T cell receptor (TCR) stimulation and cytokine cues drive the differentiation of CD4⁺ naïve T cells into effector T cell populations with distinct proinflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4⁺ T cells preferentially differentiate into FOXP3⁺ regulatory T (T_reg) cells upon TCR activation independent of exogenous cytokine signaling. This cell-intrinsic predisposition for T_reg differentiation is implicated in the generation of tolerance in utero; however, the underlying mechanisms remain largely unknown. Here, we identify epigenetic and transcriptional programs shared between fetal naïve T and committed T_reg cells that are inactive in adult naïve T cells and show that fetal-derived induced T_reg (iT_reg) cells retain this transcriptional program. We show that a subset of T_reg-specific enhancers is accessible in fetal naïve T cells, including two active superenhancers at Helios. Helios is expressed in fetal naïve T cells but not in adult naïve T cells, and fetal iT_reg cells maintain Helios expression. CRISPR-Cas9 ablation of Helios in fetal naïve T cells impaired their differentiation into iT_reg cells upon TCR stimulation, reduced expression of immunosuppressive genes in fetal iT_reg cells such as IL10, and increased expression of proinflammatory genes including IFNG. Consequently, Helios knockout fetal iT_reg cells had reduced IL-10 and increased IFN-γ cytokine production. Together, our results reveal important roles for Helios in enhancing preferential fetal T_reg differentiation and fine-tuning eventual T_reg function. The T_reg-biased programs identified within fetal naïve T cells could potentially be used to engineer enhanced iT_reg populations for adoptive cellular therapies.

T细胞受体（TCR）刺激和细胞因子提示驱动CD4 ⁺幼稚T细胞分化为具有独特促炎或调节功能的效应T细胞群体。与成年幼稚T细胞不同，人类幼稚CD4 ⁺ T细胞在TCR激活后优先于外源细胞因子信号传导而分化为FOXP3 ⁺调节性T（T _reg）细胞。T _reg分化的这种细胞内在诱因与子宫内耐受的产生有关。但是，其基本机制仍然未知。在这里，我们确定了幼稚T和定型T _reg之间共享的表观遗传和转录程序在成年幼稚T细胞中没有活性的细胞，表明胎儿诱导的T _reg（iT _reg）细胞保留了该转录程序。我们显示，T _reg特异性增强子的一个子集可在未使用过的胎儿T细胞中获得，其中包括Helios的两个活跃的超级增强子。Helios在胎儿幼稚T细胞中表达，但在成年幼稚T细胞中不表达，胎儿iT _reg细胞维持Helios表达。胎儿幼稚T细胞中Helios的CRISPR-Cas9消融破坏了TCR刺激后其分化为iT _reg细胞的能力，降低了胎儿iT _reg细胞中免疫抑制基因如IL10的表达，并增加包括IFNG在内的促炎基因的表达。因此，Helios基因敲除的胎儿iT _reg细胞具有降低的IL-10和增加的IFN-γ细胞因子产生。总之，我们的结果揭示了Helios在增强优先胎儿_Treg分化和微调最终_Treg功能方面的重要作用。在胎儿幼稚T细胞中鉴定出的T _reg偏向程序可潜在地用于工程化增强的iT _reg人群，以进行过继细胞治疗。