E3 ubiquitin ligase Cbl-b in innate and adaptive immunity

Abstract

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING finger E3 ubiquitin-protein ligase, has been demonstrated to play a crucial role in establishing the threshold for T-cell activation and controlling peripheral T-cell tolerance via multiple mechanisms. Accumulating evidence suggests that Cbl-b also regulates innate immune responses and plays an important role in host defense to pathogens. Understanding the signaling pathways regulated by Cbl-b in innate and adaptive immune cells is therefore essential for efficient manipulation of Cbl-b in emerging immunotherapies for human disorders such as autoimmune diseases, allergic inflammation, infections, and cancer. In this article, we review the latest developments in the molecular structural basis of Cbl-b function, the regulation of Cbl-b expression, the signaling mechanisms of Cbl-b in immune cells, as well as the biological function of Cbl-b in physiological and pathological immune responses in animal models and human diseases.

Introduction

Over the last decade, accumulating evidence suggests that ubiquitination of proteins by E3 ligases is a novel and crucial regulation mechanism in innate and adaptive immunity.^1,2 The gene of Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), an E3 ubiquitin-protein ligase and an adaptor protein, was initially cloned and characterized by Keane et al. in 1995.³ Cbl-b belongs to the Cbl family, which consists of c-Cbl and Cbl-3 in addition to Cbl-b and has a broad spectrum of biological functions.

Recent studies using gene-targeting approaches have yielded convincing evidence that Cbl-b negatively regulates the signaling pathways derived from the T-cell receptor (TCR),^4,5 B-cell receptor (BCR), CD40,^6,7 and FcεR1 (high affinity immunoglobulin epsilon receptor).⁸ Because of the diversities of substrates of Cbl-b in different cell types, it appears that Cbl-b regulates various signaling pathways in a cell type-dependent manner. In this review, we will summarize the most recent progress on Cbl-b-related studies in immune systems, which encompass Cbl-b structure, regulation of Cbl-b expression, and its role in innate and adaptive immune responses. We will also discuss the potential roles of Cbl-b in various diseases including autoimmune and inflammatory diseases, infection, and cancer.

Genetics, Tissue Distribution, and Subcellular Location of Cbl-b

The mammalian Cbl family of proteins is highly conserved throughout evolution from nematodes to humans and consists of c-Cbl, Cbl-b, and Cbl-3 (Fig. 1). The Cblb gene is located on chromosome 3q13.11 in humans and chromosome 16B5 in the mouse. Cbl-b is abundantly expressed in a variety of immune cells.^9,10 In T cells, Cbl-b is predominantly expressed in peripheral T cells, whereas c-Cbl is mainly expressed in thymus, suggesting a distinct role of c-Cbl and Cbl-b in T-cell development and tolerance induction.¹¹ In T cells, Cbl-b is located in the cytoplasm but can be translocated to the plasma membrane upon TCR stimulation.¹²

Figure 1. Functional domains of the Cbl family (c-Cbl, Cbl-b, and Cbl-3) in mammals. All three (3) members of the Cbl family of proteins share a highly homologous N-terminal region that serves as the structural platform for direct binding to specific pY-containing peptide motifs in activated PTKs and is accordingly referred to as the tyrosine kinase-binding (TKB) domain; this domain is assembly of a 4-helical (4H) bundle, an EF hand domain, and a variant SH2 domain. The TKB domain is followed by a highly conserved helical linker (L) domain and a RING (really interesting new gene) finger (RF) domain, which bind to ubiquitin-conjugating enzymes (E2). The proline-rich motifs (PR) bind to SH3 domain containing signaling and endocytic proteins. Induced tyrosine phosphorylation sites (major sites at Y700, Y731, and Y774 in c-Cbl) recruit SH2 domain-containing signaling proteins. The leucine zipper (LZ)/ubiquitin-associated (UBA) domain near the C-terminus is involved in ubiquitin binding and dimerization. Cbl-c lacks most of the C-terminal regions except for a short PR region for potential interactions with SH3 domain-containing proteins.

