Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules.

Regulatory T (Treg ) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and Treg instability after adoptive transfer in inflammatory conditions are major limitations to Treg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of Tregs . In this research, we evaluated Treg -like cells obtained from different Treg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4+ cells and a naive CD4+ /Treg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor β (S-TGF) and small molecules [N-acetyl puromycin and SR1555 (N-Ac/SR)]. The results showed that a high yield of a homogeneous population of Treg -like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-β, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated Treg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without Treg -specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure Treg -like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for Treg generation when compared with the other methods, including a higher purity of Tregs , easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.