初始CD4+ T细胞体外诱导分化为Th1, Th2, Th17和Treg细胞   

How to cite Favorites Q&A Share your feedback Cited by

摘要:初始CD4+ T细胞具备增殖并且分化成Th1,Th2,Th17和Treg细胞的潜能。使用细胞因子体外刺激初始CD4+T细胞的方法,用于研究不同处理条件对于初始CD4+ T 细胞分化能力的影响 (Cote-Sierra等, 2004)。

关键词: 初始CD4+ T细胞, Th1细胞, Th2细胞, Th17细胞, Treg细胞

材料与试剂

  1. 96孔板
  2. Dynabeads mons t activator CD3/CD28 (Gibco, catalog number: 11452D)
  3. Purified NA/LE hamster anti mouse CD3e (BD, catalog number: 553057)
  4. Purified NA/LE hamster anti mouse CD28 (BD, catalog number: 553294)
  5. Anti-mouse IL-4 (BD, catalog number: 554434)
  6. Murine IL-12 (Pepro Tech, catalog number: 2101210)
  7. Murine IL-2 (Pepro Tech, catalog number: 2121220)
  8. Murine IL-4 (Pepro Tech, catalog number: 214145)
  9. Anti- mouse IFN-γ (BD, catalog number: 551216)
  10. Anti- mouse IL-12 (BD, catalog number: 551219)
  11. Murine IL-6 (Pepro Tech, catalog number: 21616)
  12. Human TGFβ1 (Pepro Tech, catalog number: 10021C10)
  13. Murine TNFα (Pepro Tech, catalog number: 31501A5)
  14. Murine IL-1β (Pepro Tech, catalog number: 21111B)
  15. EasysepTM mouse naïve CD4+ T cells isolation kit (Stem cell, catalog number: 19725A)
  16. β-Mercaptoethanol (Millipore, catalog number: ES007E)
  17. PBS
  18. 1640培养基
  19. IMDM培养基 
  20. 谷氨酰胺
  21. 双抗
  22. 1640完全培养基 (见溶液配方)
  23. IMDM完全培养液 (见溶液配方)

仪器设备

  1. 细胞培养箱 (Thermo Fisher Scientific)
  2. FACSLSRII流式细胞仪 (Becton Dickinson)

实验步骤

一. 初始CD4+ T细胞分离
使用EasysepTM mouse naïve CD4+ T cells isolation kit阴选出脾脏中的初始CD4+ T细胞 (naïve CD4+ T cells)。如果在96孔板中刺激诱导,每孔铺1 x 105 naïve CD4+ T cells,以下均以1 x 105细胞为例。

二. CD3和CD28预先铺板
Th0,Th1和Treg这3种细胞使用purified NA/LE hamster anti mouse CD3e和purified NA/LE hamster anti mouse CD28预先铺板,两个的铺板工作浓度均为1 μg/ml (PBS稀释),96孔板铺板体积为50 μl。可在4 °C预铺12~16小时或者37 °C预铺2小时。
Th17细胞铺板浓度不同,purified NA/LE hamster anti mouse CD3e工作浓度 2 μg/ml,CD28工作浓度 5 μg/ml。
预铺结束,缓慢吸走上清,开始洗板子,即缓慢加入1640完全培养液室温静止5分钟,吸走。洗三次,注意最后一次在加入细胞前再吸走培养液。

三. 刺激分化

  1. Th0细胞培养: 每孔200 μl 1640完全培养液悬起细胞,其中加入细胞因子工作浓度为:anti-IL-4: 10 μg/ml, anti-IFN-γ: 10 μg/ml;
  2. Th1细胞培养: 每孔200 μl 1640完全培养液悬起细胞,其中加入细胞因子工作浓度为:IL-12: 10 ng/ml, IL-2: 10 ng/ml, anti-IL-4: 10 μg/ml;


    图1. Th1细胞流式结果示意图

  3. Th2细胞培养: 每孔200 μl 1640完全培养液悬起细胞,其中加入细胞因子工作浓度为:IL-4: 10 ng/ml, IL-2: 5 ng/ml, anti-IFN-γ: 10 μg/ml, anti-IL-12 :10 μg/ml, 加CD3/CD28 beads: 2.5 μl beads (2.5 μl beads/105 cells) (Cote-Sierra等, 2004);
    注:Beads提前用1640完全培养液洗三遍,后加入培养液中。


    图2. Th2细胞流式结果示意图

  4. Th17细胞培养: 每孔200 μl IMDM完全培养液悬起细胞,其中加入细胞因子工作浓度为:IL-6: 30 ng/ml, TGFβ: 3 ng/ml, TNFα: 10 ng/ml, IL-1β: 10 ng/ml, anti-IFN-γ: 5 μg/ml, anti-IL-4: 5 μg/ml (Kleinewietfeld等, 2013);


    图3. Th17细胞流式结果示意图

  5. Treg细胞培养: 每孔200μl 1640完全培养液悬起细胞,其中加入细胞因子工作浓度为:IL-2: 5 ng/ml, TGFβ: 3 ng/ml (Freudenberg等, 2018);


    图4. Treg细胞流式结果示意图

            72小时后轻轻吸走100 μl培养液,再次加入细胞因子种类及浓度和之前一样的100 μl培养液,再培养24小时后,PI reboost 3~4小时后检测各细胞诱导比例。

注意事项

  1. 用加入细胞因子的培养液悬起细胞后,尽量轻柔的将细胞加到板里,避免吹起之前铺好的 anti-CD3/CD28抗体。
  2. 当刺激细胞数量比较大时,48小时开始每12小时观测细胞浓度,如果细胞密度很大,则立即铺加刺激,将培养液分到新的孔中,同样在最初刺激96小时后检测。96孔板最多培养初始3 x 105细胞,更多细胞则需要按比例依次换更大的孔板。

溶液配方

  1. 1640完全培养基
    1640培养基加10% FBS
    加双抗 (100x)
    加50 μM Mercaptoethanol 
  2. IMDM完全培养液
    IMDM培养基加10% FBS
    加双抗 (100x)
    加谷氨酰胺 (100x)
    加5 μM Mercaptoethanol

致谢

此份实验操作方法是实验室多位师兄师姐查阅文献,并结合实际实验操作最终总结的实验方法,实验室后续多位同学验证可行。特此感谢实验室相关人员对此实验方法的贡献!

参考文献

  1. Cote-Sierra, J., Foucras, G., Guo, L., Chiodetti, L., Young, H. A., Hu-Li, J., Zhu, J. and Paul, W. E. (2004). Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci U S A 101(11): 3880-3885. 
  2. Freudenberg, K., Lindner, N., Dohnke, S., Garbe, A. I., Schallenberg, S. and Kretschmer, K. (2018). Critical Role of TGF-beta and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation. Front Immunol 9: 125.
  3. Kleinewietfeld, M., Manzel, A., Titze, J., Kvakan, H., Yosef, N., Linker, R. A., Muller, D. N. and Hafler, D. A. (2013). Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature 496(7446): 518-522.
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:鲍温洁. (2019). 初始CD4+ T细胞体外诱导分化为Th1, Th2, Th17和Treg细胞. Bio-101: e1010309. DOI: 10.21769/BioProtoc.1010309.
How to cite: Bao, W. J. (2019). In vitro differentiation of naïve CD4+ T cells into Th1, Th2, Th17 and Treg cells. Bio-101: e1010309. DOI: 10.21769/BioProtoc.1010309.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.