Are T_regs defective in Type 1 Diabetes and can we fix them?

Abstract

Regulatory T cells (T_regs) are critical regulators of peripheral immune tolerance. T_reg insufficiency can lead to autoimmune disorders, including Type 1 Diabetes (T1D). Increasing evidence in mouse models of T1D, as well as other autoimmune disorders, suggest that there are defects in T_reg-mediated suppression. Indeed, while T_reg frequency in the peripheral blood of T1D patients is unaltered, their suppressive abilities are diminished compared to T_regs in healthy controls. Although expression of the transcription factor Foxp3 is a prerequisite for T_reg development and function, there are many additional factors that can alter their stability, survival and function. Much has been learned in other model systems, such as tumors, about the mechanism and pathways that control T_reg stability and function. This review poses the question: can we use these findings to develop new therapeutic approaches that might boost T_reg stability, survival and/or function in T1D, and possibly other autoimmune disorders?

Introduction

T1D, also known as Juvenile Diabetes, is a chronic autoimmune disorder where a targeted immune response by both T and B cells leads to destruction of insulin producing β-cells in the islets of the pancreas (1). T1D is one of the most common chronic diseases of children. Around 70,000 children are diagnosed with T1D each year, a number that is rising by 3-5% each year in developing countries (2). Defects in the control of effector populations is a common culprit in many autoimmune disorders including T1D (3), and this may be due to dysfunctions in T_reg-mediated suppression.

T_regs are either generated within the thymus, known as thymic-derived T_regs (tT_regs), or in the periphery, known as peripherally-derived T_regs (pT_regs), where pT_reg generation requires TGFβ for their differentiation (4, 5). While pT_regs have been shown to play an important role at mucosal sites and at the fetal-maternal interface (6, 7), we will be focusing on tT_regs as they are the dominant regulatory population that are impacted in T1D. tT_regs arise in the thymus upon high-affinity T cell receptor (TCR) signals to self-antigens and have a diverse repertoire (8, 9), suggesting that they have broad antigen specificity. tTregs are typically found in lymphoid tissues and can traffic to peripheral tissues during times of inflammation.

T_regs express the transcription factor forkhead box 3 (Foxp3), which is required for their development and function. In the absence of functional Foxp3, humans succumb to a lymphoproliferative disorder known as immunodysregulation polyendocrinopathyenteropathy X-linked syndrome (IPEX). Scurfy mice, which have a point mutation in Foxp3, develop a similar phenotype and succumb to disease early in life (10, 11). Bone marrow transplantation in IPEX patients and adoptive transfer of Foxp3⁺ T_regs or T cell-enriched splenocytes into Foxp3^−/− or Scurfy mice restores normal immune homeostasis, supporting the necessity for T_regs in preventing autoimmune responses (12, 13). pT_regs arising from CD4⁺Foxp3⁻ splenocytes have also been suggested to play a role in immune homeostasis as their TCR repertoire is non-overlapping with tT_regs (14, 15). Of note, splenocyte transfer may also limit the expansion of recipient diabetogenic T cells independently of any impact of tT_regs and pT_regs (16). T_regs can suppress immune responses by both cell-cell (CTLA-4, Granzyme B) and soluble factor- (TGFβ, IL-10, IL-35, adenosine) mediated mechanisms (17, 18). These effector functions may become deficient upon T_reg instability, which may lead to the development of autoimmunity, in this case T1D.

A two-checkpoint hypothesis has been suggested in the progression of T1D from insulitis to overt diabetes where T_regs play a central role at these checkpoints based on studies performed in mice (19). During the first checkpoint, autoreactive T cells begin entering the islet but are still under T_reg-mediated control and therefore insulitic. The transition from insulitis to overt diabetes occurs when T_regs lose their ability to suppress effector cell responses. Is the loss of stability in T_regs a factor in T1D progression from insulitis to overt diabetes? While many factors, including genetics and environment, contribute to the development of T1D, this review will focus on the failure of T_regs to control autoreactive T cells and how this may relate to T_reg instability. This review will summarize the contributions from other models in understanding what factors are important for T_reg stability (Fig. 1). Can we use what has been learned toward stabilizing T_regs in T1D?

