What defines the TReg cell phenotype and is it stable?

Shimon Sakaguchi. The cardinal pheno-typical features of thymus-derived CD4+ regulatory T (TReg) cells include their con-stitutive expression of the transcription factor forkhead box P3 (FOXP3), their cell surface expression of CD25 (the interleukin-2 receptor α-chain (IL-2Rα); a component of the high affinity IL-2R) and their cell surface and cytoplasmic expression of the co-inhibitory receptor cytotoxic T lymphocyte antigen 4 (CTLA4)1. Each molecule has its own indispensable function in TReg cells: CD25 is required for TReg cell survival (and presumably for IL-2 absorption as part of TReg cell-mediated suppression); CTLA4 is involved in the suppressive function of TReg cells by down-regulating CD80 and CD86 expression on antigen-presenting cells; and FOXP3 is essential for TReg cell development and suppressive activity, partly through the maintenance of high levels of CD25 and CTLA4 expression.

Regarding phenotypical markers in mice, FOXP3 is the most specific marker for delineating TReg cells from other T cells, whereas CD25 and CTLA4 are less specific,

because they can also be expressed by conventional T cells. These molecules are stably expressed in physiological states, but can be temporally downregulated under certain conditions. For example, when FOXP3+ TReg cells are transferred to a lymphopenic environment, a fraction of FOXP3+ T cells lose the expression of FOXP3 and CD25 (REF. 2). However, such T cells appear to be capable of re-expressing FOXP3 and of reacquiring TReg cell suppres-sive activity upon T cell receptor (TCR) stimulation, presumably because they retain a TReg cell-specific epigenetic status that facilitates the expression of TReg cell-associated molecules, including FOXP3, upon antigenic stimulation3 (see below). In addition, IL-2 that is produced by con-ventional T cells can inhibit the downregu-lation of FOXP3 expression in these cells; co-transfer of conventional T cells with FOXP3+ T cells into lymphopenic hosts prevents the conversion of FOXP3+ T cells into FOXP3− T cells because the conven-tional T cells provide IL-2 (REF. 2). Thus, the phenotype of thymus-derived TReg (tTReg) cells is essentially stable even if TReg cells may occasionally and temporally down-regulate the expression of FOXP3 and other regulatory-associated molecules.

Dario A. A. Vignali. There are three key characteristics of the TReg cell phenotype. First, a potent suppressive capacity is clearly the main phenotypical characteristic of TReg cells. The diversity of suppressive mecha-nisms at their disposal is impressive and is an important feature that endows them with the capacity to influence a very broad range of cell populations and to mediate suppres-sion in a variety of anatomical locations and disease scenarios4. I would speculate that there are important suppressive mechanisms that remain to be identified. Second, it is clear that FOXP3 is an essential transcrip-tion factor required to manifest the TReg cell phenotype5,6. However, it is also clear that FOXP3 does not function alone and that the expression of additional transcription factors is required to define the TReg cell phenotype and to establish their characteristic tran-scriptional programme7. It is also becoming clear that unique epigenetic changes that are partly induced by TCR signalling are typical of the TReg cell lineage8. Collectively, this epi-genetic landscape and transcriptional profile both give rise to a unique TReg cell signature that is characterized by the expression of molecules that can be used to facilitate their identification5,6,9,10. Third, TReg cell develop-ment and survival are very dependent on a number of key factors and signals, including IL-2, transforming growth factor-β (TGFβ) and co-stimulatory molecules (such as CD28). It is possible that additional key factors remain to be identified.

