E3 ubiquitin ligase Casitas B lineage lymphoma‐b and its potential therapeutic implications for immunotherapy

Summary

The distinction of self from non‐self is crucial to prevent autoreactivity and ensure protection from infectious agents and tumors. Maintaining the balance between immunity and tolerance of immune cells is strongly controlled by several sophisticated regulatory mechanisms of the immune system. Among these, the E3 ligase ubiquitin Casitas B cell lymphoma‐b (Cbl‐b) is a newly identified component in the ubiquitin‐dependent protein degradation system, which is thought to be an important negative regulator of immune cells. An update on the current knowledge and new concepts of the relevant immune homeostasis program co‐ordinated by Cbl‐b in different cell populations could pave the way for future immunomodulatory therapies of various diseases, such as autoimmune and allergic diseases, infections, cancers and other immunopathological conditions. In the present review, the latest findings are comprehensively summarized on the molecular structural basis of Cbl‐b and the suppressive signaling mechanisms of Cbl‐b in physiological and pathological immune responses, as well as its emerging potential therapeutic implications for immunotherapy in animal models and human diseases.

The E3 ligase ubiquitin casitas B cell lymphoma‐b (Cbl‐b) is a newly identified component in the ubiquitin‐dependent protein degradation system. Cbl‐b is known as an important negative regulator of immune cells. Cbl‐b in different cell populations could pave the way for immunomodulatory therapies of various diseases, such as autoimmune and allergic diseases, infections, cancers, and other immunopathologic conditions.

Introduction

The post‐transcriptional regulation of several cellular pathways is mainly mediated through protein ubiquitination. E3 ubiquitin ligases are found in all leukocytes and are responsible for the specificity of ubiquitination [1]. One of their functions is to maintain the balance between immune activating and inhibitory signals [2, 3, 4]. It is demonstrated that various E3 ubiquitin ligases play roles in the regulation of immune responses. Casitas B‐lineage lymphoma (Cbl) is an E3 ubiquitin ligase that acts as the most important negative regulator of immune activation [5]. Manipulations of E3 ubiquitin ligases have been shown to result in alterations in the ubiquitination pattern of their substrates in immune cells, including protein tyrosine kinase 3 (Tyro3), zeta‐chain‐associated protein kinase 70 (Zap70), p85 and linker for activation of T cells (LAT) [6, 7]. Ubiquitin ligase targeting could be a novel approach to human disease therapy, such as in autoimmunity and cancer. The E3 ligase ubiquitin Casitas B cell lymphoma‐b (Cbl‐b) is associated with the establishment of the cell activation threshold and regulation of activating signals through antigen receptors or co‐stimulatory molecules. During the last decade, many studies have described the essential role of Cbl‐b in immune responses (both innate and adaptive) in healthy individuals and the context of various immunopathological conditions. Previous studies have shown that patients with Cbl family mutations and Cbl‐b knock‐out mice are more predisposed to the development of autoimmune disorders and spontaneous in‐vivo rejection of tumors. The current review focuses upon recent progress in the comprehension of the biological function of Cbl‐b and discusses potential therapeutic implications of Cbl‐b targeting for immunotherapy in various immune‐related diseases.

Protein ubiquitination as a post‐transcriptional regulatory mechanism

The ubiquitin‐dependent protein degradation system is a universal post‐transcriptional protein modification mechanism, and involves the modification of more than 80% of normal and abnormal (damaged and misfolded) intracellular proteins [8, 9] Large‐scale mapping of ubiquitination sites by mass spectrometry has demonstrated that approximately 20 000 ubiquitination events are associated with the modulation of several cellular processes, such as cell cycle progress, signal transduction, antigen presentation, transcription, protein quality control, cell stress response and inflammation [10, 11, 12, 13].

Ubiquitin is a 76‐amino‐acid polypeptide that binds to protein substrates via an enzyme complex [14]. Protein ubiquitination alters the activity and/or stability of these macromolecules, as well as their localization into different cell compartments [15, 16, 17]. A highly organized group of enzymes are involved in the covalent binding of ubiquitin to lysine residues of target proteins [9]. Herein, in a three‐step consecutive reaction process, the ubiquitin activation enzyme (E1) activates the free Ub via forming a thioester linkage to ubiquitin in an adenosine triphosphate (ATP)‐dependent mechanism. Subsequently, E3 ubiquitin ligase assists the ubiquitin‐conjugating enzymes (E2) in identifying target proteins and catalyzes the direct transfer of activated ubiquitins from E2 enzymes to the substrates (Fig. 1) [7]. Unlike E1 and E2 ligases, E3 ubiquitin ligase has an extensive and varied superfamily, and this results in a high level of control over the ubiquitination machinery [18, 19, 20]. Ubiquitin‐tagged proteins are identified by the proteasome complex for proteolysis [12].

Fig. 1.

Structure of domain in Casitas B lineage lymphoma (Cbl) family protein.

Pathways of ubiquitination

Substrates can be either mono‐ or polyubiquitinated, each undergoing different pathways [21, 22, 23]. The polyubiquitin chain linking amino acid residue lysine (K) 48 and K29 (known as the ‘molecular kiss of death’) generates a 26s proteasome delivery signal for short‐lived proteins [9, 24], whereas other polyubiquitination patterns [e.g. K6, K11, K63 and methionine (M)‐1] may result in alteration of the function of proteins, mainly through changing the subcellular localization or increasing the turnover of the cell surface receptors [24]. In the nuclear factor kappa b (NF‐κB) pathway, ubiquitination of NF‐κB essential modulator (NEMO) or the IKKϒ subunit of the IκB kinase (IKK) has been demonstrated through K63‐linked chains in response to multiple stimuli [25]. Mono‐ubiquitination of proteins on a single lysine residue affects different cellular processes such as endocytosis, membrane trafficking and signal receptor internalization [26, 27]. The E3 ubiquitin ligase Cbl‐b facilitates the mono‐ubiquitination of the downstream T cell receptor (TCR) signaling molecules and some cell surface receptors, including G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases, for lysosomal degradation [28].

Cbl‐family E3 ligases are important components of the cellular machinery

The Cbl proteins are a family of protein‐ubiquitin E3 ligases [29, 30]. V‐Cbl, a mutant form of Cbl, was found as a fusion protein in Cas NS‐1 retrovirus, which often led to the development of pre‐B lymphoma in virus‐infected mice [31, 32, 33, 34] The mammalian Cbl family contains three homologs (c‐Cbl, Cbl‐b and Cbl‐3), of which all Cbl proteins have the following parts: an N‐terminal tyrosine kinase binding (TKB) domain for ubiquitin conjugation through recognition of special phosphotyrosine residues on target proteins; an Src homology (SH2) domain and a calcium‐binding EF‐hand, followed by a linker helical region for recognizing target proteins for ubiquitin conjugation; a RING finger (RF) domain as a recruitment factor of E2 and C‐terminal proline‐rich region, with a ubiquitin‐associated domain (UBA); and potential tyrosine phosphorylation sites, as shown in Fig. 2 [30, 35, 36, 37]. All the domains are essential for the Cbl function in the modulation of cell signaling and protein degradation [37].

