Regulatory T Cells: From Discovery to Autoimmunity

Abstract

Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. Allelic variants lead to lower thresholds of T-cell activation resulting in activation of autoreactive T cells. Environmental factors, including, among others, diet, vitamin D, and smoking, in combination with genetic predispositions, play a substantial role in disease development and activation of autoreactive T cells. FoxP3⁺ regulatory T cells (Tregs) have emerged as central in the control of autoreactive T cells. A consistent finding in patients with MS is defects in Treg cell function with reduced suppression of effector T cells and production of proinflammatory cytokines. Emerging data suggests that functional Tregs become effector-like T cells with loss of function associated with T-bet expression and interferon γ (IFN-γ) secretion.

DISCOVERY OF REGULATORY T CELLS (Tregs) IN MOUSE AND HUMAN

Since the discovery of Tregs by Sakaguchi and coworkers (1995) two decades ago, our understanding of the CD4⁺ T helper (Th) cell subtype, first characterized by the expression of the interleukin (IL)-2 receptor α-chain (CD25), has vastly expanded. Another breakthrough discovery in the Treg field was the identification of FoxP3 as the main transcription factor driving and maintaining Treg phenotype and function (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). Patients with the IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome), a severe autoimmune disorder that develops early in life, carry mutations in the FoxP3 gene locus. FoxP3 mutations lead to dysfunctional FoxP3 protein expression; patients harboring FoxP3 mutations do not develop functional Tregs (Bennett et al. 2001). A similar phenotype is observed in scurfy mice, which lack functional FoxP3 (Bennett et al. 2001). FoxP3⁺ CD25⁺ Tregs can be broadly subdivided into naturally arising Tregs and peripherally induced Tregs. Naturally arising Tregs develop in the thymus. In animal models, it was first noted that those receiving postnatal thymectomy developed severe autoimmunity. Furthermore, disease development could be prevented by the transfer of CD4⁺ T cells (Sakaguchi et al. 2006). Moreover, the depletion of CD25⁺ cells from thymocytes or peripheral T cells could not prevent autoimmunity in cotransfer experiments in immune-deficient animals. This led to the terminology of “naturally arising” or “natural” Treg cells (Sakaguchi et al. 2006). Thymic development of natural Tregs is strictly related to the stable induction of FoxP3, and requires high-affinity binding of major histocompatibility complex (MHC)–self-peptide complexes from thymic antigen-presenting cells (APCs) to the T-cell receptor (TCR). As thymic Tregs are reactive against self-peptides, they are likely to be predominantly involved in controlling autoimmune reactions. Additionally, thymic Treg development requires certain costimulatory signals and cytokine environments (in particular IL-2), different from conventional effector T cells, which leads to the generation of stable FoxP3-expressing Treg cells in the periphery (Klein and Jovanovic 2011; Hsieh et al. 2012). Fate-mapping and thymic selection studies of Tregs so far have only been conducted in mouse models and it remains to be seen whether the same processes apply to human Treg development.

Stable expression of FoxP3 is essential for Treg function and is maintained through epigenetic modifications both in the FoxP3 gene locus and Treg-specific demethylated region (TSDR) (Floess et al. 2007; Huehn et al. 2009). Naïve murine FoxP3⁻ CD4⁺ T cells can express FoxP3 in the presence of transforming growth factor β (TGF-β) or retinoic acid, which gives rise to peripherally induced Tregs (iTregs). As iTregs arise from conventional CD4⁺ T cells, they are considered to play a more pronounced role in general immune regulation (de Lafaille and Lafaille 2009). Although there are phenotypic and functional overlaps to natural Tregs, iTregs show distinctive differences in stability and gene expression (de Lafaille and Lafaille 2009; Sakaguchi et al. 2010). For instance, the TSDR region of iTregs is not fully demethylated, whereas natural Treg TSDR is fully demethylated (Floess et al. 2007).

Although the term “regulatory T cell” conventionally is used to describe FoxP3⁺ CD4⁺ T cells, it has to be noted that mouse models helped to identify subtypes of Tregs that lack the expression of FoxP3. IL-10-producing Tr1 (Treg type 1) cells, and TGF-β-producing Th3 cells are the well-established FoxP3⁻ Treg populations that can exert suppressive function on effector T cells (Chen et al. 1994; Bach 2001; Roncarolo et al. 2001).

We and others were able to identify human CD4 cells expressing very high levels of CD25, which are analogous in function in vitro to mouse Tregs (Baecher-Allan et al. 2001; Stephens et al. 2001). These populations of CD25^high CD4⁺ cells were subsequently found to express high levels of FoxP3. Although FoxP3 is essential for development and function of Treg cells (Fontenot et al. 2003), we now understand that FoxP3 can be expressed by other cell populations, and thus cannot be described as a lineage-defining transcription factor for Tregs. Specifically, as T-cell subpopulations develop in the thymus, not all CD4⁺ FoxP3⁺ T cells are homogenous in gene expression, phenotype, and suppressive function. Further, effector T cells have the potential to express the FoxP3 marker on activation. Thus, human Tregs cannot be reliably identified by FoxP3 expression alone. At present, the surface expression of CD25 and CD127 is the most consistent marker, with high levels of CD25 expression and low/negative expression of CD127 being indicative of the Treg population (Liu et al. 2006; Seddiki et al. 2006).

