Neddylation-mediated activation of Cullin-RING E3 Ligases (CRLs) are necessary for the degradation of specific immune regulatory proteins. However, little is known about how these processes govern the function of regulatory T (Treg) cells. We show that mice with Treg cell-specific deletion of Rbx1, a dual E3 for both neddylation and ubiquitylation by CRLs, develop an early-onset fatal inflammatory disorder, characterized by disrupted Treg cell homeostasis and suppressive functions. Specifically, Rbx1 is essential for the maintenance of an effector Treg cell subpopulation, and regulates several inflammatory pathways. Similar but less severe phenotypes are observed in mice having Ube2m, a neddylation E2 conjugation enzyme, deleted in their Treg cells. Thus, above work demonstrates that the Ube2m-Rbx1 axis is specifically required for intrinsic regulatory processes in Treg cells; and that Rbx1 might also play Ube2m-independent roles in maintaining the fitness of Treg cells.

Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, components of a similar but distinct neddylation-CRL complex, yields no obvious phenotype. Whether these E2s or E3s compensate each other in functional regulations of Treg cells is, however, previously unknown. We further generated Foxp3^Cre;Ube2m^fl/fl;Ube2f^fl/fl or Foxp3^Cre;Rbx1^fl/fl;Sag^fl/fl double-null mice by simultaneous deletion of both neddylation E2s or E3s in Treg cells, respectively. Remarkably, Ube2m&Ube2f double-null mice developed much severe autoimmune phenotypes than did Ube2m-null mice, indicating that Ube2m markedly compensates Ube2f in Treg cells. The minor worsened autoimmune phenotypes seen at the very early stage in Rbx1&Sag double-null than Rbx1-null mice is likely due to already severe phenotypes of the later, indicating a minor compensation of Rbx1 for Sag. The RNA profiling-based analyses revealed that up- and down-regulations of few signaling pathways in Treg cells are associated with the severity of autoimmune phenotypes. Finally, severer inflammation phenotypes seen in mice with double E3-null than with double E2-null Treg cells indicate a neddylation-independent mechanism of 2 E3s, also known to serve as the RING component of CRLs in regulation of Treg cell fitness.