Developmental Plasticity of Murine and Human Foxp3+ Regulatory T Cells

CHAPTER THREE

Developmental Plasticity of Murine and Human Foxp3+ Regulatory T Cells Adrian Liston*, Ciriaco A. Piccirillo†,1

*Autoimmune Genetics Laboratory, VIB and University of Leuven, Campus Gasthuisberg, Leuven, Belgium † FOCIS Centre of Excellence, Department of Microbiology & Immunology and Medicine, McGill University and Research Institute of McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Stability and Plasticity of Regulatory T Cells 2.1 Homeostatic stability of Foxp3þ regulatory T cells 2.2 Programmed plasticity of Foxp3þ regulatory T cells 3. A Transient Flexibility Model for Regulatory T Cell Plasticity 3.1 Enhanced regulatory T cell plasticity during the initiation phase of infection 3.2 Regulatory T cell stability during the active phase of infection 3.3 Transient Treg cell development during the resolution phase of infection 3.4 Implications of the transient flexibility model beyond the context of infections 4. Differences in Foxp3/FOXP3 Between Mice and Humans 5. Significance and Future Directions Acknowledgments References

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Abstract Murine and human CD4þ regulatory T (Treg) cells expressing the Forkhead box p3 (Foxp3) transcription factor represent a distinct, highly differentiated CD4þ T cell lineage that is programmed for dominant self-tolerance and control of immune responses against a variety of foreign antigens. Sustained Foxp3 expression in these cells drives the differentiation of a regulatory phenotype and ensures the stability of their suppressive functions under a variety of inflammatory settings. Some recent studies have challenged this premise and advanced the notion that Foxp3þ Treg cells manifest a high degree of functional plasticity that enables them to adapt and reprogram into effector-like T cells in response to various inflammatory stimuli. The concept of Treg cell plasticity remains highly contentious, with a high degree of variation in measured plasticity potential observed under different experimental conditions. In this chapter, we

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propose a unifying model of Treg cell plasticity, which hypothesizes that the stable fates of regulatory and effector T (Teff ) cell lineages allow transient plasticity into the alternative lineage under a discrete set of microenvironmental influences associated with, respectively, the initiation and resolution phases of infection. This model utilizes a theoretical framework consistent with the requirements for effective immune regulation and accounts for both the extraordinary long-term stability of Treg cells and the observed fate plasticity.

1. INTRODUCTION A plethora of evidence shows that a functionally specialized Treg cell lineage in the thymus, specifically expressing the forkhead winged helix family transcription factor Foxp3, maintains dominant peripheral tolerance toward self- and non-self-antigens (Sakaguchi, 2004). Originally defined as CD4þ T cells constitutively expressing the IL-2 receptor alpha chain (CD25) (Sakaguchi, Sakaguchi, Asano, Itoh, & Toda, 1995), CD4þFoxp3þ Treg cells are critical mediators in the control of immune responses. These CD4þ Treg cells constitute 1–10% of thymic and peripheral CD4þ T cells in the naive T cell repertoire of rodents and humans. Neonatal thymectomy, antibody depletion, or other changes that compromise the development or function in Foxp3þ Treg cells break tolerance against self- and non-selfantigens, trigger multiorgan autoimmunity, and unleash immunity against tumors, transplants, infectious and commensal microbes, and allergens (Sakaguchi, 2004). Foxp3þ Treg cells are frequently subdivided into “natural” Treg cells that arise in the thymus (tTreg) and “induced” Treg cells that differentiate either in the periphery (pTreg) or under in vitro conditions (iTreg) (Abbas et al., 2013). While some phenotypic differences have been observed between tTreg and some in vitro- or in vivo-generated pTreg/ iTreg subsets, few (if any) experiments are designed to allow the division of the naturally occurring peripheral Treg pool into tTreg and pTreg. The primary difference between the naturally occurring tTreg/pTreg pool and experimentally induced pTreg/iTreg populations is in inferior lineage stability in the induced populations, with Foxp3 expression probably initiating a similar suppressive program in both lineages (Feuerer, Hill, Mathis, & Benoist, 2009). Recent studies show that stable expression of Foxp3 is required for the genetic programming of Treg cell lineage commitment, homeostasis, and function. The autoreactive T cell receptor repertoire drives Foxp3 expression in Treg cells (Hsieh et al., 2004; Hsieh, Zheng, Liang, Fontenot, & Rudensky,