Figure 1. Mechanisms of T_reg stability and instability.

IL-2 is critical for T_reg stability and maintenance where polymorphisms in both Il2 and Il2ra have been seen in diabetes. Pro-inflammatory cytokines including IFNγ and TNFα may alter the T_reg phenotype. Many T_reg-associated molecules are important for optimal suppressive function including CTLA4, GITR, and OX-40. Interestingly, agonistic antibodies to GITR are detrimental to T_reg mediated stability and suppression. Intracellular molecules including Helios, Eos, and PTEN are also key molecules in optimal T_reg function. Foxo1/3a localization into the nucleus is necessary to stabilize Foxp3 in T_regs. Green: stabilizing signal; Red: destabilizing signal

Loss of T_reg phenotype and function in T1D and autoimmune diabetes

While the majority of studies have reported no differences in the frequency of T_regs in peripheral blood isolated from T1D patients, defects in T_reg phenotype and suppressive capacity have been reported (20-24). Unfortunately, the majority of data obtained from T1D patients is from peripheral blood, due to the feasibility of obtaining pancreas samples from T1D patients. Therefore, whether T_regs are actively playing a role in limiting β cell destruction or have an altered phenotype of function in the islets during the disease course is unknown. Thus, mouse models of TID have been employed to investigate disease progression in the islet microenvironment.

The most commonly used model for T1D is the non-obese diabetic (NOD) mouse. NOD mice spontaneously develop autoimmune diabetes starting at ~10 weeks of age in females and with increasing incidence over time until ~25 weeks (25). Both diabetes onset and progression is delayed in male NOD mice. Diabetes incidence in females and males is usually ~80% and ~30%, respectively. This may be due to differences in the gut microbiome between females and males, due to hormonal differences (26). Other environmental factors, including housing conditions and diet, can also affect the development of autoimmune diabetes (25). Genetic analyses have uncovered susceptibility loci in NOD mice that are known as the insulin-dependent diabetes (Idd) loci. Over 40 Idd loci have been identified with the major histocompatibility complex (MHC) exhibiting the highest linkage with T1D incidence, (25, 27). The NOD mouse shares many similarities to T1D in humans, but with some notable differences (25). Nevertheless, the NOD mouse has proven to be a useful model to study the role of T_regs in autoimmune diabetes.

T_reg modulation studies have highlighted their importance in limiting autoimmune diabetes and controlling immune responses in the islet, despite some contradictory observations. Whereas T_reg depletion using anti-CD25 (PC61) has been shown to accelerate autoimmune diabetes development in several studies (28-30), one group observed complete protection from the development of autoimmune diabetes (31), perhaps due to the depletion of activated diabetogenic CD25⁺ effector T cells in addition to CD25⁺ T_regs as a consequence of late initiation of PC61 treatment (>9weeks). However, mice that lack T_regs, due to Foxp3 deficiency rapidly develop autoimmune diabetes (32). Indeed, temporal depletion of T_regs, due to diphtheria toxin (DT) treatment of Foxp3-DTR (diphtheria toxin receptor) mice showed strong immune infiltrates in the pancreas two weeks after DT treatment (33). Of note, NOD.Foxp3-DTR mice (Foxp3 bacterial artificial chromosome (BAC) Tg, DEREG mouse model) do not develop diabetes at an accelerated rate (34). These conflicting observations with two independently generated BAC Tg NOD.Foxp3-DTR strains may be due to differences in expression and deletional efficiency and warrant further investigation. Interestingly, mice expressing the BDC2.5 TCR transgene (expressed on CD4⁺ T cells specific for the islet antigen chromogranin A), which are immunocompetent only develop insulitis (35). However, when the BDC2.5 TCR transgene is expressed on a Rag^−/− background, in which CD4⁺ effector T cells develop but T_regs do not, mice succumb to diabetes rapidly. Indeed, diphtheria toxin treatment of NOD.Foxp3-DTR mice crossed to BDC2.5 Tg also rapidly develops diabetes (33). Collectively, these studies suggest that diabetes onset may be associated with decreased T_reg numbers or function.