Structural Components of Cbl-b and Their Functions

The Cbl family of ubiquitin ligases in mammals share highly conserved regions in their N-terminal halves, which encompass their TKB (protein tyrosine-kinase-binding), linker (L), and RING (really interesting new gene) finger (RF) domains (Fig. 1). The unique feature of the TKB domain is that it recognizes specific substrates of Cbl-b, which is achieved by binding to proteins containing specific phosphorylated tyrosine-containing motifs, such as Syk and Zap-70, and a range of receptor tyrosine kinases.^6,13 Interaction of proteins with the TKB domain of Cbl is mediated by 3 distinct subdomains consisting of a 4-helix bundle (4H), a calcium-binding EF hand, and a variant SH2 domain, all 3 of which are functionally required to form a unique PTB (phosphotyrosine-binding) module.¹⁴ SH2 domain within the TKB recognizes tyrosine-phosphorylated proteins for ubiquitin conjugation.¹⁵ A highly conserved α-helix of the L domain plays an important role in maintaining E3 activity.^16,17 The crystal structure shows that the L region contacts the TKB, RF, and E2 ubiquitin-conjugating enzymes.¹⁶ The RF domain has intrinsic E3 ubiquitin ligase activity and binds to ubiquitin-E2 for the transfer of ubiquitin to specific substrates.^18-20 Recent studies also indicate that the phosphorylation of Y363, located in the L region between TKB and RF domains, regulates the E3 activity of Cbl-b by 2 mechanisms: one is to remove the masking of the RF domain from the TKB domain, and the other is to form a surface to enhance binding affinity to E2s.^21,22 Consistent with this finding, the equivalent tyrosine in c-Cbl, i.e., Y371, has been shown to regulate its E3 ubiquitin ligase activity in a similar fashion.²³

In contrast, the C-terminal regions of this family of proteins are less conserved. The proline-rich (PR) domain in the C-terminus of c-Cbl and Cbl-b refers to a PX(P/A)XXR motif that binds to SH3 domains of the CIN85/RUK (regulator of ubiquitous kinase)/CD2AP (C2-associated protein) family of proteins.^24,25 The tyrosine residues at the C termini of c-Cbl and Cbl-b are phosphorylated by protein tyrosine kinases (PTKs) following stimulation of a diverse array of cell surface receptors.^26,27 c-Cbl can bind Vav-family guanine nucleotide exchange factors (GEFs), the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3K), and the Crk-family of adaptor proteins that link Cbl proteins to C3G through interactions with phospho-Y700, Y731, and Y774, respectively.^28-31 Likewise, Cbl-b is phosphorylated at Y655 and Y709 upon TCR stimulation. However, the phosphorylation of Cbl-b is weaker compared with that of c-Cbl in response to this stimulation.^10,32 It is noted that the Y⁷³¹EAM motif provides c-Cbl a docking site for the SH2 domains of p85, thus enabling c-Cbl to function as a positive regulator of PI3K activity.^33-36 Indeed, Akt and c-Cbl Y737 (mouse) are highly phosphorylated in lineage-negative bone-marrow (BM) cells upon stimulation with stem cell factor (SCF) or FLT3 ligand, whereas c-Cbl^−/− BM cells display defective phosphorylation of Akt, possibly due to loss of c-Cbl, which results in the uncoupling of PI3K p85 from the membrane.³⁷ The absence of this p85-binding motif in Cbl-b highlights a potentially important divergence in the role and mode of action of these 2 highly similar regulatory proteins.³³ In support of this notion, Cbl-b has been documented to inhibit Pten inactivation via Nedd4 (neuronal precursor of cell expressed developmentally downregulated gene 4) independently of its E3 ubiquitin ligase activity in T cells.¹²

Both c-Cbl and Cbl-b have an ubiquitin-associated (UBA) domain at their C terminal end, which interacts with ubiquitin and ubiquitin-like domains of proteins such as Nedd8,^38,39 and is present in a variety of proteins involved in ubiquitin-mediated processes. Moreover, c-Cbl and Cbl-b also can form homo- and hetero-dimers through interaction between their UBA domains. Cbl-b dimerization is regulated by ubiquitin binding and requires tyrosine phosphorylation of Cbl-b and ubiquitination of Cbl-b substrates.⁴⁰ However, Cbl-b, rather than c-Cbl, constitutively coimmunoprecipitates with high molecular weight ubiquitinated proteins.⁴¹ Furthermore, the UBA domain of Cbl-b has a much greater affinity for polyubiquitin chains than for monoubiquitin, and inhibits a variety of ubiquitin-mediated processes, such as degradation of ubiquitinated proteins.^41,42 This difference in ubiquitin-binding reflects distinct regulatory functions of Cbl-b from c-Cbl.