Fig. 2.

Schematic of the ubiquitin–proteasome system.

c‐Cbl and Cbl‐b homologs are expressed in hematopoietic cells, whereas the expression of Cbl‐3 is limited to epithelial tissues. Different types of stimuli such as growth factor receptors and many immune receptors trigger the tyrosine‐residues phosphorylation of Cbl family proteins [38]. Although the expression profile and structure of c‐Cbl and Cbl‐b are almost similar, their physiological functions are distinct [38, 39]. Cbl‐b was known as the primary E3 ubiquitin ligase acting as a potent negative factor of numerous signaling pathways in different hematopoietic cells [40, 41].

Modulation of innate and adaptive immunity and E3 ubiquitin ligase Cbl‐b

Protein ubiquitination has been recently shown to function as an important regulatory mechanism in innate and adaptive immune systems [42]. Cbl‐b acts as a key regulatory mediator in activating the immune cells and maintaining peripheral tolerance. Studies on murine and human B and T cells have demonstrated that Cbl‐b functions in the negative feedback of adaptive immune system through establishing the threshold for the activation of antigen receptors [41, 43, 44, 45]. Furthermore, Cbl‐b‐deficient mice represent an increase in mature T and B cell proliferation [38]. Moreover, the Cbl‐b proteins are also involved in regulating the population of leukocytes in innate immunity, including antigen‐presenting cells (APCs), monocytes and NK cells [42].

Due to the diversity of Cbl‐b substrates in a variety of cell types, it is plausible that the regulatory mechanism of Cbl‐b is a cell type‐dependent function, which occurs through controlling the signaling pathways derived from the TCR, B cell receptor (BCR), CD40 and FcεR1 [46]. A more reliable understanding of the Cbl‐b regulatory mechanisms of signaling pathways in immune cells provides opportunities for future immunotherapies of human diseases. In the following sections, we review the innate and adaptive signaling pathways regulated by Cbl‐b.

The molecular function of Cbl‐b protein in T lymphocytes

Several studies have pointed to the regulatory function of E3 ubiquitin ligases in the regulation of T cell activation [41, 47]. It has been proposed that Cbl‐b is the first E3 ubiquitin ligase directly involved in the regulation of peripheral tolerance of T cells [41]. Studies on Cbl‐b^−/− mice revealed that the presence of Cbl‐b is pivotal for peripheral immune tolerance of T lymphocytes, so‐called clonal anergy, which elicits maintenance of the balance between activation and tolerance of these cells [48]. Maintaining this balance is crucial for impeding the self‐reactive T cell development involved in immune‐related diseases [49]. Several pathways are involved in the control of co‐stimulatory signals and induction of tolerance in T lymphocytes [48, 50]. The deficiency of Cbl‐b shunts the need for CD28 co‐stimulation in the TCR‐mediated induction of T cell proliferation, production of IL‐2 and phosphorylation of Vav guanine nucleotide (Vav) [38], which is associated with a hyperproliferative T cell phenotype [41].

The TCR–CD3 thymocytes complex and the selection process of these cells are affected by the activity‐dependent ubiquitination [50]. Biochemical analyses have revealed that Cbl homologs are differentially found in thymocytes and mature T cells and play a central role in the development of thymocytes (c‐Cbl) or peripheral activation of T cells (Cbl‐b) [38, 51, 52]. The engagement of TCR on thymocytes with major histocompatibility complex (MHC) peptide results in TCRζ–Zap‐70–Cbl complex formation. Src‐like adapter protein (SLAP) has a role in bridging the interactions between Cbl and TCRζ–ZAP‐70 [53]. Furthermore, the co‐operative functions of c‐Cbl and SLAP regulate TCR expression at the surface of thymocytes [54]. Cbl proteins (primary c‐Cbl) mediate tag ubiquitination of the TCRζ chain, which can be identified by Ub‐interacting proteins (UIPs) and degraded via the lysosome. Down‐regulation of CD3/TCR and ζ degradation in thymocytes modulates the kinetics of TCR signaling during thymocyte selection [5, 55, 56]. At the molecular level, Cbl‐b accelerates the ubiquitination of p85 and results in suppression of its translocation into CD28 and TCR–CD3 complex, therefore inhibiting the activity of downstream effector molecules, such as Vav and Akt in the NF‐κB signaling pathway (Fig. 3) [5, 57].

Fig. 3.

Negative signaling pathways mediated by Casitas B lineage lymphoma (Cbl) molecules involved in the regulation of receptor signaling from thymocytes to different T cell subpopulations. SH2 = Src homology 2 domain; TKB = tyrosine–kinase‐binding domain.

Furthermore, Cbl‐b prevents NF‐kB and nuclear factor of activated T cell (NFAT)‐mediated gene transactivation in T cells by repressing the protein kinase C (PKC‐θ) [58], indicating the antagonistic role of PKC‐θ and Cbl‐b in the decision between tolerance and activation of T cells [49]. Numerous studies have illustrated that the lack of PKC‐θ induces T cell tolerance, and mice deficient in PKC‐θ are resistant to induction of experimental autoimmune encephalomyelitis (EAE) [59].

The tight regulation of Cbl‐b expression by the CTLA‐4/CD28 signaling pathway is necessary for inducing and maintaining the interleukin (IL)‐2‐producing potential of T cells on concomitant stimulation of the TCR and CD28. In the case of adequate stimulation of T cells, Cbl‐b is ubiquitinated and degraded via the proteasomal pathway due to the functional connection between CD28 signaling and anergy avoidance [49]. Moreover, CTLA‐4‐B7 interaction increases Cbl‐b expression [36], while co‐stimulation of CD28 abrogates the Cbl‐b–Src homology containing tyrosine phosphatase 1 (SHP‐1) interaction and provides the phosphorylation of Cbl‐b. It has been demonstrated that Cbl‐b phosphorylation is crucial for its E3 ubiquitin ligase function. Therefore, SHP‐1 restricts T cell responses by regulating Cbl‐b phosphorylation and ubiquitination [36].

Cbl‐b in T cell subpopulations

During antigen presentation to naive T cells several factors, including the affinity of TCR for antigen, the antigen concentration during TCR engagement and the cytokine environment, stimulate the naive CD4⁺ T helper (Th) cells to drive into functionally distinct Th cell subsets. In the absence of Cbl‐b, Th1 murine cells have revealed resistance to activation‐induced cell death (AICD) following CD3 ligation [47, 51]; also, the engagement of CD3 in Cbl‐b absence results in the mobilization of rafts and rearrangement of the cytoskeleton in Th1 cells. The presence of Cbl‐b leads to the prevention of activation‐induced apoptosis in murine Th2 cells following CD3 ligation, demonstrating a distinct cell‐type‐specific regulatory function for the molecule [47]. CD28 co‐stimulation is essential for the blockade of Cbl‐b‐negative effects on Th1 cell activation. The intensity and duration of CD28 engagement are key determinants of the degradation extent. In contrast, the TCR complex ligation in Th2 cells is presumably sufficient for inducing Cbl‐b ubiquitination and restraining its negative regulations. Unlike Th1 cells, the signals generated by the CD3/TCR complex in Th2 cells are more robust, because Th2 cells activate the PI3K in response to CD3 stimulation alone [37, 60].