If humans and mice are not T_reg deficient, why do they succumb to T1D and autoimmune diabetes, respectively? What is affecting their functionality? Interestingly, islet infiltrating T_regs in mouse models still express high levels of Foxp3, but have decreased expression of the high affinity IL-2 receptor CD25 and survival factor Bcl2 (36). Likewise in T1D patients, T_regs found in PBMCs have low expression of another T_reg-associated marker GITR (37), which will be further discussed later in the review. In children with T1D, a higher proportion of T_regs produce the pro-inflammatory cytokines IL-12 and IL-18, which are also found at increase levels in serum, compared to healthy controls (23). Consequently, this altered T_reg phenotype has been implicated in T1D pathogenesis. Thus, if altered T_reg numbers and/or function are primary contributors to the development of T1D, boosting either parameter in vivo may provide a therapeutic opportunity.

Boosting T_regs in mice and humans

Pharmacological-based therapy

Inducing T_reg proliferation via multiple pharmacological methods has been proposed and attempted in both the NOD mouse model and in clinical trials. IL-2, which is important for maintenance of T_regs, has been a potential target for T_reg therapy (38) (Fig. 1). While high dose IL-2 has been used as a therapeutic approach in the treatment of melanoma and renal cancers, low dose IL-2 in NOD mice can reverse established disease by increasing T_reg numbers and function (36, 39). IL-2/anti-IL-2 Ab complexes have also been used to preferentially promote T_reg expansion (40). Modulation of mTOR activity with rapamycin has been shown to promote T_reg expansion, survival and function (41). Although no difference in T_reg number, proliferation or cytokine production was seen with rapamycin therapy prior to islet transplantation, T_regs do have increased suppressive capabilities (42). A combinational therapy has also been assessed with the use of IL-2/anti-IL-2 Ab complexes in combination with rapamycin and islet antigen peptide treatment. T_reg expansion was observed and mice protected from diabetes development in both spontaneous and induced models of diabetes (43).

Non-activating, non-FcR binding CD3 antibodies may currently be the most promising treatment for T1D. More than 8 clinical trials have targeted this approach, with five of which are using teplizumab, a humanized non-FcR-binding anti-CD3 monoclonal antibody (44). C-peptide is a byproduct of insulin production and is produced at equimolar concentrations and thus can be used to determine the amount of insulin produced by β-cells. Short-term treatment of younger individuals and recent onset patients with teplizumab has shown promising results in four-year follow-up studies, based on C-peptide levels, with limited toxicity (45-48). While its mechanism of action is currently unclear, a two-fold tolerance induction has been suggested through depletion of pathogenic T cells and preservation of T_regs and their function (49, 50). Although the mechanisms of action of all these therapeutic approaches is different, in all cases the common denominator is increased T_reg number and function.

Cell-based therapy

As T_reg insufficiency may be a key driver of T1D and autoimmune diabetes, increasing the number of T_regs in circulation may overcome this deficiency. Repeated T_reg adoptive transfer into neonatal NOD mice can delay the onset of autoimmune diabetes (51), suggesting that T_reg number or functionality may be deficient in NOD mice over time thereby requiring supplementation. Adoptive transfer of pre-diabetic NOD splenocytes or BDC2.5 TCR Tg T_eff cells into immunodeficient NOD mice develop autoimmune diabetes ~14 days post-transfer. Interestingly, disease can be prevented following co-transfer with >10⁶ polyclonal T_regs or as few as 5×10⁴ BDC2.5 TCR Tg T_regs (34). Adoptive transfer of a low number of DC-expanded BDC2.5 TCR Tg T_regs into pre-diabetic NOD mice also blocks diabetes development and can rescue mice with overt diabetes (52). While low numbers of antigen-specific T_regs are able to reverse autoimmune diabetes, adoptive transfer of ten-fold more polyclonal T_regs is not as effective in treating NOD mice therapeutically (53), suggesting that specificity for β cell antigens is critically important for optimal T_reg functionality.