Biochemical Function and the Expression Modification of Cbl-b

The ubiquitin–protein ligase system consists of 3 classes of enzymes known as ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin–protein ligases (E3). The ubiquitination reaction is initiated when 76-amino acid ubiquitin is activated by E1. A thioester bond forms between the active cysteine residue of E1 and the C terminus of ubiquitin in an ATP-dependent reaction. Following ubiquitin activation, activated ubiquitin is transferred to E2 in another ATP-dependent reaction. As an E3 ubiquitin-protein ligase, Cbl-b transfers ubiquitin from specific E2 to the ε-amino group of a lysine (K) residue on the protein substrate. (Fig. 2). The fate of the tagged substrate depends on the number of ubiquitin molecules added (mono-ubiquitin vs. poly-ubiquitin) as well as the K residue involved in the formation of the polyubiquitination chains. Generally, proteins polyubiquitinated through K48 are degraded in the 26S proteasome,^43,44 whereas mono-ubiquitination (or multi-ubiquitination) usually marks membrane proteins for endocytosis and subsequent degradation in lysosomes.⁴⁵ Polyubiquitination through K11, 29, 63 may endow substrate proteins new functions,⁴⁶ which serves as a signal for functional modification of the substrate, including transcriptional regulation,^47-49 ubiquitination-dependent processing of precursor proteins,⁵⁰ and kinase activation.⁵¹ Much less is known about the precise function and topology of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33,⁵² which may target proteins for degradation.⁵³ Given that Cbl-b interacts with many proteins in various immune cells,^10,54-61 Cbl-b is thought to play important roles in maintaining the homeostasis of the immune system through elaborate signal transduction pathways.

Figure 2. Overview of the ubiquitin pathway utilized by RING type E3 ligases. Three types of enzyme are required for substrate ubiquitination: ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin–protein ligase (E3) enzymes. The E1-E2-E3 cascade mediates ubiquitination of the substrate with the substrate specificity provided by the E3 enzyme. The substrate protein can be tagged with just one ubiquitin, or polyubiquitin chains, which determines the fate of target proteins. –S–, thiolester bond.

The tyrosine and serine residues of Cbl-b are phosphorylated upon stimulation of an vast array of cell-surface receptors, including the TCR,^{10,40,56,59,62-66} a process that is essential for Cbl-b function. Cbl-b also undergoes ubiquitination upon CD28 costimulation in T cells, resulting in its proteasomal degradation. CD28 costimulation potentiates TCR-induced Cbl-b degradation, whereas CTLA-4-B7 interaction is required for Cbl-b re-expression.^67,68 Thus, CD28 and CTLA-4 tightly regulate Cbl-b expression, which is critical for establishing the threshold for T-cell activation and tolerance induction. The proteasomal degradation of Cbl-b may be mediated by Nedd4, which has been shown to target Cbl-b for ubiquitination and degradation,⁶⁹ and PKC-θ, which phosphorylates Cbl-b at Ser282 in the TKB domain, facilitating Cbl-b ubiquitination.⁵⁹ In keeping with this, it was reported that Nedd4 promotes adaptive T-cell responses in vitro and in vivo.^12,69

Signaling Pathway of Cbl-b in Innate and Adaptive Immune Cells

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.⁵⁷ Cbl-b plays a negative regulatory role by targeting proteins for ubiquitination or by interacting with other proteins via its PR region, TKB domain, or UBA domain. For example, Cbl-b interacts with phospho-tyrosine-containing proteins via its TKB domain,^10,13 E2-ubiquitin complexes via its RF domain,^20,70 binds to SH3 domain-containing proteins via its PR region,⁵⁷ SH2 domain-containing proteins via its C-terminal tyrosine residues,¹⁰ and polyubiquitinated proteins via its UBA domain.^40,41 It has been documented that dimerization of Cbl-b is required for the binding of Cbl-b to poly-ubiquitin but not for mono-ubiquitin.^{9,10,41,56,57,71-73} Thus, Cbl-b plays an important regulatory role in innate and adaptive immune cells through its involvement with many signaling pathways.

