Targeting ubiquitin signaling for cancer immunotherapy

Cancer immunotherapy has become an attractive approach of cancer treatment with tremendous success in treating various advanced malignancies. The development and clinical application of immune checkpoint inhibitors represent one of the most extraordinary accomplishments in cancer immunotherapy. In addition, considerable progress is being made in understanding the mechanism of antitumor immunity and characterizing novel targets for developing additional therapeutic approaches. One active area of investigation is protein ubiquitination, a post-translational mechanism of protein modification that regulates the function of diverse immune cells in antitumor immunity. Accumulating studies suggest that E3 ubiquitin ligases and deubiquitinases form a family of potential targets to be exploited for enhancing antitumor immunity in cancer immunotherapy.

Targeted therapy and immunotherapy are two advanced strategies of cancer treatment.1Strategies in targeted therapy are based on cell-autonomous mechanisms of tumor growth and survival to directly target tumor cells. While these methods generate clinical responses in most cancer patients carrying the specific genetic mutations, they often lack durability due to acquisition of resistance by tumor cells.1 Immunotherapy does not directly target tumor cells but rather acts through mobilization of patients’ own immune system to attack the tumors. A major advantage of immunotherapy is the possible generation of long-lasting antitumor effects in addition to relatively minor side effects.2 In particular, therapies based on immune checkpoint inhibitors (ICIs), monoclonal antibodies targeting the T cell coinhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1), have revolutionized cancer treatments.2 However, since the response rate of many types of cancers to single ICI therapies is still low, extensive efforts are being made to develop combination strategies and characterize new therapeutic targets, such as the costimulatory receptors of T cells. In addition, targeting intracellular signaling molecules provides an additional opportunity for improving antitumor immunity.

Ubiquitination has become a well-recognized signaling mechanism that regulates diverse aspects of immune system functions. Ubiquitination is a posttranslational mechanism of protein modification involved in diverse biological processes, including proteasomal protein degradation, receptor endocytosis, DNA repair, gene transcription, kinase activation, protein-protein interaction and assembly of signaling complexes.3 Ubiquitination involves covalent conjugation of monoubiquitin or polyubiquitin chains onto lysine (K) resides of target proteins, which is catalyzed by sequential actions of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes.4 Polyubiquitin chain formation involves connection of ubiquitin molecules via different internal K residues or the N-terminal methionine (M1) residue, leading to generation of a large variety of ubiquitin chains with distinct functions.5 For example, K48-linked ubiquitin chains target proteins to the proteasome for degradation, whereas K63-linked and M1-linked (also called linear) ubiquitin chains are best known for mediating protein-protein interaction and enzymatic activation in signal transduction. The process of protein ubiquitination is counteracted by deubiquitinases (DUBs), a large family of proteases that cleaves ubiquitin chains.3 Mammalian cells express more than 600 E3 ligases and about 100 DUBs, which display substrate specificities and regulate specific cellular functions.3,5 An increasing number of E3s and DUBs have been identified as important regulators of immune responses, providing exciting opportunities for developing novel drugs for cancer immunotherapy. In this review, we will discuss recent studies regarding the roles of ubiquitination in regulating immune cell functions and their potential in cancer immunity.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that mediate T cell activation and play a vital role in immune responses against infections and cancer.6 In tumor microenvironment, DCs uptake antigens released from dying tumor cells and migrate to draining lymph nodes, where DCs present the tumor antigens to the TCR of tumor-specific T cells, triggering T cell activation and differentiation, and the generated effector T cells traffic to the tumors to mediate tumor cell destruction.7 Among the different DC subsets, conventional DCs (cDCs) are specialized in antigen presentation for T cell activation, and the cDCs can be further divided into type 1 cDC (cDC1) and type 2 cDC (cDC2) subsets characterized by expression of CD8α and CD11b, respectively.8 The cDC1 subset is particularly efficient in presenting cancer antigens to CD8 T cells for mediating anticancer immunity. Understanding the mechanisms that regulate DC functions is crucial for rational design of cancer vaccines and other therapeutic agents to improve the efficacy of cancer immunotherapy.6,9

Antigen processing and presentation form an integral part of DC functions, which involves processing of endogenous proteins and internalized exogenous proteins into small peptides and presentation of the peptide antigens by major histocompatibility complex (MHC) molecules.10 MHC class I (MHC I) typically presents peptides generated through proteasomal degradation of endogenously synthesized cytosolic proteins. However, MHC I in professional APCs, particularly DCs, can also present peptides from internalized exogenous proteins via a process known as cross-presentation, which is crucial for CD8 T cell responses against infections and tumorigenesis.11,12 In this process, the exogenous proteins are either transported from the endosomal vesicles into the cytosol for proteasomal processing or directly processed within the endosomal compartments. MHC class II (MHC II) present peptides derived from lysosomal degradation of extracellular proteins.10 The antigens presented by MHC I and II are recognized by the TCR of CD8 and CD4 T cells, respectively. In response to infections or tumorigenesis, DCs undergo a process of maturation, involving upregulation of costimulatory molecules and chemokine receptors, which is required for their migration to the draining lymphoid organs and function in T cell priming.10 Ubiquitination plays a crucial role in regulating both antigen processing and maturation of DCs, highlighting new opportunities for DC manipulations in cancer immunotherapy.