Treg HETEROGENEITY: ONE FAMILY, DIVERSE APPEARANCES

Parallel to the discovery of diversity among T cells is the identification of diversity within the CD4⁺ CD25⁺ FoxP3⁺ Treg population. A widely used classification of human Tregs is based on the expression of CD45RA⁺ (naïve-like or resting Tregs) and CD45RO⁺ (activated effector or memory-like Tregs) (Miyara et al. 2009). Additionally, surface expression of HLA-DR can further divide Tregs into DR⁺ and DR⁻ Tregs, in which expression is correlated with higher suppressive activity (Baecher-Allan et al. 2006, 2011). The subset of CCR4⁺ memory Tregs has also been isolated as a highly suppressive population. Additionally, subsets can be defined based on chemokine receptor expression. Thus, subset-specific markers can be used in the laboratory to isolate several subpopulations of the heterogeneous Treg population for study.

The expression of CD25 is not restricted to human Tregs, as it is also up-regulated on activated conventional T cells, which can be best identified by the surface expression of CD25 in combination with CD127, the α-chain of the IL-7 receptor. This is contrary to Tregs, which display a high expression of CD25 while they are predominantly negative for CD127 (CD25^high CD127^low/neg) (Liu et al. 2006; Seddiki et al. 2006). Another method to isolate human Tregs is the combination of CD49d (the α-chain of VLA-4) and CD127. We have shown that the majority of human Tregs only express low levels of CD49d and that by depleting both CD49⁺ and CD127⁺ cells, the isolation of Tregs is achievable without the use of CD25. Importantly, because most of the cytokine-expressing activated CD25⁺ effector cells express higher levels of CD49d, this method removes contaminating cytokine-expressing cells from human Treg preparations (Kleinewietfeld et al. 2009). FoxP3⁺ Tregs further express a set of characteristic markers and molecules, of which some are direct targets of FoxP3. Tregs express, for example, high levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor (TNF) receptor–related protein (GITR) (Sakaguchi et al. 2010). Subsets of both murine and human Tregs express the TNF receptor 2 (TNFR2) (Chen et al. 2008, 2010).

TNF-α stimulation induces, synergistically with IL-2, the expression of other members of the TNFR superfamily, such as 4-1BB and OX40 (Hamano et al. 2011). Interestingly, OX40 (CD134, TNFRSF4) has recently been shown to induce Treg activation and suppressive function (Piconese et al. 2014b). Furthermore, in vitro experiments have shown an induction of human Treg proliferation and promotion of Treg stability on OX40 engagement via APC-mediated activation and stimulation with OX40 ligand (OX40L). TNF-α stimulation further increases OX40 expression and promotes human Treg-suppressive function (Piconese et al. 2014a,b).

HETEROGENOUS Tregs EXERT DIVERSE MODES OF ACTION

Cytolysis mediated by the secretion of granzymes, the method typically used by natural killer (NK) cells and CD8⁺ cytotoxic T cells, is now considered a possible mechanism of suppression by Tregs. Additionally, cell–cell contact-dependent suppression plays an important role for the cell death receptor CD95, which mediates killing of CD8⁺ T cells via CD95L interaction (Strauss et al. 2009). Mechanisms that mediate the metabolic disruption of the effector T-cell target suggest that Treg cells induce apoptosis via IL-2 deprivation (Pandiyan et al. 2007). Tregs may also modulate the function of dendritic cells (DCs), which are required for the activation of effector T cells (Bluestone and Tang 2005). However, the complexity of suppressive function by Tregs and the great heterogeneity of the Treg population continue to challenge our understanding of the molecular mechanisms and physiological context of human Tregs.

Tregs control peripheral tolerance and have the ability to suppress the activation, proliferation, and effector functions (cytokine production) of several immune cells, including CD4⁺ and CD8⁺ T cells, NK and NK T (NKT) cells, B cells, and APCs (Roncarolo et al. 2006; Sakaguchi et al. 2008). As Tregs can control a variety of cell types under differential inflammatory conditions, investigating the mode of action and mediation of suppressive function has been a main target of research. In general, human CD4⁺ CD25⁺ FoxP3⁺ Tregs can exert suppressive function in four distinct ways: release of inhibitory cytokines, mediation of cytolysis, metabolic disruption of the target cell, and modulation of APCs (Vignali et al. 2008). Some mechanisms are specific to certain Treg subpopulations and are highly dependent on the microenvironment and type of immune reaction (Vignali et al. 2008; Sakaguchi et al. 2010). Most of the suppressive mechanisms have been described in in vitro suppression assays, leaving room for speculation about whether the mechanisms are similar in vivo. In 1998, Thornton and Shevach showed that murine Tregs can inhibit T effector cell activation through cell–cell contact (Thornton and Shevach 1998).

