The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and gastrointestinal tract. To mount an effective response against these pathogens, the immune system must recruit the right cells with effector responses that are appropriate for the task at hand. Several types of CD4+ T cells can be recruited, including Th cells (Th1, Th2, and Th17), T follicular helper cells, and regulatory T cells. These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. Because these cells differentiate from a common progenitor, the composition of their intracellular milieu of proteins changes to appropriately guide their effector function. One underappreciated process that impacts the levels and functions of effector fate-determining factors is ubiquitylation. This review details our current understanding of how ubiquitylation regulates CD4+ T cell effector identity and function.
Ubiquitylation is the posttranslational addition of ubiquitin to a substrate protein. Ubiquitylation of a substrate requires the sequential action of three classes of enzymes: the E1 or ubiquitin-activating enzyme, the E2 or ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase. E1s activate ubiquitin by the formation of a thiol ester with the carboxyl group of glycine 76 on ubiquitin (1). The ubiquitin is transferred to a catalytic cysteine on an E2 (2), which associates with an E3 ubiquitin ligase that is in a complex with a substrate. The E3 may serve as a scaffold to facilitate the transfer of ubiquitin from the E2 to the substrate, as is the case for really interesting new gene (RING)-type E3s (3, 4). Alternatively, homologous to E6AP C terminus (HECT) (5) and the RING-between-RING–type E3s (6, 7) receive the ubiquitin onto a catalytic cysteine residue before transferring it to a lysine on the substrate. Thus, E3 ligases can identify the substrate, as well as dictate the formation of ubiquitin linkages, driving the monoubiquitylation, multimonoubiquitylation, or polyubiquitylation of the substrate. Ubiquitin has seven accessible lysines on its surface (K6, K11, K27, K29, K33, K48, and K63), each of which can be points of attachment for ubiquitin chains (8–11). Furthermore, the amino terminal methionine of ubiquitin can also serve as a point of attachment for linear chains in a reaction catalyzed by an E3 ubiquitin ligase complex known as the linear ubiquitination assembly complex (12). Ubiquitin chains can alter the fate of a substrate by changing its intracellular location, promoting its interactions with other proteins, or driving degradation. Ubiquitylation of protein substrates may be modified or reversed by enzymes called deubiquitinating enzymes (DUBs). The ∼100 DUBs encoded by the human genome can be divided into five main families based on their structural domains: ubiquitin-specific proteases (USPs), ubiquitin carboxyl-terminal hydrolases, ovarian tumor–related proteases (OTUs), Machado–Joseph disease domain proteases, and the JAB1/MPN/Mov34 metalloproteases (13). Given the capacity of ubiquitylation to alter protein levels and function, it is not surprising that ubiquitylation has the potential to influence the identity and function of CD4+ T cells.
When a naive T cell encounters a peptide displayed on MHC (pMHC), the naive T cell can get primed to become an effector T cell (14, 15). The sequence of events that leads to the formation of an effector requires that the T cell receive three signals: pMHC, costimulation, and cytokine (16, 17). Ubiquitylation can influence each of these signals within a T cell by altering the levels and functions of signaling intermediates or by influencing transcription factors that drive CD4+ T cell identity and function. Many E3 ligases have known roles in regulating T cell activation and costimulation. These include the RING E3 ligases, Casitas B-lineage lymphoma b (Cbl-b) (18–22), TRAC-1 (23), and Peli1 (24), and the HECT E3 ligases, Itch and Nedd4 (25–29) (reviewed in Refs. 30–33). This review explores how ubiquitylation impacts the identity and lineage stability of CD4+ Th/effector subsets.
Th1 cells are important for the clearance of intracellular pathogens. They differentiate from naive CD4+ T cells in response to pMHC and costimulation in a cytokine milieu containing IL-12 and IFN-γ. IL-12 signaling induces the phosphorylation of Jak2 and Tyk2, leading to STAT4 activation, which, in turn, drives IFN-γ production (34). In an amplification loop, IFN-γR signaling through STAT1 induces expression of the transcription factor T-box expressed in T cells (T-bet) (35). T-bet transactivates the IFN-γ gene to drive further IFN-γ cytokine production (35) and increases expression of IL-12Rβ2 to promote more IL-12 responsiveness (36). Thus, STAT1, STAT4, and T-bet help to promote Th1 cell identity.
