Ubiquitin ligases and Dubs in CD4 T cell effector fate choice and function

Abstract

The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and GI 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 T helper cells known as Th1, Th2, and Th17, T follicular helper (Tfh) cells, and regulatory T cells (Tregs). These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. As 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 both levels and functions of effector fate-determining factors is ubiquitylation. This review will detail our current understanding of how ubiquitylation regulates CD4 T cell effector identity and function.

INTRODUCTION

Ubiquitylation is the post-translational 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 ubiquitin¹. The ubiquitin is then transferred to a catalytic cysteine on an E2², which then 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 RING (Really Interesting New Gene) type E3s^3,4. Alternatively, HECT (Homologous to E6AP C-Terminus)⁵ and the RBR (RING-between-RING) type E3s^6,7 first receive the ubiquitin onto a catalytic cysteine residue before transferring it to a lysine on the substrate. E3 ligases can thus identify the substrate as well as dictate the formation of ubiquitin linkages, driving the mono-, multi-mono-, or polyubiquitylation of the substrate. Ubiquitin has seven accessible lysines on its surface, K6, K11, K27, K29, K33, K48 or K63, each of which can be points of attachment for ubiquitin chains^8–11. Furthermore, the amino terminal methionine of ubiquitin (M1) can also serve as a point of attachment for linear chains in a reaction catalyzed by an E3 ubiquitin ligase complex called the linear ubiquitination assembly complex (LUBAC)¹². Ubiquitin chains can alter the fate of a substrate by changing its intercellular 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 may be split into five main families based on their structural domains. These five families are the ubiquitin-specific proteases (USPs); the ubiquitin carboxy-terminal hydrolases (UCHs); the ovarian tumour-related proteases (OTUs); the Machado-Joseph disease domain proteases (MJDs) and the JAB1/MPN/Mov34 Metalloproteases (JAMMs)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 naïve T cell encounters an antigen presenting cell (APC) expressing a peptide antigen displayed on its MHC (pMHC), the naïve 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, co-stimulation, 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 co-stimulation. These include the RING E3 ligases: Cbl-b^18–22, TRAC-1²³, Peli1²⁴ and the HECT E3 ligases, Itch and Nedd4^25–29, as reviewed^30–33. This review will explore how ubiquitylation impacts the identity and lineage stability of CD4+ T helper/effector subsets.

TH1 CELLS

Th1 cells are important for the clearance of intracellular pathogens. They differentiate from naïve CD4+ T cells in response to pMHC and costimulation in a cytokine milieu containing IL-12 and IFNγ. IL-12 signaling induces the phosphorylation of Jak2 and Tyk2, leading to STAT4 activation which in turn drives IFNγ production³⁴. In an amplification loop, IFNγ receptor signaling through STAT1 induces expression of the transcription factor T-Box Expressed In T Cells (T-bet)³⁵. T-bet, transactivates the IFNγ gene to drive further IFNγ cytokine production³⁵ and increases expression of the IL-12Rβ2 to promotes more IL-12 responsiveness³⁶. 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 STAT 1 and STAT4 in response to IFNα or IL-12 signaling³⁷. SLIM has been shown to aid in polyubiquitylation of STAT 4 in vivo and in vitro³⁷, and STAT 1 in vitro³⁸. The CD4+ T cells in SLIM −/− mice make increased IFNγ upon in vivo challenge with heat-killed Listeria monocytogenes³⁷. It is however unclear whether the increased IFNγ production by these cells would offer enhanced resistance to pathogen or rather, enhanced immunopathology. Another E3 ligase, Smad ubiquitylation regulating factor 1 (Smurf1), has been shown to mediate K48 polyubiquitylation and degradation of STAT1 in transformed cell lines but it remains to be shown whether this happens in T cells as well³⁹. STAT1 ubiquitylation is reversed by the DUB, USP13⁴⁰. Furthermore, while T-bet can undergo ubiquitin-mediated degradation, the identity of the E3 ligase which drives this degradation is unknown⁴¹. However, a DUB, USP10, is known to reverse this ubiquitylation and stabilize T-bet⁴². Since the major driver of Th1 identity, Tbet, is regulated by ubiquitylation, it raises the question of how else ubiquitylation influences both epigenetic modifications and the stability of proteins that result in the decision to be a Th1 cell.