Studies over the following years discovered that Tregs, mostly linked to ICOS costimulation, release IL-10 and TGF-β to suppress effector T cells and APCs (Li et al. 2006; Ito et al. 2008; Vignali et al. 2008). Along this line, we have shown that subsets of human Tregs can secrete IL-10 during in vitro suppression assays to suppress effector T-cell proliferation (Baecher-Allan et al. 2006). FoxP3⁺ Tregs express high levels of CTLA-4, GITR, and other costimulatory receptors (Sakaguchi et al. 2010). Through CTLA-4 ligation of CD80 and CD86, FoxP3⁺ Tregs can modulate DC function, thereby inhibiting DC costimulation of effector T cells (Wing et al. 2008). This mechanism might work, at least in part, by trans-endocytosis, as it was shown that CTLA-4 of human Tregs was able to capture CD86 from interacting DCs (Qureshi et al. 2011). Another Treg-intrinsic strategy to modulate APC function is the induction of IDO. Induction and stabilization of this enzyme by T-cell CTLA-4 and IL-10 initiates the tryptophan catabolism, depriving the microenvironment of tryptophan. The lack of tryptophan causes production of inhibitory kynurenines, which block proliferation of effector T cells in the proximity of the DC (Fallarino et al. 2003; Mellor and Munn 2004; Munn et al. 2004a,b). Because human IDO⁺ DCs express CCR6 (Munn et al. 2002), the IL-10-producing CCR6⁺ effector memory-like Treg subset may amplify suppression by this mechanism while being attracted to similar sites through CCL20 gradients in vivo (Kleinewietfeld et al. 2005). Cytolysis mediated by granzyme A (GZMA) and perforin is another mechanism to suppress ongoing immune reactions (Grossman et al. 2004a,b). Metabolic disruption by cytokine deprivation is another mode of action Tregs can use to facilitate suppression of effector T-cell proliferation. Murine CD25⁺ Tregs can deprive the microenvironment of IL-2 by their high-affinity IL-2 receptor and thereby blocking effector T-cell growth (de la Rosa et al. 2004; Busse et al. 2010). However, it remains to be seen whether human Tregs can use a similar mechanism. The discovery that the ectonucleotidase CD39/ENTPD1 is highly expressed on a subset of human Tregs led to the identification of an additional and distinct pathway, namely, the hydrolysis of extracellular adenosine triphosphate (ATP) and the generation of immune-suppressive adenosine (Borsellino et al. 2007; Sakaguchi et al. 2010). Treg activation increases enzymatic activity of CD39, which facilitates the hydrolysis of extracellular ATP. As ATP is a classical “danger signal,” removal of ATP from the microenvironment can extend cell survival of Tregs, prevent APC activation, and generate antiproliferative adenosine through a CD39/CD73-induced signaling cascade (Borsellino et al. 2007; Deaglio et al. 2007). Joint expression of both CD39 and CD73 has only been shown on murine Tregs (Borsellino et al. 2007; Deaglio et al. 2007), so an involvement of the CD39/CD73 signaling cascade may only work in inflammatory settings with CD73 up-regulation on effector T cells (Dieckmann et al. 2002; Doherty et al. 2012). Finally, human Tregs can indirectly confer suppression by the induction of other immune-suppressive cells, a mechanism termed “infectious tolerance.” Along this line, human Tregs have the potential to induce IL-10-expressing Tr1-like cells (Dieckmann et al. 2002) through a mechanism partly mediated by TGF-β (Jonuleit et al. 2002).

REGULATORY T-CELL PLASTICITY: MIMICKING EFFECTOR T-CELL PHENOTYPES FOR SPECIFIC IMMUNE REGULATIONS

An emerging body of literature recently described the capacity of Tregs acquiring effector T-cell phenotypes to specifically regulate Th-effector T-cell subtypes in mice. Tregs can induce expression of Th-specific transcription factors T-bet, GATA3 and IRF4, RORγt and STAT3, or Bcl6 to control Th1, Th2, Th17, or T follicular helper (Tfh) effector T-cell responses, respectively (Chaudhry and Rudensky 2013; Vahedi et al. 2013). Along this line, we and others showed that human Tregs display a great magnitude of plasticity as well and, under certain inflammatory circumstances, can convert into Th1-like or Th17-like Tregs that show pathogenic phenotypes characterized by loss of suppressive function and release of proinflammatory cytokines (Kleinewietfeld and Hafler 2013).

Recent studies have been aimed at identifying underlying mechanisms. For Th17-like Tregs, it has been shown that in vitro stimulation by dectin-1-activated DCs leads to up-regulation of RORγt and IL-17 in Tregs (Osorio et al. 2008). Human Th17-like Tregs can be induced in vitro by stimulation in the presence of IL-6 and IL-1β, and could have pathogenic potential (Beriou et al. 2009; Voo et al. 2009). Nevertheless, human Th17-like Tregs keep their suppressive phenotype despite IL-17 production (Beriou et al. 2009; Voo et al. 2009). Further, Th17-like Tregs have been observed in humans and mice in the context of a variety of pathological conditions such as colon cancer (Kryczek et al. 2011; Blatner et al. 2012), inflammatory bowel disease (Hovhannisyan et al. 2011), skin lesions of psoriasis patients (Bovenschen et al. 2011), as well as arthritis (Komatsu and Takayanagi 2015).

