Summary

激活的B细胞CD40的小鼠模型

Published: March 05, 2010
doi:

Summary

在这个视频中,我们展示的CD40从C57BL / 6小鼠的脾细胞,它可以用来作为模型抗原提呈细胞(APC)的研究免疫诱导小鼠的B细胞的活化和扩大的过程。

Abstract

有研究表明,B细胞CD40的激活提高其抗原提呈能力。当白细胞介素4和CD40配体(CD40L)的刺激下,没有非常大量高纯度的CD40 – B细胞(14天之内的困难,从少量外周血的可扩展人类B细胞> 10<sup> 9</sup>细胞每名患者),从健康的捐赠者以及癌症患者<sup> 1-4</sup>。 CD40 – B细胞表达重要的淋巴结归巢的分子,可以吸引T细胞<em>在体外</em<sup> 5</sup>。此外,他们采取有效,过程和目前的T细胞抗原<sup> 6,7</sup>。 CD40 – B细胞被证明不仅总理天真,但也扩大记忆性T细胞<sup> 8,9</sup>。因此,CD40 -激活的B细胞(CD40 – B细胞)进行了研究,作为一种替代源免疫刺激抗原提呈细胞(APC),基于细胞immunotherapy1,5,10。为了进一步研究是否CD40 – B细胞诱导有效的T细胞在体内的反应和我们建立了小鼠CD40激活的B细胞产生的细胞培养系统研究的基础机制。使用脾细胞或纯化C57BL / 6小鼠的B细胞CD40的激活,确定了最佳工艺条件如下:开始的C57BL / 6小鼠淋巴细胞(单体型H – 2B)的脾细胞密度梯度离心法和HeLa细胞共同培养纯化重组鼠CD40配体(tmuCD40L的HeLa细胞表达)<sup> 11</sup>。细胞是再培养每隔3-4天,和关键部件,如CD40L,白细胞介素- 4 -巯基乙醇和环孢素A的补充。在这个协议中,我们演示了如何获得类似的APC -表型的人CD40 B细胞(图1A,B)完全激活的小鼠的CD40 – B细胞(mCD40B)。 CD40的刺激会导致快速的生长和细胞培养在14天的高纯度(> 90%),CD19 + B细胞的扩张(图1C,D)​​。为了避免污染与非转染的细胞,小鼠CD40的配体的转染表达经常被控制(图2)。小鼠CD40激活的B细胞可用于研究B细胞的活化和分化,以及调查他们的潜能作为APC,在体内和体外的功能。此外,他们代表了建立抗肿瘤治疗或预防接种有前途的工具,将有助于回答问题,这种方法的安全性和免疫原性<sup> 12</sup>。

Protocol

协议分为两个部分:第一部分为一代小鼠CD40激活的B细胞(脾mCD40B)一个演示小鼠CD40配体(CD40L)表达HeLa细胞(tmuCD40L HeLa细胞),这将被用作板准备束缚饲养层细胞。 B部分介绍了实际的小鼠CD40 – B的文化。 A.制备饲养细胞(HeLa细胞tmuCD40L) tmuCD40L HeLa细胞是一个附着的人类上皮细胞系,这不应该成为完全融合。因此,这些细胞被劈裂每周两次。超过6周以…

Discussion

本协议的修改是可能的,尤其是关于CD40的刺激和使用的小鼠品系类型的起源,也高产高效代小鼠CD40激活的B细胞。艾哈迈迪等人。表明小鼠CD40的配体转染小鼠成纤维细胞类似的结果。可以肯定地排除在这种异种抗原的背景下演示,但在体内的安全性和毒性的深入研究了在此设置使用人类转染的HeLa细胞的炎症或自身免疫反应的迹象。然而,最好的替代代表与激活和增殖活动在同一时间,这?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们诚恳地感谢克莱门斯Wendtner博士提供tmuCD40配体HeLa细胞株。

Materials

A. Preparation of Media:

Feeder cell wild type medium Feeder cell selection medium  
Reagent Concentration Reagent Concentration
RPMI 1640   RPMI 1640  
L-Glutamine 300 μg/mL L-Glutamine 300 μg/mL
FBS 10 % FBS 10 %
HEPES 10 mM HEPES 10 mM
Gentamycin 15 μg/mL Gentamycin 15 μg/mL
   Hygromycin B 0.2 mg/mL
mCD40-B washing medium mCD40-B culture medium
Reagent Concentration Reagent Concentration
D-MEM
D-MEM
L-Glutamine 580 μg/mL L-Glutamine 580 μg/mL
Glucose 4.5 mg/mL Glucose 4.5 mg/mL
HEPES 10 mM HEPES 10 mM
Gentamycin 15 μg/mL Gentamycin 15 μg/mL


MEM 0.1mM (1x)


FBS 10%

B. Miscellaneous Reagents:

Reagent Company Order No
RPMI 1640 Invitrogen Gibco® REF 21875
D-MEM Invitrogen Gibco® REF 41965
Dulbecco´s PBS (10x) PAA Cat No H15-011
Gencin® Delta Select Art No 7395800
FBS LONZA Cat No DE14-802C
HEPES Buffer PAA Cat No S11-001
MEM NEAA Invitrogen Gibco® REF 11140
Hygromycin B PAA Cat No P02-015
Recombinant Murine Interleukin-4 Immunotools Cat No 12340045
Cyclosporin A Novartis Cat No NDC 0078-0109-01
Trypsin/EDTA (10x) Invitrogen Gibco® REF 15400-054

C. Miscellaneous Supplies:

Reagent Company Order No
Sterile pipette tips Sarstedt  
6-well plate NUNC Cat No 140675
50mL conical tube BD Falcon™ Cat No 352070
Tissue culture flask Sarstedt Cat No 83.1813.002

Referencias

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Citar este artículo
Liebig, T. M., Fiedler, A., Klein-Gonzalez, N., Shimabukuro-Vornhagen, A., von Bergwelt-Baildon, M. Murine Model of CD40-activation of B cells. J. Vis. Exp. (37), e1734, doi:10.3791/1734 (2010).

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