Cbl‐b also plays a critical role in regulating the progress and function of pro‐allergic Th2 and Th9 cells [61]. After IL‐4 ligation, Cbl‐b selectively interacts with signal transducer and activator of transcription 6 (STAT‐6) and makes it a target for ubiquitination and degradation. Interestingly, the lack of Cbl‐b facilitates the differentiation of Th2 and Th9 cells, which leads to expanded airway inflammation (Fig. 3). The Cbl‐b–TKB domain is required for Cbl‐b–STAT‐6 association induced by IL‐4. Following stimulation of IL‐4, attachment of the Cbl‐b–TKB region to STAT‐6 phosphotyrosine residues prepares a precisely regulated interaction which can induce STAT‐6 ubiquitination [37]. Modulation of the TCR activation threshold, and similarly the induction of TGF‐β sensitivity as a crucial factor for Th9 differentiation, could be modulated by Cbl‐b‐induced‐T cell activation. Knock‐down of Runt‐related transcription factor 1 (RUNX1) by siRNA in naive CD4⁺ T cells and subsequent differentiation of these cells into Th9 cells provokes increased IL‐9 mRNA and protein expression in RUNX1‐depleted Th9 cells in comparison to control Th9 cells. Cbl‐b is reported to limit Th9 differentiation, and so may be a possible target to alter the Th9 cell development in allergy and cancer [37].

Cbl‐b deficiency leads to in‐vitro and in‐vivo multi‐functional defects in the responsiveness of T cells to transforming growth factor (TGF)‐β, showing that Cbl‐b might target various molecules associated with TGF‐β signaling [49]. Current publications have confirmed that the enzymatic function of Cbl‐b is needed for expressing forkhead box protein 3 (FoxP3) in TGF‐β‐induced regulatory T cells (T_regs) by investigating the same Cbl‐b–C373A ligase‐deficient mice [2, 21, 62, 63, 64]. Wolfert et al. reported that T_regs in Cbl‐b‐deficient miceshow normal function, but CD4⁺CD25⁻ T cells are resistant to T_regs or soluble TGF‐β inhibition [63]. Mothers against decapentaplegic homolog 8 (SMAD‐7), a negative factor of TGF‐β receptor signaling, is the main Cbl‐b target, which is ubiquitinated for degradation resulting in TGF‐β signaling in T cells [65].

As an interesting finding, Cbl‐b deficient CD4⁺ T cells can be applied to improve the anti‐tumor function of effector T cell and their resistance to suppressive functions of T_reg cells. In other words, Cbl‐b knock‐out–CD4⁺FoxP3⁻ T cells induce up‐regulation of IL‐2Ra and the production of IL‐2. IL‐2 renders effector T cell escape from the negative regulation of T_regs, their resistance to T_regs and promoting their proliferation and survival, which results in efficient functions against tumors [66]. Of interest, TCR engineering‐based immunotherapy, known as chimeric antigen receptor (CAR) T cell therapy, has been developed to promote anti‐tumor immune responses; for example, by over‐producing the IL‐2 cytokine via targeting Cbl‐b [67, 68, 69]. TGF‐β, also incorporated by IL‐6, has the major role in the induction of Th17 cells [70].

Additionally, Cbl‐b is known as a negative regulator of CD8⁺ T cells by interacting with programmed cell death 1/PD ligand 1 (PD‐1)/PD‐L1) in dendritic cells (DC). A recent study demonstrated that PD‐L1 silencing in DCs causes the down‐regulation of Cbl‐b in CD8⁺ T cells, leads to hyperproliferation of CD8⁺ T cells and improves anti‐tumor responses [71]. Cbl‐b regulates down‐regulation of TCR after antigen induction and the production of IFN‐γ by the recruitment of phosphatases (SHPs) [72] (Fig. 3). Decreased levels of clonal expansion, function and the elimination of virus‐specific CD8⁺ T cells have been reported in Cbl‐b‐deficient mice infected by lymphocytic choriomeningitis virus (LCMV) [51].

The molecular function of Cbl‐b in B lymphocytes

B cell receptor (BCR) signaling is critical for B cell activation in response to foreign antigens, as well as the induction of tolerance to self‐derived antigens in the bone marrow and periphery [56, 73]. BCR is composed of membrane immunoglobulins as antigen receptors and a heterodimer of immunoglobulin (Ig)α and Igβ, which contains the immunoreceptor tyrosine‐based activation motifs (ITAMs) in its cytoplasmic domain. BCR cross‐linking induces ITAMs phosphorylation by Src family kinase, named Lyn, which results in the recruitment and activation of the non‐receptor protein tyrosine kinase (Syk). Given the fundamental role of Syk in B cell activation, modulation of Syk is potentially required for producing the correct signaling thresholds for the beginning and termination of immune responses [2, 74]. However, the regulators of BCR signaling during B cell activation, including Src homology 2 (SH2) domain‐containing inositol 5–phosphatase (SHIP) and SHP‐1, do not appear to target the Syk [4].

Recent investigations have suggested that Cbl‐b functions as a negative regulator of BCR signaling during the ordinary course of the response, mainly by targeting the Syk [4] or together with Ig‐α [56] for ubiquitination. In wild‐type mice, BCR cross‐linking results in the prompt ubiquitination of Syk, whereas in Cbl‐b‐deficient B cells Syk is not ubiquitinated following BCR engagement. Cbl‐b is reported to ubiquitinate active phosphorylated Syk, promoting the attenuation of B cell signal transduction after the initiation of BCR signaling. Thus, it plays a central role in the down‐regulation of BCR signaling [4]. Continued phosphorylation of Syk, associated with its reduced ubiquitination displaying Cbl‐b, negatively modulates BCR signaling through targeting the Syk for ubiquitination. In Cbl‐b‐deficient mice, cross‐linking of BCRs has led to the continued phosphorylation of Igα, Syk and phospholipase C‐γ2 (PLC‐γ2). Consequently, this results in prolonged calcium (Ca2⁺) mobilization, as well as up‐regulation of c‐Jun N‐terminal kinases (JNK) and extracellular signal‐regulated kinase (ERK) phosphorylation accompanied by expression of the activation marker, CD69 [4].

Cbl is a crucial factor in defining cellular fate during peripheral B cell development. Cbl^−/− mice exhibit the development of marginal zone (MZ) B cells and a reduced population of follicular B cells in the spleen, with significant development in the B1 cell population in the peritoneal cavity [75]. Cbl‐b and c‐Cbl‐negative cells indicate slight B cell developmental alterations, such as B1 cell and marginal B cell increment, but BCR signaling and mature B cell anergy are impaired [56].