STAT1, STAT4, and T-bet are degraded via ubiquitin-mediated networks. An E3 ligase enzyme called STAT-interacting LIM (SLIM) protein (also known as PDLIM2 or mystique) drives rapid nuclear degradation of STAT1 and STAT4 in response to IFN-α or IL-12 signaling (37). SLIM was shown to aid in polyubiquitylation of STAT4 in vivo and in vitro (37), as well as STAT1 in vitro (38). The CD4+ T cells in SLIM−/− mice make increased IFN-γ upon in vivo challenge with heat-killed Listeria monocytogenes (37). However, it is unclear whether the increased IFN-γ production by these cells offers enhanced resistance to pathogen or rather, enhanced, immunopathology. Another E3 ligase, Smurf1, was shown to mediate K48 polyubiquitylation and degradation of STAT1 in transformed cell lines; however, it remains to be shown whether this happens in T cells as well (39). STAT1 ubiquitylation is reversed by the DUB USP13 (40). Furthermore, although T-bet can undergo ubiquitin-mediated degradation, the identity of the E3 ligase that drives this degradation is unknown (41). However, one DUB, USP10, is known to reverse this ubiquitylation and stabilize T-bet (42). Because the major driver of Th1 identity, T-bet, is regulated by ubiquitylation, it raises the question of how else ubiquitylation influences epigenetic modifications and the stability of proteins that result in the decision to be a Th1 cell.
Th2 cells mediate immunity against extracellular microbes, such as worms, and facilitate clearance of allergens and toxins. Th2 cells differentiate from naive CD4+ T cells in response to pMHC and costimulation in the presence of the cytokine IL-4. Th2 cells may secrete a variety of cytokines, including IL-4, IL-5, and IL-13. IL-4 drives Th2 cell generation in a positive feed-forward loop (43). IL-4 binds to its receptor, resulting in the phosphorylation and activation of STAT6, which translocates to the nucleus and drives transcription of GATA3. GATA3 drives Th2 cell identity via Notch-dependent and Notch-independent mechanisms (44–47). Although IL-4 and STAT6 may be dispensable for in vivo generation of Th2 cells (46–48), GATA3 is required for the generation of Th2 cells (44).
GATA3 and STAT6 are regulated by ubiquitylation, either directly or indirectly. The catalytic ubiquitin ligase, Itch, regulates Th2 cell differentiation and identity by regulating IL-4 production. As with most catalytic E3 ligases, Itch enzymatic activity is restrained by a closed conformational state known as autoinhibition (49). Upon T cell activation, a small membrane-bound adaptor known as Nedd4 family-interacting protein 1 (Ndfip1) is expressed, which activates Itch, allowing it to polyubiquitylate targets, including JunB (25, 50) This results in JunB degradation and prevents its localization to the nucleus where it would otherwise pair with c-Maf to drive IL-4 transcription (26, 51, 52). In mice lacking Ndfip1 or Itch, CD4+ T cells accumulate high levels of JunB and produce excessive quantities of IL-4, resulting in a preponderance of Th2 cells (25, 26). Supporting this model, transgenic mice that overexpress JunB to levels found in Th2 cells show a specific increase in Th2 cytokines, such as IL-4 and IL-5 (51).
STAT 6 levels are regulated by two E3 ligases: gene related to anergy in lymphocytes (GRAIL) and Cbl-b. GRAIL drives polyubiquitylation and degradation of STAT6 and, therefore, limits the generation of Th2 cells. GRAIL is highly expressed in Th2 cells, and its knockdown results in an increase in IL-4, IL-5, and IL-13 from T cells. GRAIL-knockout animals are highly susceptible to allergic inflammation, and their naive CD4+ T cells fail to appropriately degrade STAT6 after in vitro TCR stimulation (53). Cbl-b also drives STAT6 polyubiquitylation and degradation (54). Similar to Grail−/− animals, Cbl-b−/− animals are highly susceptible to induced allergic inflammation due to a Th2 and Th9 bias in their T cells.
