Summary

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

Published: October 29, 2015
doi:

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 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

在这项研究中,成年(6-8周)雌性C57BL / 6小鼠。小鼠根据根特大学动物饲养的指导原则安置和饲养。所有动物的程序获得批准的机构动物护理和伦理委员会。 1.准备单核从小鼠脾细胞(跨国公司)的通过颈脱位牺牲鼠标。 把鼠标放在解剖板背下来,并修复后和前肢与引脚。 消毒皮肤表面,用70%(体积/体积)乙醇/ H 2 O 切割沿腹部中线到?…

Representative Results

使用密度梯度脾的单核细胞的分离需要大约1小时,并消除了使用以裂解红血细胞(红细胞)所需的试剂的。使用这种方法和碎屑的脏器应变被去除期间产生得到一种高收率的活细胞。通常,iNKT细胞的脾淋巴细胞池内的频率总T淋巴细胞的1至5%的范围内然而,这可以根据所使用的动物的年龄,性别和健康状况而变化。大约10 6 iNKT细胞可以从3只小鼠的合并脾和产量最高iNKT细胞来获取从6-8周?…

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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Cite This Article
Govindarajan, S., Elewaut, D., Drennan, M. An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen. J. Vis. Exp. (104), e53256, doi:10.3791/53256 (2015).

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