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2006), although the local antigenic milieu can also contribute (Lathrop et al., 2011). Mutation of the Foxp3 gene in Scurfy mice results in a loss in Foxp3 protein and Treg cells, leading to the development of the Scurfy syndrome, a fatal, multiorgan autoimmune condition, and reminiscent to the outcome of Foxp3-deficient (/), day 3 thymectomized or Treg-depleted mice, which can be corrected by transfer of CD4þFoxp3þ Treg cells (Brunkow et al., 2001; Sakaguchi et al., 1995). Expression of Foxp3 in conventional CD4þCD25 T cells leads to the acquisition of suppressive activity in vitro and in vivo. Together, this demonstrates that Foxp3 expression is both necessary and sufficient for Treg cell suppressive function in mice. Moreover, inheritable mutations of the FOXP3 gene lead to immune dysregulation polyendocrinopathy enteropathy X-linked, a rare, Scurfy-like, multiorgan autoimmune disorder in humans, demonstrating the conserved function of FOXP3 (Bennett et al., 2001; d’Hennezel et al., 2009).

2. STABILITY AND PLASTICITY OF REGULATORY T CELLS 2.1. Homeostatic stability of Foxp31 regulatory T cells As Foxp3þ Treg cells are indispensable for maintaining dominant selftolerance, the global suppressive function of Treg cells must be both durable and stable. This issue of cell lineage stability is particularly critical for Treg cells as they readily undergo robust cell expansion upon T cell activation and possess a TCR repertoire capable of recognizing self-antigens (Hsieh et al., 2004). As such, a stable and committed cell lineage has been viewed as a necessary precondition to ensure self-tolerance under homeostatic and inflammatory conditions (Piccirillo, d’Hennezel, Sgouroudis, & Yurchenko, 2008). The identification of Foxp3 as the master switch of Treg cell lineage identity and programming of regulatory functions has promoted this concept (Fontenot, Gavin, & Rudensky, 2003; Hori, Nomura, & Sakaguchi, 2003; Khattri, Cox, Yasayko, & Ramsdell, 2003). Experimental data also support this conceptual framework of Treg cell stability. Foxp3þ Treg cells labeled by an inducible Cre–Lox system show a high degree of lineage stability, with cells retaining Foxp3 expression for at least several months (Rubtsov et al., 2010). The mechanisms of stable Foxp3 expression in Treg cells are known to act at three levels. First, there are classical feedback loops initiated, whereby Foxp3 expression drives further Foxp3 expression, possibly via CD25 upregulation and Stat5 activation (Antov, Yang, Vig, Baltimore, & Van Parijs, 2003; Zheng et al., 2007), with

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Foxp3 induction in Foxp3-deficient T cells resulting in lower transcription of the locus (Gavin et al., 2007). Likewise, Foxp3 expression negatively regulates pathways leading to alternative fates, as Foxp3þ Treg cells have tighter control over cytokine expression than Foxp3low or Foxp3-deficient “Treg” cells (Gavin et al., 2007; Wan & Flavell, 2007). Second, following Foxp3 expression, an epigenetic modification of the locus is initiated (Floess et al., 2007). Epigenetic feedback loops are mediated by demethylation of a noncoding, evolutionarily conserved element of the Foxp3 locus in Foxp3þ Treg cells (Floess et al., 2007). When the Foxp3 locus is demethylated through azacytidine treatment, even basic activation is sufficient to induce Foxp3 expression (Polansky et al., 2008). These epigenetic changes in turn promote direct feedback loops, as Foxp3 is able to directly bind to a conserved noncoding sequence (CNS2) in the Foxp3 gene when it is demethylated in stable Foxp3þ T cells, amplifying its own expression (Zheng et al., 2010). The full extent of how epigenetic variables induce, maintain, or modulate Foxp3 expression, or its downstream targets in the Treg differentiation program, is not fully understood and warrants further investigation at the whole-genome level. The third stability mechanism is less well understood but is mediated by the microRNA network, as Dicer/ Treg cells have normal suppressive capacity during homeostasis but lose suppressive identity when challenged by an inflammatory context (Liston, Lu, O’Carroll, Tarakhovsky, & Rudensky, 2008). This effect may be mediated by a cluster of five microRNA (miR-7, miR-18a, miR-21, miR-34a, and miR-155) that suppress SATB1 (Beyer et al., 2011) and miR-146a-mediated suppression of Stat1 (Lu et al., 2010). Together, these three mutually dependent feedback processes likely serve to lock Treg cells into a Foxp3-expressing profile under homeostatic conditions.