Cbl-b in innate immune responses

The responses of innate immune cells to extracellular matrix proteins, cytokines, pathogens, cellular damage, and many other stimuli are regulated by a complex network of intracellular signal transduction pathways, most of which are either initiated or modulated by Src-family or Syk tyrosine kinases present in innate cells.⁷⁴ Cbl-b has been implicated in the major signaling pathways of macrophages, dendritic cells, natural killer (NK) cells, and NKT cells and mast cells in innate immunity.

Integrins are critical for the migration and function of macrophages during inflammation, and Cbl-b plays an important role in integrin signal transduction. Cbl-b deficiency facilitates activation of β2-integrin leukocyte function-associated antigen-1 (LFA-1) and LFA-1-mediated inflammatory cell recruitment. Cblb^−/− mice display increased macrophage recruitment in thioglycollate-induced peritonitis, and Cblb^−/− bone marrow-derived mononuclear phagocytes (BMDMPs) show increased adhesion to endothelial cells resulting from activation of LFA-1, which mediates adhesion of BMDMPs to ICAM-1. Cbl-b deficiency also results in increased phosphorylation of T758 in the β2-chain, thereby enhancing the association between 14–3-3β protein and the β2-chain, leading to activation of LFA-1.⁷⁵ Cbl-b is also implicated in the Toll-like receptor (TLR)-triggered PI3K-RapL-integrin-α(M), CD11b activation pathways.⁷⁶ The initial study that suggests that Cbl-b is involved in innate immune responses came from evidence that Cbl-b participates in acute lung injury by negatively regulating TLR4 signaling in mouse monocytes. Loss of Cbl-b markedly aggravates acute lung inflammation and leads to 100% lethality upon polymicrobial sepsis induction.⁷⁶ However, no additional studies by other investigators verified this finding. Subsequently, Cbl-b was shown to target MyD88 and TRIF, which is potentiated by activating the tyrosine kinases Src and Syk in macrophages upon TLR stimulation including TLR4.⁷⁷ However, Nrdp1 has also been shown to ubiquitinate MyD88 and TBK-1 in macrophages upon TLR4 ligation.⁷⁸ Since these studies did not examine macrophages from mice lacking Cbl-b, or expressing a Cbl-b RF mutation, it is currently unknown whether Cbl-b is indeed the E3 ubiquitin ligase for MyD88 in a physiological setting. Therefore, the physiological substrates for Cbl-b in innate immune cells are largely unknown.

Cbl-b also plays an important regulatory role in dendritic cells, NK, NKT cells, and mast cells. Cbl-b functions not only as a negative regulator of signaling, but also as a positive modulator of TNF receptor superfamily signaling. TRANCE and CD40L-mediated Akt activation is defective in Cblb^−/− dendritic cells,³⁵ suggesting that Cbl-b positively regulates these pathways. It was reported that Cbl-b may target CARMA1, a critical signaling molecule in NF-κB activation, for mono-ubiquitination in NKT cells.⁷⁹ Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability, suggesting that this process is mediated by a proteolysis-independent mechanism.⁷⁹ It has been recently reported that a novel inhibitory role of Cbl-b in the regulation of NK cell functions via TAM receptors. Releasing the inhibition imposed by this TAM/Cbl-b pathway would render NK cells capable of rejecting tumor metastases.⁸⁰ Furthermore, Cbl-b negatively regulates IgE-mediated activation of mast cells as well as the activation and tissue infiltration of macrophages.^81,82 The molecular mechanism by which Cbl-b inhibits IgE-mediated mast cell activation remains to be defined.