The substantial role of CD40 for B cell function is well documented. Cbl‐b is involved in regulating the CD40L interaction on T cells with CD40 on B cells, playing a main role in the homeostatic regulation of B cell activity [76]. Studies have demonstrated that CD40 stimulation augments the proliferation and survival of B cells and T cell‐dependent antibody production in Cbl‐b‐negative B cells. Cbl‐b selectively down‐regulates the c‐Jun N‐terminal kinase (JNK) and CD40‐induced‐activation of NF‐κB. TNF receptor‐associated factor (TRAF), as an adaptor molecule, plays a key role in CD40‐ and BCR‐mediated signaling pathways. Cbl‐b suppresses the recruitment of TRAF‐2 to CD40 and subsequently weakens the JNK and CD40‐mediated NF‐κB activation in B cell responses [46, 77]. The degradation of TRAF‐3 is impaired in Cbl‐b^−/− B cells following ligation of CD40. It has been demonstrated that the degradation of CD40‐induced TRAF‐3 is dependent upon TRAF‐2. Ligation of CD40 also results in different protein tyrosine kinase (PTK) activation, such as Btk and Syk in B cells. Cbl‐b inhibits the TRAF‐2 recruitment to CD40 and offers Igα, Igβ and Syk targets for ubiquitination, resulting in the suppression of both BCR‐ and CD40‐signaling pathways. CD40 ligation improves BCR‐induced ubiquitination and degradation of Cbl‐b, which evokes the elimination of Cbl‐b from the BCR‐and CD40‐signaling pathways and provides optimal stimulation of B cells.

The function of Cbl‐b in NK cells

As well as the regulation of adaptive lymphocyte populations, Cbl proteins crucially regulate the function of NK cells as innate immune cells [23]. NK cells possess cytolytic functions and act as the first line of defense at the site of the viral infection or tumor, modulating the innate and adaptive immune responses [78]. Cbl‐b has been recognized as the central gatekeeper limiting NK cell activation. Increased expression of Cbl proteins during NK cell inhibition has been reported in recent studies. Cbl proteins are activated following the suppressory receptor engagement and subsequent NK cell inhibition, resulting in ubiquitination of LAT by c‐Cbl and Cbl‐b. This process eventually elicits proteasomal degradation and abrogated NK cell cytotoxicity. These observations suggest that inhibitory KIR receptors control the stability of the Cbl proteins, thereby enabling the Cbl‐mediated suppression of NK cell cytotoxicity [79]. Following the long‐term exposure of NK cells to MHC class I chain‐related protein A (MICA)‐expressing targets, Cbl‐b mediates the regulation of the natural killer group 2 member D (NKG2D) pathway [3].

NK cells lacking Cbl‐b exhibit strong anti‐tumor immune responses, which are related to the crucial role of Cbl‐b in modulating TAM receptors (Tyro‐3, Axl and Mer). Studies have shown that Cbl‐b regulates TAM receptor internalization at the plasma membrane via ubiquitination [80]. This increase of anti‐tumor function in Cbl‐b^−/− NK cells is associated with their resistance to TAM receptor‐mediated inhibition [81]. Additionally, the effector function of NK cells may be attenuated by TAM receptors mediated by post‐translational modification of the E3 ubiquitin ligase Cbl‐b. In this manner, Cbl‐b elicits LAT1 degradation, a key signaling molecule downstream of NK cell activation, and subsequent NK cell activation following the phosphorylation by TAM receptors [82].

Stimulation of Cbl‐b silenced NK cells with IL‐2 in the absence of tumor cells, either as a single agent or in combination with IL‐12, leads to enhanced NK cell activation and IFN‐γ expression, as well as up‐regulation of an early T cell activation marker, CD69 [83]. These data confirm that the down‐modulating Cbl‐b could be considered as the potential target for cancer immunotherapy [80].

Cbl‐b in DCs

DCs as innate immune cells play a key role in adaptive immune recognition of foreign pathogens. DCs may also sense self‐cues and prevent autoimmune diseases under steady state [84, 85]. Given the importance of DCs in immune regulation, it is conceivable that understanding how differentiation, homeostasis and functional quiescence of DCs are regulated in physiological and pathological conditions not only increases our knowledge of controlling immune regulation, but also helps to find novel strategies for the therapy of infectious and autoimmune diseases and cancers [86, 87]. Currently, the role of the E3 ligase Cbl‐b in DCs biology has not been adequately discussed.

Numerous studies have demonstrated that among the Cbl E3 ligase family c‐Cbl has a central mediator in modulating the DC activation and the induction of proinflammatory cytokine response. It is reported that ovalbumin (OVA)‐pulsed DC vaccines enhance the proliferation of IFN‐γ‐secreting T cells and peptide‐specific cytotoxicity of splenocytes in c‐Cbl knock‐out mice. Therefore, c‐Cbl‐deficient DCs are considered as a key inducer of Th1 polarization [88].

Interaction of PD‐L1 on DCs with PD‐1 receptor on CD8 T cells contributes to ligand‐induced TCR down‐regulation. Prohibition of PD‐L1/PD‐1 signaling inhibits TCR down‐regulation, which leads to hyperactivated proliferative CD8 T cells. PD‐L1 blockade in DCs inhibits the expression of Cbl‐b in CD8⁺ T cells and prevents CD8 TCR down‐modulation, leading to hyperactivated TCR^high CD8 T cells [71].

In macrophages, ubiquitination of myeloid differentiation factor 88 (MyD88), FcεRIγ and SYK alter the outcome of pattern recognition receptors (PRRs) signaling in human plasmacytoid DCs (pDCs). It has been shown that upon CD303 cross‐linking (a DC‐specific type II C‐type lectin), CD2AP (CD2‐associated protein) forms a Cbl‐b/SHIP1 complex with decreased Cbl ubiquitin ligase function compared to the SHIP1 or CD2AP knock‐down human pDC cells. Afterwards, the CD2AP–SHIP1–Cbl complex is recruited to the cell membrane and co‐localizes with the cross‐linked FcεR1γ–BDCA2 complex. Repression of the Cbl‐b‐induced degradation of SYK and FcεR1γ by the SHIP1–CD2AP complex causes BCR‐like signaling pathway up‐regulation and suppression of the TLR‐7 and TLR‐9 signaling pathways [89].

In the tumor setting, the combination of DC vaccines and the adoptive transfer of Cbl‐b^−/− CD8 T cells delays tumor growth [90, 91]. Therefore, it was tempting to understand whether mice with Cbl‐b‐deficient DC cells are protected from tumor formation [92]. Some observations revealed that various surface proteins on immature and lipopolysaccharide (LPS)‐stimulated DCs were not altered in Cbl‐b‐deficient DC, but the expression of DEC‐205 increased on their surface [92]. The expression of DEC‐205 induces T_regs [93], which explains the enhancement of the tumor‐infiltrating regulatory T cells in Cbl‐b‐deficient mice [21].