2.2. Programmed plasticity of Foxp31 regulatory T cells Despite the theoretical and experimental underpinnings of a stable and committed Treg cell lineage, recent studies have challenged this basic concept and have provided the alternative model that Treg cells instead display the intrinsic potential to deconvert or reprogram into various Teff cell lineages in response to a distinct array of environmental cues (Duarte, Zelenay, Bergman, Martins, & Demengeot, 2009; Komatsu et al., 2009). For example, Foxp3þ Treg cells differentiate into Th17 cells in the presence of exogenous IL-6 and TGFb1, suggesting a certain level of functional plasticity at least in vitro (Yang et al., 2008). In vivo, Foxp3þ Treg cells can readily

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downregulate Foxp3 expression upon transfer into lymphopenic environments, in the course of an infection, in the context of organ-specific autoimmunity, or in tumor microenvironments (Komatsu et al., 2009; Sharma et al., 2010). An alternative Cre–Lox fate-mapping study to the one described above came to a mixed conclusion—with both a high level of stability in existing Treg cells and clear signs of transient Foxp3 expression in non-Treg cells (Miyao et al., 2012). Together, these studies suggest that, in certain contexts, Foxp3þ Treg cells manifest a high degree of functional plasticity; lose their expression of Foxp3, and hence suppressor phenotype; become functionally unstable; and “reprogram” or differentiate into Teff cells that secrete proinflammatory cytokines. In particular, but not exclusively, those subsets of in vitro- and in vivo-generated iTreg/pTreg cells display a transient nature of Foxp3 expression. A lingering, controversial question concerns the cellular origin of newly generated reprogrammed Foxp3þ T cells, and in some cases, it is unclear whether they developed from bona fide Foxp3þ Treg cells or from residual Foxp3 T cells. While some researchers have suggested that the potential for reprogramming is a feature of all Foxp3þ Treg cells, others have suggested that an unstable subpopulation found within the Foxp3þ Treg cell pool is uniquely reprogrammable. As an example of the latter, Komatsu and colleagues proposed that an unstable subpopulation found within the Foxp3þ Treg cell pool, low for CD25 expression, selectively retains developmental plasticity, in contrast to the Foxp3þCD25þ cell subset representing a stable Treg population (Komatsu et al., 2009). Furthermore, which different inflammatory signals affect Foxp3 expression and Treg cell plasticity in vivo remains poorly defined, as does the molecular process by which the stability mechanisms are bypassed. The conditions under which a Treg cell loses its lineage identity may depend both on the origin of the cell and on the nature and robustness of the microenvironmental signals in select anatomical tissues.

3. A TRANSIENT FLEXIBILITY MODEL FOR REGULATORY T CELL PLASTICITY With experimental data favoring both high stability and potential plasticity, a model is required to accommodate these divergent cellular outcomes. Fortunately, two observations can aid in uniting an otherwise fragmented literature. First, Foxp3þ Treg cells show a diversity in functional stability and therefore in their susceptibility to lineage reprogramming,

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based on intrinsic genetic and epigenetic history. The division between tTreg and iTreg cells is a formal demonstration of this variation, but diversity within the tTreg, pTreg, and iTreg populations is equally likely to exist. Second, the plasticity of Foxp3þ Treg cells can be influenced by external microenvironmental cues. These two factors, intrinsic variation and responsiveness to external factors, can interact synergistically to influence fate decisions (Fig. 3.1). We propose a “transient flexibility model” to unite the disparate observations on stability and plasticity (Fig. 3.2). In this model, Foxp3 neither dictates an irreversible fate decision, as in CD4-CD8 lineage determination, nor does it merely ensure a transient lineage, as in CD4þ Th cell differentiation. Under this model, not all Foxp3þ Treg cells have an equal flexibility potential: both the mode of Foxp3 activation (thymic induction in CD4þ singlepositive thymocytes or peripheral induction in Foxp3 precursors such as through TGFb1 or retinoic acid) and the strength and duration of Foxp3 expression may alter the flexibility potential that cells bear. In addition to this intrinsic variation in stability is the effect of the local microenvironment, which can either increase or decrease the stability of Treg cells, based on the

Figure 3.1 Microenvironmental conditioning of Foxp3þ regulatory T cell plasticity. The timing and duration of Foxp3 imprinting during Treg cell development determine the degree of plasticity and lineage reprogramming. The local microenvironment exposes Treg cells to a variety of stabilizing and destabilizing signals, which influence the induction and stability of Foxp3 expression threshold and consequential potential for plasticity.