The involvement of ubiquitination in MHC I-restricted antigen processing was initially suggested by the finding that fusion of viral proteins with ubiquitin enhances antigen presentation to CD8 T cells.13 A more direct evidence came from a study using cells with a temperature-sensitive defect in ubiquitin conjugation.14 Similarly, overexpression of a dominant-negative ubiquitin mutant lacking all of the lysine residues in mammalian cells abrogates polyubiquitination and potently inhibits MHC I-restricted processing of endoplasmic reticulum (ER)-targeted proteins.15 Accumulating evidence suggests that MHC I-restricted processing of exogenous proteins for cross-presentation involves ER-associated degradation (ERAD),16,17,18,19 a mechanism mediating retrotranslocation of misfolded proteins from the ER back to the cytoplasm for proteasomal degradation.20 ERAD also mediates processing of extracellular proteins involved in antigen cross-presentation.19 Thus, approaches to enhance ER entry of protein antigens, such as Grp170 chaperone-based vaccine adjuvant system, improve the efficiency of cross-presentation and induction of CD8 T cell responses in cancer immunotherapy.19,21

Ubiquitination has a vital role in ERAD-dependent protein processing. It is thought that ubiquitination of ERAD substrates facilitates their binding by the ERAD molecular machinery required for their retrotranslocation to the cytoplasm.22 A major E3 ubiquitin ligase participating in the process of ERAD is the ER-resident transmembrane protein HRD1, which forms a complex with several partner proteins, including SEL1L, HERP, DERLIN-1, OS-9 or XTP3, and BiP, to mediate ubiquitination of ERAD substrates.15,22 The E3 action of HRD1 in ERAD is counteracted by a DUB, USP25, which deconjugates the ubiquitin chains from specific ERAD substrates to prevent their retrotranslocation and proteasomal degradation.23,24 Another mechanism of HRD1 regulation is through controlling its steady-state levels by ubiquitin-dependent proteasomal degradation, a process that is protected by a DUB USP19.25 While HRD1 has been implicated in the regulation of ERAD and MHC I-restricted antigen processing, a recent gene-targeting study suggests that HRD1 also plays an important role in promoting MHC II-restricted antigen presentation to CD4 T cells.26 In this function, HRD1 promotes transcriptional expression of the MHC II gene via a mechanism that involves ubiquitin-dependent degradation of B lymphocyte-induced maturation protein 1 (BLIMP1), a transcriptional suppressor known to inhibit expression of the MHC class II transactivator (CIITA).26 These studies suggest that manipulating HRD1 expression and ERAD events may be an approach to improve antigen presentation and T cell responses in cancer immunotherapy.

Another E3 ubiquitin ligase regulating ERAD is the ER-resident ring finger protein RNF5.27RNF5, together with the E2 Ubc13, mediates ubiquitination of JNK-associated membrane protein (JAMP), an ER membrane protein facilitating ERAD-dependent degradation of unfolded proteins.27,28 RNF5-mediated JAMP ubiquitination inhibits the association of JAMP with ERAD molecular components, thereby limiting ERAD-mediated protein degradation.27Thus, unlike the positive role of HRD1, RNF5 negatively regulates ERAD, although it remains to be examined whether RNF5-mediated ERAD regulation has a role in regulating antigen processing and T cell activation. Nevertheless, a role of RNF5 in regulating antitumor immunity has been linked to a function of RNF5 in regulating another ER-protective process, unfolded protein response (UPR).29 UPR is a mechanism that restores normal function of the ER in response to ER stress and has been linked to important functions of immunity and inflammation.30 RNF5 deficiency in mice attenuates UPR and reduces antimicrobial peptide expression in intestinal epithelial cells, which in turn alters gut microbiota composition and, thereby, promotes the recruitment and activation of DCs for T cell activation.29 Consequently, the RNF5-deficient mice display stronger antitumor immunity in a microbiota-dependent manner. Another immunoregulatory function of RNF5 is to mediate ubiquitin-dependent degradation of stimulator of interferon genes (STING) and negatively regulate antiviral innate immune responses.31 Of note, STING is a central DNA sensing component that mediates DC activation in tumor microenvironment and has been actively exploited in cancer immunotherapy.32,33 Whether RNF5 deletion in DCs promotes DC activation and T cell priming in antitumor immunity is yet to be investigated.

Ubiquitination also regulates the surface expression and recycling of MHC molecules. Early studies suggest that proteins encoded by some viruses, such as HIV, KSHV, downregulate surface MHC I by inducing its endocytosis and degradation.34,35 Ubiquitin provides a signal for MHC I incorporation into intralumenal vesicles of multivesicular bodies (MVBs), a subpopulation of late endosomes fusing with lysosomes.36 Members of the membrane-associated RING-CH (MARCH) E3 ubiquitin ligases have been shown to mediate endosomal trafficking and degradation of immune receptors, including MHC molecules.37 MARCH4 and MARCH9 mediate MHC I ubiquitination to promote its endocytosis and lysosomal degradation, whereas MARCH1 and MARCH8 mediate ubiquitin-dependent degradation of MHC II.38,39 Studies using MARCH1-deficient DCs reveal that although MARCH1-mediated MHC II ubiquitination does not affect MHC II endocytosis, it prevents recycling of the internalized MHC II/peptide complexes and promotes their degradation.40,41 Thus, MARCH1-deficient DCs have drastically increased levels of MHC II/peptide complexes on the cell surface. Interestingly, lipopolysaccharide (LPS)-induced DC maturation is associated with downregulation of MARCH1 expression and attenuation of MHC II ubiquitination, coupled with increased MHC II surface stabilization.41,42 Conversely, the immunosuppressive cytokine IL-10 induces the expression of MARCH1 and, thereby, downregulates MHC II surface expression, contributing to IL-10-mediated suppression of antigen presentation.43,44,45 The role of ubiquitination in MHC regulation has also been demonstrated through the study of DUBs. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been shown to positively regulate MHC I recycling and antigen cross-presentation to CD8 T cells, although it is unclear whether UCH-L1 directly deubiquitinates MHC I.46 Another DUB, USP14, facilitates MHC I-restricted direct antigen presentation, particularly the peptides derived from defective ribosomal products (DRiPs).47 These findings suggest that targeting E3 ubiquitin ligases and DUBs represents a potential strategy to modulate MHC expression and antigen presentation in vaccine development and other approaches of cancer immunotherapy.

