Chen Kelvin Y, Kibayashi Tatsuya, Giguelay Ambre, Hata Mayu, Nakajima Shunsuke, Mikami Norihisa, Takeshima Yusuke, Ichiyama Kenji, Omiya Ryusuke, Ludwig Leif S, Hattori Kunihiro, Sakaguchi Shimon
Laboratory of Experimental Immunology, Immunology Frontier Research Center, Osaka University, Osaka, Japan.
Joint Research Chair of Innovative Drug Discovery in Immunology, Immunology Frontier Research Center, Osaka University, Osaka, Japan.
Nature. 2025 Mar 26. doi: 10.1038/s41586-025-08795-5.
Regulatory T (T) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases. Although stimulation of naive CD4 T cells in the presence of TGFβ and IL-2 can induce FOXP3 T cells in vitro (iT cells), the resulting cells are often unstable and have thus far hampered translational efforts. A systematic approach towards understanding the regulatory networks that dictate T differentiation could lead to more effective iT cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalogue gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCOR repressor complex as a novel, context-specific negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iT differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iT cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iT cells more effectively suppressed xenogeneic graft-versus-host disease than control iT cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.
调节性T（Treg）细胞特异性表达主转录因子FOXP3，在维持免疫耐受和体内平衡中起关键作用，并且有可能彻底改变自身免疫性疾病的细胞疗法。尽管在转化生长因子β（TGFβ）和白细胞介素-2（IL-2）存在的情况下刺激初始CD4 T细胞可在体外诱导产生FOXP3 Treg细胞（诱导性Treg细胞，iTreg细胞），但所产生的细胞通常不稳定，迄今为止阻碍了转化研究工作。一种系统的方法来理解决定Treg细胞分化的调控网络可能会带来更有效的基于iTreg细胞的疗法。在这里，我们进行了全基因组CRISPR功能丧失筛选，以编目原代人T细胞中FOXP3诱导的基因调控决定因素，并使用Perturb-icCITE-seq在单细胞分辨率下表征它们的作用。我们确定RBPJ-NCOR抑制复合物是FOXP3表达的一种新的、上下文特异性负调节因子。靶向RBPJ的基因敲除增强了iTreg细胞的分化和功能，独立于经典的Notch信号通路。体外反复进行细胞因子和T细胞受体信号刺激表明，与对照细胞相比，RBPJ缺陷的iTreg细胞通过FOXP3增强子CNS2的DNA去甲基化表现出更高的表型稳定性，从而加强了FOXP3的表达。相反，RBPJ的过表达通过HDAC3直接调节FOXP3组蛋白乙酰化而有力地抑制了FOXP3的诱导。最后，在人源化小鼠模型中，RBPJ缺失的人iTreg细胞比对照iTreg细胞更有效地抑制了异种移植物抗宿主病。总之，我们的研究结果揭示了FOXP3的新调节因子，并指出了提高自身免疫性疾病过继性细胞疗法疗效的新途径。
