Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated...

Regulatory T (T_reg ) 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 T_reg instability after adoptive transfer in inflammatory conditions are major limitations to T_reg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of T_regs . In this research, we evaluated T_reg -like cells obtained from different T_reg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4⁺ cells and a naive CD4⁺ /T_reg 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 T_reg -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 T_reg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without T_reg -specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure T_reg -like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for T_reg generation when compared with the other methods, including a higher purity of T_regs , easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.