The E3 ligase, murine double minute 2 (Mdm2), drives GATA3 polyubiquitylation. Mdm2 is well known for its role in ubiquitin-driven degradation of the tumor suppressor p53 (55, 56), but it has several other substrates, including NFAT2c (57, 58) and GATA3 (59). Upon TCR stimulation, activation of the ERK-MAPK pathway leads to the association of Mdm2 with GATA3 and consequent polyubiquitylation and proteasomal degradation of GATA3 (59). A DUB named USP15 deubiquitinates Mdm2 and prevents its proteasomal degradation (57). Therefore, ubiquitylation of GATA3 and STAT6, as well as of factors that regulate IL-4 production, contribute to the decision of a CD4+ naive cell to become a Th2 cell.
Th17 cells are important for the clearance of extracellular pathogens, such as fungi. Th17 cells differentiate in the presence of a cytokine milieu containing TGF-β and IL-6 (60–62). Other cytokines, such as IL-1β and IL-23, drive differentiation and maintenance of Th17 cells and may even drive Th17 cells in the absence of TGF-β (63). STAT3 is induced downstream of IL-6, IL-21, and IL-23 and cooperates with IRF4 (induced downstream of IL-1β) to elevate levels of RAR-related orphan receptor γ T (RORγT) (64, 65). RORγT and RAR-related orphan receptor α are sufficient to drive Th17 differentiation (66, 67).
Several enzymes in the ubiquitin-conjugation pathway can influence the differentiation or maintenance of Th17 cells. For example, mice lacking Ndfip1 and Itch, which, as discussed in the previous section, have increased frequencies of Th2 cells, also have increased frequencies of Th17 cells. Although this may be due to cell-extrinsic effects of IL-4–mediated inflammation driving increased proinflammatory cytokines and tissue damage (68), there may also be more direct roles for these factors in Th17 generation or function. Similarly, mice lacking a RING E3 ligase, Ro52/TRIM21, show increased production of inflammatory cytokines, such as IL-23, IL-6, and IL-21, and these mice have increased frequencies of Th17 cells (69). Crossing Ro52−/− mice to IL-23p19−/− mice abrogates tissue damage and excessive cytokine production and lowers the frequencies of Th17 cells, indicating that the increase in Th17 cells is indeed driven by the dysregulated IL-23/IL-17 axis. Ro52 acts downstream of IFN (predominantly type II) signaling to polyubiquitylate IRF3, IRF5, and IRF8 and to target them for degradation. This limits the production of cytokines, including IL-6 and IL-23, which would otherwise drive Th17 generation. A third example of an E3 ubiquitin ligase that influences the Th17 cell fate choice is SLIM. As described above, SLIM can drive degradation of STAT4 in Th1 cells but can also limit Th17 differentiation by promoting the proteasomal degradation of STAT3 (70, 71). Supporting this, SLIM-deficient animals show increased frequencies of Th17 and Th1 cells and are very susceptible to experimental autoimmune encephalitis, a mouse model of multiple sclerosis (71).
Several DUBs are important for Th17 differentiation. USP4 regulates Th17 cells in two ways. First, USP4 deubiquitylates Ro52 in transformed cell lines (72) and, thus, limits the expression of cytokines needed for Th17 generation (69). Second, it directly interacts with RORγT in primary human Th17 cells and deubiquitylates RORγT in transformed cell lines (73). USP17 (also known as DUB-3) is a second DUB that maintains RORγT stability (74). Knockdown of USP17 in primary Th17 cells results in a decrease in endogenous RORγT levels (74). Because IL-4 and IL-6 signaling can induce USP17 (75), it appears that, in response to IL-6 signaling, two responses occur: RORγT levels are increased downstream of STAT3, and RORγT protein is stabilized downstream of USP17. How USP17 influences the timing and integration of these two events downstream of IL-6 signaling needs further evaluation in primary CD4+ T cells.