DCs normally exist in an immature state in peripheral tissues, and their T cell-priming function requires a process of maturation characterized by upregulation of MHC II, costimulatory molecules such as CD80 (also called B7-1) and CD86 (also called B7-2), and chemokine receptors.48 DC maturation is stimulated by signals from different immune receptors, including pattern-recognition receptors (PRRs) that respond to pathogen-associated molecular patterns (PAMPs) or host-derived damage-associated molecular patterns (DAMPs).5,48 Exposure of DCs to PRR ligands also activate DCs to secrete cytokines that regulate the differentiation of activated T cells to generate specific subsets of effector T cells. DCs and other innate immune cells express several families of PRRs, which include toll-like receptors (TLRs), cytosolic DNA sensors, RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs).49 PRR signals function by triggering cascades of signaling events, including activation of MAP kinase (MAPKs), IkB kinase (IKK), and the IKK-related kinase TBK1, which mediate activation of transcription factors AP1, NF-κB, and IRF3, respectively.5 The MAPK and IKK pathways are crucial for PRR-stimulated DC activation and proinflammatory cytokine production involved in T cell activation and differentiation, whereas the TBK1 pathway mediates induction of type I interferons (IFNs). In addition to regulating antiviral innate immunity, type I IFNs have a pivotal role in inducing DC maturation and anticancer immunity.50,51 DCs lacking type I interferon receptor 1 (IFNAR1) are defective in polyI:C-stimulated maturation in vivo, as shown by impaired induction of costimulatory molecules and MHC II.50 On the other hand, type I IFN production in DCs is not required for DC maturation, since nonhematopoietic cells are the main source of type I IFN stimulated by polyI:C.50 In fact, DC-conditional TBK1 deficiency promotes DC maturation and anticancer T cell responses due to a negative role of TBK1 in regulating IFNAR1 signaling.52 TBK1 acts by phosphorylating and promoting the function of STAT3, a negative regulator of IFNAR1 signaling. Thus, the PRR-stimulated signaling pathways play important, although distinct, roles in the regulation of DC maturation and activation involved in antitumor immunity.

Ubiquitination regulates both the upstream common signaling steps and the downstream specific pathways of the PRR signaling.5 Several E3 ubiquitin ligases, including cIAP1, cIAP2, Peli1, TRAF6, TRIM31, and TRIM56, have been shown to conjugate K63-linked polyubiquitin chains to various PRR signaling adapters, such as RIP1, RIP2, TRAF6, MAVS, and STING, thereby facilitating the recruitment and activation of downstream kinases.5,53,54,55 K63 ubiquitination also has a more direct role in regulating downstream signaling events, particularly IKK activation. An early study suggests that ubiquitination of the regulatory subunit of IKK, NF-κB essential modulator (NEMO), is required for DC maturation and activation.56 DCs derived from patients with ectodermal dysplasia with immune deficiency (EDI), which carries a point mutation in the C-terminal zinc finger domain of NEMO (C417R), display a defect in CD40-stimulated ubiquitination of NEMO and activation of an NF-κB member, c-Rel, associated with attenuated induction of costimulatory molecules and a key immunostimulatory cytokine, IL-12, as well as T cell-priming function.56 A20, an ovarian tumor (OTU) family DUB known to regulate IKK/NF-κB signaling and TNF-induced cell death,57,58 has been shown to negatively regulate the maturation, proinflammatory cytokine production, and immunostimulatory functions of DCs59,60 (Fig. 1). A20 knockdown in murine and human DCs enhances the induction of costimulatory molecules and proinflammatory cytokines by ligands of TLR3 and TLR4, rendering DCs hyper-active in priming T cell responses (Fig. 1). In line with these studies, gene targeting studies demonstrate that A20 functions as a checkpoint in DC activation and survival, which involves inhibition of LPS-stimulated IKK and MAPK signaling pathways.61,62The A20-deficient DCs undergo spontaneous maturation under homeostatic conditions, characterized by aberrant expression of the costimulatory molecules. Consistently, the peripheral lymphoid organs of DC-conditional A20 knockout mice have an increased frequency of effector/memory-like T cells indicative of spontaneous T cell activation under steady conditions.61,62 Signal transduction from TLRs, with the exception of TLR3, relies on a common signaling adapter, MyD88, whereas TLR3 signals through another adapter protein, TRIF.5 Deletion of MyD88 in DC-conditional A20 knockout mice does not prevent spontaneous DC maturation or aberrant T cell activation, suggesting that A20 restricts MyD88-independent signals to prevent spontaneous DC maturation and aberrant T cell activation under homeostatic conditions.62 However, MyD88 deletion prevents A20-deficient DCs from hyper-production of cytokines, including IL-6 and TNF, that drive T cell proliferation. Therefore, A20 restricts MyD88-dependent signals in DCs to suppress IL-6 and TNF production, thereby preventing aberrant T cell expansion.62 A20 knockdown studies reveal that A20 downregulation in DCs impairs their function in stimulating regulatory T (Treg) cells, thereby further enhancing effector T cell responses59 (Fig. 1). The potential therapeutic value of targeting A20 has been demonstrated using a mouse model of DC-based cancer therapy.59When adoptively transferred into tumor-bearing mouse, the A20-silenced DCs are much more potent than wildtype DCs in suppressing growth of the established tumors, associated with increased infiltration of effector T cells with enhanced resistance to Treg-mediated suppression59 (Fig. 1). These studies suggest that A20 controls DC functions in T cell priming and effector function as well as in Treg regulation, implicating A20 targeting as a potential approach for improving the efficacy of cancer immunotherapy, particularly DC-based therapy (Fig. 1).

