞然后最初在体外培养的小鼠重组IL-7存在下被扩展之前使用的重组鼠细胞因子和板结合的T细胞受体（TCR）的刺激的组合。使用这种技术，大约可以在18-20天的培养，之后可用于功能测定法在体外 ，或在小鼠体内转移实验，他们会产生10 8 iNKT细胞。
Introduction
鼠不变的自然杀伤T（iNKT细胞）细胞是由CD1d的表达皮质胸腺细胞1,2在胸腺选择的先天T淋巴细胞的独特群体。 iNKT细胞表达由一个不变的Vα14-Jα18TCR链配对任Vβ8，Vβ7或Vβ2TCR的3，其能够识别内源性以及外国脂质抗原的CD1d的上下文中的T细胞受体（TCR）。例如，鼠iNKT细胞识别并通过内源性脂质抗原称为isoglobotrihexosylceramide（iGb3）4，以及α-半乳糖神经酰胺（αGalCer）5,6-，糖脂自海绵中分离被激活。 TCR依赖性iNKT细胞的活化促进适应性免疫应答的引发，并作为其结果，iNKT细胞已被证明是功能上参与的范围病状包括风湿性疾病7和癌症的改善或发展<SUP> 8。目前，合成的iNKT细胞的配体构成有希望的新的疫苗佐剂可以是能够调节的一些免疫病理状况。
先前已经证明，iNKT细胞可以在体外从以下然而小鼠组织分离来产生;许多研究中采用的使用主要抗原呈递细胞（APC）和/或细胞系9，Vα14TCR转基因（Tg）为10只小鼠，或胸腺瘤为的iNKT细胞衍生的杂交瘤11,12的产生。此外，大量的老鼠，大批量试剂如αGalCer加载CD1d的二聚体，和漫长的文化时代使一些出版协议不太道德和经济上的吸引力9,13。
在本报告中，我们描述了用于分离和在 iNKT细胞的小鼠脾脏体外扩增的方法适于。更具体地，协议描述了一种方法富集从小鼠脾脏这减少了所需的用于FACS细胞分选小鼠，试剂和时间，并提出了在体外扩展排序脾iNKT细胞的优化方法iNKT细胞。
Protocol
Representative Results
Discussion
在当前协议关键步骤包括分离和CD5 +淋巴细胞（第1和2）的随后的富集，FACS分选（第3节）和iNKT细胞的初始接种（第4节）。第1节执行的步骤，切记要仔细层中的脾细胞悬浮液密度梯度介质，使得不同的细胞相间的离心后产生的。随后的富集CD5 +淋巴细胞通过磁性细胞分离（第2部分）最大程度地减少染色所需的试剂和时间和FACS分类iNKT细胞（第3节），这有助于增加的iNKT细胞产量后?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.
Materials
| Material | |||
| recombinant murine IL-2 | eBiosciences | 14-8021 | |
| recombinant murine IL-12 | eBiosciences | 39-8122-65 | |
| recombinant murine IL-7 | eBiosciences | 14-8071 | |
| purified anti-mouse CD3e (145-2C11) | eBiosciences | 16-0032-86 | |
| purified anti-mouse CD28 (37.51) | eBiosciences | 16-0281-85 | |
| e450-conjugated anti-mouse CD19 (eBio1D3) | eBiosciences | 48-0193-82 | |
| e450-conjugated anti-mouse CD8α (53-6.7) | eBiosciences | 48-0081-82 | |
| FITC-conjugated anti-mouse CD5 (53-7.3) | eBiosciences | 11-0051-81 | |
| V500-conjugated anti-mouse CD3ε (500A2) | BD biosciences | 560771 | |
| anti-mouse CD5 (Ly-1) microbeads | Macs Miltenyi Biotec | 130-049-301 | |
| purified anti-mouse CD16/32 (2.4G2) | Macs Miltenyi Biotec | 130-092-575 | |
| MACS MS Columns | Macs Miltenyi Biotec | 130-042-201 | |
| Trypan Blue, 0.4% (wt/vol) | Gibco | 15250-061 | |
| RPMI 1640 medium | Gibco | 12633-020 | |
| 1x PBS | Gibco | 10010-056 | [Ca2+/Mg2+ – free] |
| Fetal calf serum | Gibco | 10270 | |
| L-Glutamine | Gibco | 25030-123 | |
| Penicillin-streptomycin | Sigma-Aldrich | P4458-100ML | |
| Bovine serum albumin | Sigma-Aldrich | A7906-100MG | |
| β-Mercaptoethanol | Sigma-Aldrich | M3148 | |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | EDS-1KG | |
| Ficoll-Paque plus | GE Healthcare | 71-7167-00 AF | |
| Equipment | |||
| 96-well plate F-bottom | Greiner-bio one | 657-160 | |
| 24-well plate | Greiner-bio one | 665-180 | |
| 6-well plate | Greiner-bio one | 655-180 | |
| 15 ml falcon tube | Greiner-bio one | 188-271 | |
| 50 ml falcon tube | Greiner-bio one | 227-261 | |
| 70 µm filter | Greiner-bio one | 542-070 | |
| 30 µm filter | Millipore | SVGP01050 | |
| MiniMACS separator | Macs Miltenyi Biotec | 130-042-102 | |
| MS Columns | Macs Miltenyi Biotec | 130-042-201 | |
| Water-jacketed CO2 incubator | VWR | ||
| Hemocytometer | VWR | ||
| Dissection Kit | VWR | ||
| BD FACSAria III | BD Biosciences |
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