We generated Th1-like iTregs by culturing iTregs with IL-12, then assessed their suppressive capacity, expression of iTreg suppression markers, and enzymatic activity of PRMT5 using histone symmetric arginine di-methylation (H3R2me2s) as a read out. We used ChIP sequencing on Th1 cells, iTregs, and Th1-like iTregs to identify H3R2me2s-bound genes unique to Th1-like iTregs, then validated targets using CHiP-qPCR. We knocked down PRMT5 to validate its contribution to Th1-like iTreg lineage commitment. Finally we tested the therapeutic potential of Th1-like iTregs using a Th1-mediated mouse model of AA.
Exposing iTregs to the Th1 cytokine, interleukin-12 (IL-12), during early events of differentiation conveyed increased suppressive function. We observed increased PRMT5 enzymatic activity, as measured by H3R2me2s, in Th1-like iTregs, which was downregulated in iTregs. Using ChIP-sequencing we discovered that H3R2me2s is abundantly bound to the Sirt1 promoter region in Th1-like iTregs to negatively regulate its expression. Furthermore, administering Th1-like iTregs to AA mice provided a survival benefit.
Knocking down PRMT5 in Th1-like iTregs concomitantly reduced their suppressive capacity, supporting the notion that PRMT5 is important for the superior suppressive capacity and stability of Th1-like iTregs. Conclusively, therapeutic administration of Th1-like iTregs in a mouse model of AA significantly extended their survival and they may have therapeutic potential.
■我们通过用IL-12培养iTregs来产生Th1样iTregs,然后评估它们的抑制能力,iTreg抑制标记的表达,和PRMT5的酶活性,使用组蛋白对称精氨酸二甲基化(H3R2me2s)作为读数。我们在Th1细胞上使用了ChIP测序,iTregs,和Th1样iTregs来鉴定Th1样iTregs特有的H3R2me2s结合基因,然后使用CHiP-qPCR验证靶标。我们取消了PRMT5,以验证其对Th1样iTreg谱系承诺的贡献。最后,我们使用Th1介导的AA小鼠模型测试了Th1样iTregs的治疗潜力。
■将iTregs暴露于Th1细胞因子,白细胞介素-12(IL-12),在分化的早期事件中,表达了抑制功能的增加。我们观察到PRMT5酶活性增加,用H3R2me2s测量,在类似Th1的iTregs中,在iTregs中被下调。使用ChIP测序,我们发现H3R2me2s与Th1样iTreg中的Sirt1启动子区域大量结合,从而负向调节其表达。此外,向AA小鼠施用Th1样iTregs提供了生存益处。
■在Th1样iTregs中击倒PRMT5的同时降低了它们的抑制能力,支持PRMT5对于Th1样iTregs的优异抑制能力和稳定性很重要的观点。最后,Th1样iTregs在AA小鼠模型中的治疗性给药可显着延长其生存期,并且它们可能具有治疗潜力。