In vitro-expanded polyclonal T_regs are currently in clinical trials as a promising alternative to pharmacological-based therapies. Phase 1 clinical trials have been performed in both children and adults with no safety concerns thus far (54-56). Interestingly, some potential efficacy has been observed in children at 4-5 week follow-up based on C-peptide levels. However, while C-peptide levels were increased initially at one- and two-year follow-ups, they declined over time. Approximately 25% of the transferred T_regs with a naïve/memory-like phenotype were still present in patients at one-year follow-up based on deuterium incorporation. A similar trial has also been conducted in Poland with promising results. At a one-year follow-up of 12 children with T1D, increased C-peptide levels and diminished use of insulin was observed in 8 of 12 patients and, remarkably, complete insulin independence was achieved in 2 of 12 patients (55). Whether these observations are durable and can be replicated in Phase 2 clinical trials remains to be determined.

While these initial observations are encouraging, the key challenge is likely to focus on understanding what the primary limitations are for successful, durable responses and can these be overcome with [i] increased T_reg numbers, [ii] islet antigen specificity, and/or [iii] approaches that increased stability, survival, functionality and longevity. There is a growing consensus that future clinical trails need to focus on the development of T_regs with β cell antigen-specificity to maximize [i] islet homing and therapeutic index, and [ii] retention of T_regs over time to endure a durable response. Also, is the adoptive transfer of more T_regs the only viable therapeutic approach or could the T_regs that are already present in the patient be ‘reinvigorated’? Clearly, gaining a greater understanding of the mechanisms and factors that control T_reg stability and function will greatly inform future clinical development.

Loss of T_reg stability and function

What is T_reg stability, how does this differ from plasticity, and what drives instability? T_reg plasticity and stability have been used interchangeably in the past, but represent two distinct T_reg fates. A stable T_reg expresses the transcription factor Foxp3, is suppressive, produces anti-inflammatory cytokines (such as IL-10 and IL-35), and a minimal amount of effector cytokines (eg. IFNγ, TNFα, IL-2) (57). When T_regs exhibit plasticity, they still express Foxp3 and remain functionally suppressive but gain distinct migratory and functional programs that can enhance their capacity to suppress certain Th subsets (58). In contrast, destabilized T_regs lose their suppressive abilities and gained effector functions, while either retaining Foxp3 expression (59) and eventually losing Foxp3 expression and becoming pathogenic “ex-T_regs” in inflammatory environments (60). In both the NOD mice and humans with T1D, T_regs are identified based on Foxp3 expression, yet exhibit defective suppressive activity suggesting that they may be destabilized. Further analysis of T_reg stability has recently been extended to include epigenetic modifications by assessing the methylation pattern of the conserved noncoding sequence 2 (CNS2 or TSDR) in the 5’ UTR of the Foxp3 gene, where tT_regs are demethylated at this locus and loss of stability has been associated with re-methylation at this locus (61). Foxp3 CNS2 hypomethylation appears to be important for the binding of key transcription factors including NF-kB, CREB/ATF, Ets1 and STAT5 (62-64). Methylation studies have expanded to other T_reg associated genes Il2ra (CD25), Ikzf4 (Eos), Ctla4 (cytotoxic T-lymphocyte-associated protein 4), and Tnfrsf18 (GITR), which are also hypomethylated (65).