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Figure 3.2 The transient flexibility model of regulatory T cell stability during infection. Under the transient flexibility model, the capacity to induce flexibility during infection increases the rate of clearance and inflammation resolution. While the Treg cell lineage is stable under homeostasis, during infection initiation, the effect of TLR ligands and early cytokines increases Treg cell plasticity, allowing reprogramming into ex-Treg cells and a more rapid escalation of immunity. In the active phase of infection, where specific Teff are available, the shift in the local environment allows ex-Tregs to spontaneous revert, reducing the risk of induced autoimmunity. In the resolution phase of infection, where the key pressure is on preventing inflammation, even antigen-specific Teff may transiently enter the Treg cell lineage to quench inflammation and restrict tissue damage. As these induced pTreg cells are unstable, they revert during the postinfectious period, allowing normal recall responses.

combination of factors present. Thus, our flexibility model establishes different thresholds for the induction/stability of Foxp3þ Treg cell subsets depending on history of the cell and the nature of the microenvironment. The “transient flexibility model” builds upon the “heterogeneity model” proposed by Hori and colleagues (Miyao et al., 2012), by asking the underlying question of the evolutionary advantage in allowing Treg cell plasticity, that is, unifying the observations of transient flexibility which creates a heterogeneous pool of Tregs with various levels of plasticity into a context where heterogeneity would have clear evolutionary advantage. This question of why is quite critical, as the existence of fate flexibility poses obvious immunological disadvantages to the host: first, deconversion of self-reactive

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Treg cells to Teff cells may be potentially harmful to the host by increasing the autoimmune risk, and second, Teff to Treg cell cellular conversion risks limiting future legitimate responses to a variety of antigens and shutting down immune responses against cancers. In the transient flexibility model, we make use of the context of different stages of infection to illustrate the basis on which the evolutionary advantage of transient flexibility may be achieved (i.e., to outline a plausible scenario under which Treg cell plasticity may have evolved, without making the claim that this model has been verified). The context of infection represents one of the most challenging microenvironments for Treg cells. Treg cells must not only permit an immune response to the infectious agent and control the infection-related pathology but also maintain lineage identity. The loss of a regulatory phenotype in an inflammatory environment has potential to unleash the autoimmunity encoded in the Treg cell repertoire (Gavin et al., 2007; Williams & Rudensky, 2007). Despite the general requirement for Treg cell stability during infection, the relative costs and benefits of unstable Treg cell identity are theoretically likely to vary across the different stages of infection, where a theoretical basis for decreased and increased Treg cell stability can be found during the initiation and resolution phases of infection, respectively.

3.1. Enhanced regulatory T cell plasticity during the initiation phase of infection During the initiation phase of an infection, the primary selective pressure of the host is to generate a rapid response and recruit inflammatory mediators into infected sites. In principle, the temporary suspension of Treg cell function or identity (including within the otherwise stable tTreg population) may be tolerated in this context, as the cost of short-term autoimmunity may be outweighed by the benefit of more rapid escalation of immunity (Oldenhove et al., 2009; Sharma et al., 2010). In support of this supposition is the evidence that various pathogen-associated molecular patterns (PAMPs) create instability in Treg cells in vitro, with signaling through TLR2, TLR4, TLR7, or TLR9 reducing Foxp3þ T cell suppressive activity and stability, potentiating reprogramming Treg cells into an effector lineage (Hackl, Loschko, Sparwasser, Reindl, & Krug, 2011; Liu, Komai-Koma, Xu, & Liew, 2006; Nyirenda et al., 2011; Sharma et al., 2010; Zhu et al., 2011). These effects may be synergistic with an enhanced resistance of Teff cells to Treg cell suppression, or altered Teff/Treg balance due to differential homing, expansion, or survival in situ, in turn, creating a temporal phase

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where Treg cell suppression is suspended and Treg cells may contribute to the immune response (Oldenhove et al., 2009; Zhang et al., 2011). Cytokines induced early in the infection response may have a similar effect to TLR signaling, as IFNa (Golding, Rosen, Petri, Akhter, & Andrade, 2010; Yan et al., 2008), IL-1 (Deknuydt, Bioley, Valmori, & Ayyoub, 2009; Koenen et al., 2008; Raffin, Raimbaud, Valmori, & Ayyoub, 2011), nitric oxide (NO), TNF-a, and IL-6 (Yang et al., 2008) can reduce Treg cell function, either directly or through dendritic cell-mediated effects. The molecular mechanisms by which these signals reduce Foxp3 expression or regulatory function remain unknown. One intriguing possibility for the IFNa effect is the observation that IFNa can downregulate Dicer expression in several cell types (Wiesen & Tomasi, 2009), which may reduce the microRNA stability program and result in transient loss of regulatory identity (Liston et al., 2008). Thus, while microbial-derived signals may directly trigger immune cell recruitment and APC function, and subsequently polarize T cell responses, these same mediators may help drive antipathogen immunity by reprogramming Treg into Teff cells. What remains unclear is whether reprogrammed effector-like Treg cells manifest a stable Teff cell phenotype following activation, undergo apoptosis following a transient fate as Teff cell, or retain the potential to revert to their parental origin, particularly after extensive cell division in vivo. One study has shown that reprogrammed Foxp3þ Treg cells can reacquire Foxp3 expression under TGFb-inducing conditions in vitro (Floess et al., 2007). If such is the case, this may suggest that TCR reactivation in these cells reestablishes the “memory” of original Foxp3 expression in parent cells by enabling de novo active transcription at the remodeled Foxp3 locus.

