CD4+ T-Cell Differentiation In Vitro

  • Wenyong Yang
  • Xueying Chen
  • Hongbo HuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)


CD4+ T helper cells play crucial roles in adaptive immune response against pathogens, as well as in host immune homeostasis. Upon TCR activation, naïve CD4+ T cells differentiate into one of several lineages of Th cells, with hallmark transcription factors, cytokine production, and functions in vivo, according to the particular cytokine milieu. To study the regulating mechanism and function of Th cells, in vitro CD4+ T-cell differentiation is crucial. The following protocols describe the methods to induce naïve CD4+ T-cell differentiate into Th1, Th2, Th17 and Treg by activating TCR, together with the different cytokines and blocking antibodies in vitro. The efficiency of T helper cell differentiation is examined by detecting the expression of hallmark cytokines and transcription factors.

Key words

CD4+ T cells T-cell differentiation Cytokines TCR activation Flow cytometry 



This study was supported by grant from the Ministry of Science and Technology (the National Key Research and Development Program 2016YFA0502203) and National Natural Science Foundation of China (no. 91740111 and 81871232).


  1. 1.
    Zhu J, Yamane H, Paul WE (2010) Differentiation of effector CD4 T cell populations (∗). Annu Rev Immunol 28:445–489CrossRefGoogle Scholar
  2. 2.
    Wu W, He C, Liu C et al (2015) miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD. Gut 64:1755–1764CrossRefGoogle Scholar
  3. 3.
    Wang D, Liu Y, Li Y et al (2017) Galphaq regulates the development of rheumatoid arthritis by modulating Th1 differentiation. Mediat Inflamm 2017:4639081Google Scholar
  4. 4.
    Bradley LM, Dalton DK, Croft M (1996) A direct role for IFN-gamma in regulation of Th1 cell development. J Immunol 157:1350–1358PubMedGoogle Scholar
  5. 5.
    Hu H, Wang H, Xiao Y et al (2016) Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination. J Exp Med 213:399–414CrossRefGoogle Scholar
  6. 6.
    Yamane H, Paul WE (2012) Memory CD4+ T cells: fate determination, positive feedback and plasticity. Cell Mol Life Sci 69:1577–1583CrossRefGoogle Scholar
  7. 7.
    Mowen KA, Glimcher LH (2004) Signaling pathways in Th2 development. Immunol Rev 202:203–222CrossRefGoogle Scholar
  8. 8.
    Pulendran B, Artis D (2012) New paradigms in type 2 immunity. Science 337:431–435CrossRefGoogle Scholar
  9. 9.
    Hirose K, Iwata A, Tamachi T et al (2017) Allergic airway inflammation: key players beyond the Th2 cell pathway. Immunol Rev 278:145–161CrossRefGoogle Scholar
  10. 10.
    Hwang ES, Szabo SJ, Schwartzberg PL et al (2005) T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 307:430–433CrossRefGoogle Scholar
  11. 11.
    Yang J, Sundrud MS, Skepner J et al (2014) Targeting Th17 cells in autoimmune diseases. Trends Pharmacol Sci 35:493–500CrossRefGoogle Scholar
  12. 12.
    Ivanov II, Mckenzie BS, Zhou L et al (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133CrossRefGoogle Scholar
  13. 13.
    Veldhoen M, Hocking RJ, Atkins CJ et al (2006) TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24:179–189CrossRefGoogle Scholar
  14. 14.
    Mangan PR, Harrington LE, O'quinn DB et al (2006) Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 441:231–234CrossRefGoogle Scholar
  15. 15.
    Mcgeachy MJ, Chen Y, Tato CM et al (2009) The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol 10:314–324CrossRefGoogle Scholar
  16. 16.
    Hu H, Sun SC (2016) Ubiquitin signaling in immune responses. Cell Res 26:457–483CrossRefGoogle Scholar
  17. 17.
    Sakaguchi S, Yamaguchi T, Nomura T et al (2008) Regulatory T cells and immune tolerance. Cell 133:775–787CrossRefGoogle Scholar
  18. 18.
    Rudensky AY (2011) Regulatory T cells and Foxp3. Immunol Rev 241:260–268CrossRefGoogle Scholar
  19. 19.
    Vignali DA, Collison LW, Workman CJ (2008) How regulatory T cells work. Nat Rev Immunol 8:523–532CrossRefGoogle Scholar
  20. 20.
    Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336CrossRefGoogle Scholar
  21. 21.
    Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Rheumatology and Immunology, State Key Laboratory of Biotherapy and Collaborative Innovation Center for Biotherapy, West China HospitalSichuan UniversityChengduChina

Personalised recommendations