激活的B细胞CD40的小鼠模型
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
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 |
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