Maintenance of regulatory T cells CD4+CD25highFOXP3+ (Treg) stability is vital for proper Treg function and controlling the immune equilibrium. Treg cells are heterogeneous and can reveal plasticity, exemplified by their potential to express IL-17A. TNFα-TNFR2 signaling controls IL-17A expression in conventional T cells via the anti-inflammatory ubiquitin-editing and kinase activity regulating enzyme TNFAIP3/A20 (tumor necrosis factor-alpha-induced protein 3). To obtain a molecular understanding of TNFα signaling on IL-17 expression in the human effector (effTreg, CD25highCD45RA-) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve (naïveTreg, CD25highCD45RA+) and effTreg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; effTreg (TNFαlow/IL-17Ahigh) and naïveTreg (TNFαhigh/IL-17Alow). In effTreg, TNFα-TNFR2 signaling prevented IL-17A expression, whereas inhibition of TNFα signaling by clinically applied anti-TNF antibodies led to increased IL-17A expression. Inhibition of TNFα signaling led to reduced TNFAIP3 expression, which, by using siRNA inhibition of TNFAIP3, appeared causally linked to increased IL-17A expression in effTreg. Kinome activity screening of CD3/CD28-activated effTreg revealed that anti-TNF-mediated neutralization led to increased kinase activity. STRING association analysis revealed that the TNF suppression effTreg kinase activity network was strongly associated with kinases involved in TCR, JAK, MAPK, and PKC pathway signaling. Small-molecule-based inhibition of TCR and JAK pathways prevented the IL-17 expression in effTreg. Together, these findings stress the importance of TNF-TNFR2 in regulating the kinase architecture of antigen-activated effTreg and controlling IL-17 expression of the human Treg. These findings might be relevant for optimizing anti-TNF-based therapy and may aid in preventing Treg plasticity in case of Treg-based cell therapy.

中文翻译：

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TNFα信号调节人类效应器Treg的激酶活性并调节IL-17A表达。

维持调节性T细胞CD4 + CD25highFOXP3 +（Treg）的稳定性对于正常的Treg功能和控制免疫平衡至关重要。Treg细胞是异质的，可以显示可塑性，以其表达IL-17A的潜力为例。TNFα-TNFR2信号传导通过抗炎性泛素编辑和激酶活性调节酶TNFAIP3 / A20（肿瘤坏死因子-α诱导的蛋白3）控制常规T细胞中IL-17A的表达。为了获得对TNFα信号传导对人类效应子（effTreg，CD25highCD45RA-）Treg亚群中IL-17表达的分子理解，我们在这里研究了通过TNFα信号传导调节的激酶活性。使用FACS排序的纯真（naïveTreg，CD25highCD45RA +）和effTreg亚群，我们证明了TNFα与IL-17A表达之间的相互关系。effTreg（TNFαlow/ IL-17Alow）和朴素的Treg（TNFαhigh/ IL-17Alow）。在effTreg中，TNFα-TNFR2信号传导阻止了IL-17A表达，而临床应用的抗TNF抗体对TNFα信号的抑制导致IL-17A表达增加。TNFα信号的抑制导致TNFAIP3表达降低，这通过使用siRNA抑制TNFAIP3表现出与effTreg中IL-17A表达增加有因果关系。CD3 / CD28激活的effTreg的激酶组活性筛选显示抗TNF介导的中和导致激酶活性增加。STRING关联分析表明，TNF抑制effTreg激酶活性网络与参与TCR，JAK，MAPK和PKC途径信号传导的激酶密切相关。基于小分子的TCR和JAK途径抑制作用阻止effTreg中的IL-17表达。一起，这些发现强调了TNF-TNFR2在调节抗原激活的effTreg的激酶结构和控制人Treg的IL-17表达中的重要性。这些发现可能与优化基于抗TNF的疗法有关，并且在基于Treg的细胞疗法中可能有助于防止Treg可塑性。