Cbl‐b has a role in MyD88 degradation and consequent restraint of the MyD88‐mediated inflammation [94]. Cbl‐b knock‐out DCs secrete higher levels of proinflammatory cytokines and chemokines and induce the potent responses of allogeneic T cells, but responses of antigen‐specific T cells are not changed [92]. Compared to wild‐type murine bone‐marrow‐derived DCs (BMDCs), Cbl‐b^−/− BMDCs were equally efficient in the induction of antigen‐specific T cell activation and differentiation, both in vitro and in vivo [92]. As a result, in contrast to c‐Cbl, Cbl‐b targeting is not a promising approach for augmenting the capacity of T cell priming in the DC vaccine.

Cbl‐b in other myeloid/monocytic cells

Microbial infections and host‐derived ligands are recognized by associated receptors of innate immune cells, such as regulatory receptors (RRs) and Toll‐like receptors (TLRs). Cbl‐b is involved in the modulation of different TLR signaling pathways, such as LPS‐induced TLR‐4 signaling (regulated by MyD88 and TRIF ubiquitination) [94] and saturated fatty acid‐induced TLR‐4 signaling (controlled by TLR‐4 ubiquitination and degradation). Thus, the regulation of TLR and RR signaling in myeloid cells depends upon the type of stimulus [95]. Therefore, Cbl‐b may biochemically operate as both a multivalent adapter protein and E3 ligase, depending on the cellular context within each myeloid or monocytic cell type [41].

Recent reports have pointed to the Cbl‐b role in the inhibition of TLR‐4 signaling in macrophages and neutrophils. Cbl‐b negatively controls infiltration and activation of macrophages, and Cbl‐b deficiency in mice was shown to lead to macrophage infiltration into the adipose tissue, peripheral insulin resistance and glucose intolerance [96]. It has been demonstrated that Eritoran‐induced blockade of TLR‐4 signaling in macrophages diminishes the serum IL‐6 levels and fasting blood glucose in obese Cbl‐b^−/− mice. Deficiency of Cbl‐b could overstate a resistance of high‐fat diet (HFD)‐induced insulin by saturated fatty acid‐mediated macrophage activation [95]. Cbl‐b contributes to the suppression of LPS signaling in neutrophils, mainly through inhibiting the TLR4–MyD88 complex formation and subsequent TLR4‐mediated acute inflammatory responses induced by sepsis [97].

Cbl‐b plays an essential role in the phagocyte recruitment to the sites of inflammation through regulation of the interaction between intercellular adhesion molecule 1 (ICAM‐1) on endothelial cells and lymphocyte function‐associated antigen 1 (LFA‐1) on leukocytes. In vitro, Cbl‐b‐deficient BMDMs represent the elevated LFA‐1–ICAM‐1 interaction of leukocytes and endothelial cells, but not vascular cell adhesion molecule 1/very late antigen 4 (VCAM‐1/VLA‐4)‐mediated adhesion. Cbl‐b deficiency results in the elevated phosphorylation of β2‐chain in the structure of LFA‐1, which provides the more crucial association of the 14‐3‐3 beta protein, with the β2‐chain heading the LFA‐1 activation [98].

Cbl‐b also regulates the osteoclast activity, as Cbl‐b knock‐out mice demonstrate osteopenia [99]. Recent studies have pointed to the Cbl‐b function in the controlling of bone resorption, which has a predominant role in osteoclast survival through the interaction with phosphoinositide 3‐kinase (PI3K) [100]. Therefore, Cbl‐b could be a promising target for modulating bone metabolism in diseases such as osteoporosis and cancer. Cbl‐b negatively regulates the FcɛRI‐induced degranulation of bone marrow‐derived mast cells (BMMCs) [101]. It was shown that lack of enzymatic function of Cbl‐b leads to the elevated FcɛRI induced Ca²⁺ response, histamine release and production of inflammatory cytokines, mainly independent of the RING finger [38, 101]. Cbl‐b deficiency also increases the tyrosine phosphorylation of phospholipase C‐γ (PLC‐γ) and Syk induced by FcɛRI [38].

Cbl‐b in immune‐related diseases

Cbl‐b in autoimmune diseases

Failure of immunological tolerance and anergy due to the environmental and genetic factors may increase autoimmune disease predisposition [26]. Cbl‐b has appeared as a gatekeeper that regulates the threshold of activation in precursor and mature T and B lymphocytes [37]. Experimental evidence shows that Cbl‐b depletion can cause different autoimmune diseases characterized by the activated T and B lymphocyte infiltration, the production of autoantibodies and the destruction of several tissues [43, 64]. Further studies on the Cbl‐b pathway may help to elucidate the pathogenesis of autoimmune diseases and suggest new therapeutic targets. Studies of an animal model of type 1 diabetes (T1D), the Komeda diabetes‐prone rat, demonstrate that the Cbl‐b gene has been proposed as the main susceptible gene in the progress of diabetes and other autoimmune conditions [102, 103]. Because of the suppressor effects of Cbl‐b on CD28‐dependent T cell activation, Cbl‐b could play a role in T1D pathogenesis in human patients [104].

Previous studies have reported that the majority of Cblb^−/− mice exhibit both IgG and IgA anti‐nuclear antigen (ANA) and anti‐double‐stranded DNA (anti‐dsDNA) in the sera. The experimental data suggest that mutant mice develop spontaneously manifest systemic lupus erythematosus (SLE)‐like autoimmune disorders [56]. Recent findings reveal that polymorphic variation affecting the threshold of TCR activation plays a role in SLE development in both humans and mice [105]. Moreover, SLE patients showed a significant decrease in the expression of Cbl‐b in T follicular helper (Tfh) cells. The findings of another study of SLE patients described that Tfh development is suppressed through targeting B cell lymphoma 6 (BCL6) for degradation by Cbl‐b. This also indicates that the deregulated expression of Cbl‐b causes the aberrant development of Tfh, favoring the production of pathogenic autoantibody by B cells [106]. Cbl‐b regulates resistance to immunological suppression by modulating the profile of K63 polyubiquitination in T_regs of lupus patients. Defects in the polyubiquitination of STAT‐3 has been related to enhanced STAT‐3 expression and may contribute to the lack of suppressive function of T_regs in SLE cases [107]. Similarly, increased frequency of peripheral Tfh and down‐regulated Cbl‐b were found in SLE patients with lupus nephritis (LN) who complained of abnormal renal clinical symptoms [108]. Based on this, it was found that Tfh cells were elevated in lpr Cbl‐b knock‐out (Cbl‐b^−/−) mice compared to B6‐lpr mice, which elicited the aggravated neuropathological changes. Therefore, Cbl‐b as a negative regulator of Tfh can considerably prevent the occurrence of lupus nephritis [109].

Experiments in animal models have pointed to the regulatory function of Cb1‐b in stimulating antigen‐induced arthritis. It was also indicated that Cbl‐b ablation exacerbates autoimmune arthritis, even in the absence of mycobacterial adjuvant. Lack of Cbl‐b rescues decreased mobilization of calcium in anergic T cells, which was assigned to the Cbl‐b‐mediated modulation of PLCγ‐1 phosphorylation [50].

