Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.

Itch is an E3 ubiquitin ligase that is disrupted in nonagouti-lethal or itchy mice. Itch deficiency leads to severe immune and inflammatory disorders and constant itching of the skin. Here we show that Itchminus sign/minus sign T cells show an activated phenotype and enhanced proliferation. Production of the type 2 T helper (TH2) cell cytokines interleukin 4 (IL-4) and IL-5 by Itchminus sign/minus sign T cells was augmented upon stimulation, and the TH2-dependent serum concentrations of immunoglobulin G1 (IgG1) and IgE in itchy mice were also increased. Molecularly, Itch associated with and induced ubiquitination of JunB, a transcription factor that is involved in TH2 differentiation. These results provide a molecular link between Itch deficiency and the aberrant activation of immune responses in itchy mice.

Genetic studies identified

Transforming growth factor-β (TGF-β) signaling in naive T cells induces expression of the transcription factor Foxp3, a 'master' regulator of regulatory T cells (T reg cells). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch −/− T cells were resistant to TGF-β treatment and had less Foxp3 expression. The E3 ubiquitin ligase Itch associated with and promoted conjugation of ubiquitin to the transcription factor TIEG1. Itch cooperated with TIEG1 to induce Foxp3 expression, which was reversed by TIEG1 deficiency. Functionally, 'TGF-β-converted' T reg cells generated from TIEG1-deficient mice were unable to suppress airway inflammation in vivo. These results suggest TIEG and Itch contribute to a ubiquitindependent nonproteolytic pathway that regulates inducible Foxp3 expression and the control of allergic responses.Allergic asthma is a chronic inflammatory disease of the airways characterized by higher serum immunoglobulin E (IgE) concentrations, airway hyper-responsiveness, excessive airway mucus production, lung eosinophilia and airway remodeling. Differentiation of CD4 + T cells into T helper type 2 (T H 2) cells renders them able to produce the cytokines interleukin 4 (IL-4), IL-5 and IL-13, which are critical for driving asthmatic pathogenesis 1,2 . The induction of T H 2 differentiation is regulated by many factors, including the properties of the antigens, the duration of stimulation, and genetic factors. In addition, the T H 2-mediated immune responses are counterbalanced by tolerogenic mechanisms that prevent excessive reactions. One of the tolerance mechanisms involves regulatory T cells (T reg cells), which limit allergic responses 3 . In mouse models of allergic asthma, repeated delivery of antigen to the respiratory tract results in inhibition of allergic responses to the same antigen after subsequent challenges 4 . Published studies have shown that repeated treatment with a low dose of antigen induces the generation of Foxp3 + CD4 + CD25 + T reg cell population, which accounts for the attenuation of T H 2-mediated airway inflammation 5,6 . T reg cells represent a unique subpopulation of CD4 + T cells that suppress the effector function of other types of T cells and

The transcription factor Foxp3 is essential for optimal regulatory T (T reg) cell development and function. Here, we show that CD4+ T cells from Cbl-b RING finger mutant knockin or Cbl-b–deficient mice show impaired TGF-β–induced Foxp3 expression. These T cells display augmented Foxo3a phosphorylation, but normal TGF-β signaling. Expression of Foxo3a rescues Foxp3 expression in Cbl-b–deficient T cells, and Foxo3a deficiency results in defective TGF-β–driven Foxp3 induction. A Foxo3a-binding motif is present in a proximal region of the Foxp3 promoter, and is required for Foxo3a association. Foxo1 exerts similar effects as Foxo3a on Foxp3 expression. This study reveals that Foxo factors promote transcription of the Foxp3 gene in induced T reg cells, and thus provides new mechanistic insight into Foxo-mediated T cell regulation.

Foxp3+ regulatory T (Treg) cells play a critical role in immune homeostasis; however, the mechanisms to maintain their function remain unclear. Here, we report that the E3 ubiquitin ligase VHL is essential for Treg cell function. Mice with Foxp3-restricted VHL deletion displayed massive inflammation associated with excessive Treg cell interferon-γ (IFN-γ) production. VHL-deficient Treg cells failed to prevent colitis induction, but converted into Th1-like effector T cells. VHL intrinsically orchestrated such conversion under both steady and inflammatory conditions followed by Foxp3 downregulation, which was reversed by IFN-γ deficiency. Augmented hypoxia-inducible factor 1α (HIF-1α)-induced glycolytic reprogramming was required for IFN-γ production. Furthermore, HIF-1α bound directly to the Ifng promoter. HIF-1α knockdown or knockout could reverse the increased IFN-γ by VHL-deficient Treg cells and restore their suppressive function in vivo. These findings indicate that regulation of HIF-1α pathway by VHL is crucial to maintain the stability and suppressive function of Foxp3+ T cells.

