Preferential Expansion of CD4+Foxp3+ Regulatory T Cells (Tregs) In Vitro by Tumor Necrosis Factor

  • Chon-Kit Chou
  • Xin ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)


CD4+Foxp3+ regulatory T cells (Tregs) are a distinct subset of CD4 T cells that play indispensable role in the maintenance of immune homeostasis and prevention of deleterious immune responses to self-antigens. Tumor necrosis factor (TNF) is a key cytokine in the autoimmune inflammatory responses. The effect of TNF on Treg activity was extensively studied in the past decade. We for the first time reported that TNF through TNFR2 preferentially activates and expands Tregs. Our discovery is increasingly supported by the research community; however, some controversial results were reported. The differential results are likely caused by different experimental condition. A standard experiment protocol can help researchers to obtain more consistent results. In this chapter, we detail methods used to examine in vitro effect of exogenous TNF on the proliferative expansion of Tregs in unfractionated mouse CD4+ T cells. The related technic issues are analyzed and discussed.

Key words

CD4+FOXP3+ regulatory T cells (Tregs) Tumor necrosis factor (TNF) TNF receptor 2 (TNFR2) Selective expansion CFSE cell division assay Flow cytometry 



We thank Ms. Tianzhen He, Mr. Shuoyang Liu, and Ms. Jingbin Zheng at Institute of Chinese Medical Sciences, University of Macau, for the help in the preparation of manuscript.

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding: This work was supported by University of Macau under Grants MYRG2016-00023-ICMS-QRCM and MYRG2017-00120-ICMS and the Science and Technology Development Fund of Macao S.A.R. (FDCT) under grant 201/2017/A3 and 0056/2019/AFJ. 


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

Authors and Affiliations

  1. 1.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical SciencesUniversity of MacauMacau SARChina

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