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

Summary 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.