Th1-like Tregs have been observed in both mice and human diseases and are characterized by the expression of the Th1 signature transcription factor T-bet as well as the type I cytokine interferon γ (IFN-γ) (Dominguez-Villar et al. 2011). Interestingly, studies performed in mice identified two distinct Th1-Treg subtypes: Th1-supressing Tregs and Th1-like Tregs, with the latter losing suppressive function and acquiring a pathogenic phenotype. During type I immune responses, NK cells and Th1 effector T cells release the proinflammatory cytokine IFN-γ. IFN-γ signaling, in turn, increases T-bet and IL12RB2 expression through STAT4, a mechanism used both by murine and human Tregs (Koch et al. 2012; Piconese et al. 2014b). Up-regulation of IL12RB2 renders Tregs susceptible to IL-12 signaling (Koch et al. 2009). We and others observed that culturing Tregs with IL-12 in vitro renders them dysfunctional, and induces a pathogenic phenotype characterized by IFN-γ release and up-regulation of typical Th1 markers, including T-bet and CXCR3 (Wei et al. 2009; Lu et al. 2010; Dominguez-Villar et al. 2011). Previous studies were unable to detect IL-12RB2 expression in Tregs ex vivo, leading to the assumption that a preexposure to IFN-γ is necessary for Tregs to become susceptible to IL-12. However, we and others showed that exposure of ex vivo Tregs to IL-12 alone is sufficient to increase IFN-γ expression in mouse and human Tregs (Oldenhove et al. 2009; Wei et al. 2009; Dominguez-Villar et al. 2011; McClymont et al. 2011). IL-12-induced IFN-γ-producing Th1-like Tregs show defects in proliferation, suppression, and reduced expression of IL-2RA (Zheng et al. 2009; Dominguez-Villar et al. 2011; McClymont et al. 2011). Supporting this observation, Bhairavabhotla and coworkers were able to detect IL12RB2 expression at the RNA level in the steady state of human Tregs ex vivo (Bhairavabhotla et al. 2016).

LOSS OF REGULATORY T-CELL FUNCTION IN AUTOIMMUNE DISEASE

The loss of dominant peripheral tolerance, which is normally controlled by Tregs, can lead to spontaneous autoimmune disease, immunopathology, metabolic disease, allergy, and loss of fetal–maternal tolerance during pregnancy. Therefore, defective Treg cells have a crucial role in regulating autoimmune responses. Dysfunctional Th1-like Tregs have been observed in a variety of autoimmune settings, including multiple sclerosis (MS) (Dominguez-Villar et al. 2011; Kitz et al. 2016), type 1 diabetes (T1D) (McClymont et al. 2011), de novo autoimmune hepatitis in patients with liver transplant (Arterbery et al. 2016), and in inflammatory bowel disease (IBD) (Holmen et al. 2006; Feng et al. 2011; Cao et al. 2014).

We examined the frequency and function of Tregs in a series of patients with MS using in vitro suppression assays, and observed a marked decrease in function (Fig. 1A), which has been widely replicated (Viglietta et al. 2004; Haas et al. 2005; Huan et al. 2005; Kumar et al. 2006; Feger et al. 2007; Venken et al. 2008a,b; Frisullo et al. 2009), whereas there was no significant difference in the frequency of Tregs in peripheral blood when compared with healthy control subjects (Putheti et al. 2003; Viglietta et al. 2004). However, MS patients did show an enrichment of Tregs with effector-memory-like phenotypes in their cerebrospinal fluid (CSF) compared with peripheral blood (Feger et al. 2007; Venken et al. 2008b).

Figure 1.

T regulatory cells (Tregs) from multiple sclerosis (MS) patients are less suppressive and produce more proinflammatory interferon (IFN)-γ. (A) Tregs from MS patients are significantly less suppressive compared with healthy controls. (From Viglietta et al. 2004; modified, with permission, from the authors.) (B) Tregs isolated from MS patients produce significantly increased level of IFN-γ but not interleukin (IL)-17A compared with healthy controls (Dominguez-Villar et al. 2011).

Although a proinflammatory cytokine expression in human CD25⁺ Treg cells was noticed from early on (Dieckmann et al. 2001), these data were complicated owing to the limitation of valid Treg markers and, thus, by potential contamination with effector T cells in Treg preparations. The presence of IFN-γ-producing cells was also observed in FoxP3-expressing cells after PMA/ionomycin stimulation (Kleinewietfeld et al. 2009) or after prolonged in vitro expansion of FoxP3⁺ Treg cells (Hoffmann et al. 2006, 2009). However, because FoxP3 can also be transiently up-regulated on human effector T cells (Gavin et al. 2006), it is unclear whether the IFN-γ-producing cells derived from Tregs or contaminated effector T cells.