The function of NF-κB signaling in DCs is also subject to ubiquitin-dependent regulation at the epigenetic level, as demonstrated through the study of a A20-related DUB, Trabid (also called Zranb1).63 Trabid is required for TLR-stimulated expression of two proinflammatory cytokines, IL-12 and IL-23, which in turn are crucial for generation of Th1 and Th17 subsets of CD4 effector T cells. Although Trabid is dispensable for the induction of NF-κB nuclear translocation, Trabid is required for recruitment of NF-κB members, particularly c-Rel and p50, to the promoters of Il12a, Il12b, and Il23a genes. Ubiquitination targets the degradation of Jmjd2d, a histone methylase mediating histone modifications at the Il12/Il23 promoters required for binding by p50 and c-Rel, and the ubiquitin-dependent Jmjd2d degradation is counteracted by Trabid-mediated deubiquitination.63 Trabid-mediated DC regulation plays an important role in autoimmune responses. Since IL-12 family of cytokines have an important role in regulating antitumor T cell and natural killer (NK) cell responses,64 it is important to examine whether Trabid also plays a role in regulating antitumor immunity.

T cells form a central component of adaptive immune responses against infections and cancer. T cell activation occurs in lymphoid tissues that drain the sites of infections or tumorigenesis, where naïve CD4 and CD8 T cells encounter APCs displaying antigens on MHC II and MHC I, respectively. Upon engagement by the antigen/MHC complex, TCR delivers a primary signal for T cell activation. T cell activation also requires costimulatory signals, which in naïve T cells is primarily mediated through ligation of the costimulatory receptor CD28 by its ligands, CD80 or CD86, on APCs.65 T cells also express costimulatory receptors of the TNF receptor (TNFR) superfamily, many of which are induced along with T cell activation and are required for long-lasting T cell responses and generation of effector and memory T cells.66 In addition, signals stimulated by cytokines and growth factors contribute to the activation and differentiation of T cells. The TCR and costimulatory signals are opposed by signals from a variety of inhibitory receptors, most notably members of PD1 family of co-inhibitory receptors.67 Ubiquitination regulates different aspects of signaling events involved in the activation and function of T cells.

Upon engagement by an antigen, TCR initiates receptor-proximal signaling events, including activation of the protein tyrosine kinase Lck and phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3 and ζ chains of the TCR complex. Phosphorylation of the ITAMs leads to recruitment and activation of an ITAM-binding protein tyrosine kinase, ZAP70, which in turn amplifies the TCR signal by phosphorylating scaffold proteins, including LAT and SLP76.68 These phosphorylated scaffold proteins mediate recruitment and activation of key signaling components that target different downstream signal cascades, leading to activation of transcription factors of the NFAT, AP1 and NF-κB families that cooperatively induce the expression of genes involved in T cell activation, survival, proliferation, and differentiation (Fig. 2).

Regulation of T cell signaling by E3 ligases and DUBs. Grail and Cbl-b mediate ubiquitin-dependent degradation of TCR ζ chain and upstream signaling factors, VAV and PI3K, to negatively regulate TCR and CD28 signaling. Non-degradative ubiquitination of ZAP70, a negative mechanism of its regulation, is catalyzed by the E3 Nrdp1 and counteracted by the DUB Otud7b. MDM2 and Peli1 mediates ubiquitin-dependent degradation of NFAT1 and NF-kB c-Rel, respectively, to control these downstream pathways. The DUB USP15 stabilizes MDM2 in activated T cells and functions together with MDM2 in NFAT1 regulation. The CBM complex, composed of CARMA1, BCL10, and MALT1, associates with the K63-specific E2 Ubc13 and an E3 and catalyze K63 ubiquitination required for activation of IKK. This signaling step is negatively regulated by the DUBs CYLD and A20. The DUB Otub1 inhibits K63 ubiquitination and activation of AKT and controls a major metabolic signaling pathway of T cells. E3 ligase Fbxo38 mediates ubiquitin-dependent degradation of the coinhibitory receptor PD1, thereby promoting T cell responses in cancer immunity

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The TCR-proximal signaling events are tightly controlled by ubiquitination, which mediates degradation or functional inactivation of key components of the TCR signaling pathway, such as CD3ζ, ZAP70, phospholipase C gamma 1 (PLCγ1), PI3 kinase (PI3K), and protein kinase C theta (PKCθ).5 A well-characterized E3 ligase involved in the regulation of TCR signaling is Cbl-b, which targets PI3K and the guanine nucleotide exchange factor VAV and is crucial for maintaining T cell tolerance and preventing autoimmunity69,70 (Fig. 2). Cbl-b deficiency reduces the activation threshold of T cells and renders T cells hyper-responsive to TCR stimulation even in the absence of CD28 ligation, causing development of autoimmune diseases in mice.71,72 Consistently, Cbl-b-deficient CD8 T cells display markedly enhanced activity in rejecting transplanted and spontaneous tumors.73,74 Cbl-b deficiency also renders T cells resistant to inhibition by Treg cells and the immune checkpoint PD-L1.75,76,77 Because of the enhanced activation and antitumor responses of Cbl-b-deficient T cells, Cbl-b has become a focus for understanding the mechanisms of antitumor immunity and an attractive cancer immunotherapy target being exploited in preclinical studies and clinical trials.78,79 Another E3 ubiquitin ligase Grail (RNF128) has been shown to negatively regulate T cell activation and maintain T cell anergy and tolerance.80,81 Grail inhibits TCR-proximal signaling by targeting CD3ζ for ubiquitin-dependent degradation of CD3ζ, and Grail deficiency impairs T cell tolerance and sensitizes mice for autoimmune diseases82 (Fig. 2). In CD8 T cells, Grail also mediates ubiquitin-dependent degradation of IL-21 receptor (IL-21R), and Grail-deficient CD8 T cells show enhanced antitumor reactivity and functionality, involving enhanced IL-21R expression and signaling.83

