Abstract：
Induced regulatory T cell (iTregs) can be generated in vitro. Thus, iTregs-based therapeutics are receiving increased attention for their potential to treat autoimmune diseases and prevent transplant rejection. However, iTregs fail to maintain FoxP3 expression and suppressive activity, which limits their clinical application. Increasing lines of evidence suggest that methyltransferase-like 14 (METTL14), a critical component of the m6A writer complex, regulates the stability and function of the Treg cells. However, beyond meeting the epigenetic modification of Treg cells, whether Mettl14 plays a role in the fate determination of iTregs is unclear. Here, we systemically investigated the potential function of METTL14 in iTregs differentiation and regulatory activity. In our study, iTregs were generated from CD4+ naïve T cells under iTreg-polarizing conditions, we found that the expression of METTL14 was increased in iTregs compared with CD4+naïve T cells. Subsequently, the expression of METTL14 was knocked down by siRNA-METTL14 interference in CD4+ naïve T cells and cultured under iTreg-polarizing conditions. According to the results, Mettl14 deficiency resulted in the disruption of iTregs differentiation evidenced by the limited FoxP3 expression. Meanwhile, inflammatory cytokines such as IFN-γ and IL-17a were upregulated in cultured iTregs. We next determined the functional change in METTL14-deficient iTregs. The results of the colitis development in Rag1-/- mice and CFSE assays revealed that loss of METTL14 significantly compromised the suppressive function of iTregs in vivo and in vitro. We further checked the altered signaling pathway in METTL14-deficient iTregs. We found that reduced METTL14 leads to activation of the mTOR pathway with increased p-mTOR and p-p70S6K, which are known to modulate the suppressive function of iTregs. In conclusion, our study revealed that Mettl14 plays a critical role in the development and suppressive function of iTregs in vitro and could thus serve as a regulatory element for stabilizing iTregs in cell-based therapy.
摘要：
诱导的调节性T细胞(iTregs)可以在体外产生。因此，基于iTregs的疗法因其治疗自身免疫性疾病和预防移植排斥的潜力而受到越来越多的关注。然而,iTregs无法维持FoxP3表达和抑制活性，这限制了它们的临床应用。越来越多的证据表明甲基转移酶样14(METTL14)，M6A作家综合体的关键组成部分，调节Treg细胞的稳定性和功能。然而,除了满足Treg细胞的表观遗传修饰，Mettl14是否在iTregs的命运决定中起作用尚不清楚。这里,我们系统地研究了METTL14在iTregs分化和调节活性中的潜在功能。在我们的研究中,iTreg在iTreg极化条件下从CD4+初始T细胞产生，我们发现，与CD4+初始T细胞相比，iTregs中METTL14的表达增加。随后，在CD4+初始T细胞中通过siRNA-METTL14干扰敲低METTL14的表达,并在iTreg极化条件下培养。根据结果,Mettl14缺陷导致iTregs分化的破坏,这由有限的FoxP3表达证明。同时,炎性细胞因子如IFN-γ和IL-17a在培养的iTregs中上调。接下来，我们确定了METTL14缺陷型iTreg的功能变化。Rag1-/-小鼠和CFSE测定中结肠炎发展的结果显示,METTL14的缺失显著损害了体内和体外iTregs的抑制功能。我们进一步检查了METTL14缺陷型iTregs中信号通路的改变。我们发现，减少的METTL14导致mTOR途径的激活与增加的p-mTOR和p-p70S6K，已知它们可以调节iTregs的抑制功能。总之,我们的研究表明,Mettl14在体外iTregs的发育和抑制功能中起关键作用,因此可以在基于细胞的治疗中作为稳定iTregs的调节元件.
