In vitro generation of regulatory T cells: A challenging tool for studying immune aging in humans

Regulatory T (Treg) cells play a key role in immune tolerance and homeostasis. They prevent exaggerated immune responses, autoimmunity, and are crucial in graft-versus-host responses. On the other hand, their presence in many tumors is associated with a poor prognosis. In aged individuals, Treg populations are modified with a strong bias toward an effector phenotype and a reduced number of naïve Treg cells, but little is known about the functionality of human Treg cells in old age. The low frequency of Treg cells in human peripheral blood is the main limitation to studying them. Therefore, the development of induced Treg (iTreg) cells has become a valuable tool for in vitro research on peripheral Treg cells. Following a gold-standard protocol to generate iTreg in vitro, we aimed to investigate age-related differences in the generation of iTreg cells. However, we detected some unexpected results regarding Foxp3 induction and iTreg functionality. We found no differences in Foxp3⁺ CD4⁺ generated cells between young and older individuals. Surprisingly, the presence of the cytokine TGF-β did not play a role in either Foxp3 expression levels or in the suppressive activity of the resulting cells, regardless of the age of the donors. Therefore, we hypothesize that Foxp3 alone does not adequately define bona fide iTreg cells, and the presence of TGF-β is not essential for the in vitro differentiation of human conventional CD4⁺ T cells into suppressive Foxp3⁺ cells.