E3 Ubiquitin Ligase Cbl-b Research Articles

Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine

AbstractAbstract 957 Introduction:Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy. Material and Methods:We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine. Results:Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo. Conclusions:In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients. Disclosures:No relevant conflicts of interest to declare.

Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b

Integrins are critical for the migration and function of leukocytes in inflammation. However, the interaction between integrin alpha(M) (CD11b), which has high expression in monocytes and macrophages, and Toll-like receptor (TLR)-triggered innate immunity remains unclear. Here we report that CD11b deficiency enhanced TLR-mediated responses in macrophages, rendering mice more susceptible to endotoxin shock and Escherichia coli-caused sepsis. CD11b was activated by TLR-triggered phosphatidylinositol 3-OH kinase (PI(3)K) and the effector RapL and fed back to inhibit TLR signaling by activating the tyrosine kinases Src and Syk. Syk interacted with and induced tyrosine phosphorylation of MyD88 and TRIF, which led to degradation of these adaptor molecules by the E3 ubiquitin ligase Cbl-b. Thus, TLR-triggered, active CD11b integrin engages in crosstalk with the MyD88 and TRIF pathways and subsequently inhibits TLR signaling in innate immune responses.

K33-Linked Polyubiquitination of T Cell Receptor-ζ Regulates Proteolysis-Independent T Cell Signaling

Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

Resistance of Cbl-b-/- Teff to Tregs is infectious (143.21)

Abstract The E3 ubiquitin ligase Cbl-b regulates T cell activation, and dysregulation of this protein in both mice and humans has been associated with autoimmunity. Our lab previously proposed resistance to CD4+CD25+ Tregs as one mechanism for the autoimmunity observed in Cbl-b-/- mice. Recently, we confirmed the in vivo resistance of Cbl-b-/- CD4+CD25- T effectors (Teff) to wild type (WT) Tregs using a model of GVHD. Examining the cells ex vivo, we observed a resistance of Cbl-b-/- Teff to suppression of cytokine production. To further characterize this resistance, WT Teff were examined for their ability to be suppressed by Tregs when mixed with Cbl-b-/- Teff in vitro. We found that WT Teff became resistant to Treg-mediated cytokine suppression when co-cultured with Cbl-b-/- Teff. This is the first evidence that resistance to Tregs is “infectious” and can be transmitted to WT Teff. We next determined that in vitro resistance of Cbl-b-/- Teff is only partially reversed by antibodies to IL-2 and IL-4, suggesting that other mechanisms, in addition to increased production of these cytokines, mediate this resistance. The ability of one subpopulation of Teff to induce Treg resistance in another population may have significant implications for mechanisms underlying autoimmune disease. Finally, using Cbl-b-/- Teff to identify mechanisms of infectious resistance may provide important clues in understanding Treg function and in developing novel strategies for treatment of autoimmune disease.

Cbl-b in T-cell activation

Peripheral activation of antigen-specific T cells is stringently controlled to prevent immune responses against self-antigens. Only after a T cell is presented with two signals, an antigen and a co-stimulatory signal, can they be fully activated. In case antigen presentation occurs without co-stimulation, T-cell receptor (TCR) signaling pathways are regulated to prevent T-cell activation and induce T-cell tolerance. Thus, for a productive T-cell response to occur, co-stimulatory receptors need to serve the dual role of amplifying the TCR signaling while concomitantly releasing T cells from suppression. Biochemical and genetic studies during the last 10 years have documented the critical role of the E3 ubiquitin-ligase Cbl-b in this fundamental two-signal modulation of T-cell responses. In this review, we will discuss our current understanding on how Cbl-b controls T-cell activation and tolerance, its in vivo implications, as well as mechanisms for tuning T-cell-mediated immune responses by this essential E3 ligase.

The E3 Ubiquitin Ligase Cbl-b Regulates Expansion but Not Functional Activity of Self-Reactive CD4 T Cells

Cbl-b is an E3 ubiquitin ligase that limits Ag responsiveness in T cells by targeting TCR-inducible signaling molecules. Cbl-b deficiency thus renders T cells hyperresponsive to antigenic stimulation and predisposes individuals toward developing autoimmunity. In part because Cbl-b(-/-) T cells do not require CD28 costimulation to become activated, and insufficient costimulation is a critical parameter that confers anergy induction over effector differentiation, it has been hypothesized that Cbl-b(-/-) T cells are resistant to anergy. This possibility has been supported in models in which anergy is normally induced in vitro, or in vivo following exposure to soluble Ag boluses. In the current study, we characterized the response of Cbl-b(-/-) CD4 T cells in an in vivo system in which anergy is normally induced by a constitutively expressed peripheral self-Ag. Cbl-b expression increased in self-Ag-induced anergic wild-type CD4 T cells, and Cbl-b(-/-) CD4 T cells underwent more robust proliferation and expansion upon initially encountering cognate self-Ag compared with wild-type counterparts. Nevertheless, both wild-type and Cbl-b(-/-) CD4 T cells ultimately developed the same impaired ability to respond to antigenic restimulation. The more extensive expansion that occurred during the initial induction of anergy did, however, allow the anergic CD4 T cells to expand to greater numbers when they were functionally resuscitated following replacement of the initial source of tolerizing self-Ag with a viral form of the same Ag.