It has recently been suggested that findings from the genome‐wide association study (GWAS) of multiple sclerosis (MS) patients show a polymorphic variation within the Cbl‐b gene that is associated with an increased risk of developing the disease [110]. Mice deficient in Cbl‐b are prone to develop EAE, the animal model for MS [111], and this is mediated mainly by over‐activated Th17 cells. It has been documented that Cbl‐b‐expressing macrophages can repress the Th17 responses through inhibition of IL‐6 (a critical differentiation factor for the generation of Th17 cells) secretion from macrophages. Hence, the regulation of Th17 cells by Cbl‐b could prevent EAE development [112, 113].

Regulation of peripheral T cell tolerance by Cbl‐b could serve as a barricade in the clinical onset of autoimmune polyendocrinopathy syndrome type 1 (APS1) in autoimmune regulator (AIRE) deficiency [114]. Cbl‐b^−/− mice are susceptible to autoimmunity, which is thought to be mediated by impaired T_reg cell differentiation. Recently, investigations revealed that Cbl‐b is involved in the naive CD4⁺CD25⁻ T cell conversion into inducible CD4⁺CD25+Foxp3⁺ T cells (iT_regs) via a forkhead box protein O1 (FoxO1)/3a‐dependent pathway [62] and Akt‐2‐dependent mechanism [115]. Moreover, Cbl‐b controls the development of thymically derived FoxP3⁽⁺⁾ regulatory T cells (tT_regs) by targeting FoxP3 for ubiquitination [116]. Thus, these findings might provide opportunities to design novel therapeutic interventions in autoimmune diseases.

Cbl‐b in infections

More reliable recognition of the Cb1‐b role in regulating the signal transduction in cells of the adaptive and innate immune system is a critical determinant for competent Cbl‐b manipulation which contributes to developing the new immunotherapies for infectious diseases. Comprehensive studies on the profiles of T helper cells have shown a bias towards the multi‐bacillary stage of leprosy [117]. Cbl‐b over‐expression could contribute to the hyporesponsiveness of T cells, probably through the degradation of the major T cell signaling components in progressive stages of leprosy. The over‐expression of Cb1‐b was reported to rely upon TGF‐β levels, as found in anti‐TGF‐β monoclonal antibody (mAb) treatment for severe multi‐bacillary leprosy. The increased proliferation of T cells and enhanced secretion of IL‐2 in treated PBMCs by anti‐TGF‐β or shRNA‐mediated cytotoxic T lymphocyte antigen (CTLA)‐4 knock‐out mice suggest a strategy to return the hyporesponsiveness of T cell via down‐regulating the expression of Cbl‐b in leprosy. Therefore, this attitude modulates the Th2 bias and confirms a functional cross‐talk between Cbl‐b, TGF‐β and CTLA‐4, which ultimately contributes to the persistence of Mycobacterium leprae [118].

In a polymicrobial sepsis model with TLR‐4‐mediated inflammation, the loss of Cbl‐b expression increases acute lung inflammation. It was shown that the association of MyD88 and TLR‐4 is regulated by Cbl‐b [97]. The nucleotide‐binding and oligomerization domain (NOD)‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome is a multi‐protein complex that plays a crucial role in inflammation in the course of systemic inflammatory response syndrome (SIRS). NLRP3 inflammasome activation is crucial for the IL‐1β‐induced inflammatory response in LPS‐induced endotoxemia [119]. It has been documented that NLRP3 activation can be negatively regulated by Cbl‐b via targeting the NLRP3 LRR domain for K63‐linked polyubiquitination and subsequent proteasomal degradation [120].

Pseudomonas aeruginosa exploits a number of pathways to deliver proteins into the cytoplasm of the host cell that plays various functions during infection [121]. The experimental data show that Cbl‐b degrades the type III‐secreted effector exotoxin T (ExoT) and plays a crucial role in limiting bacterial dissemination [122].

Cbl‐b serves as a negative regulator of the innate anti‐fungal immunity through C‐type lectin (CLR) receptors. The Syk‐coupled CLR dectin‐2 and ‐3 are critical CLRs involved in the recognition of fungal pathogens and initiation of anti‐fungal immunity [123]. Recent studies have reported that Cbl‐b mediates the ubiquitination and degradation of dectin‐1 and ‐2, as well as their downstream kinase (SYK), consequently suppressing the dectin‐1/2‐mediated innate immune responses. In‐vivo delivery of Cbl‐b siRNA resulted in the protection of C57BL/6 mice from lethal systemic infection with Candida albicans [124] and Aspergillus fumigatus infection [123], suggesting a potential therapeutic target for fungal infections.

There is evidence to show that Cbl‐b plays an essential role in T cell exhaustion (induction of tolerance) during chronic viral infections. Cbl‐b participates in the secretion of IFN‐γ from effector CD8 T cells and down‐regulation of antigen‐induced TCR. The Cbl‐b (^−/−) mice infected with low doses of LCMV displayed a substantial cytotoxic T cell response that induces the quick clearance of the infection [51]. This approach is a crucial perspective of immunotherapeutic strategies that have been developed to reduce such persistent infections and enhance the anti‐viral effector mechanisms which promote the management of viral infections.

Cbl‐b in allergic diseases

Cbl‐b is a critical mediator in the induction of tolerance and maintenance of the immunological homeostasis in the airways. Qiao and colleagues discovered that the absence of Cbl‐b in cell cultures and an asthma mouse model promotes in‐vitro Th2 and Th9 cell differentiation, leading to severe allergic airway inflammation and strong responses of Th2 and Th9 cells. Cbl‐b specifically interacts with STAT‐6 upon IL‐4 stimulation and targets STAT‐6 for ubiquitin‐mediated degradation [37]. STAT‐6 is predominantly essential for the activation of transcription, causing Th2 differentiation and inhibiting FoxP3 expression in Th9 cells [125]. Accordingly, this claim was consistent with the increased production of both Th2 and Th9 cytokines, together with higher airway inflammation in bronchoalveolar lavage fluid (BAL) of Cbl‐b‐deficient mice, as well as elevated levels of IgE in the serum [70]. These findings indicate that Cbl‐b can strikingly suppress the development of proallergic Th2 and Th9 cells and inflammation of the allergic airway. This is mediated by Cbl‐b binding to STAT‐6 selectively and targeting it for polyubiquitination (at K108 and K398), which is subsequently destroyed in the proteasome. It was found that Th2 and Th9 responses were strongly abolished in STAT‐6 deficiency with a Cbl‐b^−/− background [70]. These data support the idea that the responses of Th2 and TH9 cells can be inhibited by Cbl‐b in a STAT‐6‐dependent mechanism with both STAT‐6‐dependent and ‐independent regimens, respectively [37, 125].