There are some features of the TReg cell phenotype that have been proposed that I feel may be misleading and, thus, are worth discussing here. First, TReg cell suppression is not predominantly ‘contact dependent’ as has been suggested. While TReg cells can certainly suppress other cells via contact-dependent mechanisms, cytokines clearly have a key role4. This misunderstanding may be partly explained by our observation that it is the potentiation or ‘boosting’ of TReg cell function that has a crucial contact-depend-ent requirement, rather than the suppression itself 11. Second, the notion that TReg cells suppress in a ‘non-antigen-specific manner’ is misleading. It is certainly true that TReg cells can suppress T cells that recognize a different antigenic epitope, but TReg cells still

V I E W P O I N T

The plasticity and stability of regulatory T cellsShimon Sakaguchi, Dario A. A. Vignali, Alexander Y. Rudensky and Rachel E. Niec, and Herman Waldmann

Abstract | Regulatory T (TReg

) cells are crucial for the prevention of fatal autoimmunity in mice and humans. Forkhead box P3 (FOXP3)+ T

Reg cells are

produced in the thymus and are also generated from conventional CD4+ T cells in peripheral sites. It has been suggested that FOXP3+ T

Reg cells might become

unstable under certain inflammatory conditions and might adopt a phenotype that is more characteristic of effector CD4+ T cells. These suggestions have caused considerable debate in the field and have important implications for the therapeutic use of T

Reg cells. In this article, Nature Reviews Immunology asks several

experts for their views on the plasticity and stability of TReg

cells.

PERSPECTIVES

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 1

Nature Reviews Immunology | AOP, published online 17 May 2013; doi:10.1038/nri3464

© 2013 Macmillan Publishers Limited. All rights reserved

recognize antigen and require TCR signal-ling for optimal activation and function. Third, the notion that TReg cells have a ‘low proliferative capacity’ is inaccurate and is merely an in vitro characteristic owing to the lack of IL-2 in the culture medium. In fact, TReg cells proliferate robustly in inflamma-tory environments (such as in tumours and in the islets of NOD mice), often to a greater extent than other T cell subsets.

The question of TReg cell stability is more complex. There are two FOXP3+ TReg cell populations: tTReg cells and peripherally derived ‘induced’ TReg (pTReg) cells12. Subtle differences in these two populations, such as the methylation status of conserved non-coding sequence 2 (CNS2; also known as the TReg cell-specific demethylated region (TSDR)) in the FOXP3 locus, may underlie much of the contentious debate over the issue of stabil-ity5,6. Most researchers agree that under nor-mal circumstances tTReg cells are very stable and long-lived13. However, it has been argued that under inflammatory or pathogenic con-ditions some TReg cells may become ‘unstable’, losing FOXP3 expression and developing fea-tures that are more characteristic of effector T cell populations (termed ‘ex-TReg cells’)14. However, the prevalence of this instability

remains contentious, as does the origin of this ‘unstable’ population. There are three possibilities. First, it is possible that a ‘genu-ine’ tTReg cell population becomes unstable. Second, it is possible that this perceived instability only affects pTReg cells, which cannot yet be unambiguously distinguished from tTReg cells. Third, it has been suggested that transient FOXP3 expression in a small T cell population may inadvertently define these cells as ex-TReg cells but these cells may never have been destined to become a stable bona fide TReg cell population3. Although more analysis is required, the bottom line is that FOXP3+ TReg cells are predominantly stable. However, it is possible that a small percentage may become unstable under unique and/or extreme circumstances.

Alexander Y. Rudensky and Rachel E. Niec. TReg cells are defined by having a suppres-sive phenotype endowed by high and sus-tained expression of the transcription factor FOXP3, as well as by other TReg cell signature genes. The TReg cell lineage is highly stable owing to the multiple mechanisms that maintain high levels of FOXP3 expression after the establishment of the TReg cell transcriptional programme13 (see below).

FOXP3 is considered to be the lineage specification factor for this T cell subset. Indeed, forced FOXP3 expression in CD4+ T cells results in a regulatory phenotype and function, whereas experimental deletion of the Foxp3 gene in differentiated TReg cells results in the loss of their suppressive capac-ity15–17. However, FOXP3 cooperates with other nuclear factors, the activity of which must precede or coincide with the expres-sion of FOXP3 to establish the molecular signature and function of TReg cells. FOXP3 binds to a set of pre-existing regulatory elements that become accessible in pre-cursor cells in response to TCR signals of a particular strength, as well as in response to other cues8,18. Thus, although it is required, FOXP3 expression alone does not impart the characteristic suppressive and homeostatic features of TReg cells and, therefore, its expres-sion does not unequivocally mark the cells to be TReg cells.