Non-degradative ubiquitination of ZAP70 is a mechanism that negatively regulates its activation and TCR signaling84,85 (Fig. 2). The E3 ubiquitin ligase NRDP1 conjugates K33-linked polyubiquitin chains to ZAP70, which facilitates recruitment of ubiquitin-binding tyrosine phosphatases, Sts1 (Ubash3b) and Sts2 (Ubash3a),84 known to dephosphorylate and inactive ZAP70.86 A DUB, Otud7b, deubiquitinates ZAP70 to prevent its association with Sts1 and Sts2, thereby positively regulating TCR signaling.85 NRDP1 deficiency selectively promotes CD8 T cell activation and cytokine production.84 Since CD8 T cells play a central role in tumor immunity, it will be important to examine whether targeting NRDP1 promotes antitumor immunity and can be exploited as an approach for cancer immunotherapy.

Ubiquitination also regulates signaling steps involved in activation of individual downstream pathways.5 In this regard, the NF-κB signaling pathway has been extensively studied for ubiquitin-dependent regulation. Non-degradative ubiquitination, particularly K63 ubiquitination, positively regulates TCR-stimulated activation of IKK and its downstream transcription factor NF-κB. TCR-stimulated activation of PKCθ triggers the assembly of an intermediate signaling complex, composed of the scaffold protein CARMA1 (CARD11), the adapter Bcl10, and the paracaspase MALT1; this so-called CBM complex activates an E3 ubiquitin ligase that functions together with a dimeric E2 enzyme, Ubc13/Uev1A, to catalyze conjugation of K63-linked polyubiquitin chains onto BCL10, MALT1, as well as components of IKK and its activating kinase TAK1.5,87,88 The ubiquitin chains conjugated to BCL10 and MALT1 serve as a platform that facilitate the recruitment and activation of TAK1 and IKK.5 The ubiquitin-dependent activation of IKK/NF-κB signaling is subject to tight regulation by DUBs, including CYLD and A2089,90,91 (Fig. 2). CYLD deficiency causes spontaneous IKK/NF-κB activation, associated with aberrant T cell activation and development of intestinal inflammation.89 T cell-conditional deletion of A20 sensitizes CD8 T cells for activation, and the A20-deficient CD8 T cells display increased production of effector cytokines and heightened antitumor activity.92,93 In addition to controlling NF-κB signaling, A20 also functions in a DUB-independent manner to prevent cell death induction by TNF.57

NF-κB signaling is also subject to negative regulation by K48 ubiquitination. In response to TCR/CD28 costimulation, the E3 ubiquitin ligase Peli1 catalyzes K48 ubiquitination and proteasomal degradation of an NF-κB member, c-Rel, thereby controlling T cell activation and preventing autoimmunity94 (Fig. 2). The Peli1-deficient CD4 and CD8 T cells are hyper-responsive to TCR/CD28 stimulation for production of IL-2 and IFNγ and display reduced sensitivity to inhibition by Treg cells and TGFβ.94 The Peli1-deficient mice display stronger tumor-suppressive ability compared to wildtype control mice, associated with enhanced tumor infiltration with CD8 effector T cells (unpublished data). Another E3 ligase, MDM2, negatively regulates TCR-stimulated NFAT activation through targeting NFATc2 (NFAT1) for ubiquitin-dependent degradation.95 MDM2 deletion or pharmacological inhibition promotes NFATc2 activation and IFNγ production in T cells. Interestingly, MDM2 itself is also mediated by ubiquitin-dependent degradation in activated T cells, and this process is counter-regulated by an MDM2-binding DUB, USP15. By deubiquitinating and stabilizing MDM2, USP15 facilitates the function of MDM2 in NFATc2 regulation95 (Fig. 2). Therefore, USP15 deficiency promotes NFATc2 activation, causes increased T cell responses and function in antitumor immunity.95Several other E3 ubiquitin ligases and DUBs have been shown to regulate TCR signaling.5,96