Caspase-8 Is Involved in Neovascularization-Promoting Progenitor Cell Functions

Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits alpha5 and beta1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells.

Nedd4 augments the adaptive immune response by promoting ubiquitin-mediated degradation of Cbl-b in activated T cells

Nedd4 and Itch are E3 ubiquitin ligases that ubiquitinate similar targets in vitro and thus are thought to function similarly. T cells lacking Itch show spontaneous activation and T helper type 2 polarization. To test whether loss of Nedd4 affects T cells in the same way, we generated Nedd4(+/+) and Nedd4(-/-) fetal liver chimeras. Nedd4(-/-) T cells developed normally but proliferated less, produced less interleukin 2 and provided inadequate help to B cells. Nedd4(-/-) T cells contained more of the E3 ubiquitin ligase Cbl-b, and Nedd4 was required for polyubiquitination of Cbl-b induced by CD28 costimulation. Our data demonstrate that Nedd4 promotes the conversion of naive T cells into activated T cells. We propose that Nedd4 and Itch ubiquitinate distinct target proteins in vivo.

T-Cell Receptor-Induced NF-κB Activation Is Negatively Regulated by E3 Ubiquitin Ligase Cbl-b

It has previously been shown that E3 ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) negatively regulates T-cell activation, but the molecular mechanism(s) underlying this inhibition is not completely defined. In this study, we report that the loss of Cbl-b selectively results in aberrant activation of NF-kappaB upon T-cell antigen receptor (TCR) ligation, which is mediated by phosphatidylinositol 3-kinase (PI3-K)/Akt and protein kinase C-theta (PKC-theta). TCR-induced hyperactivation of Akt in the absence of Cbl-b may potentiate the formation of caspase recruitment domain-containing membrane-associated guanylate kinase protein 1 (CARMA1)-B-cell lymphoma/leukemia 10 (Bcl10)-mucosa-associated lymphatic tissue 1(MALT1) (CBM) complex, which appears to be independent of PKC-theta. Cbl-b associates with PKC-theta upon TCR stimulation and regulates TCR-induced PKC-theta activation via Vav-1, which couples PKC-theta to PI3-K and allows it to be phosphorylated. PKC-theta then couples IkappaB kinases (IKKs) to the CBM complex, resulting in the activation of the IKK complex. Therefore, our data provide the first evidence to demonstrate that the down-regulation of TCR-induced NF-kappaB activation by Cbl-b is mediated coordinately by both Akt-dependent and PKC-theta-dependent signaling pathways in primary T cells.

Control of Virus-Specific CD8+T-Cell Exhaustion and Immune-Mediated Pathology by E3 Ubiquitin Ligase Cbl-b during Chronic Viral Infection

A characteristic feature in the immune response to many persistent viral infections is the dysfunction or deletion of antigen-specific T cells (exhaustion). This down-regulation of virus-specific T-cell response represents a critical control mechanism that exists within T-cell activation pathways to prevent lethal disease by inappropriate responses against disseminating virus infections. However, the molecular mechanisms by which the immune system determines whether to mount a full response to such infections remain largely unexplored. Here, we have established that in the murine lymphocytic choriomeningitis virus (LCMV) model, induction of the T-cell receptor signaling inhibitor molecule E3 ligase Cbl-b is critically involved in this decision. In particular, our data revealed that Cbl-b controls the program responsible for T-cell tolerance (exhaustion) induction during a chronic viral infection. Thus, Cbl-b(-/-) mice infected with a low dose of LCMV Docile mount a strong CD8(+) T-cell response that rapidly clears the infection, and the animals remain healthy; in contrast, down-regulation of the epitope-specific CD8(+) T-cell population in persistently infected Cbl-b(-/-) mice, compared to that in chronically infected B6 mice, was significantly delayed, and this was associated with increased morbidity and eventual death in nearly 20% of the animals. Interestingly, infection of Cbl-b(-/-) mice with a moderate virus dose resulted in rapid death with 100% mortality by 7 to 8 days after infection, caused by a dysregulated antiviral T-cell response, whereas the infected B6 mice survived and remained healthy. In conclusion, our results suggest that Cbl-b is critically involved in T-cell exhaustion and prevention of lethal disease.