In addition to epigenetic modifications of target genes, other mechanisms including microRNAs (miRNAs) may also modulate disease development. These short non-coding RNAs are transcribed and processed via the RNAses Drosha and Dicer to generate mature miRNAs that silence genes either through repressing translation or accelerating transcript degradation (66). Mice with a T_reg-restricted deletion of Dicer or Drosha possess unstable T_regs with poor suppressive ability, diminished expression of T_reg-associated molecules, increased effector cytokines, and succumb to a Scurfy-like disease (67-69). Similar results have also been seen upon gene silencing of miR-126 in a breast cancer tumor model leading to increased anti-tumor immunity by altering activation of the PI3K/AKT pathway (70). While miR-155 is not necessary for T_reg homeostasis or its suppressive function, its role in downregulating SOCS1, which increases responsiveness of STAT5, can make T_regs better responders to IL-2, even under suboptimal conditions (71). T_reg-specific ablation of the miR-17-92 cluster results in exacerbated EAE with decreased IL-10 producing T_regs (72), but is not required for thymic generation of T_regs (73). miR-10a is selectively expressed in T_regs and expression has been correlated to autoimmune disease susceptibility as the autoimmune-resistant C57BL/6 strain expresses high levels of miR-10a while the autoimmune-susceptible NOD strain expresses lower levels (74). These studies suggest that certain miRNAs may be important in maintaining T_reg stability and function. Indeed, miR-342, miR-191, and miR-510 are differentially expressed in T_regs of patients with T1D, but whether these are biomarkers or contribute to disease still needs to be further elucidated (75).

Understanding the factors and pathways that control T_reg stability would clearly facilitate their therapeutic utilization in T1D, as well as other autoimmune and inflammatory diseases, and potentially in transplantation. While Foxp3 is the master transcription factor that is required for T_reg development and functionality, a variety of external signals from cytokines and surface receptors, via intracellular signaling molecules impinge on T_regs and impact their stability.

Factors that impact T_reg stability

Cytokines

Several cytokines have a substantive impact on T_reg development and function (Fig. 1). IL-2, produced by effector T cells, is necessary for the maintenance and function of T_regs, as they do not make their own autocrine IL-2 (76-78). The majority of T_regs express the high affinity IL-2 receptor (Il2ra, CD25) that signals via STAT5 (79). Genetically manipulated mice deficient in Il2 or Il2ra phenocopy Foxp3-deficient or T_reg-ablated mice, yet still harbor T cells that express diminished levels of Foxp3 (80, 81). Humans with CD25 deficiency also have many of the same symptoms as seen in patients with IPEX (82). IL-2 reverses anergic, non-proliferative phenotype of T_regs in vitro and promotes their capacity to suppress immune responses (83). IL-2 withdrawal has been shown in vitro to limit T_reg suppressive ability (84). Under sub-optimal IL-2 conditions, the CNS2 element sustains Foxp3 expression, while in its absence, actively proliferating T_regs lose Foxp3 expression at an accelerated rate (85). Genome wide association studies have identified IL-2 pathway polymorphisms in both T1D (Il2ra) and autoimmune diabetes (Il2) (86-88). Indeed, reduced IL-2 signaling, via pSTAT5 analysis, has been documented in T1D patients with diminished T_reg suppressive capabilities (89, 90). In NOD mice, T_regs have decreased Bcl2 and CD25 expression only in inflamed islets. This may be due to decreased levels of IL-2 in the islet as low-dose IL-2 treatment increases T_reg survival and protection (36). These studies highlight the importance of IL-2 in T_reg function and possible defects that might lead to the development of T1D.

Inflammatory environments have been shown to destabilize T_regs in many models due to their interaction with or production of pro-inflammatory cytokines. While several cytokines may destabilize T_regs, we will focus here on those that may be relevant to T1D. IFNγ is highly expressed in many inflammatory conditions and may limit T_reg function. Upon stimulation with IFNγ in vitro, T_regs downregulate CD25, lose Foxp3 expression, and exhibit limited expansion (91). Under high salt conditions, T_regs can begin to produce IFNγ and lose suppressive activity, which can be restored upon antibody blockade of IFNγ (92). Whether this T_reg-derived IFNγ acts on T_regs or T_effs still needs to be further elucidated (92). In T1D patients, increases in IFNγ⁺Foxp3⁺ T_regs has been observed in peripheral blood. These cells are predominantly hypermethylated at the CNS2 locus but still exhibit suppressive function (93).