3.2. Regulatory T cell stability during the active phase of infection During the active phase of immunity to pathogens, Treg cells are exposed to cytokines that promote entry into the Th1, Th2, and Th17 effector lineages. Despite this, during this stage Foxp3þ T cells are remarkably stable, with those Foxp3þ Treg cells labeled during homeostasis having a very high retention of Foxp3 expression at the peak of the immune response against Listeria monocytogenes (Rubtsov et al., 2010). Thus, the lineage instability provoked by microenvironmental signals during the initiation phase of infection must be regained during the active phase of infection, unless Listeria infection ends up being atypical in this regard. The ability of Foxp3þ T cells to maintain Foxp3 expression and suppressive function under these

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conditions is dependent on the microRNA stability network, as it is lost in Dicer/ cells (Liston et al., 2008). Specifically, the resistance of Treg cells to entering the Th1 lineage is enforced by miR-146a suppression of SOCS1 (Lu et al., 2010), although other microRNA contribute to the phenotype (e.g., through suppression of SATB1; Beyer et al., 2011), as miR-146a/ Treg cells have a less severe phenotype than Dicer-deficient Treg cells. Interestingly, IFNg has been reported to enhance Dicer expression (Wiesen & Tomasi, 2009), leading to the possibility that Foxp3þ T cells respond to active inflammation by increasing the buffering capacity of the microRNA stability network. Other positive feedback loops that may be present during the active phase of immunity include enhanced expression of CD28 ligands and IL-2, both of which are important for Foxp3 induction (Fontenot, Rasmussen, Gavin, & Rudensky, 2005; Tai, Cowan, Feigenbaum, & Singer, 2005). Consistently, we recently showed in a model of Plasmodium chabaudi AS malaria infection that IL-2 maintains a tight balance between Treg and effector CD4þ Th1 cells and consequently influences the host ability to eliminate pathogens (Berretta, St-Pierre, Piccirillo, & Stevenson, 2011). On the reverse side of the equation, cytokines associated with the active phase of injection (IFNg, IL-4, IL-6, IL-21) suppress the induction of Foxp3 in Teff cells (Miyao et al., 2012), providing feedback loops to both Treg and Teff cells to maintain cell fate during this crucial period.

3.3. Transient Treg cell development during the resolution phase of infection In contrast to the initiation phase of infection, the selective pressure during the resolution phase of an infection is to rapidly reduce local inflammation. In the context of a resolved infection, the conversion of Teff cells into a regulatory lineage may be beneficial, as it would drive a more rapid suppression of immunity and consequent pathology than the recruitment and expansion of preexisting Treg cells alone in infected sites. Such conversion would pose a detrimental risk of reducing the immunogenicity of a recall response but would be ameliorated if the induction of Foxp3 and suppressive capacity were transient. The primary support for this hypothesis stems from the function of TGFb as a transient stabilizing or amplifying strategy for the induction of Foxp3þ T cells. TGFb is a classical resolution phase cytokine which is frequently associated with Treg cell activity: either direct TGFb production or indirectly as a consequence of protection in situ. While TGFb has been associated with the effector phase of Treg cell function in vitro and in vivo, TGFb has a strong capacity to induce Foxp3 expression during in vitro