We first discovered that untreated relapsing-remitting MS (RRMS) patients harbor an increased frequency of Th1-like Tregs in peripheral blood compared with healthy controls (Dominguez-Villar et al. 2011). Focusing on the Treg phenotype, Th1-like Tregs from MS patients showed increased expression of the classical Th1 markers CXCR3, T-bet, CCR5, and IFN-γ (Fig. 1B), whereas the expression level of TGF-β and CTLA-4 are decreased. The distinct phenotype translates into defects in function, with decreased proliferation and reduced suppressive capacity. Further, IFN-γ-producing Tregs displayed an almost fully demethylated TSDR region in the FoxP3 locus, similar to IFN-γ-negative Tregs, which was indicative of functional natural Tregs (Baron et al. 2007). IFN-γ-producing Tregs strongly contribute to the observed Treg dysfunction in RRMS patients, and blocking IFN-γ in coculture-suppression assays with RRMS-derived Tregs restored their suppressive capability (Dominguez-Villar et al. 2011).

Interestingly, an increased frequency of Th1-like Tregs could also be observed in humans with T1D (McClymont et al. 2011), indicating that Th1-like Tregs are associated with several autoimmune diseases in humans. It remains to be seen, however, whether the Th1-Treg plasticity plays a role for disease pathogenesis. Moreover, the question of whether this Treg subset has a physiological role under nonautoimmune conditions remains unanswered. In the context of intestinal inflammation, increased Treg frequencies, accompanied by increase ratios of potentially pathogenic IFN-γ and IL17-producing Tregs, have been observed in animal models and IBD patients (Holmen et al. 2006; Feng et al. 2011). Several animal models of colitis reported a potentially protective role of IFN-γ-producing Th1-Tregs, which suggests the presence of a Th1-supressing Treg subtype in this particular setting (Holmen et al. 2006; Feng et al. 2011).

Several observations provide possible mechanisms leading to the loss of Treg-suppressive capacity, and even a trend toward their aberrant proinflammatory activity in MS patients. Loss of suppression correlates with a decrease in FoxP3 and CTLA-4 expression (Huan et al. 2005; Venken et al. 2008b; Sellebjerg et al. 2012). Additionally, it has been seen that a lower thymic output and decreased CD58/CD2-dependent costimulation contributes to Treg loss of function (De Jager et al. 2009; Baecher-Allan et al. 2011). An altered distribution of distinct Treg subsets was also seen in RRMS patients (Haas et al. 2007; Venken et al. 2008a), and we specifically observed a lower frequency of CD39⁺ Tregs in these patients (Borsellino et al. 2007). The CD39⁺ Treg population is of particular importance for the suppression of Th17 cells, but is functionally impaired in MS patients (Fletcher et al. 2009).

The critical involvement of regulatory CD4⁺ CD25⁺ T cells in MS was initially indicated by studies in experimental autoimmune encephalomyelitis (EAE), a murine animal model of MS. The most significant findings show that the adoptive transfer of CD4⁺ CD25⁺ Tregs was sufficient to ameliorate disease, whereas the depletion of Tregs worsened disease in models of EAE (Randolph and Fathman 2006; Lowther and Hafler 2012). Interestingly, Th1-like Tregs have been observed in the central nervous system (CNS) of mice after EAE induction using myelin oligodendrocyte glycoprotein (MOG) (Korn et al. 2007). MOG-specific Tregs isolated from mice at different time points during EAE could not suppress MOG-specific effector T cells neither in vivo nor in vitro, and produced significant levels of IFN-γ at different disease stages (Korn et al. 2007).

It is interesting to note that FoxP3⁺ Tregs and IL-17-producing Th17 cells are reciprocally related; whereas Tregs are anti-inflammatory, Th17 cells are proinflammatory. The balance between these two cell populations is critical in the development of human autoimmunity (Sakaguchi et al. 2008; Korn et al. 2009), and TGF-β is a key determinant of this balance between Th17 and FoxP3⁺ Treg differentiation. Whereas TGF-β induces the differentiation of Treg cells, the combination of TGF-β with IL-6 or IL-21 induces Th17 cells while simultaneously blocking Treg differentiation (Korn et al. 2009). At a molecular level, FoxP3 can physically bind to Rorγt and Rorα to antagonize each other’s function (Du et al. 2008; Zhou et al. 2008). Furthermore, in the presence of retinoic acid, which enhances TGF-β signaling and blocks expression of the IL-6 receptor, Tregs are preferentially induced over Th17 cells (Mucida et al. 2007).

The notion that Tregs play a critical role in MS is further supported by the genetic changes seen in conventional FoxP3⁺ Tregs of MS patients. Disease susceptibility is highly associated with genes that are strongly implicated in Treg function. Of importance are the IL2RA and IL7RA loci (Hafler et al. 2007; Nylander and Hafler 2012). Genome-wide association studies (GWAS) on MS patients uncovered an association with the CD58 gene encoding for the costimulatory molecule lymphocyte function–associated antigen 3 (LFA-3). As previously described, CD58/CD2 costimulation is important in normal Treg function. After analyzing this variant in more detail, we saw that the allelic variant of CD58 leads to increased CD58 expression, which could potentially lead to FoxP3 up-regulation and enhanced Treg function through CD2 engagement. Interestingly, MS patients in clinical remission showed increased messenger RNA (mRNA) expression of CD58, suggesting restored Treg function, potentially because of increased CD58/CD2 costimulation (De Jager et al. 2009). Additionally, the transcription factor BTB and CNC homology 2 (BACH2), which is essential for Treg cell development and function in mice, was recently discovered as a new risk variant for MS, and is also associated with type 1 diabetes, asthma, Crohn’s disease, celiac disease, and vitiligo (Sawcer et al. 2011; Roychoudhuri et al. 2013).