A recent parallel study described the significant elevation of orosomucoid 1‐like protein 3 (ORMDL3) expression, while Cbl‐b expression was down‐regulated in patients with recurrent wheeze compared to healthy subjects. These data documented that ORMDL3 expression is controlled by Cbl‐b, which targets the STAT‐6 for ubiquitination and subsequent degradation via phosphorylation [126].

In addition to Th2 cells, group II innate lymphoid cells (ILC2) are prominently involved in allergic airway inflammation and subsequent allergic asthma [127]. It has been demonstrated that Cbl‐b can negatively regulate ILC2 responses and prevents ILC2‐derived inflammation. As a consistent result, Guo et al. investigated the Cbl‐b effect on Rag1 Cbl‐b mice, hypersensitive to inhalation of A. fumigatus, which elicits allergic asthma due to the elevated levels of ILC2, eosinophils and type 2 cytokines. Findings revealed that Cbl‐b targeted ST2 for ubiquitination, suppressed ILC2 immunity and reduced inflammation in the lungs [128].

Studies show that the inactivation of Cbl‐b leads to Ca⁺⁺ influx and FcɛRI receptor‐mediated histamine release. The antigen‐induced tyrosine phosphorylation of cellular proteins such as PLC‐γ and SYK is augmented in Cbl‐b protein deficiency [38, 129]. A point mutation in the Cbl‐b RING finger domain results in the FcεRI‐mediated degranulation of mast cells and allergic reactions, indicating that Cbl‐b negatively regulates FcεRI receptor‐mediated signaling [130]. It parallels a 2012 Yale whole‐exome sequencing study in humans, suggesting that a mutation in the Cbl‐b produced gene is correlated with a higher risk of asthma in children [131].

Cbl‐b^−/− T cells are partially resistant to T_reg cell‐mediated suppression and unable to develop T cell anergy [126]. It is well established that maintaining the expression of FoxP3 in T_regs is dependent upon Cbl‐b [36]. Cbl‐b‐deficient mice manifest an impaired generation of iT_reg cells that could also contribute to intense proallergic Th2 responses and exacerbate allergic airway inflammation [62, 64]. Intriguingly, Cbl‐b^−/− C57BL/6 mice show an elevated inflammatory response to OVA‐induced allergic airway inflammation, including exacerbations in outcomes at both allergic airway disease (AAD) and local inhalational tolerance (LIT) time‐points and raised levels of Th2 proinflammatory cytokines and chemokines in the BAL [132]. These data indicate that the Cbl‐b dependent regulatory mechanism of T cell differentiation may serve as a noteworthy therapeutic target for allergic diseases.

Cbl‐b protein, a target for tumor immunotherapy

Novel therapeutic approaches for blocking the immune system suppressory pathways have revived hopes for the use of such therapies [133]. Targeting the inactivation of Cbl‐b results in increased adaptive and innate anti‐tumor immunity and implies that Cbl‐b inhibition might act as an anti‐tumor immunity booster in various tumors [134].

Targeting the Cbl‐b as a negative regulator downstream of the TCR represents a novel strategy to improve cancer immunotherapy. Functional responses of CD8⁺ T cells to the tumor, as key players in the immune surveillance of tumors, may be impaired due to the lack of co‐stimulatory signals and the presence of negative cues of the tumor microenvironment, including tumor‐associated immunosuppressive factors such as like T_reg cells, TGF‐β and IL‐10 [21, 26, 34]. CD8⁺ T cell activation upon TCR engagement is inhibited by the PD‐1/PD‐L1 pathway in the absence of CD28 co‐stimulation [135]. Anti‐PD‐1/PD‐L1 therapies have shown noticeable clinical outcomes in treating various cancers. For the first time, Fujiwara and colleges reported that Cbl‐b (^−/−) T cells were resistant to inhibition by PD‐L1/PD‐1 in vitro and in vivo [136]. Upon treatment of non‐small‐cell lung carcinoma (NSCLC) with atypical protein kinase C (aPKCs) inhibitors, so‐called DNDA (3,4‐amino‐2,7 naphthalene disulfonic acid), DNDA interrupts the link between PKC‐ι/FAK and subsequently leads to FAK ubiquitination and degradation mediated by Cbl‐b. Finally, this process results in the induction of apoptosis in lung cells, as well as the prevention of cancer cell proliferation, migration and invasion [137]. The more elevated expression level of miR‐1323 foretells poor prognosis in lung adenocarcinoma (LUAD) and NSCLC patients, whereas enhanced expression of Cbl‐b is associated with better prognosis [138].

Deletion of Cbl‐b resulted in a substantial up‐regulation in CD8⁺ T cell functional responses, including proliferation, granzyme B production and infiltration into the tumor site by increasing the LFA‐1 when compared with wild‐type T cells [139]. Stromness et al. showed that knock‐down of the Cbl‐b mediated by RNAi in effector CD8⁺ T cells enhanced the anti‐leukemia efficacy of these cells in a mouse model of adoptive transfer of T cells [140]. Robles‐Valero et al. found that Vav1 functions as a tumor suppressor in immature T cells through controlling and promoting the degradation of active Notch1 fragment (ICN1). The Cbl‐b E3 ubiquitin ligase mediates this non‐catalytic function of Vav1. Vav1‐deficient mice were highly prone to T cell acute lymphoblastic leukemia (T‐ALL) by enhancing the signaling of ICN1, further underscoring the inhibitory role of this pathway.

Mutations in the RING finger domain of the Cbl family or Cbl‐b linker sequence comprise significant pathogenic lesions correlated not only to juvenile myelomonocytic leukemia (JMML), pre‐leukemic chronic myelomonocytic leukemia (CMML) and other myeloproliferative neoplasms (MPN), but also progression to acute myeloid leukemia (AML). These data propose that degradation impairment of activated TKs composes a significant cancer mechanism [141, 142].

In adoptively transferred immune cells, siRNA‐mediated Cbl‐b‐depleted autologous CD8⁺ T cells could function as an effective adjuvant for DC vaccination and presents a reasonable approach to increase the effectiveness of adoptive cell therapies (ACT) [143]. It has been determined that a Cbl‐b‐defective mutation in a mice model of CD8⁺ T cells or recovering an adoptive transfer of Cbl‐b siRNA‐transfected CD8⁺ T cells promotes a higher rate of spontaneous rejection of ultraviolet B radiation‐induced skin cancer [144], human papillomavirus antigen‐expressed tumor cells [41, 144], B16 melanoma model [91], prostate cancer [145] and gallbladder cancer cells [146].

Interestingly, an enhanced anti‐tumor function of CD8⁺ T cells resulted from the lack of Cbl‐b, even in the CD4⁺ T cell absence, proposes a potential regimen for improving the anti‐tumor function of T cells in vivo [144]. Cbl‐b^−/− CD8⁺ T cells are mechanistically resistant to T_reg‐mediated suppression and display robust activation and tumor infiltration [144]. In the setting of T cell anti‐tumor activity, CD4⁺FoxP3⁻Cbl‐b^−/− T cells exhibit both hypersecretion of and hypersensitivity to IL‐2, which serve as significant mechanisms to escape T_reg cell suppression. [147]. These results suggest that Cbl‐b, as a key signaling molecule, is involved in the control of the spontaneous anti‐tumor function of cytotoxic T lymphocytes in various cancer models. Cbl‐b blocking could be regarded as a new therapeutic approach to induce long‐lasting immunity against cancers. Transferring the naive Cbl‐b silenced CD8⁺ T lymphocytes are adequate for the rejection of established primary tumors. It has been demonstrated that up to 1 year after the first exposure to the tumor cells, Cbl‐b knock‐out mice display an ‘anti‐cancer memory’ [144].