T_regs constitutively express TNFRII, which upon signaling leads to diminished Foxp3 mRNA and protein levels, and reduced suppressive actvity. Not surprisingly, patients with active rheumatoid arthritis (RA) possess T_regs that express lower Foxp3 expression and suppressive ability, and this could be reversed with anti-TNF (infliximab) treatment (94). In contrast, others have shown the requirement for TNF signaling in the generation of functional T_regs within the thymus and their function in inflammatory settings. In colitis models, expression of TNFRII expression is critical for T_reg function (95, 96). Likewise, in NOD mice, TNF receptor deficiency protects mice from autoimmune diabetes and increases T_reg mediated suppression (97).

The role of IL-27 in T_reg stability has been quite conflicting. IL-27 has been shown to antagonize pT_reg generation (98), but has been shown to enhance tT_reg function in a T-cell mediated colitis model via a Lag3-mediated mechanism (99). In a tumor model, IL-27Rα-deficient mice have decreased T_regs in the tumor microenvironment suggesting IL-27 may act indirectly on T_regs via suppressing IL-2 generation by effector T cells (100). Nevertheless, the role of IL-27 specifically on T_regs has yet to be clarified. Increased IL-27 has been documented in autoimmune diabetes and blockade of IL-27 in NOD mice delays the onset of autoimmune diabetes (101).

Extensive studies still need to be performed to assess whether these cytokines directly impact T_regs before conclusions can be drawn regarding their role in modulating T_reg function in T1D and autoimmune diabetes.

Surface molecules

Several cell surface molecules have been shown to impact T_reg stability and function (Fig. 1). OX40 (Tnfrsf4, CD134) is part of the tumor necrosis factor superfamily receptors (TNFRs) and is expressed on T_regs (102), yet its role in T_reg-mediated suppression has led to conflicting results both in vitro (103-105) and in vivo. OX40 expression on T_regs may play a role in suppressing inflammatory responses in vivo as mice with a T_reg-restricted deficiency in OX40 retain Foxp3 expression yet develop gut inflammation in a T cell-mediated gut inflammation model (106). Indeed, use of an agonist anti-OX40 (OX86) protects NOD mice from the development of autoimmune diabetes (107). However, disease is inhibited in Ox40l^−/− mice and neutralizing anti-OX40L-treated NOD mice (108). Whether T_regs are the primary subset responding to OX40L has not been fully addressed as CD4⁺ and CD8⁺ T cells also express OX40 during autoimmune diabetes progression (109).

GITR (Tnfrsf18, CD357) is another TNFR family member that is found at high levels on the surface of T_regs (102). Paradoxically, use of an agonist anti-GITR (DTA-1) undermines T_reg-mediated suppression and tolerance in tumor models. Both a decrease in T_reg frequency and expression of Foxp3 in intratumoral T_regs has been seen (110). This loss of Foxp3, and Helios, expression is mediated by the c-Jun N-terminal kinase (JNK) pathway. Treatment of lung allergy mice with a JNK inhibitor led to reversal of GITR-induced changes in phenotype and function; and therefore was rescued from disease (111). Indeed, accelerated development of autoimmune diabetes has also been seen using a different agonistic anti-GITR antibody (2F8) (112), suggesting that activation of this pathway may be detrimental to T_reg stability.

Cytotoxic T lymphocyte antigen 4 (Ctla4, CD152) is highly expressed on T_regs and has extensively been studied as an inhibitory molecule important for T cell homeostasis and tolerance (113). Ctla4^−/− mice succumb to fatal lymphoproliferative disease (114), while T_reg numbers are increased (115, 116). Results from in vivo models of autoimmunity have been quite conflicting, where Ctla4^−/− T_regs are suppressive in some instances but not in others (115, 117). Ctla4 is a susceptibility gene in autoimmune diseases, including T1D, where many polymorphisms have been identified (118-120). Costimulation blockade using anti-CTLA4 (Abatacept) has recently been shown in phase II clinical trials to delay the progression of T1D (121), but whether T_regs are playing a direct role still needs to be assessed further.