Tr1 REGULATORY T CELLS

In 1990, Bachetta and coworkers described a suppressive CD4⁺ T cell type that was not dependent on FOXP3 expression, termed Tr1 cells. Tr1 Tregs are induced in the periphery and differ from Foxp3⁺ Tregs, namely, releasing IL-10, TGF-β, IL-5, granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, and low levels of IL-4, IL-17, and IL-2 (Bacchetta et al. 1990, 1994; Groux et al. 1997). Interestingly, IL-10 secretion is a rapid process in Tr1 cells already starting 4 h after activation and reaching a maximum release 12–24 h after activation (Bacchetta et al. 1994). Tr1 cells were also found to express coinhibitory molecules like CTLA4 (Bacchetta et al. 2002; Akdis 2008), PD-1 (Akdis 2008), and ICOS (Haeringer et al. 2009), as well as CD39 and CD73, indicating that suppressive function might be reached through generation of adenosine and disrupting the metabolic state of effector T cells (Bergmann et al. 2007). On stimulation, FoxP3⁻ Tr1 cells start to up-regulate FoxP3 expression, but the level of expression remains lower than in naturally arising or peripherally induced FOXP3⁺ Tregs (Levings et al. 2005; Brun et al. 2009, 2011).

Similar to FoxP3⁺ Tregs, IL-10-secreting Tr1 cells can be induced by CD58/CD2 costimulation (Wakkach et al. 2001), indicating that MS-associated protective allelic variant of CD58 is related to Tr1 cells as well. Higher expression levels of CD58 could potentially trigger TR1 cell induction, and subsequently impact IL-10 secretion in humans. Along this line, increased CD58 mRNA levels in MS patients during remission might be correlated with increased IL-10 levels, as well as enhanced Tr1 cell frequency and function (De Jager et al. 2009).

However, when comparing MS patients with healthy control subjects, we observed a remarkable reduction in IL-10 secretion and suppressive function in Tr1 cells (Astier et al. 2006). Notably, this observation only held true in Tr1 cells induced by anti-CD46 costimulation, but not under anti-CD28 stimulatory conditions; this only impacted IL-10 secretion but not CD46 surface expression, proliferation, or IFN-γ production (Astier et al. 2006). To investigate the effect of IFN-β treatment on Tr1 cells, we analyzed CD46-induced IL-10 secretion in RRMS patients with IFN-β therapy. Although IFN-β therapy was reported to increase IL-10 levels associated with remissions in RRMS patients, we did not observe restored Tr1 cell function in IFN-β-treated patients (Astier and Hafler 2007), although another study could identify slight changes in a subset of patients receiving IFN-β (Chiarini et al. 2012). Focusing on CD46, we were able to correlate the impaired Tr1 function in RRMS patients with altered isoform expressions of CD46. RRMS patients showed increased expression of the isoform Cyt2 (CD46.2), which carries a cytoplasmic tail that leads to proinflammatory responses and blocks IL-10 secretion (Marie et al. 2002; Astier et al. 2006; Choileain et al. 2011). The defective Tr1 response in RRMS was replicated in a larger cohort of patients, linking the reduced IL-10 induction to abnormal IL-10 signaling in MS patients (Martinez-Forero et al. 2008). In line with the functionally impaired Tr1 population in RRMS patients, similar findings were observed in a model of MS in cynomolgus monkeys (Ma et al. 2009). A recent study indicated that the defective CD46-triggered Tr1 response of RRMS patients could be partly restored in vitro in the presence of vitamin D3 (Kickler et al. 2012).

Th1-LIKE TREGS: MANY PATHS LEAD TO IFN-γ SECRETION

In terms of Treg functionality, we have observed several stimuli and pathways rendering Tregs dysfunctional, promoting IFN-γ secretion, and destabilizing Treg phenotype through Foxo1 phosphorylation. Although IFN-γ is clearly involved in decreased Treg function, suppressive capacity can be restored by blocking of IFN-γ (Dominguez-Villar et al. 2011), and different stimuli use distinct pathways to induce IFN-γ release.

We observed Th1-like Tregs in RRMS patients, as discussed above, but also observed this in vitro through IL-12 induction (Fig. 2A,B), and in culture conditions with sodium chloride (Fig. 2C), as well as in GBM (Sarbassov et al. 2005; Gonzalez and McGraw 2009; Kerdiles et al. 2010; Ouyang et al. 2010, 2012; Dominguez-Villar et al. 2011; Hernandez et al. 2015; Huynh et al. 2015; Lowther et al. 2016).

Figure 2.

In vitro induction of defective T helper (Th)1-like T regulatory cells (Tregs) from healthy donors using interleukin (IL)-12 or salt. (A) IL-12 induces interferon γ (IFN-γ) release in Tregs from healthy controls and multiple sclerosis (MS) patients, and (B) decreases suppressive function (Dominguez-Villar et al. 2011). (C) 40-mm sodium chloride significantly reduces Treg-suppressive function in Tregs isolated from healthy donors (Hernandez et al. 2015).