Signaling pathways of Cbl-b in B cells

Direct target molecules of Cbl-b such as tyrosine kinase Syk, phospholipase C-gamma2 (PLC-γ2), p85/PI3K, Rho-family GTP-GDP exchange factor Vav, and growth-factor receptor-bound protein-2 (Grb2) are involved in BCR signaling during the normal response course.⁸³ Syk and its substrate BLNK (also called SLP65) and Cbl-interacting protein of 85 kDa (CIN85) are key components of the BCR-associated primary transducer module required for the onset and progression phases of BCR signal transduction. Syk-mediated complex formation consisting of Vav, Btk, BLNK, and PLC-γ2 is required for effective downstream signaling including MAPKs, Akt, Ca²⁺ influx, and NF-κB activation.⁸³ CIN85 also interacts with SH2-containing inositol phosphatase 1 (SHIP-1), an inositol 5-phosphatase expressed in hemopoietic cells, which acts by hydrolysing the 5-phosphates from PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4), thereby negatively regulating the PI3K pathway.⁸⁴ Thus, multiple signaling pathways of Cbl-b are coordinating in response to BCR stimulation.

Studies using Cblb^−/− mice have yielded more definitive results that support the notion that Cbl-b is a negative regulator of BCR signaling.⁶ Cblb^−/− B cells display sustained phosphorylation of Igα, Syk, and PLC-γ2 in response to BCR stimulation, which leads to prolonged Ca²⁺ mobilization and increases extracellular signal-regulated kinase (ERK) and JNK phosphorylation, and surface expression of the activation marker, CD69.⁶ This heightened BCR signaling is possibly mediated by ubiquitination and proteasomal degradation of Syk and Igα by Cbl-b. In accordance with these data, B cell-specific ablation of both c-Cbl and Cbl-b (Cbl^−/−Cblb^−/−) results in enhanced tyrosine phosphorylation of Syk, PLC-γ2, and Vav, and Ca²⁺ mobilization and substantial attenuation of tyrosine phosphorylation of adaptor protein BLNK.⁷

Cbl-b is also involved in the germinal center formation. Loss of Cbl-b restores Ig class switching and germinal center formation in Vav1 mutant mice in response to an in vivo viral challenge.⁸⁵ Genetic inactivation of Cbl-b also rescues impaired antiviral IgG production rather than germinal center formation in Cd28^−/− mice.⁸⁵ It has been shown that Grb2 is degraded in a Cbl-b-dependent fashion and plays an important role in germinal center formation in the spleen.⁵⁵ Ablation of Grb2 in B cells results in enhanced BCR signaling, and Grb2^−/− B cells do not form germinal centers in the spleen after antigen stimulation.⁸⁶ Therefore, it is assumed that the Cbl-b/Grb2 signaling pathway might play an important role in germinal center formation. In addition, we have previously shown that Cblb^−/− mice display enhanced thymus-dependent antibody responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects.⁸⁷ Cbl-b selectively downmodulates CD40-induced activation of NF-κB and JNK. Cbl-b associates with TRAF-2 upon CD40 ligation and inhibits the recruitment of TRAF-2 to CD40. These data suggest that Cbl-b attenuates CD40-mediated NF-κB and JNK activation, thereby suppressing B-cell responses.⁸⁷

Signaling pathways of Cbl-b in T cells

Cbl-b acts as a gatekeeper that prevents excessive T-cell activation initiated by the engagement of TCR, thus setting the threshold for T-cell activation and regulating peripheral T-cell tolerance. The signaling pathways that are regulated by Cbl-b in T cells have been more extensively studied than those of other immune cells (Fig. 3).

Figure 3. Model of Cbl-b action on T-cell activation Upon TCR stimulation, Pten is inactivated via Nedd4, which targets Pten for K63-linked polyubiquitination, and this process is inhibited by Cbl-b. Inactivation of Pten leads to the accumulation of PtdIns(3,4,5)P₃, which recruits PDK-1, Vav-1, and Akt to the plasma membrane via its interaction with the PH domains within these molecules. Therefore, Cbl-b inhibits Vav-dependent activation of WASP, which leads to actin reorganization and TCR clustering. In addition, Vav1 links PKC-θ to PDK-1, the former coupling IKKs, to the CBM complex. Activated Akt also facilitates the formation of the CBM complex possibly by phosphorylating CARMA1. Thus, Cbl-b inhibits NF-κB activation via PKC-θ and Akt. One of the important outcomes for Akt is that Akt can phosphorylates Foxo1/3a, which excludes them from the nucleus, thus inhibiting Foxp3 expression. In anergic T cells, Cbl-b targets PLC-γ1 and PKC-θ for ubiquitination, thus promoting T-cell anergy induction. The expression of Cbl-b in T cells is controlled by CD28 and CTLA-4. CD28 costimulation induces Cbl-b ubiquitination and proteasomal degradation, which is possibly mediated by Nedd4 and PKC-θ. In contrast, CTLA-4-B7 interaction induces Cbl-b expression.