A third DUB involved in Th17 differentiation is DUBA (also known as OTUD5), an OTU family DUB. DUBA facilitates the degradation of RORγT by removing a regulator of RORγT degradation known as UBR5 (76). UBR5 polyubiquitylates and targets RORγT for proteasomal degradation. UBR5 itself is regulated via poylubiquitylation. When DUBA deubiquitylates UBR5 and rescues it from degradation, UBR5 is free to polyubiquitylate RORγT, driving its degradation and consequently limiting Th17 differentiation. Therefore, T cell–specific loss of DUBA results in reduced levels of UBR5, stabilization of RORγT, and increased IL-17A production in response to TCR stimulation (76).
It is becoming clear that Th17 cells are heterogeneous; some are more pathogenic than others (63, 77, 78). GM-CSF secretion, for example, which occurs downstream of IL-23 signaling, marks pathogenic Th17 cells that cause experimental autoimmune encephalitis (79–82). Further work needs to be done to determine the extent to which ubiquitylation that occurs downstream of cytokines, such as TGF-β or IL-23, or downstream of TCR signaling, may influence the pathogenic potential of a differentiating Th17 cell, as well as its ultimate function.
T follicular helper cells.
T follicular helper (Tfh) cells are CD4+ T cells that provide costimulatory help to B cells in germinal centers to enable B cell functions (83–85). Tfh cells exist in an interdependent relationship with B cells, wherein B cells are required for appropriate Tfh differentiation and function, and, reciprocally, Tfh cells promote the generation of high-affinity Ab-producing B cells. Tfh cells differentiate from naive precursors in response to pMHC interactions and under the influence of IL-6, ICOS, and IL-21 (86–90). Tfh cells can secrete cytokines, such as IL-4 and IL-21, as well as chemokines, such as CXCL13 (reviewed in Ref. 91). The differentiation of a naive CD4+ T cell into a Tfh cell occurs in a step-wise fashion, which requires the transcription factor achaete-scute homolog 2 to generate a BCl6lo CXCR5+ Tfh-intermediate cell. This intermediate undergoes maturation and complete differentiation in the presence of Bcl6 to generate a Bcl6hi CXCRhi Tfh cell (92). Thus, Bcl6 is essential for Tfh identity (87, 93, 94).
Several E3 ligases affect Tfh differentiation and function by regulating Bcl6 directly or indirectly. Four of these are RING-type E3 ubiquitin ligases. First, Bcl6 may be repressed by interaction with Cul3 E3 ligase, a ligase that is known, among other things, to ubiquitinate histone proteins. In thymocytes, complexes of Cul3 and Bcl6 directly bind and lay down repressive epigenetic marks on two genes important for Tfh identity: Batf and Bcl6 (95). Intriguingly, this repression is epigenetically carried over into the periphery when the T cells encounter Ag. Therefore, in mice that lack Cul3 in T cells, Tfh cells are increased in secondary lymphoid tissues, and these cells drive germinal center B cell expansion (96). Exactly how the Cul3 RING ligase complex represses Bcl6 expression remains unresolved. Does the complex directly ubiquitylate Bcl6, leading to its degradation? Or does the complex regulate Bcl6 indirectly by ubiquitylating other proteins, such as histone modifiers, which are associated with Bcl6? Second, the E3 ligase Roquin reduces expression of ICOS, upstream of Bcl6, to limit Tfh differentiation. Roquin is a RING E3 ligase with an RNA-binding domain that binds and silences target genes, including ICOS mRNA. Supporting this model, mice that bear a mutation in the gene encoding Roquin (also called sanroque mice) have increased ICOS levels in naive and activated T cells. This leads to a T cell–intrinsic increase in Tfh differentiation, large numbers of germinal centers, and increased serum Abs of various IgG isotypes, among other defects (97). Bcl6 is regulated by two other E3 ligases in diffuse large B cell lymphomas (98, 99). Pellino1 E3 ligase (PELI1) directs K63 (nondegradative) chains on Bcl6, leading to Bcl6 stabilization in transgenic mice overexpressing human PELI1 (98). Furthermore, in these lymphoma cell lines, another E3 ligase, FBXO11, which normally marks Bcl6 for degradation, undergoes loss-of-function mutations (99). Follow-up of these observations in primary T cells will be crucial in determining whether PELI1 and FBXO11 have roles in regulating Bcl6 and, consequently, in Tfh differentiation in vivo.