Th1-like Tregs generated through IL-12 stimulation, as well as in RRMS patients, are highly correlated with activation of the PI3K/AKT/Foxo1/3 pathway (Kitz et al. 2016). Activation of this pathway starts with different stimuli that trigger a variety of receptors such as the TCR (Ward et al. 1992), costimulatory molecules such as CD28 (Di Cristofano et al. 1999), cytokine receptors (Migone et al. 1998), G protein–coupled receptors (GCPRs) (Schwindinger and Robishaw 2001), and insulin (Knight et al. 2006), among others. The AKT family is highly multifunctional and the PI3K/AKT pathway is involved in various cell functions. PI3K has recently been shown to be crucial in regulating Treg homeostasis and stability (Huynh et al. 2015). The AKT family further consists of three protein isoforms, AKT1, AKT2, and AKT3, each mediating differential downstream signaling events (Gonzalez and McGraw 2009). After the initial stimulation, PI3K is activated through phosphorylation, causing it to catalyze the formation of the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) (Okkenhaug 2013). PIP3 binds to PDK1 and AKT, allowing PDK1 to phosphorylate the Thr 308 residue on AKT (Stokoe et al. 1997). For full activation, AKT also has to be phosphorylated at Ser 473 by the mTROC2 complex (Sarbassov et al. 2005). Activated AKT then has the ability to further phosphorylate different downstream targets involved in a variety of cell functions, including the transcription factors Foxo1 and Foxo3 (Brunet et al. 1999), which are involved in Treg development and phenotype stabilization (Kerdiles et al. 2010; Ouyang et al. 2010, 2012). On activation, these transcription factors are excluded from the nucleus and thus can no longer regulate their transcription targets (Kitz et al. 2016). Treg-specific deletion of Foxo1 in the nucleus of mice resulted in a loss of Treg function and ectopic expression of IFN-γ (Ouyang et al. 2012). Consistent with these results, we show that the PI3K/AKT/Foxo1/3 pathway plays a pivotal role in obtaining a Th1-like phenotype in human Tregs, and, interestingly, the AKT1 isoform specifically contributes to Th1 polarization (Kitz et al. 2016).

The critical result of the activation of this major pathway is the induction of IFN-γ secretion. This is directly correlated with reduced suppressive capacity of Tregs. In other words, increased frequency of IFN-γ⁺ FoxP3⁺ Tregs leads to severe impairment of suppressive capacity (Kitz et al. 2016). IFN-γ secretion by Tregs is a signature defect in immune regulation observed in patients with multiple sclerosis. After blockade of this major pathway in Tregs from MS patients, IFN-γ secretion was inhibited and the suppressive ability of these Tregs was restored. Thus, this experiment suggests that the PI3K/AKT/Foxo1/3 pathway was activated in vivo in RRMS patients. On PI3K or AKT1 inhibition, we saw inhibited IFN-γ secretion in RRMS Tregs, but no effect on Tregs from healthy controls (Kitz et al. 2016). This further supports that the Th1-Treg phenotype observed in Tregs from RRMS is, at least in part, the result of an in vivo activation of this major pathway, which can be corrected by PI3K or AKT1 blockade.

Dietary salt has recently been linked to a variety of diseases and is discussed as an environmental factor contributing to the increase and clinical manifestation of multiple sclerosis (Farez et al. 2015; Hucke et al. 2016). The increase of dietary salt intake has been linked to increased sodium accumulation in skin, muscle, and lymphoid tissues (Go et al. 2004; Kopp et al. 2013; Wiig et al. 2013). The impact of increased tissue sodium levels on immune cells has been the focus of research. In recent studies, we and others showed that physiologic sodium concentrations directly favor the induction of Th17 T cells and initiate an inflammatory signature in human T cells in vitro (Kleinewietfeld et al. 2013), as well as in EAE in vivo where a high salt diet induced aggravated disease pathogenesis (Wu et al. 2013). Both human salt-induced Th17 T cells and Th17 T cells obtained from mice with EAE showed elevated expression of the serum- and glucocorticoid-regulated kinase-1 (SGK1), a serine/threonine protein kinase involved in the regulation of the natrium channel ENaC (Kuntzsch et al. 2012; Lang and Shumilina 2013). We then investigated the effect of sodium chloride treatment on Tregs and discovered that sodium chloride renders Tregs dysfunctional (Fig. 2C) and drives them toward a Th1-like phenotype both in vitro and in a mouse model of GvHD (Hernandez et al. 2015). Sodium-treated FoxP3⁺ Tregs up-regulated T-bet expression, released higher amounts of IFN-γ, and displayed higher levels of Foxo1 phosphorylation. Fitting our observations in salt-induced Th17 effector T cells, sodium-treated FoxP3⁺ Tregs expressed elevated levels of SGK1, emphasizing its role in salt-induced Treg dysfunction. Interference of the SGK1-IFN-γ axis by pharmacological inhibitors, or by lentiviral transmitted short hairpin RNA (shRNA) against either SGK1 or IFN-γ, could, in turn, restore Treg-suppressive function (Go et al. 2004; Kuntzsch et al. 2012; Kopp et al. 2013; Lang and Shumilina 2013; Wiig et al. 2013; Farez et al. 2015; Hucke et al. 2016; Jorg et al. 2016).