While the TCR provides a primary and antigen-specific signal for T cell activation, costimulatory signals are also required for initial activation and recall responses of T cells. CD28 is a major costimulatory receptor for naïve T cell activation, which potentiates the TCR signaling and lowers the threshold of T cell activation.65 CD28 has two ligands, CD80 (B7-1) and CD86 (B7-2), which are expressed on activated APCs particularly DCs. Both CD28 and B7 form a large family of structurally related proteins with costimulatory or coinhibitory functions.97 CTLA4 and PD1 are members of the CD28 family that serve as key coinhibitory receptors and are also known as immune checkpoints.2 Major breakthrough in cancer immunotherapy came from the successful treatment of advanced cancer patients with the ICIs anti-CTLA4 and anti-PD-1.67 Recent studies have also provided new insight into the mechanisms that regulate the fate and function of these immunosuppressive receptors. In particular, an E3 ubiquitin ligase, Fbxo38, has been reported to interact with PD1 and mediate its K48 polyubiquitination and subsequent proteasomal degradation.98 T cell-conditional knockout of Fbxo38 results in increased surface expression of PD1 in T cells and impairs antitumor immunity, leading to more rapid tumor growth98 (Fig. 2). Another E3 ligase, c-Cbl, has been shown to interact with the cytoplasmic tail of PD1 and mediate ubiquitin-dependent PD1 degradation in the tumor microenvironment.99 Ubiquitination and deubiquitination also regulate the stability of PD1 ligand 1 (PD-L1) in tumor cells, which contributes to the regulation of T cell function and antitumor immunity.100,101

Another family of T cell costimulatory/coinhibitory receptors includes members of the TNFR superfamily, many of which are induced along with T cell activation and play a particularly important role in regulating effector and memory T cell responses.102,103,104 Among the costimulatory receptors of the TNFR superfamily are OX40 (TNFRSF4), 4-1BB (CD137, TNFRSF9), GITR (CD357, TNFRSF18), CD27 (TNFRSF7), and TNFR2 (TNFRSF). Engagement of these receptors by their ligands from the TNF superfamily enhances effector T cell generation and functionality and facilitates memory development and recall responses. Most, if not all, of the TNFR family of costimulatory receptors also trigger the activation of noncanonical NF-κB pathway, which is integral for the effector and memory T cell responses.105 Agonistic monoclonal antibodies for these receptors, particularly OX40 and 4-1BB, promote antitumor T cell responses either alone or in combination with other cancer immunotherapy strategies, such as immune checkpoint blockade, cytokine therapy, and radiation therapy.106,107 In addition to extensive preclinical studies, several early phase cancer immunotherapy clinical trials are being conducted to test the efficacy of anti-OX40 and anti-4-1BB in combination therapies.106,107

Signal transduction of the TNFR costimulatory receptors involves recruitment of TNFR-associated factors (TRAFs), which in turn target activation of NF-κB and MAPK pathways.103 A hallmark of TRAF-mediated signaling is the critical involvement of protein ubiquitination, particularly the non-degradative K63 and linear ubiquitination.108 Two functionally redundant E3 ubiquitin ligases, cIAP1 and cIAP2, catalyze K63 ubiquitination in the TNFR signaling complex, which cooperates with the linear ubiquitin ligase LUBAC to mediate canonical NF-κB activation and prevent cell death induction.57 The role of K63 ubiquitination in mediating T cell costimulation by TNFRs has been well demonstrated in the 4-1BB pathway. T cell co-stimulation with a 4-1BB agonist antibody triggers K63 ubiquitination of 4-1BB-associated TRAF2, which is crucial for NF-κB activation and induction of antitumor immunity.109Conversely, the 4-1BB signaling is negatively regulated by two DUBs, CYLD and A20, which physically associate with the 4-1BB/TRAF2 complex.110 Overexpression of either CYLD or A20 inhibits NF-κB activation by the 4-1BB agonist antibody, whereas silencing these DUBs enhances NF-κB activation mediated by 4-1BB costimulation in human CD8 T cells.110 The role of K63 ubiquitination in 4-1BB-mediated CD8 T cell costimulation is further supported by the finding that CD8 effector and memory T cells expressing a dominant-negative mutant of the E3 ligase cIAP2 (cIAP2H570A) have attenuated 4-1BB signaling and impaired survival, demonstrated using a viral infection model.111

The expression or signaling function of TNFRs is also negatively regulated by degradative ubiquitination. The E3 ligase NEDD4 mediates ubiquitination and degradation of GITR and negatively regulates T cell-mediated antitumor immunity.112 Roquin 1 (Rc3h1) and its paralog Roquin 2 (Rc3h2), E3 ubiquitin ligases and RNA binding proteins, repress the expression of OX40, as well as the CD28 family costimulatory receptor ICOS, by promoting degradation of their mRNAs.113,114,115 Roquins play a crucial role in regulating differentiation of Tfh cells and pathogenesis of autoimmune and inflammatory diseases, but it is unclear whether they also play a role in regulating antitumor immunity.116 Ubiquitination also negatively regulates the noncanonical NF-κB pathway. In contrast to its K63-specific E3 function in facilitating TNFR-mediated canonical NF-κB activation, cIAP (cIAP1 or cIAP2) negatively regulates noncanonical NF-κB pathway by catalyzing K48 ubiquitination and degradation of the noncanonical NF-κB-inducing kinase NIK.105 This function of cIAP also requires TRAF2 and TRAF3, which function in the assembly of the cIAP-TRAF2-TRAF3 complex and recruitment of NIK. TNFR-stimulated noncanonical NF-κB activation involves ubiquitin-dependent degradation of TRAF3 or TRAF2.105 Importantly, pharmacological inhibition of cIAPs or genetic ablation of TRAF2 or TRAF3 causes constitutive activation of noncanonical NF-κB.117 Consistently, small molecule inhibitors of cIAPs (or smac mimetics) promote T cell activation and effector function and augment antitumor immunity.118,119,120 These studies implicate cIAP inhibitors as new therapeutic agents in combinatorial cancer immunotherapy.