The HECT-type E3 ligase Itch also regulates the differentiation of Tfh cells. Mice lacking Itch globally or only in their T cells are unable to generate Tfh cells following infection with vaccinia virus (100). In Itch-deficient T cells, Foxo1 is not appropriately degraded, and Tfh development can be rescued in Itch-deficient animals by knockdown of Foxo1 or by forced expression of Bcl6, suggesting that the defect in Itch-deficient animals is upstream of Bcl6 (100). ICOS signaling converges on this pathway by transiently inactivating Foxo1 to relieve Foxo1 repression of Bcl6 and to allow Tfh differentiation downstream of short-term ICOS signals (101). Surprisingly, complete knockout of Foxo1 in T cells prevents the formation of germinal centers or Tfh cells (101). This suggests that Foxo1-mediated regulation of germinal centers and Tfh cells is complex, differentiation stage dependent, and must be intricately regulated by precise timing of the expression of proteins, such as Foxo1. Given that Foxo1 is important for regulatory T cell (Treg) generation and function (102), and Itch-deficient mice have decreased Tfh cells, increased Foxo1 levels, and defective Treg numbers and function (100, 103), it will be important to determine whether Foxo1 levels are changed in Itch−/− Tregs and to what extent the Tfh defects in Itch−/− mice are related to the Treg dysfunction. Further work will be needed to explore the role of ubiquitin-mediated pathways in other proteins that contribute to Tfh identity.
Tregs are a subset of CD4+ T cells that are capable of suppressing the actions and functions of other immune cell types (104, 105). Several distinct subsets of Tregs have been described, including Foxp3− Tr1 cells (type 1 Tregs), Foxp3+ Th3 cells, and Foxp3+ thymic-derived Tregs. Foxp3+ Tregs develop in the thymus, when TCR/CD28 and IL-2 signaling drives expression of the transcription factor Foxp3 (106). Foxp3 is central to the function of this major subset of Tregs, and mutations in foxp3 in mice and men leads to nonfunctional Tregs and autoimmunity (107). Foxp3+ Tregs may also be induced in the periphery through the action of TGF-β and IL-2. TGF-β signals through the complementary proteins Smad2 and Smad3 to drive Foxp3 transcription (108). IL-2 binding to the IL-2R complex leads to JAK1 and JAK3 recruitment and eventual recruitment and activation of STAT5, which, in turn, drives expression of Foxp3 (109, 110).
Two E3 ligases can regulate Foxp3 stability: Stub1 and Cbl-b. Stub1 (also known as CHIP or C terminus of Hsc70-interacting protein) interacts with Hsp70 and Foxp3 to drive K48-linked polyubiquitylation of Foxp3 (111). Exposure of the Jurkat T cell line to inflammatory cues, such as LPS and IL-1β, drives the translocation of Stub1 into the nucleus, where it interacts with Hsp70, and drives the ubiquitin-mediated proteasomal degradation of Foxp3. Overexpression of Stub1 in Tregs and the subsequent cotransfer of these cells with naive T cells into a lymphoreplete host resulted in loss of Foxp3 in the Tregs and subsequent conversion of these cells into IFNγ+ Th1-like effector cells.
Cbl-b was shown to ubiquitylate Foxp3 by working together with Stub1. Specifically, Cbl-b binds ubiquitylated Foxp3 downstream of TCR/CD28 signaling and recruits it to Stub1, allowing additional ubiquitylation of Foxp3 and increased proteasomal degradation (112).
E3 ligases may also influence Tregs by affecting their function. One such example is the regulation of Treg function by the E3 ligase von Hippel–Lindau (VHL) (113). Under normal oxygen levels (normoxia), an oxygen sensor named hypoxia-inducible factor α (HIF1α) is hydroxylated, recognized, and polyubiquitylated by VHL. This results in proteasomal degradation of HIF1α. Under conditions of low oxygen stress (hypoxia), HIF1α is not degraded by VHL and is free to drive the transcription of genes necessary for surviving hypoxia. HIF1α also serves as a switch between Th17 and Treg fates (114, 115). HIF1α can directly bind RORγT and p300 to drive transcription of IL-17A and promote Th17 identity (114) while simultaneously binding and targeting Foxp3 for degradation to repress Treg differentiation (114, 115). Interestingly, mice lacking VHL only in their Tregs develop a disease characterized by large numbers of IFNγ+ Tregs that infiltrate tissue and fail to suppress conventional T cells or prevent colitis (113). In VHL-deficient Tregs, stabilized HIF1α drives the IFN-γ promoter, resulting in large amounts of secreted IFN-γ and poor in vivo function of these Tregs (113).