Cbl-b Regulates Antigen-Induced TCR Down-Regulation and IFN-γ Production by Effector CD8 T Cells without Affecting Functional Avidity

The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-gamma production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-gamma by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.

Regulation of T Cell Signaling and Function by Cbl-b

The activation of intracellular signaling pathways by the engagement of ligands with cell-surface receptors is a key event in determining the fate and function of cells. While these outcomes are primarily determined by the nature of the ligand and its receptor, proteins that negatively regulate the strength and duration of these signals are also critical components in this process. In recent years the E3 ubiquitin ligases c-Cbl and Cbl-b have emerged as prominent negative regulators of signaling responses initiated from antigen receptors on thymocytes and T cells respectively. In this review we focus on the role of Cbl-b in regulating T cell signaling and function and update new and exciting findings relating to Cbl-b deficient T cells that promote the rejection of tumors.

E3 ubiquitin ligase Cblb regulates the acute inflammatory response underlying lung injury

The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. Here we show that Cblb also has an unexpected function in acute lung inflammation. Cblb attenuates the sequestration of inflammatory cells in the lungs after administration of lipopolysaccharide (LPS). In a model of polymicrobial sepsis in which acute lung inflammation depends on the LPS receptor (Toll-like receptor 4, TLR-4), the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Loss of Cblb significantly increases sepsis-induced release of inflammatory cytokines and chemokines. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of wild-type Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for the transcription factor nuclear factor-kappaB (NF-kappaB) in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of neutrophils is impaired in the absence of Cblb. Our data reveal that Cblb regulates the TLR4-mediated acute inflammatory response that is induced by sepsis.

Negative Regulation of CD40-mediated B Cell Responses by E3 Ubiquitin Ligase Cbl-b (87.21)

Abstract CD40 is a TNF receptor (TNFR) superfamily member which is essential for B cell function. Cbl-b, an adapter protein and ubiquitin ligase, has been shown to regulate activation of T and B cells through their antigen receptors. In this study, we report that CD40-induced proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b−/− mice display enhanced thymus–dependent (TD) Ab responses and germinal center (GC) formation, whereas introduction of CD40 deficiency abolishes the hyper-Ab responses and heightened GC formation in response to TD Ag challenge. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-κB and JNK. Cbl-b associates with TNFR-associated factor 2 (TRAF-2) upon CD40 ligation, and inhibits the recruitment of TRAF-2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-κB and JNK activation, thereby suppressing B cell responses.

T Cell Receptor-induced NF-KB Activation Is Negatively Regulated by E3 Ubiquitin Ligase Cbl-b via Akt- and PKC-theta-dependent Pathways (87.10)

Abstract Previously, we demonstrated that Cbl-b tunes the threshold for T cell activation mediated by CD28 and CTLA-4. Cbl-b negatively regulates Vav-1 via a PI3-K-dependent mechanism. However, it is unknown whether Cbl-b also targets other signaling pathway(s). In this study, we found that loss of Cbl-b selectively results in aberrant activation of TCR-induced NF-κB which is mediated by PI3-K/Akt and PKC-𝛉. The absence of Cbl-b also results in enhanced TCR-induced activation of PLC-γ1 which may selectively induce activation of PKCs including PKC-𝛉 but not Ca2+ signaling pathway. Cbl-b associates with PKC-𝛉 upon TCR stimulation and regulates TCR-induced PKC-𝛉 activation through Vav-1 which couples PKC-𝛉 to PI3-K and allows it to be phosphorylated possibly by PDK-1. PKC-𝛉 then couples IKKs to CBM complex, resulting in activation of IKK complex. Therefore, our data first demonstrate that down-regulation of TCR-induced NF-κB activation by Cbl-b is coordinately mediated by both Akt- and PKC-𝛉dependent signaling pathways in primary T cells.

The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T–mediated virulence

Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III-secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III-secreted effector.

Cutting Edge: Deficiency in the E3 Ubiquitin Ligase Cbl-b Results in a Multifunctional Defect in T Cell TGF-β Sensitivity In Vitro and In Vivo

Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b-/- CD4+CD25- T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-beta. We have now asked whether the resistance noted in Cbl-b-/- T cells is restricted solely to TGF-beta's antiproliferative effects, whether the TGF-beta resistance has in vivo relevance, and whether a defect can be identified in the TGF-beta signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-beta-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b-/- mice show a significantly enhanced response to a tumor that is strictly TGF-beta regulated; and 3) Cbl-b-/- T effector cells have defective TGF-beta-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-beta signaling, and that its absence results in multifunctional TGF-beta-related defects that have important disease-related implications.