Indeed, FOXP3 can be expressed by non-regulatory T cells; cells can initiate and then discontinue FOXP3 expression or they can express FOXP3 in a ‘non-permissive’ chromatin landscape. Multiple studies have demonstrated transient activation-induced upregulation of FOXP3 expression in both mouse and human T cells19,20. These obser-vations of transient FOXP3 expression do not necessarily indicate TReg cell lineage instability, as it is possible that these cells might never have fulfilled the strict pheno-typical and molecular criteria necessary to be defined as bona fide TReg cells. Indeed, the examples of transient expression of FOXP3 refer to cells that never fully engaged in the positive auto-feedback loop at the Foxp3 locus (discussed below) and thus have not committed to the TReg cell lineage. The con-ditions of cellular activation might induce a state of functional ‘adolescence’ — an inter-mediate maturation stage in which newly generated TReg cells might engage in some eclectic behaviour — before establishing the full TReg cell character and commitment.

Herman Waldmann. The errant description of suppressor T cells in the 1970s has been followed by a general acceptance in the early 1990s that some CD4+ T cells can and do regulate immune responses. Among these suppressive cells is a population of T cells that express the transcription factor FOXP3. Even this population of cells has been shown to exhibit significant heterogeneity, some of which is related to a physiological need for these regulators to express certain features of the different T cell subsets that they control21, while a further source of

The contributors*

Alexander Y. Rudensky is Chairman of the Immunology Program at the Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York, USA, and Investigator with the Howard Hughes Medical Institute, USA. His research is focused on investigating the development of T cells, as well as their function and role in the regulation of immune responses to infection and inflammation and in the prevention of autoimmunity. His research is also focused on developing novel methods of tumor immunotherapy and intervention in a wide range of autoimmune and inflammatory diseases, as well as in chronic infections. As a member of the Rudensky laboratory, Rachel E. Niec has focused her research on regulatory T cell lineage stability and peripherally generated T

Reg cells. Rachel is a

member of the Tri-institutional M.D./Ph.D. programme.

Shimon Sakaguchi obtained his M.D. and Ph.D. in Japan, and trained as a pathologist and immunologist in Japan and the United States. He is currently the Vice Director for human immunology and is Professor at the World Premier International Immunology Frontier Research Center, Osaka University, Japan. His main research interest is in immunological tolerance and immune regulation.

Dario A. A. Vignali received his Ph.D. in 1988 from the London School of Hygiene & Tropical Medicine, University of London, UK. He then held postdoctoral positions at the German Cancer Research Center, Heidelberg, Germany, and then at Harvard University, Cambridge, Massachusetts, USA. He is currently Vice Chair and Member (Full Professor equivalent) of the Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. His research focuses on the molecular and cellular aspects of regulatory T cell function, immune regulation by inhibitory receptors and inhibitory cytokines (interleukin-35) in tumour immunity, mucosal immunity and type 1 diabetes. He also studies proximal events in T cell receptor–CD3 signalling.

Herman Waldmann is Emeritus Professor of Pathology at the University of Oxford, UK, after he recently retired as Head of the Sir William Dunn School of Pathology, University of Oxford. His main research interests have been to therapeutically harness the body’s own tolerance processes to enhance the acceptance of transplants and the reversal of autoimmune diseases. His laboratory was responsible for the discovery and academic development of the CAMPATH-1 antibodies that culminated in the licensing of alemtuzumab (Lemtrada). His current research focuses on the mechanisms by which regulatory T cells mediate dominant (infectious) tolerance.