The HECT-type E3 ubiquitin ligase Itch also regulates Treg differentiation and function. Mice encoding a Treg-specific deletion of the E3 ligase Itch develop a Th2-mediated disease characterized by infiltration of activated T cells into mucosal sites and show particularly severe inflammation in the airways (103). The inability of Itch-deficient Tregs to suppress Th2-mediated inflammation supports other published studies showing that mice that lack Ndfip1, an important adaptor and activator of Itch, express an inactive form of Itch that fails to degrade JunB and limit IL-4 production (25). Thus, T cells from mice that lack Ndfip1 are defective in induced Treg generation as a result of high IL-4 production (116). Two other E3 ligases, Smurf2 (117) and β-TrCP (FBXW1) (118), regulate Treg function indirectly by mediating ubiquitylation and degradation of EZH2 in neurons and transformed cells, respectively. EZH2 protein forms part of the polycomb repressive complex 2 (PRC2) that trimethylates histone H3 to repress gene transcription. EZH2-deficient Tregs are unable to suppress inflammation in vivo and show an increased ability to lose Foxp3 expression (119). Whether these E3 ligases stabilize EZH2 and reinforce Treg identity in primary T cells will be interesting to explore in future studies.
Relatively little is known about the role of DUBs in Treg differentiation. The USP family member cylindromatosis (CYLD) plays a role in the generation of Tregs. In response to TGF-β signaling, K63-linked polyubiquitylation of Smad7 results in stabilized Smad7, which activates TAK1, increases binding of AP-1 to the Foxp3 promoter, and increases Foxp3 transcription (120). CYLD opposes this process by removing K63 chains on Smad7. In CYLD−/− T cells, unopposed K63 ubiquitination upon TGF-β signaling stabilizes Smad7 and drives increased differentiation of Tregs (120). These examples show that, in addition to regulating Treg abundance, ubiquitin enzymes can regulate Treg differentiation and function.
Guiding CD4+ T cell effector fate and lineage identity.
Maintaining CD4+ Th cell identity is important for proper immune function. The flexibility to transition from an initial CD4+ T effector cell into a more relevant effector cell may be important for quickly tailoring the immune response as infection progresses. However, recent work suggests that CD4+ effector cells have the potential to lose stability and express transcription factors and cytokines that are typically ascribed to other lineages. Although it remains possible that this helps to promote pathogen clearance, in many instances this is associated with ineffective immune responses or correlates with inappropriate immune responses that are seen in autoimmune diseases.
Data from patients with Crohn’s disease suggest that dual Th1/Th17 cells may play a pathogenic role in disease (121). These cells appear to retain aspects of Th1 and Th17 identity, expressing T-bet and RORγT and secreting IFNγ and IL-17A (121, 122). This coexpression of T-bet and RORγT is intriguing because T-bet is thought to repress Th17 identity by binding Runx1, to prevent Runx1 from transactivating Rorc (123–125). Runx1 also represses the Th2 fate by binding directly to GATA3 (126). Runx1 is ubiquitylated and degraded by the E3 ligase Stub1 (127), placing ubiquitylation of Runx1 squarely at the center of decisions of CD4+ effector fate. Furthermore, in vitro–derived Th1 cells bear activating H3K4me3 marks at the gene loci for IFN-γ and T-bet, as expected, but also unexpectedly at the locus for GATA3 (128), suggesting that Th1 cells may be poised to take on other Th identities. This corroborates data by other groups showing that Th1 cells may convert to IL-4–producing cells in response to infection with Nippostrongylus brasiliensis (129). Conversely, in the absence of GATA3, CD4+ T cells enter into the Th1 cell, rather than Th2 cell, lineage (47, 130). Continued research is needed to further elucidate the role of ubiquitylation in these Th1/Th2 cell fate decisions.