In naϊve T cells, Cbl-b regulates CD28-dependent T-cell activation by selectively restraining the TCR-mediated Vav1–Wiscott Aldrich syndrome protein (WASP) signaling pathway.^4,88-90 Loss of Cbl-b in T cells frees TCR-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. The Rho family GDP/GTP exchange factor, Vav1, GTPases Rac1, CDC42, and RhoA, and CDC42-associated WASP constitute a signaling pathway that links antigen receptor engagement to cytoskeletal reorganization, receptor clustering and cap formation, and effective T-cell activation.⁹¹ Vav1 is optimally tyrosine phosphorylated by co-stimulation of TCR/CD28. Loss of Cbl-b results in hyper-activation of Vav1 upon TCR stimulation and uncouples the requirement for optimal Vav activation from CD28 costimulation. Further study suggests that Cbl-b suppresses the activation of Vav, thus attenuating the extent of actin reorganization and TCR clustering via a CDC42/WASP-dependent mechanism.⁹² Introduction of the Cbl-b deficiency into a Vav1^−/− background relieves the functional defects of Vav1^−/− T cells and causes spontaneous autoimmunity.⁹⁰ In further support of the notion that Cbl-b regulates the CD28 dependence of T-cell activation, T cells deficient for Cbl-b do not require CD28 costimulation for T-cell proliferation and IL-2 production, and Cbl-b deficiency fully restores defective T-cell proliferation, IL-2 production, and T cell-dependent antibody responses in Cd28^−/− mice.⁸⁸ WASP is a key regulator of actin dynamics during cell motility and adhesion. The studies using Cblb^−/−Wasp^−/− and Cblb^−/−Vav1^−/−Wasp^−/− mice reveal that WASP deficiency abrogates hyper-T-cell responses and TCR clustering.⁹⁰ WASP phosphorylation at tyrosine 291 results in recruitment of Cbl-b, which, together with c-Cbl, ubiquitinates WASP at lysine residues 76 and 81, located at the WASP WH1 domain. Disruption of WASP ubiquitination causes WASP accumulation and alters actin dynamics and the formation of actin-dependent structures.⁹³ Taken together, these data suggest that Cbl-b negatively regulates the Vav–WASP signaling pathway downstream of CD28 but upstream or at the level of WASP. In addition to deregulated actin reorganization and TCR clustering, the loss of Cbl-b selectively results in aberrant activation of NF-κB upon TCR ligation, which is mediated by Akt and PKC-θ ⁹⁴.

In an effort to define the molecular mediator(s) that regulates Vav activation in T cells, Cbl-b was suggested to promote the ubiquitination of p85, the regulatory subunit of PI3K, through an interaction with the C-terminal PR domain, resulting in the inhibition of the binding of p85 to TCRζ and CD28, thus attenuating the activation of the downstream targets Vav and Akt.⁵⁷ However, this finding, although well-cited, has not been independently verified by other investigators. Rather, our recent study revealed that Cbl-b does not regulate PI3K but rather inhibits the ubiquitin ligase activity of Nedd4, which targets Pten for K63-linked polyubiquitination, thus suppressing inactivation of Pten. Cbl-b may exert its effect on Pten by impeding the binding of Pten to Nedd4, which is independent of its E3 ubiquitin ligase activity.¹²

Cbl-b also plays a negative role in Crk-L-C3G-mediated Rap1 and LFA-1 activation in T cells. Cbl-b affects the association between Crk-L and C3G, rather than the stability of Crk-L by ubiquitinating Crk-L. In Cblb^−/− T cells, the interaction between Crk-L and C3G, and the activity of the small GTPase Rap1, are increased. Cblb^−/− T cells also display increased adhesion and cell surface binding to ICAM-1 by the enhanced clustering of LFA-1 in response to TCR stimulation.⁹⁵ By contrast, ICOS upregulation, germinal center (GC) formation, and production of IFN-γ and IL-4 are under the control of signaling pathways independent of Cbl-b-regulated Vav1 activity.⁸⁵

In addition to the above signaling pathways regulated by Cbl-b in primary naïve T cells, Cbl-b also ubiquitinates PLCγ 1 and PKC-θ in anergic T cells, attenuating the activation of PLCγ 1 and PKC-θ, which suppresses calcium mobilization and the activation of transcription factors that lead to IL-2 production.^59,69,70 Therefore, Cbl-b appears to be crucial for the induction of T-cell anergy which we will discuss below.