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T cell activation (Chen et al., 2003), and peripheral numbers of Foxp3þ T cells are reduced, although they retain some functionality, in TGFb/ mice (Marie, Letterio, Gavin, & Rudensky, 2005), demonstrating the critical role of this cytokine in the peripheral maintenance of the Treg pool. However, TGFb has little apparent role in thymic induction of Foxp3 (Marie et al., 2005), and Foxp3þ Treg cells induced in vitro by TGFb exposure have poor stability when transferred in vivo (Lal et al., 2009) or when assessed by cell fate methods (Miyao et al., 2012). While TGFb can promote Treg cell survival and expansion, these features suggest that the TGFbmediated Foxp3 induction pathway is specialized for rapid and transient induction of Foxp3, rather than stable long-term lineage commitment. The molecular mechanism for transient entry into the Treg cell lineage may be the capacity of TGFb to induce high amounts of Foxp3 protein without creating the demethylation of the Foxp3 locus that stabilizes Foxp3 expression over an extended duration (Miyao et al., 2012; Polansky et al., 2008). Without demethylation of CNS2, Foxp3 is unable to bind the Foxp3 locus (Zheng et al., 2010), removing one of the feedback loops that ensure stable expression. Notably, TGFb induction utilizes a distinct conserved region in the Foxp3 locus (CNS1) (Zheng et al., 2010) but is not required for tTreg differentiation (Schlenner, Weigmann, Ruan, Chen, & von Boehmer, 2012), further supporting a model where TGFb can drive Foxp3 expression without inducing the standard stability mechanisms. In some instances of chronic inflammation, in contrast to acute forms, committed stable tTreg or pTreg cells may promote the TGFb expression in inflammatory sites to convert some local responders into short-term-induced pTreg cells. Once pathology/inflammation has been resolved, these induced pTreg cells would display greater plasticity and can either die or default back to being Teff cells. This would endow Treg cells with the potential to rapidly shut down a response and could explain why TGFb uses a distinct and less stable regulatory element to activate Foxp3 expression. Thus, TGFb is an ideal candidate as a molecular mediator for the transient conversion of Teff cells into Treg cells during the resolution phase, enabling a rapid restriction of inflammation while preserving the long-term capacity of these cells to act as antimicrobial effectors in subsequent infection challenges. Additional mediators associated with the resolution of infection may also contribute to pTreg induction. For example, IL-10, a potent, immunomodulatory cytokine frequently produced during resolving or chronic infections, has multifunctional roles: while IL-10 can induce the generation of Foxp3 Treg cells, it can also mediate many effector suppressive functions

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of Treg cell subsets and stabilize Foxp3 expression in committed Treg cells, particularly in mucosal immunity. In addition, angiogenic and tissue remodeling factors would be key candidates for further testing. Another possibility is that damage-associated molecular patterns (DAMPs) could contribute to the process, as the PAMP:DAMP ratio would be inverted between the initiation and resolution phases. The shared components of PAMP and DAMP signaling pathways, such as TLR4, could be responsible for some of the contradictory in vitro data about the effect of TLR4 antagonists on Treg cell fate (Jia et al., 2012; Manfredi, Capobianco, Bianchi, & Rovere-Querini, 2009; Milkova et al., 2010).

3.4. Implications of the transient flexibility model beyond the context of infections The transient flexibility model is intended as a conceptual framework by which the current literature can be integrated and interpreted, rather than an absolute explanation of Treg cell behavior. While the purpose of this model is to unify most divergent observations, some examples of conditions that do not fit this model can be found in the literature (e.g., contradictory effects have been reported for TLR4 ligands either stabilizing or destabilizing Foxp3 expression; de Kleer et al., 2010; Zhu et al., 2011). Nevertheless, this model, as applied to the salient case of microbial infections, not only fits the majority of research on Treg cell stability and plasticity but also generates hypotheses on Treg cell behavior well beyond the classical context of infection, namely, autoimmunity and cancer. During typical autoimmune responses, dominant inflammatory stimulators consist primarily of DAMPs rather than PAMPs, due to the absence of an infectious organism. Exposure to DAMPs has been demonstrated to directly drive the autoimmune reaction in mouse models (Ehrchen, Sunderkotter, Foell, Vogl, & Roth, 2009; Loser et al., 2010). As these conditions mimic those associated with chronic rather than acute stages of infection (Rubartelli & Lotze, 2007), the transient flexibility model would predict an increase in transient Treg cell induction in the local, autoinflammatory sites. Indeed, autoimmunity is frequently accompanied by an increase in local (as opposed to systemic) Treg cell numbers, such as in the synovial fluid of rheumatoid arthritis (RA) patients (Appel et al., 2011; Cao et al., 2006; de Kleer et al., 2004; Han, O’Neil-Andersen, Zurier, & Lawrence, 2008; Jiao et al., 2007; Mottonen et al., 2005; Nistala et al., 2008). In a mouse model of diabetes, the autoimmunity driven

