Corcoran K et al. (PD-1, BTLA, or TIM3) receptors on T cells (reviewed in ). For example TCR/MHC-peptide engagement paired with CD28/B7 costimulation initiates calcineurin, Ras, and PKC- signaling that activates NFAT, AP1, and NF-B transcription factors required to induce IL-2 production, T cell proliferation and differentiation (Figure 1B). In contrast, T cells with TCR/MHC-peptide engagement in the absence of CD28/B7 costimulatory signals do not upregulate IL-2 and enter anergy MT-7716 free base  (Figure 1B). Several E3s are transcriptionally upregulated under anergic conditions, among them Cbl-b, Itch, and Grail (also known as RNF128) [20C22], which function to limit TCR signaling output. This is achieved through proteolysis-dependent mechanisms, such as ubiquitin-dependent TCR/CD3 receptor downmodulation by Cbl-b  and Grail  or downregulation of downstream TCR signaling components including PLC- and PKC- by Itch and MT-7716 free base GLUR3 Cbl-b [20, 25]. Itch further regulates AP1 by proteasomal degradation of junB, a mechanism involved in CTLA-4-mediated T cell inhibition [26, 27]. Cbl-b and Grail also exhibit proteolysis-independent functions that promote anergy: Cbl-b inhibits recruitment of the phosphoinositide 3-kinase subunit p85 to CD28 and TCR , preventing TCR clustering , and Grail ubiquitination of Rho GDP-dissociation inhibitor stabilizes it, preventing IL-2 expression . Thus, by modulating the abundance/activity of critical TCR signaling molecules, E3s serve as T cell intrinsic checkpoints to limit T cell activation. These molecular mechanisms partially explain spontaneous autoimmunity or enhanced experimentally-induced autoimmunity observed in mice deficient for Cbl-b, Itch, or Grail [24, 31C34] (Table 1) and may serve to develop targeted therapeutics for patients suffering multisystem autoimmune disease, mediated e.g. by polymorphisms in the human homolog of Itch . Interestingly, overstimulated of MT-7716 free base T cells by the effector cytokine IL-2 MT-7716 free base results in co-expression of the apoptosis-related factors Fas (CD95) and Fas ligand (FasL). Engagement of Fas by FasL induces T cell apoptosis, thereby providing an additional safety mechanism to prevent T cell overactivation and mice with defects in Fas, FasL or IL-2R develop autoimmunity due to failure of T cells to undergo AICD [15, 36]. Among E3s that either stimulate or inhibit AICD are WWP2 , c-Cbl , and A20 . WWP2 limits AICD in T cells by ubiquitinating and destabilizing the transcription factor EGR2, thereby limiting EGR2-mediated FasL upregulation to promotes T cell survival . As A20 has been implicated in the control of RIPK and NF-B signaling, the role of A20 in AICD  and its contribution to the phenotypes observed in A20 knockout mice, which exhibit multi-organ inflammation  and in men with A20 polymorphisms that are associated with systemic lupus erythematous, Crohns disease or psoriasis  remain to be determined. In addition to nTregs that are selected in thymus , immunosuppressive Treg can also emerge from na?ve CD4+ T cells following TCR activation in the presence of the suppressor cytokine TGF- [41C43] secreted by tolerogenic DCs ; this Treg subset is called induced (i)Treg. The immunosuppressive function of Treg is crucial for tolerance induction, and decreased Treg number or function is associated with a variety of autoimmune pathologies . Interestingly, E3s are implicated in both Treg development and function. For example, Stub1 and Cbl-b activity destabilizes Foxp3, which is required for Treg identity, by ubiquitin-dependent degradation [46, 47]. The E3s von Hippel-Lindau (VHL) and Itch support maintenance of functional Treg [48, 49]. Treg-specific loss of VHL results in HIF1-dependent conversion of Treg into TH1-like, IFN-producing effector T cells, which culminates in multi-organ lymphocyte infiltration and early mouse mortality . Additionally, in an adoptive transfer murine colitis model, VHL-deficient Tregs were unable to prevents colitis, further exemplifying their role in autoimmunity . Similar, Treg-specific Itch deficiency in mice results in severe airway inflammation, mediated by increased TH2 cytokine production by Itch-deficient Tregs . Furthermore, Grail is critical for MT-7716 free base Treg function, as Grail?/? Treg are less immunosuppressive, and express TH17 cell-related genes . Given the crucial role of E3s in maintaining Treg homeostasis, along with their ability to induce anergy in self-reactive T cells, they serve as critical T cell checkpoints to maintain T cell tolerance, thereby preventing autoimmunity. We note that B cells play an equally important role in autoimmunity (recently reviewed ) and point to the fact that E3s are crucial regulators of B cell reponses (Table 1; Box 3). Box 3: E3 ubiquitin ligases in B cell tolerance While.