In cancer therapy, immunotherapies targeting T-cell surface markers have recently shown promising results. Monoclonal antibodies targeting CTLA-4 (Leach et al. 1996; Phan et al. 2003) and PD-1 (Brahmer et al. 2012; Topalian et al. 2012) are successful in modulating tumor immune responses in malignant tumors and improve clinical outcomes (Sznol and Chen 2013). PD-1 is expressed on a subset of tumor-infiltrating Tregs in the context of cancers in lung (Zhang et al. 2010) and brain (Jacobs et al. 2009). However, the function of these Tregs is largely unpredicted, as PD-1 can be involved both in Tregs activation and exhaustion. We were recently able to further characterize tumor-infiltrating Tregs in the context of glioblastoma multiforme (GBM) and observed that PD-1⁺ FoxP3⁺ Tregs from the circulation of both GBM patients and healthy controls show a reduction in suppressive capacity (Lowther et al. 2016) and release higher levels of IFN-γ (Fig. 3A,B). As we have seen in other circumstances, dysfunctional PD-1⁺ FoxP3⁺ Tregs produced elevated levels of IFN-γ (Fig. 3C,D), and blocking of IFN-γ could partially restore suppressive function. Additionally, PD-1⁺ FoxP3⁺ Tregs showed increased phosphorylation of Foxo1, indicating an increased activity of the PI3K/AKT pathway and subsequent destabilization of the Treg phenotype. On the other hand, circulating PD-1⁺ FoxP3⁺ Tregs expressed elevated level of exhaustion markers. Further, transcriptional profiling of tumor-infiltrating PD-1⁺ FoxP3⁺ Tregs identified even higher mRNA levels of exhaustion markers and coexpression of IFN-γ. PD-1 is usually a suppressor of the PI3K/AKT pathway so that increased PD-1 expression and exhaustion may occur to regulate and stabilize the PI3K/AKT pathways. Additionally, Foxo1 phosphorylation was recently shown to be associated with migration of Tregs into tissues, arguing that tumor-infiltrating PD-1⁺ FoxP3⁺ Tregs recirculate from the tumor tissue (Luo et al. 2016).

Figure 3.

Dysfunctional T helper (Th)1-like T regulatory cells (Tregs) in tumors. (A) Tregs isolated from tumor biopsies release high levels of interferon γ (IFN-γ), and (B) are less suppressive (Lowther et al. 2016). (C) PD-1^hi Tregs isolated from healthy controls produce higher levels of IFN-γ, which correlates with (D) lower suppressive capacity (Lowther et al. 2016).

CONCLUDING REMARKS

In summary, there is clear evidence that CD4⁺ CD25⁺ Treg cells play a crucial role in autoimmune disease, specifically MS. The genetic factors related to MS are directly or indirectly linked to Treg cell phenotype and function. Plasticity of Tregs toward Th1 and Th2 phenotypes is a significant factor in MS pathogenesis. During Th1-Treg reprogramming, the PI3K/AKT/Foxo1/3 pathway is activated, triggering IFN-γ secretion. This consequentially reduces the suppressive capacity of Tregs. Environmental factors can also contribute to defective immune function. Specifically, we have seen that salt can enhance proinflammatory Th17 activity (Fig. 4) (Kleinewietfeld et al. 2013), while disrupting Treg-suppressive activity (Hernandez et al. 2015). Factors that regulate plasticity in these cell populations will be critical for developing immunotherapies for autoimmune diseases and, reciprocally, for cancer.

Figure 4.

The balance between tolerance and autoimmunity. Differentiation of regulatory T cells (Tregs) into dysfunctional T helper (Th)1-like Tregs enables autoimmunity. Treg cells express coinhibitory molecules glucocorticoid-induced tumor necrosis factor (TNF) receptor–related protein (GITR), LAG3, and cytotoxic T-lymphocyte antigen 4 (CTLA4) and release, among others, interleukin (IL)-10 to regulate effector T cells and maintain peripheral tolerance. Environmental factors (i.e., salt or the proinflammatory cytokine IL-12) can trigger Treg differentiation into IFN-γ-producing Th1-like Tregs, which up-regulate other Th1 effector markers like CXCR3, T-bet, and ICOS and lose capacity to suppress autoreactive effector T cells, leading to autoimmunity.

The available data on Treg cells in MS is complex, indicating that several mechanisms and subset populations could contribute to the observed effects of defective Treg function and its association to the disease. There is an exponential combination of genetic and environmental factors that can interact to contribute to disease development. The role of the gut microbiota in the induction, maintenance, and function of Treg cells is under preliminary investigation. It was shown that both Bacteroides fragilis and Clostridial species, which can be found in the microbiome, produce short-chain fatty acids, inducing Treg responses (Round and Mazmanian 2010; Rudensky and Chervonsky 2011). Additionally, future research to study the Treg-cell phenotype and function in the CNS or CSF would be necessary for further clarity on the role of CD4⁺ CD25⁺ Treg cells in MS.