Egr-2 and Egr-3 are negative regulators of T cell activation

T cell receptor engagement in the absence of proper accessory signals leads to T cell anergy. E3 ligases are involved in maintaining the anergic state. However, the specific molecules responsible for the induction of anergy have yet to be elucidated. Using microarray analysis we have identified here early growth response gene 2 (Egr-2) and Egr-3 as key negative regulators of T cell activation. Overexpression of Egr2 and Egr3 was associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Conversely, T cells from Egr3(-/-) mice had lower expression of Cbl-b and were resistant to in vivo peptide-induced tolerance. These data support the idea that Egr-2 and Egr-3 are involved in promoting a T cell receptor-induced negative regulatory genetic program.

The E3 Ubiquitin Ligase TRIM36, a Transcriptional Target of Tob, Is Expressed in Anergic T Cells and Mediates Unresponsiveness through Proteolysis of Signaling Proteins PLC- γ1 and PKC-#.

Ligation of the T cell receptor (TCR) and costimulatory receptors leads to cytokine secretion and clonal expansion, whereas ligation of TCR alone leads to anergy. We have previously determined that anergic cells express Tob, a member of the novel APRO gene family, which inhibits T cell activation. The precise molecular mechanisms via which Tob mediates its effects in T cells are not fully understood. Tob functions as transcriptional coactivator and enhances DNA binding of Smads. Therefore, Tob may regulate de novo mRNA synthesis or gene transcription. To identify genes that are induced by Tob, Jurkat T cells that lack endogenous Tob, were transfected with Tob cDNA or empty vector and differential gene expression was determined by suppression subtractive hybridization. TRIM36 was one of the genes induced by Tob. TRIM36 is a RING finger E3 ubiquitin ligase. It belongs to a recently identified tripartite motif (TRIM) gene family which also includes Pyrin/Marenosrtin, MID1, MUL, PML, RFP and TIF1, proteins implicated in familial human diseases and cancer. E3 proteins confer substrate specificity to the ubiquitin system. Previous studies have shown that the trancriptional profile of anergic cells includes the E3 ubiquitin ligases Cbl-b, GRAIL and Itch. Therefore, the finding that Tob, a transcriptional regulator expressed in anergic cells, induces expression of TRIM36 E3 ubiquitin ligase is very intriguing. Northern blot analysis confirmed that TRIM36 mRNA was selectively upregulated in anergic T cells. To determine the role of TRIM36 on IL-2 gene transcription, Jurkat T cells were transfected with full-length TRIM36 cDNA along with the IL-2 promoter/enhancer cDNA (2kb) linked to the luciferase gene. TRIM36 inhibited CD3+CD28-mediated IL-2 transcription by 90%. Interestingly, when cells were stimulated with PMA+Ionomycin, which bypass the TCR proximal signals, IL-2 transcription was almost unaffected. These results prompted us to search for candidate ubiquitination substrates among signaling molecules that have a critical role on TCR-mediated T cell activation and IL-2 transcription. Previous studies have shown that among T cell signaling molecules, TCRζ, ZAP70, PLC-γ1 and PKC-# undergo ubiquitin-targeted degradation. For this reason, we investigated whether any of these proteins might be substrates for TRIM36-mediated ubiquitination. V5-tagged TRIM36 or empty vector was expressed in Jurkat T cells followed by stimulation with anti-CD3+anti-CD28 mAbs in the presence of ubiquitin aldehyde that prevents substrate deubiquitination. Immunoblot with antibodies specific for TCR ζ, ZAP70, PLC-γ1 and PKC-# showed that expression of PLC-γ1 and PKC-# was selectively reduced in the presence of TRIM36. Immunoprecipitation with V5 mAb followed by immunoblot with substrate-specific antibodies revealed that PLC- γ1 and PKC-# coprecipitated with TRIM36. Immunoblot with ubiquitin-specific antibody revealed that PLC-γ1 and PKC- 0 were substrates for ubiquitination by TRIM36. Our results show that at least one molecular mechanism via which Tob mediates its inhibitory effect on T cell activation involves the induction of TRIM36 ubiquitin ligase, which mediates degradation of two key signaling proteins, PLC- γ1 and PKC-#. Moreover, these results suggest that TRIM36 may represent a novel target of molecular intervention for induction of transplantation tolerance.

Essential Role of the E3 Ubiquitin Ligase Cbl-b in T Cell Anergy Induction

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 PLCγ-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.