TH2 CELLS

Th2 cells mediate immunity against extracellular microbes, such as worms, and facilitate clearance of allergens and toxins. Th2 cells differentiate from naïve CD4+ T cells in response to pMHC and co-stimulation 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 loop⁴³. 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 both Notch-dependent and Notch-independent mechanisms^44–47. While IL-4 and STAT6 may be dispensable for in vivo generation of Th2 cells^46–48, GATA3 is required for the generation of Th2 cells⁴⁴.

IL-4, 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 autoinhibition⁴⁹. 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 which overexpress JunB to levels found in Th2 cells, show a specific increase in Th2 cytokines such as IL-4 and IL-5⁵¹.

STAT 6 levels are regulated by two different E3 ligases: gene related to anergy in lymphocytes (GRAIL) and Casitas B-lineage lymphoma b (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 naïve CD4+ T cells fail to appropriately degrade STAT6 after in vitro TCR stimulation⁵³. Cbl-b also drives STAT6 polyubiquitylation and degradation⁵⁴. 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 Mdm2 has several other substrates including NFAT2c^57,58 and GATA3⁵⁹. Upon TCR stimulation, activation of the ERK-MAPK pathway leads to the association of Mdm2 with GATA3, and consequent polyubiquitylation and proteasomal degradation of GATA3⁵⁹. A DUB named USP15 deubiquitinates Mdm2 and prevents its proteasomal degradation⁵⁷. Therefore ubiquitylation of GATA3 and STAT6, as well as of factors that regulate IL-4 production, all contribute to the decision of a CD4 naïve cell to become a Th2 cell.

TH17 CELLS

Th17 cells are important for the clearance of extracellular pathogens such as fungi. Th17 cells differentiate in the presence of a cytokine milieu containing transforming growth factor β (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 Th17s in the absence of TGF-β⁶³. STAT3 is induced downstream of IL-6, IL-21 and IL-23, and cooperates with IRF4 (induced downstream of IL-1β) to elevate RORγT levels^64,65. RAR-related orphan receptor gamma T (RORγT) and alpha (RORα) 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 that, as discussed in the prior section, have increased frequencies of Th2 cells, also have increased frequencies of Th17 cells. While this may be due to cell-extrinsic effects of IL-4 mediated inflammation driving increased pro-inflammatory cytokines and tissue damage⁶⁸, 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 cells⁶⁹. Crossing the Ro52−/− mice to IL-23p19−/− mice abrogates tissue damage, excessive cytokine production and lowers the frequencies of the 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 that 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 (EAE), a mouse model of multiple sclerosis⁷¹.

Several DUBs are important for Th17 differentiation. USP4 regulates Th17s in two ways. First, USP4 deubiquitylates Ro52 in transformed cell lines⁷² and thus limits the expression of cytokines needed for Th17 generation⁶⁹. Second, it directly interacts with RORγT in primary human Th17 cells and deubiquitylates RORγT in transformed cell lines⁷³. USP17 (also known as DUB-3) is a second DUB that maintains RORγT stability⁷⁴. Knockdown of USP17 in primary Th17 cells results in a decrease of endogenous RORγT levels⁷⁴. Since IL-4 and IL-6 signaling can induce USP17⁷⁵, it appears that in response to IL-6 signaling, two responses occur: first, RORγT levela are increased downstream of STAT3 and second, 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 UBR5⁷⁶. 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. T-cell specific loss of DUBA therefore results in reduced levels of UBR5, stabilization of RORγT, and increased IL-17A production in response to TCR stimulation⁷⁶.

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 Th17s which cause EAE^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.

TFH CELLS

Tfh (T follicular helper) cells are CD4+ T cells that provide co-stimulatory 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 antibody producing B cells. Tfh cells differentiate from naïve precursors in response to pMHC interactions and under the influence of cytokines such as 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, as reviewed⁹¹. The differentiation of a naïve CD4+ T cell into a Tfh cell occurs in a step-wise fashion, which requires the transcription factor, achaete-scute homologue 2 (Ascl2), to generate a BCl6^lo CXCR5⁺ Tfh-intermediate cell. This intermediate undergoes maturation and complete differentiation in the presence of Bcl6 to generate a Bcl6^hi CXCR^hi Tfh cell⁹². Bcl6 is thus essential for Tfh identity^93–95.

