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Updated: Jun 9, 2026
In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol
Published on: December 30, 2016
In vitro generation of RORγt+ regulatory T cells reveals enhanced immunosuppressive function and OXPHOS-dependent
Marcella Cipelli1, Eloísa Martins da Silva2, Lais Cavalieri Paredes1
1Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Background:
RORγt+ regulatory T cells (Treg) play a crucial role in immune regulation, particularly in the gut. However, most current knowledge about this subset derives from in vivo studies, as in vitro investigation has been limited by the lack of protocols capable of preserving their phenotype.
Methods:
Here, we developed and optimized an in vitro differentiation protocol to efficiently generate RORγt+ Treg cells. The protocol was evaluated based on the frequency of RORγt+ Treg cells generated, their suppressive function compared to conventional induced Treg (iTreg), and their metabolic profile.
Results:
The optimized protocol increased the frequency of RORγt+ Treg cells in vitro by up to 70%, providing a robust system for their study. Functionally, in vitro-differentiated RORγt+ Treg cells displayed enhanced immunosuppressive activity compared to conventional iTreg, effectively inhibiting effector CD4⁺ T cell proliferation. Metabolic analyses further revealed a reliance on oxidative phosphorylation (OXPHOS) in this subset.
Conclusion:
This protocol enables the efficient in vitro generation of RORγt+ Treg cells, facilitating functional and metabolic studies of this population and opening new avenues for potential therapeutic applications in immune-mediated diseases.