NK cells as innate immune cells have an important role in malignant cell rejection. It has been demonstrated that Cbl‐b co‐operates with the major regulatory mediators of NK cell receptors and co‐stimulatory signal transduction pathways. Recent results showed that intervening with Cbl‐b activity in NK cells facilitates synergistic effects of the main cytokines of the innate (IFN‐γ and TNF‐α) and adaptive immune systems (IL‐12 and IL‐2) [83]. Cbl‐b‐deficient NK cells display more powerful anti‐tumor immune responses and spontaneously reject metastatic tumors. Thus, improving Cbl‐b inhibitors in the context of cancer immunotherapy should not be restricted to the T cell population, but include NK cells [134].

Clinical trials show that the Cbl‐b‐mediated ubiquitination could prevent multi‐drug resistance (MDR) during the chemotherapy of different cancers. This is due to the attenuation and disruption of target molecules of the Akt/PI3K downstream signaling pathway and a decreased expression of drug transport pumps [5, 148, 149]. The reduced Cbl‐b expression has been demonstrated in MDR gastric and breast cancer cells (but not in immune cells), and Cbl‐b over‐expression was decreased in both in‐vitro and in‐vivo cell migration in MDR cell cultures [150, 151, 152]. Over‐expression of the Cbl‐b also prevented the epithelial–mesenchymal transition (EMT) through the induction of epidermal growth factor receptor (EGFR) ubiquitination and degradation, which resulted in the EGFR–ERK/protein kinase B–microRNA‐s00c–zinc finger enhancer binding protein (Akt–miR‐200c–ZEB1) axis inhibition. Similarly, it was indicated that β‐element, an anti‐tumor natural agent of traditional Chinese herbal medicine, played an anti‐metastatic role in MDR gastric cancer by suppressing the expression of matrix metalloproteinase (MMP)2 and MMP9 following suppression of the EGFR–ERK/AKT pathways mediated by Cbl‐b up‐regulation. It was found that β‐element can suppress miR‐1323 and subsequent up‐regulate Cbl‐b expression, which paves the way to EGFR ubiquitination and degradation by Cbl‐b [153]. Consequently, these data clearly illustrate the Cbl‐b role in the prevention of tumor metastasis, mainly through keeping the epithelial phenotype in MDR breast and gastric cancer cells [150, 152]. As consistent findings in lung and gastric cancers, suppression of cell proliferation was applied by Cbl‐b through ubiquitination of the EGFR pathway [154]. Similarly, inhibition of tumor cell proliferation by Cbl‐b was also shown in breast cancers [155]. Of interest, it was shown in a report [155] that the positive expression of Cbl‐b can be considered an appropriate prognostic predictor in breast cancer, mainly in cases with lymph node metastasis and stages II–III.

In a murine model of colon cancer, strong anti‐tumorigenic immune responses were observed in Cbl‐b‐silenced animals in vivo, providing pre‐clinical proof for the effectiveness of Cbl‐b silencing by siRN as a practical approach for cellular immunotherapy [156]. Thus, modulating the Cbl‐b E3 ligase activity together with other immune‐activating approaches, such as DNA‐based vaccines, DC vaccines and anti‐checkpoint mAbs, could be introduced as a novel immunotherapy strategy, which boosts the immunity against tumors without predisposing the signs of autoimmunity [91, 134, 143].

Polymorphisms associated with Cbl‐b

The human Cbl‐b gene contains 19 exons on chromosome 3q11–13.1 and is the largest of all three Cbl genes (~220 kb). The severity of the disease and the way the body responds to treatments are also manifestations of genetic variations. Due to the various functions of Cbl‐b, polymorphisms or mutations in the gene sequence in different diseases have been considered by researchers. Cbl‐b polymorphisms linked to immunological diseases have been presented as a list in Table 1.

Table 1.

Casitas B lineage lymphoma (Cbl‐b) variants in various diseases157

Disease	Exon	Variant	Association	Population	No. of subjects	References
Type 1 diabetes	Exon 1	rs1503922 T/G	No	Danish	253 family	[104,157]
		New C/T	No	Danish	253 family
	Exon 6	New T/C	No	Danish	253 family
	Exon 10	rs2305035 C/T	No	Danish	253 family
		rs2305036 A/C	No	Danish	253 family
	Exon 11	rs2305037 A/G	No	Danish	253 family
	Exon 12	rs3772534 A/G	Yes	Danish	480 family
	Exon 18	New C/T	No	Danish	253 family
	Exon 19	rs1042852 A/G	No	Danish	253 family
Systemic lupus erythematosus	Exon 12	rs3772534 A/G	Yes	Mexican	150/163	[105]
Graves’ disease	Exon 12	rs2305035 C/T	No	Taiwanese	158/237	[158]
Multiple sclerosis (MS)	Intron 1	rs9657904 T/C	Yes	Italian	1435 cases	[159]
	Intron 1	rs9657904 T/C	No	Germany	Not mentioned	[160]
	‐	rs12487066	Yes
	Intron 1	rs9657904 T/C	Yes	Sardinian	882	[110]
	–	rs12487066 C>G	No	Iranian	410	[161]
Myeloid malignancies	AML	Mutations detected by Sequencing	Yes	American	110	[141]
	MPN				22
	MDS/MPN				98
Non‐small‐cell lung cancer	Exon 19	rs1042852 C>T	No	American	393
	‐Exon 10	rs2305035 G>A	Yes
	–	rs7649466 C>G	No
	–	rs3772534 C>T	No	Chinese	200	[162]
	Exon 10	rs2305035 G>A	Yes
	–	rs9657904 C>T	Yes

MDS = myelodysplastic neoplasms; MPN = myeloproliferative neoplasms; MPN = myeloproliferative neoplasms.

Final comments and perspectives

Although little is known concerning the mechanisms of the regulatory function of Cbl‐b in human immune cells, evidence suggests that Cbl‐b exerts its inhibitory role on the cells of hematopoietic origin, favoring a balanced immune function. In autoimmune diseases, degradation inhibition of Cbl‐b targets could be regarded as a rational approach. Moreover, anti‐cancer immune responses might be increased by direct or indirect inhibition of the Cbl‐b activity. It has been shown that the inhibition of Cbl‐b by small molecules or Cbl‐b‐targeting siRNA could be considered as a promising approach for the biological therapy of immune‐mediated diseases, such as infectious autoimmune diseases, allergic reactions and tumors.

Disclosures

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.

Data Availability Statement

Not applicable.