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.

中文翻译：

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使用小分子从初始 T 细胞中定向分化调节性 T 细胞并防止其炎症介导的不稳定性。

调节性 T (T _reg ) 细胞疗法是一种在自身免疫病和细胞/器官移植中诱导免疫耐受的有前途的方法。期间下游工艺和T不足隔离产量和杂质_REG在炎性病症过继转移后的不稳定性是至T主要限制_REG疗法，并指示在寻找一有效的，可靠的方法的重要性从头生成的T_个REG。在本研究中，我们评估了从不同 T _reg分化方案中获得的T _reg样细胞的产量、纯度和活性。分化是在幼稚的 CD4 ^{+ 上进行的}细胞和幼稚CD4 ⁺ / T _REG共培养通过使用三个不同的协议-叉头框蛋白P3（E-的FoxP3）可溶性转化生长因子β（S-TGF）和小分子[N-乙酰嘌呤霉素和异位表达SR1555 (N-Ac/SR)]。结果表明，在 T 辅助型 17 (Th17) 极化条件下，特别是白细胞介素 (IL)-6 和 TGF，通过 N-Ac/SR 方法可以获得高产的 T _reg样细胞群-β，与 E-FoxP3 和 S-TGF 方法相比。令人惊讶的是，SR 完全抑制了产生 IL-17 的细胞的分化并促进了炎症条件下的T _reg生成，并且在没有 T 的情况下对 T 细胞增殖具有高度抑制活性。_reg特异性去甲基化酶区 (TSDR) 去甲基化。据我们所知，我们首次报道了在体外炎症条件下通过使用小分子产生高效、纯_Treg样细胞。我们的结果表明，与其他方法相比，N-Ac/SR 方法在 T _reg生成方面具有几个优势，包括更高的 T _regs纯度、更容易的操作、在炎症条件下具有优异的抑制活性和降低成本。