Foxp3+ regulatory T cells (Tregs) are potent immunoregulatory cells, prompting strong interests in manipulating them for therapeutic purposes. However, significant challenges remain, including their heterogeneity and functional instability. Here we focused on the inducible Tregs (iTregs) and studied whether the Foxp3 locus can be epigenetically edited ex vivo to produce stable therapeutic iTregs. Under iTreg‐inducing condition where activated CD4⁺ T effector cells were converted to Foxp3+ Tregs, we tested approximately 30 compounds and identified 3 chromatin‐modifying chemical compounds (3C) consisting of sodium butyrate (a broad histone deacetylase inhibitor), UNC0646 (a histone methyltransferase inhibitor), and vitamin C (a TET dioxygenase co‐activator), that together produced complete demethylation at the conserved noncoding sequence 2 (CNS2) region of Foxp3 locus. We found that iTregs induced in the presence of 3C (3C‐iTregs) are stable, even after exposure to inflammatory cytokines. They expressed high levels of Foxp3 and exhibited potent suppressive activities both in vitro and in vivo. We showed that in models of autoimmunity and transplant rejection, adoptive transfer of antigen‐specific 3C‐iTregs prevented the induction of experimental autoimmune encephalitis and enabled long‐term skin allograft survival. Our data demonstrate that the Foxp3 locus can be epigenetically edited ex vivo to generate stable therapeutic iTregs.

中文翻译：

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表观遗传修饰 Foxp3 基因座以生成稳定的抗原特异性 Treg 作为细胞疗法。

Foxp3+ 调节性 T 细胞 (Treg) 是有效的免疫调节细胞，促使人们对出于治疗目的操纵它们产生浓厚兴趣。然而，仍然存在重大挑战，包括它们的异质性和功能不稳定性。在这里，我们专注于诱导型 Tregs (iTregs)，并研究了Foxp3基因座是否可以在体外进行表观遗传编辑以产生稳定的治疗性 iTregs。^{在激活 CD4 +}的 iTreg 诱导条件下T 效应细胞转化为 Foxp3+ Tregs，我们测试了大约 30 种化合物并鉴定了 3 种染色质修饰化合物 (3C)，包括丁酸钠（一种广泛的组蛋白脱乙酰酶抑制剂）、UNC0646（一种组蛋白甲基转移酶抑制剂）和维生素 C（一种TET 双加氧酶共激活剂），它们一起在Foxp3的保守非编码序列 2 (CNS2) 区域产生完全去甲基化轨迹。我们发现在 3C 存在下诱导的 iTregs (3C-iTregs) 是稳定的，即使在暴露于炎性细胞因子后也是如此。它们表达高水平的 Foxp3 并在体外和体内表现出有效的抑制活性。我们表明，在自身免疫和移植排斥模型中，抗原特异性 3C-iTregs 的过继转移阻止了实验性自身免疫性脑炎的诱发，并使皮肤同种异体移植物能够长期存活。我们的数据表明Foxp3基因座可以在体外进行表观遗传编辑以产生稳定的治疗性 iTregs。