Abstract
In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using tumor cells conditioned media from mouse naïve T cells and to identify them based on their specifics markers (Dalotto-Moreno et al., 2013).
Keywords: Regulatory T cell, Isolation, Breast cancer, T cells, Naive T cells
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol is based in the original work published in Dalotto-Moreno et al. (2013). This work was supported by grants from Agencia Nacional de Promoción Científica y Técnica Argentina (ANPCyT; PICT 2007-093 to M.S. and 2010-870 to G.A.R. and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; PIP 2010-2012 to M.S. and G.A.R.), Fundación Sales to G.A.R. The authors wish to express special thanks to María Rosa Morales for animal technical help.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
That is correct! This protocol is carried out in serum free medium. Treg differentiation can be carried out like this but it is NOT OK for effector T cells as they will die unless serum is present. You can also differentiate Tregs in medium with serum. We have done it in 2%FCS containing medium and has given us great results. The proteins present in the serum can however retain TGFb and can thus impact negatively on the differentiation efficiency. Write if you have any other doubts!