Neuropilin-1 (Nrp1) is an important factor in axonal guidance during embryonic development, but its role in the immune system has only recently been appreciated. Nrp1 is highly expressed on tT_regs but is expressed at lower levels in pT_regs (122-124). A role for Nrp1 in promoting the stability, survival and function of T_regs has been suggested (125). Nrp1 on T_regs has been shown to interact with both Semaphorin-4a (Sema4a) and VEGF. Mice with a T_reg-specific Nrp1 deletion had substantially reduced tumor growth in multiple models suggesting that T_reg-mediated suppression of anti-tumor immunity has been lost (125, 126). Interestingly, these mice did not succumb to autoimmunity and inflammatory disease and the frequency of Foxp3⁺ T_regs was not altered (125). Stabilization via the Nrp1:Sema4a pathway enhances expression of the survival factor Bcl2, effector molecules IL-10 and CD73, limits expression of lineage-associated transcription factors, including T-bet, IRF4 and RORγt, and the pro-inflammatory cytokine IFNγ (125). Boosting T_reg function by engaging the Nrp1:Sema4a pathway may be a possible therapeutic approach to stabilize T_regs in vivo or prior to adoptive transfer.

Intracellular signaling molecules

There are also several intracellular proteins that appear to modulate T_reg stability and function by dependently or indirectly modulating Foxp3 function or stability (Fig. 1). Eos (Ikzf4), a zinc-finger transcription factor, is a member of the Ikaros family of transcription factors and is highly expressed in T_regs. Eos interacts directly with Foxp3 and is necessary for gene silencing (Il2, Ifng, etc.) while maintaining expression of key T_reg-associated genes including Ctla4 and GITR (127). Silencing of Eos using siRNA does not result in loss of Foxp3 expression but does result in the loss of T_reg suppression in a T cell-mediated colitis model and induction of effector cytokines, such as IFNγ and IL-2 (127). Downregulation of Eos expression is required for the reprograming of T_regs into helper-like cells that retain Foxp3 expression (128). These Eos⁻Foxp3⁺ T_regs (Eos-liable) exhibit reduced regulatory function and enhanced expression of CD40L, IL-2 and IL-17 (128). Of note, global deletion of Eos in mice does not affect the function or phenotype of T_regs in vivo and in vitro, but does result in the development of more severe EAE. This observation was attributed to the function of Eos in T_eff populations (129).

Helios (Ikzf2), another member of the Ikaros transcription factor family, was once thought to distinguish tT_regs from pT_regs, however, it now appears that Helios expression is highly dependent on antigen stimulation via the TCR (130, 131). While Helios does not form a complex with Foxp3 or bind to the Foxp3 locus, Helios plays an indirect role in supporting T_reg stability (132, 133). In mice with a T_reg-specific Helios deficiency develop autoimmunity development and appear to possess unstable T_regs with diminished Foxp3 expression, increased effector cytokine expression and reduced suppressive activity (133). ChIP-Seq and pathway analysis of Helios targeting genes in T_regs highlighted deficiencies in the IL-2Ra/STAT5b pathway suggesting that Helios may be important in regulating IL-2 signaling and T_reg survival (133).

Foxo1 and Foxo3, which are also forkhead box transcription factors, pay a key role in maintaining Foxp3 expression in T_regs (134). Mice deficient in Foxo1 in T_regs succumb to a Scurfy-like phenotype by 5 weeks. This lymphoproliferative disease is not due to the loss of T_reg number but rather their loss of function (135, 136). This phenotype can be rescued by expression of Foxo1^AAA, where Foxo1 is insensitive to 14-3-3-mediated cytosolic restriction and is this confined to the nucleus where it can facilitate Foxp3 function. Autoimmunity is further exacerbated by dual deletion of Foxo1 and Foxo3 (136). Foxo1/3 binds directly to the Foxp3 locus and controls promoter activity (136, 137).