Noncanonical NF-κB activation by T cell costimulation via TNFRs also plays a role in CD4 T cell differentiation, as demonstrated for the induction of Th9 cells by OX40 costimulation.121 Th9 cells form a subset of CD4 effector cells characterized by production of the cytokine IL-9. Although Th9 cells have been extensively studied for their involvement in autoimmunity and inflammation, recent evidence suggests that they also serve as a major subset of CD4 effector T cells mediating antitumor immunity.122,123,124,125 OX40 ligation potently induces the expression of TRAF6, which is required for noncanonical NF-κB activation and Th9 cell generation.121 Since TRAF6 is a K63-specific E3 ligase, it is possible that TRAF6 may activate cIAP via K63 ubiquitination, which allows cIAP to mediate K48 polyubiquitination and degradation of TRAF2 or TRAF3 leading to NIK stabilization and noncanonical NF-κB activation. Ubiquitination also regulates the differentiation of antitumor Th9 cells via autophagy, a catabolic mechanism that removes unnecessary and dysfunctional cellular components.126 PU.1, an important transcription factor involved in Th9 cell differentiation, undergoes K63 ubiquitination and degradation through selective autophagy. Selective autophagy employs specific cargo adapters to target proteins, protein complexes, or organelles for degradation.127 One major cargo adapter of selective autophagy is p62 (also called SQSTM1), which targets ubiquitinated cargos for autophagic degradation.127 Silencing of p62 or genetic deletion of major autophagy components in CD4 T cells enhances PU.1 expression and promotes Th9 differentiation and antitumor immunity in different mouse cancer models.126

In addition to the TCR and costimulatory signals, cytokines play an important role in regulating the nature, magnitude, and persistence of T cell responses. In particular, the common gamma chain (γc or CD132) family of cytokines, especially IL-2, IL-7, IL-15, and IL-21, have been actively explored for improving the efficacy of cancer immunotherapies.128 The receptors of these cytokines share the γc subunit that forms dimeric or trimeric receptor complexes mediating signaling that promotes the survival, proliferation, effector/memory generation and maintenance of T cells.128,129 Among the γc family cytokines, IL-15 is unique in that it requires a transpresentation mechanism to engage its receptor for signal stimulation.130 IL-15 receptor (IL-15R) is composed of three submits: IL-15Rα, IL-15Rβ (also called IL-2Rβ or CD122), and γc. When produced in supporting cells, such as monocytes and dendritic cells, IL-15 forms a complex with IL-15Rα on the cell surface and is presented to the IL-15Rβγ heterodimer on target cells, particularly CD8 T cells and NK cells, to trigger signaling.130 Major signaling events stimulated by IL-15 include activation of the transcription factors STAT5 and the PI3K/AKT survival pathway.131 Exogenously administered IL-15 sensitizes the TCR for antigen stimulation, promotes CD8 T cell proliferation and effector function, and also rescues tolerant CD8 T cells in cancer immunotherapy.132,133,134 Recombinant IL-15, particularly IL-15 superagonists (IL-15/IL-15Rα complexes), have shown promise in preclinical experiments and clinical trials of cancer immunotherapies when used in combination with other agents, such as ICIs.135

The role of ubiquitination in regulating signal transduction stimulated by the γc cytokines is poorly studied. Nevertheless, a recent study demonstrated a ubiquitin-dependent mechanism that mediates IL-15-stimulated activation of AKT signaling and CD8 T cell responses in antitumor immunity.136 AKT activation is known to be mediated by PI3K, which catalyzes the conversion of the membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), the latter of which recruits AKT to the membrane compartment for activation by upstream kinases, PDK1 and mTORC2. The PI3K/AKT pathway integrates the TCR/CD28 and IL-15R signals and is negatively regulated by the DUB Otub1 (Fig. 2). IL-15 not only activates PI3K but also induces K63 ubiquitination of AKT, and the ubiquitination enables AKT to bind PIP3 for membrane translocation and activation136 (Fig. 3a). Notably, AKT ubiquitination occurs in its pleckstrin homology (PH) domain, which is known to mediate PIP3 binding. Since inactive AKT exists in a closed conformation involving intramolecular interaction between its kinase domain and PH domain,137 it is likely that ubiquitination causes AKT conformational changes leading to exposure of the PH domain for PIP3 binding (Fig. 3a). IL-15-stimulated AKT activation is negatively regulated by Otub1, which physically interacts with AKT and prevents its K63 ubiquitination and membrane translocation.136 Interestingly, in IL-15-exposed cells, Otub1 is relocated to the membrane compartment, a mechanism that enables Otub1 to inhibit AKT ubiquitination and activation induced by both IL-15 and TCR signals (Fig. 3b). Otub1 deficiency greatly enhances antigen-specific CD8 T cell responses and promotes their metabolic reprogramming and effector function in antitumor immunity.136 These studies suggest that IL-15 primes CD8 T cells for antigen-specific responses, which is controlled by a checkpoint, Otub1 (Fig. 3b). The potential for targeting Otub1 in cancer immunotherapy is demonstrated by the finding that inducible deletion of Otub1 in tumor-bearing mice profoundly enhances tumor rejection through unleashing the activity of CD8 T cells as well as NK cells.136 Furthermore, analysis of human skin cutaneous melanoma database reveals a remarkable inverse correlation between Otub1 expression levels and the abundance of CD8 effector T cell gene signature and patient survival.136