Th17 cells can exhibit plasticity in vivo. In a NOD/SCID autoimmune model of diabetes, transfer of Th17 cells led to acquisition of T-bet and IFN-γ secretion by these cells in vivo (131). Furthermore, in another model, Th17 cells could transdifferentiate into Tr1 cells via a TGF-β/Smad3 pathway, both at steady-state and during immune responses to worms or bacterial infections (132). Smad3 degradation in transformed cell lines is mediated by the RING E3 ligase ROC1 (133) and may be reversed by the DUB OTUB1 (134). Thus, ubiquitylation may be involved in plasticity of Th17 cells.
Bcl6 is crucial in driving Tfh lineage identity. Bcl6 levels may also be increased downstream of STAT1, STAT3, and STAT4 (135, 136). During Th1 differentiation, there is a Tfh-like transition stage during which Bcl6 and T-bet are expressed. However, as T-bet expression increases, Bcl6 levels decrease, resulting in a bias toward Th1 cell identity (135, 136). Interestingly, although the E3 ligase SLIM degrades STAT1 and STAT4 in Th1 cells (37) and STAT3 in Th17 cells (71), a role for SLIM E3 ligase in regulating Tfh differentiation has not been reported. This connection remains to be explored.
Stability of Tregs continues to be a controversial topic. Some studies showed remarkable stability of these cells in lymphoreplete and lymphopenic hosts (137, 138). Other studies showed that Tregs can lose Foxp3 upon transfer into lymphopenic hosts and gain the capacity to express effector cytokines (139, 140). A consensus may be that, although most thymically derived Tregs may be stable and committed to the Treg lineage, inflammatory conditions exist that can drive some loss of Foxp3 protein and, thus, Treg instability. Alternatively, there may be conditions in which a small subset of Tregs that are not fully committed to the Treg fate lose their Foxp3 expression (141, 142). In autoimmune arthritis, for example, “exTregs” that acquire the capacity to secrete IL-17A can play a pathogenic role in the disease (143). Bcl6−/− Tregs are also more likely to lose Foxp3 and to express GATA3, as well as to secrete Th2 cytokines and IL-17A (144). Because Bcl6 may be degraded by ubiquitination, as discussed in the Tfh cell section, it will be interesting to determine, in future studies, how ubiquitylation and degradation of Bcl6 regulate Treg stability under steady-state and inflammatory conditions.
With continued use of modern genetic technology, such as reporter mice for many of the known lineage-defining transcription factors and their key cytokines, future work may continue to reveal conditions under which Tregs may be unstable. Further work focusing on understanding how ubiquitylation regulates the human proteome should also help to shed more light on the role of ubiquitylation in regulating Treg stability.
With >600 E3 ligase enzymes encoded in the human genome, our knowledge of the substrates and functions of all of these enzymes is in its infancy. Relatively little is known about the contribution of these ligases to CD4+ effector fate and function (Fig. 1). As gene targeting becomes more efficient, such as with the advances of CRISPR technology, our understanding of how these ligases function in vivo will be more fully explored. Additionally, as whole-exome sequencing becomes more commonplace, mutations in E3 ligases are likely to be found to associate with immune-mediated disease, thus providing a more complete understanding of how these ligases regulate immune function. Therefore, it is easy to predict that our current knowledge is just the tip of the iceberg.
This work was supported by the American Asthma Foundation (Grant 13-0020) and the National Institutes of Health (Grants R01AI093566, R01AI114515, and T32CA009140-39).
Abbreviations used in this article:
Casitas B-lineage lymphoma b
gene related to anergy in lymphocytes
homologous to E6AP C terminus
hypoxia-inducible factor α
murine double minute 2
Nedd4 family-interacting protein 1
ovarian tumor–related protease
Pellino1 E3 ligase
peptide displayed on MHC
really interesting new gene
RAR-related orphan receptor γ T
T-box expressed in T cells
T follicular helper
regulatory T cell
The authors have no financial conflicts of interest.