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by pathogenic Teff cells results in a local increase in Treg cell number (Grinberg-Bleyer et al., 2010), indicating that the direct or indirect effects of DAMPs can positively modulate Treg cell function in inflammatory sites. This model likely does not provide a universal mechanism for all autoimmune diseases, and variations across different disease conditions, disease stage, sampling site, and Treg cell identity make cross-study comparisons problematic. While a transient flexibility in T cell responses may be beneficial during typical autoimmune responses, it does allow the potential for maladaptive “loopholes” for the host through two molecular routes. First, the signaling differences between PAMPs and DAMPs are subtle and the outcome on the Teff/Treg cell balance in inflammatory sites is strongly influenced by combinatorial factors which include the capacity of TGFb to induce Teff or Treg cell subsets depending on the presence of IL-6 (Bettelli et al., 2006). Thus, autoimmunity arises when a skewed cytokine microenvironment promotes a cycle of pathogenicity and pathology, during which Teff cell activity dominates that of Treg cells. Second, the flexibility of Treg cells transiently reconverting into Teff cells during infection initiation may enable the development of autoimmunity during this critical period. An interesting example of this scenario may be reactive arthritis, where autoimmunity is triggered by infection (Sieper, 2004). The contrast between RA and reactive arthritis may provide a natural experiment in which to test the transient flexibility model. This model would predict that analysis of synovial T cells from chronic RA, driven by DAMPs, would demonstrate the presence of induced pTreg cells with low stability, while synovial T cell from acute reactive arthritis, initiated by PAMPs, would show molecular signs of ex-Treg cells producing inflammatory cytokines. Measurement of Treg cell numbers in these two diseases would be insufficient, as inflammation can drive increased Treg cell numbers via homeostasis, independent of conversion (Franceschini et al., 2009). In the context of cancer, there is strong evidence that the transient flexibility model is maladaptive. Tumor tissues replicate the molecular signature of the infection resolution stage, with prominent TGFb expression, angiogenesis factor production, and tissue remodeling (Atanackovic et al., 2008). The TGFb expression, in particular, is responsible for the induction of Treg cells in the tumor environment and subsequent dampening of antitumor immunity (Conroy, Galvin, Higgins, & Mills, 2012; Jarnicki, Lysaght, Todryk, & Mills, 2006; Liyanage et al., 2006; Moo-Young et al., 2009). It is therefore likely that tumors are taking advantage of a molecular loophole

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in the evolved T cell fate flexibility, by “tricking” the immune system into acting as though the infection resolution phase was in place.

4. DIFFERENCES IN Foxp3/FOXP3 BETWEEN MICE AND HUMANS In mice, the evidence of Foxp3 as a self-sustaining mediator of the Treg cell fate is unambiguous, at least in the homeostatic context (under alternative microenvironments, there may be an underappreciated degree of plasticity, as described above). In humans, by contrast, the function of FOXP3 as a stable lineage identifier is debatable. As in mice, human CD25þ Treg cells express FOXP3, and transfection of naive T cells with FOXP3 is sufficient to confer suppressive capacity (Yagi et al., 2004). However, even conventional human T cells transiently express FOXP3 upon activation (Walker et al., 2003), albeit to lower levels than bona fide Treg cells (Gavin et al., 2006). While frequently described as a major difference between murine and human Foxp3/FOXP3, this observation may be the result of subtle differences in regulation or experimental heterogeneity rather than any profound change in function. Ultimately, the difference between Foxp3 regulation in mice and FOXP3 regulation in humans could simply depend on the relative importance of different mechanisms of stabilization. For example, murine Foxp3 may have greater direct feedback loops, such that initial expression needs to be tightly regulated to prevent activated T cells from forming stable Treg cells. By contrast, human FOXP3 may initiate fewer direct feedback loops so that leaky expression during activation does not need to be tightly controlled, as only prolonged expression resulting in epigenetic modifications will result in the formation of stable Treg cells. In this regard, it is notable that even low expression of murine Foxp3, insufficient to grant suppressive capacity, results in relatively stable Foxp3 expression (Wan & Flavell, 2007), while low FOXP3 expression in humans is associated with unstable expression patterns and subsequent FOXP3 loss (d’Hennezel, Yurchenko, Sgouroudis, Hay, & Piccirillo, 2011). The physiological significance of this transient expression in the absence of an associated suppressive phenotype in humans is unclear. It is possible that the transient upregulation of FOXP3 within human T cells represents a mechanism by which recently activated CD4þ T cell sensitizes themselves to Treg induction but requires additional stabilization pathways in order to achieve complete regulatory conversion. Similarly, if transient FOXP3 upregulation occurs in the absence of these

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stabilization factors, activated T cells default to the effector lineage. The nature of the local inflammatory environment likely influences the FOXP3 expression threshold and consequential influence of T cell responses. Alternatively, conclusions drawn from both the murine and human studies may be inaccurately reflecting the plasticity of the system. On the mouse side of the equation, ex vivo Treg cells used for stability analysis are almost invariably extracted from unmanipulated mice. The very high degree of lineage specificity and stability (Rubtsov et al., 2010) may therefore reflect a level of distinction between naive effector and unactivated Treg cells that is uncomplicated by the rich diversity of various activation states present in an adult human. This diversity was revealed in a clonal analysis of FOXP3þ cells, which demonstrated a heterogeneous population of stable and unstable expressers (d’Hennezel et al., 2011). In human T cells, while detection of FOXP3 is not limited to cells with regulatory function, high levels of expression (Gavin et al., 2006) and epigenetic modification of the FOXP3 locus (Baron et al., 2007) are very strong indicators of regulatory status. Thus, part of the interspecies discrepancy may lie in the complexity of mixed activation statuses present in humans and absent in mice under specific pathogen-free conditions. A rich diversity of Foxp3 plasticity may equally be present in mice under particular conditions, including inflammatory, metabolic, nutritional, and other environmental conditions that can modulate Treg cell stability. Indeed, recent research suggests that activation of T cells in mice can induce low levels of transient unstable Foxp3 induction (Miyao et al., 2012), similar to that reported in humans. Rather than using stable Treg cells from unmanipulated mice as the reference group to human Treg cells, a better comparison may be the diversity of Treg cell fates present at homeostasis, and during initiation and resolution phases of infection. Such a combination, described in the transient flexibility model, would include stable Foxp3 expressers, unstable Foxp3 expressers, and even ex-Foxp3 expressers, a cocktail that corresponds well to the heterogeneity observed in humans (d’Hennezel et al., 2011).