Roles of Cbl-b in Immune-Related Diseases

Cbl-b in tolerance induction

E3 ubiquitin ligase Cbl-b is involved in maintaining a balance between immunity and tolerance by functioning as a gatekeeper.^88,89 It has been demonstrated that CD28 and CTLA-4 may regulate the threshold for T-cell activation by controlling Cbl-b expression.^67,68 In support of this notion, Cbl-b has been shown to be a key mediator involved in T-cell anergy induction in vitro and in vivo.^70,96 In addition, CD4⁺CD25⁻ effector T cells from Cblb^−/− mice are resistant to TGF-β, Cblb^−/− and wild-type CD4⁺CD25⁺ regulatory T cells.⁹⁷ Furthermore, Cbl-b has been shown to facilitate the conversion of naïve CD4⁺CD25⁻ T cells into inducible CD4⁺CD25⁺Foxp3⁺ T cells (iTregs) via a Foxo1/3a-dependent mechanism.⁹⁸ Using both in vitro and in vivo approaches, we demonstrated that the T-cell activation threshold regulated by Cbl-b determines the fate of iTregs, and that this process is mediated by an Akt-2-dependent mechanism.⁹⁹ These results suggest that Cbl-b regulates peripheral T-cell tolerance by multiple mechanisms.

Cbl-b in autoimmunity and allergic airway inflammation

Cbl-b has been implicated in various diseases in a range of animal models. Cblb^−/− mice,⁸⁹ Cbl-b RF mutant mice,²⁰ C-terminal-truncated Cbl-b in rats,¹⁰⁰ and c-Cbl/Cbl-b double mutant mice (Cbl^−/−Cblb^−/−) mice⁴ all develop spontaneous autoimmunity or are highly susceptible to experimental autoimmune encephalomyelitis (EAE) (a model of a human demyelinating disease, multiple sclerosis [MS])¹⁰¹ and murine collagen-induced arthritis (CIA) (a mouse model of rheumatoid arthritis).^102,103 Mice with B cell-specific Cbl^−/−Cblb^−/− mutations also develop a systemic lupus erythematosus (SLE)-like autoimmune disease,⁷ further indicating that Cbl-b is essential for promoting immune tolerance. The importance of Cbl-b in peripheral T-cell tolerance is further supported by the fact that Cbl-b deficiency exacerbates disease development (exocrine pancreatitis) in mice deficient for AIRE (autoimmune regulator), which is essential for clonal deletion in the thymus.¹⁰⁴ In further support of this notion, Cbl-b deficiency subsequently precipitates type 1 diabetes in most 3A9 TCR:insHEL double transgenic mice.¹⁰⁵ In a mouse model of allergic asthma, we recently found that Cblb^−/− mice display increased airway inflammation upon OVA/alum immunization, which is due to aberrant Th2 and Th9 responses. At the molecular level, Cbl-b was found to target Stat6, a transcription factor involved in both Th2 and Th9 cell differentiation, for ubiquitination and proteasomal degradation.¹⁰⁶