Cbl-b and its close mammalian homologue, Cbl, consist of an amino-terminal variant SH2 1 domain, a RING finger, and a carboxyl-terminal proline-rich domain with potential tyrosine phosphorylation sites (1, 2). Previous studies have shown that Cbl-b and Cbl function as adaptor proteins by interacting with othercriticalsignalmolecules,includingthevariantSH2domain-dependent interaction with cell surface receptor tyrosine kinases or intracellular protein tyrosine kinases such as Syk and Zap-70 and the carboxyl-terminal region-dependent interaction with Grb2, 14-3-3, phosphatidylinositol 3-kinase (PI3-K), Vav, and Crk-L (3, 4). Genetic and biochemical studies have shown that Cbl family proteins including those from Drosophila and Caenorhabditis elegans attenuate intracellular signaling induced by the engagement of cell surface receptors. One mechanism for this negative role is Cbl-mediated ubiquitination of receptor tyrosine kinases (5-7). It is now understood that Cbl functions as an E3 ubiquitin (Ub) ligase whose RING finger recruits a Ub-conjugating enzyme, E2, and whose SH2 domain recognizes activated receptor tyrosine kinases for Ub conjugation (7-10).Ubiquitination is an important cellular process that involves ligation of a protein substrate with Ub, thereby marking it for degradation by the 26S proteasome (11-13), and it involves a cascade of reactions including E1, E2, and E3 enzymes. Ub is first activated by an activating enzyme (E1) to form a high energy thiolester bond between Ub and E1 and is then transferred to a conjugating enzyme (E2). The E3s or Ub protein ligases are the components responsible for specific substrate recognition and for promoting Ub ligation to the target protein.Therefore, the E3s can provide specificity to the Ub system. Two recent genetic studies using Cbl-b gene-targeted mice showed that Cbl-b deficiency can change the signaling thresholds: Cbl-b deficiency uncouples T-cell proliferation and interleukin 2 production from the costimulation of CD28, and the gene-targeted mice develop spontaneous autoimmunity or become highly susceptible to exogenous antigen-induced autoimmune diseases (14, 15). These studies suggest a critical role of Cbl-b in the regulation of T-cell activation thresholds and hence in the maintenance of a balance between immunity and tolerance. In Cbl-b-deficient T cells, the tyrosine phosphorylation and/or activation of Vav, a GDP/GTP exchange factor, are significantly enhanced (14, 15), suggesting that Cbl-b negatively regulates T-cell signaling by inhibiting Vav activation. However, the molecular mechanism underlying Cbl-b-mediated negative regulation of Vav remains to be determined.Previous studies have shown that PI3-K, which phosphorylates PI lipid at the D3 position of the inositol ring to form active lipid second messengers, regulates the exchange activity of Vav through the binding of the active lipid products to the pleckstrin homology (PH) domain of Vav (16,17). We have investigated whether Cbl-b acts as an E3 Ub ligase to promote ubiquitination of PI3-...

Stimulation of the T cell antigen receptor (TCR)⅐CD3complex induces rapid tyrosine phosphorylation of Cbl, a protooncogene product which has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. We found recently that Cbl associates directly with a member of the 14-3-3 protein family (14-3-3) in T cells and that the association is increased as a consequence of anti-CD3-mediated T cell activation. We report here that phorbol 12-myristate 13-acetate stimulation of T cells also enhanced the interaction between Cbl and two 14-3-3 isoforms ( and ). Tyrosine phosphorylation of Cbl was not sufficient or required for this increased interaction. Thus, cotransfection of COS cells with Cbl plus Lck and/or Syk family protein-tyrosine kinases caused a marked increase in the phosphotyrosine content of Cbl without a concomitant enhancement of its association with 14-3-3. Phorbol 12-myristate 13-acetate stimulation induced serine phosphorylation of Cbl, and dephosphorylation of immunoprecipitated Cbl by a Ser/Thr phosphatase disrupted its interaction with 14-3-3. By using successive carboxyl-terminal deletion mutants of Cbl, the 14-3-3-binding domain was mapped to a serine-rich 30-amino acid region (residues 615-644) of Cbl. Mutation of serine residues in this region further defined a binding motif distinct from the consensus sequence RSXSXP, which was recently identified as a 14-3-3-binding motif. These results suggest that TCR stimulation induces both tyrosine and serine phosphorylation of Cbl. These phosphorylation events allow Cbl to recruit distinct signaling elements that participate in TCR-mediated signal transduction pathways.