Otub1 serves as a checkpoint of IL-15-mediated CD8 T cell priming by deubiquitination of AKT. a, IL-15 stimulates PI3K activation and AKT K63 ubiquitination, both being required for AKT activation. AKT ubiquitination facilitates its binding to PIP3, probably through a conformational change leading to exposure of the PH domain of AKT, which recruits AKT to the membrane compartment, where AKT is phosphorylated and activated by the kinases PDK1 and mTORC2. Otub1 negatively regulates AKT activation through inhibiting its ubiquitination. b, IL-15 signal primes CD8 T cells for antigen stimulation, which is controlled by Otub1. In response to IL-15 stimulation, Otub1 is recruited to the membrane compartment, where it inhibits AKT activation induced by both IL-15 and TCR/CD28 signals. Otub1 thus functions as a checkpoint molecule regulating IL-15-mediated CD8 T cell priming. Deficiency in Otub1 causes increased AKT signaling and enhanced CD8 T cell responses in cancer immunity

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Treg cells are a subset of CD4 T cells that express the lineage transcription factor Foxp3 and have potent immunosuppressive functions.138 The majority of Treg cells are developed in the thymus (thymus-derived Treg or tTreg), but Treg cells can also be generated in the periphery (pTreg) or induced in vitro from naïve CD4 T cells (iTreg) cells.138 While Treg cells maintain peripheral immune tolerance to prevent the development of autoimmunity, they also serve as a major cell population that suppresses antitumor immunity in the tumor microenvironment.139 Treg depletion has been shown to promote antitumor immune responses in preclinical studies, and additional Treg-targeting methods have been actively explored for improving the efficacy of cancer immunotherapy.139 Rapid progresses have also been made in understanding of the molecular basis of Treg regulation, providing insight for further improving therapeutic approaches. Ubiquitination has been established as a crucial mechanism that regulates the development and immunosuppressive function of Treg cells.5,96

A role for nondegradative ubiquitination in regulating Treg development was initially suggested by the finding that mice deficient in CYLD, a DUB cleaving K63 and linear ubiquitin chains, have profoundly increased frequencies of thymic and peripheral Treg cells.140,141Similar results were obtained with mice expressing a nonfunctional CYLD splice variant, CYLD(ex7/8).142 This function of CYLD involves, in part, inhibition of ubiquitin-dependent NF-κB signaling,140 which is known to promote tTreg development.143 In addition, CYLD regulates signaling from IL-2R and TGFβR known to be crucial for Treg generation.140,141 A20, another DUB that controls NF-κB signaling via inhibition of K63 ubiquitination, also regulates thymic development of tTreg cells.144 Nondegradative ubiquitination also regulates the function of transcription factors involved in induction of Foxp3 expression. One example is ubiquitination of TGFβ-inducible early gene 1 (TIEG1), a Foxp3 transactivator involved in iTreg cell development.145 Itch-mediated monoubiquitination of TIEG1 appears to promote its transcriptional activity in Foxp3 gene induction.

K63 ubiquitination plays a critical role in mediating the stability and immunosuppressive function of established tTreg cells. Treg-specific deletion of a K63-specific E2 ubiquitin-conjugating enzyme, Ubc13, impairs in vivo immunosuppressive function of Treg cells and causes aberrant T cell activation and autoimmune symptoms.146 The Ubc13-deficient Treg cells are sensitized for acquiring Th1- and Th17-like inflammatory T cell phenotypes under lymphopenic and inflammatory conditions. The function of Ubc13 in Treg cells is mediated through ubiquitin-dependent activation of IKK and involves induction of SOCS1, a pivotal suppressor of proinflammatory cytokine receptor signaling.146 TRAF3, a potential K63 ubiquitin ligase, is also involved in the regulation of Treg function. Treg-specific TRAF3 deletion in mice partially impairs the immunosuppressive function of Treg cells, causing an increase in the frequency of Th1-like effector T cells under homeostatic conditions. TRAF3 is also important for antigen-stimulated generation of follicular Treg cells, a subset of Treg cells that functions in germinal centers to suppress Tfh and B cells and, thereby, inhibit antibody responses.147 TRAF3 is required for ERK MAPK activation, although it is unclear whether this function involves its K63 E3 ligase activity. Another TRAF family member, TRAF6, also plays an important role in regulating Treg function.148 TRAF6 conjugates K63 ubiquitin chains to Foxp3, which mediates proper localization of Foxp3, thereby facilitating its transcriptional function in Treg cells.148Importantly, Treg-conditional TRAF6 knockout mice display increased antitumor immunity, although it remains to be examined whether deletion of Ubc13 or TRAF3 in Treg cells also promotes antitumor immunity.

In addition to K63 ubiquitination, linear ubiquitination regulates signal transduction by nondegradative mechanisms.149 Linear ubiquitination is specifically catalyzed by linear ubiquitin chain assembly complex (LUBAC), a multi-subunit E3 composed of HOIL-1L (also called RBCK1), HOIP (also called RNF31), and SHARPIN. A role for linear ubiquitination in regulating Treg function is suggested by a study employing Treg-conditional RNF31 knockout mice.150 Treg-specific deletion of RNF31 causes massive loss of Treg cells and development of severe autoimmune and inflammatory pathology. Despite the demonstrated function of LUBAC in preventing TNF-induced apoptosis and necroptosis,149 the massive loss of Treg cells in Treg-conditional RNF31 knockout mice cannot be rescued by antibody-mediated TNF neutralization, suggesting a yet-to-be defined mechanism in RNF31-mediated Treg maintenance.150 A more recent study shows that RNF31 is also required for maintaining human Treg cell stability and function.151 Treg cells with RNF31 knockdown acquires a Th1-like phenotype, characterized by IFNγ production. RNF31 interacts with and stabilizes Foxp3 and catalyzes conjugation of multi-monoubiquitination chains onto Foxp3, suggesting an atypical function of RNF31.151 It remains to be investigated how monoubiquitination stabilizes Foxp3 and how LUBAC-mediated linear ubiquitination regulates Treg cell survival and function.