5. SIGNIFICANCE AND FUTURE DIRECTIONS Here, we propose a model of transient fate flexibility to unify the contradictory observations regarding the sustained, long-term stability (Rubtsov et al., 2010) and high degree of plasticity (Duarte et al., 2009; Komatsu et al., 2009) in the Treg cell lineage. To date, the majority of research on Treg cell stability has focused on lineage entry and stability primarily during the

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homeostatic state. Collectively, the results of these studies have provided us with the basic conceptual framework of Treg cell biology and the tools for further study. Our transient flexibility model is proposed here as a call for future experiments to focus on challenging the predictions the model makes. In addition to studies on Treg cell homeostasis, future studies should be directed at Treg cell lineage stability during immunological contexts that mimic the initiation and resolution phases of infectious challenge. Formal testing is required to determine whether, as we propose, the microenvironmental conditions during inflammatory initiation are able to temporally deactivate Treg cell activity, while the conditions during infection resolution are able to transiently induce a Treg cell function in normal Teff cells. Fate-mapping experiments assessed during homeostatic conditions may blur otherwise highly dynamic, but temporally restricted, lineage plasticity. The model described here can be studied by fate-mapping experiments of Treg and Teff lineages, specifically to test the functional dynamics and fate of ex-Treg cells in tissues during the initiation phase of infection, and the induction of Treg cells in similar sites during the resolution phase of infection, with both cell types retaining the potential to revert to their original status when the system returns to homeostasis. The concept of Treg cell plasticity described here raises questions about the safety of human Treg cell therapy in current and future immunotherapeutic regimes. Specifically, isolated Treg cells from PBMC likely represent a heterogeneous population that includes stable and unstable tTreg cell and pTreg cell populations. Moreover, in vitro induction of FOXP3 expression in non-Treg cell precursors may be insufficient to ensure stable expression in vivo. Immune monitoring of FOXP3þ Treg cell function in various clinical settings is critical to our understanding of their role in the pathogenesis of many human diseases and therapies. Considering the dynamics of FOXP3 expression in committed FOXP3þ Treg cells, and the nature and magnitude of inflammation as critical factors modulating the plasticity of FOXP3þ Treg cells, great caution should be taken in designing future treatments involving Treg cell therapies. As Treg cell-based therapy is proposed as a treatment in autoimmune and transplantation settings, it is critical to first determine the nature of the inflammatory factors that alter the stability of FOXP3þ Treg cells. If the molecular cues that provide flexibility to Treg cell fate during infection initiation and resolution can be dissected, the potential capacity to alter the regulatory threshold will be greatly increased in therapeutic

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settings. Unlike homeostatic conditions, which are difficult to alter due to their stable and durable properties, these conditions provide transient contexts where the number or function of Treg cells can be reduced or amplified accordingly. For example, in the context of cancer treatment, triggering the “infection initiation” program may suspend the function of tumor-protecting Treg cells, while in the context of autoimmunity, replication of a “resolution phase” context may succeed in inducing regulatory status in pathogenic cells. Unlike proposed interventions which directly modulate Treg cell number (i.e., by cell therapy or depletion) and would therefore be moderated by homeostatic population controls, therapeutics based on such molecular cues would have the potential for greater success as they exploit pathways of preexisting plasticity in Treg cell fate.

ACKNOWLEDGMENTS The authors would like to acknowledge the contribution of Steven Josefowicz, Ste´phanie Humblet-Baron, Susan Schlenner, Ekaterina Yurchenko, Eva d’Hennezel, Maria da Silva Martins, Khalid Bin Dhuban, and Mara Kornete in developing the ideas presented in this chapter. We acknowledge grant support from VIB (A. L.) and IAP (VII/39) (A. L.), the Canadian Institutes for Health Research (CIHR) (MOP 67211) (C. A. P.), and from the Canada Research Chair (C. A. P.) program.

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