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 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, namely Batf and Bcl6⁹⁶. Intriguingly, this repression is epigenetically carried over into the periphery when the T cells encounter antigen. Therefore, in mice which lack Cul3 in T cells, Tfh cells are increased in secondary lymphoid tissues and these cells drive germinal center B cell expansion⁹⁷. 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 that 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 both naïve and activated T cells. This leads to a T-cell-intrinsic increase in Tfh differentiation, large numbers of germinal centers, and increased serum antibodies of various IgG isotypes, among other defects⁹⁸. Bcl6 is regulated by two other E3 ligases in diffuse large B cell lymphomas (DLBCLs)^99,100. Pellino1 E3 ligase (PELI1) directs K63 (non-degradative) chains on Bcl6 leading to Bcl6 stabilization in transgenic mice overexpressing human PELI1⁹⁹. Furthermore, in these lymphoma cell lines, another E3 ligase, FBXO11, that normally marks Bcl6 for degradation, is found to undergo loss of function mutations¹⁰⁰. 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 virus¹⁰¹. 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 Bcl6¹⁰¹. ICOS signaling converges on this pathway by transiently inactivating Foxo1 in order to relieve Foxo1 repression of Bcl6 and to allow Tfh differentiation downstream of short-term ICOS signals¹⁰². Surprisingly, complete knockout of Foxo1 in T cells prevents the formation of germinal centers or Tfh cells¹⁰². 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 generation and function¹⁰³ and Itch-deficient T cells have decreased Tfh cells, increased Foxo1 levels, and defective Treg numbers and function^101,104, it will be important to determine whether Foxo1 levels are changed in Itch−/− Treg cells 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 other factors in Tfh identity.

REGULATORY T CELLS (Tregs)

Tregs are a subset of CD4+ T cells that are capable of suppressing the actions and functions of other immune cell type types^105,106. Several distinct subsets of Tregs have been described including the Foxp3- Tr1 cells (Type 1 regulatory T cells), Foxp3+ Th3 cells, and the Foxp3+ thymic-derived Tregs. Foxp3+ regulatory T cells develop in the thymus, when TCR/CD28 and IL-2 signaling drives expression of the transcription factor Foxp3¹⁰⁷. Foxp3 is central to the function of this major subset of Tregs and mutations in foxp3 in both mice and men leads to non-functional Tregs and autoimmunity¹⁰⁸. 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 transcription¹⁰⁹. IL-2 binding to the IL-2R complex leads to JAK1 and JAK 3 recruitment and eventual recruitment and activation of STAT5 which in turn drives expression of Foxp3^110,111.

Two E3 ligases can regulate Foxp3 stability: Stub1 and Cbl-b. Stub1 (also known as CHIP or carboxyl terminus of Hsc70-interacting protein), interacts with both Hsp70 and Foxp3 to drive K48-linked polyubiquitylation of Foxp3¹¹². 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 subsequent cotransfer of these cells together with naïve T cells into a lymphoreplete host resulted in loss of Foxp3 in the Tregs and subsequent conversion of these cells into IFNγ+ Th-1 like effector cells. Cbl-b, has been 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 degradation¹¹³.

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, VHL¹¹⁴. Under normal oxygen levels (normoxia), an oxygen sensor named Hypoxia-inducible Factor alpha (HIF1α) is hydroxylated, recognized and then 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^115,116. HIF1α can directly bind RORγT and p300 to drive transcription of IL-17A and promote Th17 identity¹¹⁵ while simultaneously binding and targeting foxp3 for degradation to repress Treg differentiation^115,116. Interestingly, mice lacking VHL only in their Tregs develop a disease characterized by large numbers of IFNγ+ Tregs which infiltrate tissue and fail to suppress conventional T cells or prevent colitis¹¹⁴. In VHL-deficient Tregs, stabilized HIF1α drives the IFNγ promoter resulting in large amounts of secreted IFNγ and poor in vivo function of these Tregs¹¹⁴.

