Human regulatory T (T_reg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains T_reg cell identity, yet the complete set of key transcription factors that control T_reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human T_reg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from T_reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in T_reg cell function, coregulates another gene network with SATB1 and is important for T_reg cell–mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human T_reg cells that could be targeted for immunotherapies.

人类调节性 T (T _reg ) 细胞对于免疫稳态至关重要。转录因子 FOXP3 维持 T _{reg细胞身份，但控制 T reg}细胞基因表达的整套关键转录因子仍然未知。在这里，我们使用汇集和排列的 Cas9 核糖核蛋白筛选来鉴定在基础和促炎条件下调节原代人 T _reg细胞中关键蛋白质的转录因子。_{然后我们从 T reg}生成了 54,424 个单细胞转录组受到遗传扰动和细胞因子刺激的细胞，除了由 FOXO1 和 IRF4 共同调节的网络外，还揭示了由 FOXP3 和 PRDM1 单独调节的不同基因网络。我们还发现，据我们所知，HIVEP2与 SATB1 共同调节另一个基因网络，据我们所知，之前并未涉及 T _{reg细胞功能，并且对 T reg}细胞介导的免疫抑制很重要。通过整合 CRISPR 筛选和单细胞 RNA 测序分析，我们发现了人类 T _reg细胞中的转录调节因子和下游基因网络，可以作为免疫疗法的靶点。