Prominent autoimmune phenotypes in mice with Cbl-b (or Cbl plus Cbl-b) deletion have prompted analyses of polymorphisms/mutations of Cbl-b in animal models and human patients with autoimmune diseases. Polymorphisms of Cbl-b have been found in some autoimmune diseases, such as rat type 1 diabetes (T1D),^100,107,108 human MS,¹⁰⁹ SLE,¹¹⁰ and asthma.¹¹¹ A nonsense mutation in Cbl-b has been identified from the Komeda diabetes-prone (KDP) rat, and wild-type Cbl-b significantly suppresses development of the KDP phenotype.^100,107 Furthermore, one SNP in exon 12 of the Cbl-b gene was significantly demonstrated to be associated with T1D in a large Danish T1D study of 480 families,¹⁰⁸ although further verification should be performed using large, well-characterized populations. A recent genome-wide associated study (GWAS) indicated an association of CBLB gene variants with MS, which was confirmed in 1775 cases and 2005 controls.¹⁰⁹ These data together with data that mice lacking the ortholog are prone to EAE⁸⁸ strongly support the involvement of Cbl-b in MS development. Consistent with this finding, a significant association between the 2126(A/G) SNP of Cbl-b gene and SLE was detected,¹¹⁰ suggesting that Cbl-b may contribute to the deregulated activation of T lymphocytes observed in SLE. A Cbl-b D454A variant associated asthma was found the in asthmatic children by whole-exome sequencing.¹¹¹

Cbl-b, a target for tumor immunotherapy

Although genetic inactivation of Cbl-b clearly has detrimental consequences, e.g., sensitizing the mice to develop autoimmunity, these mice do have the enviable ability to spontaneously reject various types of solid and hematopoietic tumors and viruses. Cbl-b deficiency in mice elicits an efficient and spontaneous rejection of xenografted TC1, EL4, and E.G7 tumorigenic cell lines,^92,112 and shows a markedly lower incidence of skin cancer than the wild-type control cohort upon chronic exposure to UV-B light.¹¹² In support of these observations, Cblb^−/− mice crossed to an ataxia telangiectasia mutated-deficient background (Atm^−/−) exhibits a significantly reduced incidence and delayed onset of spontaneous T-cell lymphomas compared with Cblb^+/+Atm^−/− controls.⁹² The enhanced anti-tumor immunity in Cblb^−/− mice has been ascribed to increased activity of CD8⁺ T cells.¹¹³ Indeed, transfer of siRNA Cbl-b-silenced CD8⁺ T lymphocytes augments tumor vaccine efficacy in a B16 melanoma model.^114,115 Thus, abrogating Cbl-b expression in effector T cells may improve the efficacy of adoptive therapy of some human malignancies. The recent report that an inhibitory role of Cbl-b on rejecting tumor metastases of NK cell functions would give rise to potential therapeutic effect specific for Cbl-b to tumor metastases.⁸⁰

Perspective

Although the roles of Cbl-b in adaptive immunity have been extensively studied, the involvement of Cbl-b in innate immunity and infection has only recently been appreciated. Studies using various animal models of immune diseases will unveil the potential cellular and molecular mechanisms for Cbl-b in these disease processes, and determine whether Cbl-b is a drug target for the treatment of immune-related diseases, such as autoimmune/inflammatory diseases, infectious diseases and tumors.

Glossary

Abbreviations:

Cbl-b

Casitas B-lineage lymphoma proto-oncogene-b

TCR

T-cell receptor

BCR

B-cell receptor

FcεR

high affinity immunoglobulin epsilon receptor

TKB

protein tyrosine-kinase-binding

RF

really interesting new gene finger

PTB

phosphotyrosine-binding

PTKs

protein tyrosine kinases

GEFs

guanine nucleotide exchange factors

PI3K

phosphoinositide 3-kinase

BM

bone-marrow

Nedd4

neuronal precursor of cell expressed developmentally down-regulated gene 4

UBA

ubiquitin associated region

NK

natural killer

TLR

Toll-like receptor

LFA-1

β2-integrin leukocyte function-associated antigen-1

BMDMPs

bone marrow-derived mononuclear phagocytes

PLC-γ2

phospholipase C-gamma2

Grb2

growth-factor receptor-bound protein-2

CIN85

Cbl-interacting protein of 85 kDa

SHIP-1

SH2-containing inositol phosphatase

1 WASP

Wiscott Aldrich Syndrome protein

iTregs

inducible CD4⁺CD25⁺Foxp3⁺ T cells

EAE

experimental autoimmune encephalomyelitis

MS

multiple sclerosis

CIA

murine collagen-induced arthritis

SLE

systemic lupus erythematosus

AIRE

autoimmune regulator

T1D

type 1 diabetes

GWAS

genome-wide associated study