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 airways¹¹⁷. 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 production²⁵. Thus T cells from mice that lack Ndfip1 are defective in induced Treg (iTreg) generation due to high IL-4 production¹¹⁸. Two other E3 ligases, Smurf2¹¹⁹ and β-TrCP (FBXW1)¹²⁰, 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 in order to repress gene transcription. EZH2-deficient Tregs are unable to suppress inflammation in vivo and show an increased ability to lose Foxp3 expression¹²¹. 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 CYLD (cylindromatosis), 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 transcription¹²². 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 Tregs¹²². 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 T helper cell identity is important for proper immune function. On one hand, 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. While it remains possible that this helps to promote pathogen clearance, in many instances this is associated with an ineffective immune responses or correlates with inappropriate immune responses that are seen in autoimmune diseases.

Data from patients with Crohn’s disease suggests that dual Th1/Th17 cells may play a pathogenic role in disease¹²³. These cells appear to retain aspects of both Th1 and Th17 identity, expressing T-bet and RORγT and secreting both IFNγ and IL-17A^124,125. This co-expression of T-bet and RORγT is intriguing since T-bet is thought to repress Th17 identity by binding Runx1, to prevent Runx1 from transactivating Rorc^126–128. Runx1 also represses the Th2 fate by binding directly to GATA3¹²⁹. Runx1 is ubiquitylated and degraded by the E3 ligase Stub1¹³⁰, 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 IFNγ and T-bet, as expected, but also unexpectedly at the locus for GATA3¹³¹, suggesting that Th1 cells may be poised to take on other T helper identities. This corroborates data by other groups showing that Th1 cells may convert to IL-4 producing cells in response to infection with Nippostrongylus brasiliensis¹³². Conversely, in the absence of GATA3, CD4 T cells enter into the Th1 rather than Th2 cell lineage^47,133. 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 Th17s cells led to acquisition of T-bet and IFNγ secretion by these cells in vivo¹³⁴. 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 infections¹³⁵. Smad3 degradation in transformed cell lines is mediated by the RING E3 ligase, ROC1¹³⁶ and may be reversed by the DUB, OTUB1¹³⁷. 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 STAT4^138,139. During Th1 differentiation, there is a Tfh-like transition stage during which both Bcl6 and T-bet are expressed. However as T-bet expression increases, Bcl6 levels decrease, resulting in a bias towards Th1 cell identity^138,139. Interestingly, although the E3 ligase, SLIM, degrades STAT1 and STAT4 in Th1 cells³⁷ and STAT3 in Th17 cells⁷¹, a role for SLIM E3 ligase in regulating Tfh differentiation has not yet been reported. This connection remains to be explored.

Stability of Tregs continues to be a controversial topic. Some studies have shown remarkable stability of these cells in both lymphoreplete and lymphopenic hosts^140,141. Other studies have shown that Tregs can lose foxp3 upon transfer into lymphopenic hosts and gain the capacity to express effector cytokines^142,143. A consensus may be that while 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, may lose their Foxp3 expression^144,145. In autoimmune arthritis, for example, “exTregs” which acquire the capacity to secrete IL-17A can play a pathogenic role in the disease¹⁴⁶. Bcl6 −/− Tregs are also more likely to lose Foxp3 and to express GATA3, and to secrete Th2 cytokines and IL-17A¹⁴⁷. Since Bcl6 may be degraded by ubiquitination, as discussed in the Tfh section, it will be interesting in future studies to determine how ubiquitylation and degradation of Bcl6 regulates Treg stability under steady state and under 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 shed more light on the role of ubiquitylation in regulating Treg stability.

CONCLUSIONS

With over 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. To date, relatively little is known about the contribution of these ligases to CD4 effector fate and function (Figure 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. It is therefore easy to predict that our current knowledge is just the tip of the iceberg.

Figure 1.

E3 ubiquitin ligases and Deubiquitinating enzymes (DUBs) involved in CD4+ Tcell identity. After T cell activation by an activated APC, a naïve T cell can differentiate into any of the CD4+ effector cell fates diagramed. All of the key transcriptional factors that regulate these effector fates can be regulated directly or indirectly via ubiquitylation.