TNFα-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression

Maintenance of regulatory T cells CD4⁺CD25^highFOXP3⁺ (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, CD25^highCD45RA^-) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve (_naïveTreg, CD25^highCD45RA⁺) and _effTreg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; _effTreg (TNFα^low/IL-17A^high) and _naïveTreg (TNFα^high/IL-17A^low). 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.