The phosphatase, PTEN, has recently been shown to paly a pivotal role in mediating T_reg stability. PTEN is an upstream inhibitor of the PI(3)K-Akt pathway and therefore inhibits mechanistic target of rapamycin complex (mTORC)1 and mTORC2 activity (138). Upon genetic deletion of PTEN in T_regs, mice have increased levels of auto-antibodies, renal pathology and ongoing age-related autoimmunity. Nevertheless, T_regs are found in high numbers and readily proliferate compared to PTEN-sufficient T_regs. These T_regs are highly activated and express higher levels of ICOS, PD-1 and IFNγ, decreased levels of CD25, and have a higher proportion of “ex-T_regs” based on the use of lineage-tracing experiments (61, 139). The mechanism of T_reg-mediated loss of suppression is via upregulation of mTORC2 activity upon PTEN loss (139). Indeed, inhibition of mTOR in T_regs leads to heightened stability of Foxp3 expression (140) and T_reg specific loss of mTOR inhibitor tuberous sclerosis 1 (TSC1) results in loss of Foxp3 expression, suppressive functionality, and increased expression of IL-17 (141). Interestingly, Nrp1, which as discussed above promotes T_reg stability and function, has been shown to signal via PTEN that in turn limits Akt activity, reduces Foxo phosphorylation and thus nuclear exclusion, thereby promoting Foxp3 activity (125). Taken together, these observations provide a potential causal link between Nrp1, PTEN and Foxo in mediating T_reg stability and function.

Conclusions

In summary, many factors impinge on T_regs to either promote or undermine their stability, survival and function (Fig. 1). Some of these pathways are inherent, while others are induced or selectively utilized in inflammatory environments (59). We postulate that a primary driver of autoimmunity may be T_reg insufficiency caused by a failure to promote pathways that enforce their stability survival and function. In tumors, where T_reg activity is arguably at its most robust, T_reg stability is enforced by an Nrp1:PTEN:Foxo axis, and potentially other mechanisms, to prevent effective anti-tumor immunity. This also appears to protect T_regs from destabilizing forces that may be quite severe given the hostile intratumoral microenvironment, which is hypoxic, acidic, and nutrient and glucose starved. Thus, under normal circumstances T_regs seem to be well adapted to respond to cues from diverse microenvironments to maintain T_reg stability and function. However, we posit that genetic, environmental or contextual factors conspire to undermine these programs that ultimately leads to T_reg insufficiency and autoimmunity.

This hypothesis and the information outlined above raise several key questions. (1) Can we boost T_regs that are already present but appear to exhibit insufficiency? This could be achieved by developing therapeutics that promote utilization of the Nrp1:PTEN:Foxo axis. For example, Sema4a-Ig fusion proteins may act as Nrp1 agonists thereby promoting T_reg stability and function. Alternatively, intracellular delivery of therapies that promote Foxo stability and nuclear translocation may produce a similar T_reg stabilizing effect. (2) Can we inhibit pathways that lead to instability? While we need to gain a greater understanding of the factors that promote T_reg instability, approaches that limit the factors that are known to drive these processes may be beneficial. The use of blocking antibodies against cytokines that can destabilize T_regs may be useful in a manner analogous to TNFα blockade in RA. We could also develop antibodies to block OX40L from interacting with OX-40 on T_regs. (3) Is a combinatorial therapy possible and necessary? Given that there may be a two-fold defect in T_reg number and function in T1D, combinatorial therapy may be most useful. One could combine T_reg adoptive transfer with approaches that promote T_reg stability, prior to and/or following transfer. Of course, these approaches may also be combined with current therapies that are in clinical trials for T1D, such as teplizumab (non-FcR binding anti-CD3). Indeed, one might argue that as combinatorial approaches are the mainstay of effective cancer therapy it is likely that combinatorial approaches will be required for the treatment of T1D, with perhaps the inclusion of therapies that promote T_regs stability and function.