*Listed in alphabetical order

P E R S P E C T I V E S

2 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/immunol

© 2013 Macmillan Publishers Limited. All rights reserved

heterogeneity is the so-called ‘stability and plasticity’ of their functional status. The cur-rently used terms stability and plasticity do not distinguish between epigenetic stability within individual TReg cells and competition with cellular contaminants, uncommitted cells or cells that have reverted from more committed cells in the particular sample population under investigation. In addition, although we are really concerned with ‘func-tional’ stability, what is often measured is the stability of FOXP3 expression, as though that alone were the basis for suppressive function. However, it is becoming clear that the post-translational state of FOXP3 may be important to its function and that pro-inflammatory cytokines can affect this22. Not surprisingly, these types of uncertain-ties all contribute to the current confusion regarding the stability and plasticity of TReg cells.

Some of the controversies about TReg cell stability may not be as irreconcilable as they might appear at first, but could well reflect the limitations of the read-out systems that are used; indeed, in these systems the con-text (for example, inflammatory rather than quiescent environments, or lymphopenic rather than replete immune systems) deter-mines the extent of the epigenetic changes that are associated with increasing com-mitment to the TReg cell phenotype3,13,23–25. Moreover, as most studies investigating the stability of TReg cells have used bulk popula-tions rather than single cells26, the seemingly robust interpretations of such studies may not completely reflect the characteristics of individual cells at different stages of develop-ment. The most ‘stable’ TReg cell, even when it is fully differentiated, may still have the capacity for destabilization if given the appropriate stimulus.

How might TReg cell stability or plastic-ity be regulated? To what extent does

the context of antigen exposure determine the stability of TReg cells?

D.A.A.V. I prefer to view ‘stability’ and ‘plasticity’ as different terms, with stability determined by whether a FOXP3+ TReg cell becomes a FOXP3− ex-TReg cell, and plasticity determined by whether a TReg cell changes its migratory or functional capabilities but still maintains its fundamental FOXP3+ TReg cell identity.

Although FOXP3+ TReg cells are pre-dominantly stable, I feel it is very probable that there are several external signals and factors (such as Toll-like receptor (TLR)-induced IL-6) that could induce TReg cell

instability27 and, thus, that there are several counteractive mechanisms that are required to maintain TReg cell stability, particularly in inflammatory environments. Determining the factors and mechanisms on both sides of this equation will be important. These factors and mechanisms could act by impinging on the expression and function of FOXP3 (or other transcription factors that are required for the TReg cell phenotype), by modulating the expression of effector T cell programmes that need to be repressed in TReg cells, or by modulating the expression of survival and/or quiescence factors that need to be enhanced in TReg cells.

TReg cell plasticity is probably regulated by a complex network of cooperative and counteractive transcription factors. While much still remains to be clarified, it does appear that certain lineage-specific tran-scription factors can be expressed by TReg cells, endowing them with an enhanced capacity to regulate effector T helper (TH) cell populations that use the same transcrip-tion factors (such as interferon-regulatory factor 4 (IRF4), signal transducer and activa-tor of transcription 3 (STAT3) or T-bet)28–30. The extent to which this is a selective pro-cess as opposed to a regulated process is unclear. There is also the important issue of functional plasticity and whether all TReg cell-associated suppressive mechanisms can be used by every TReg cell, or whether there are subpopulations that have different subspecialities that allow them to adapt to different environments. It seems probable that TReg cells have mechanisms that allow them to adapt to highly varied targets and microenvironments, either on a single-cell basis or as a population.

The context of antigen exposure or, more specifically, the extent of TCR and/or co-stimulatory signalling is likely to affect TReg cell stability. Indeed, TReg cells in inflammatory sites are probably exposed to three sets of signals that will determine their stability: one set of signals from the TCR and co-stimulatory molecules, another set of signals that drive instability and a final set of signals (both cell-intrinsic and cell-extrinsic) that actively maintain stability. Indeed, the latter may constitute the primary basis for the TReg cell stability observed. However, the molecular details of these signals and how they might interact remain unknown.

A.Y.R. and R.E.N. Once the TReg cell tran-scriptional programme is established, there are multiple mechanisms in place to endow TReg cells with remarkable stability;

that is, the capacity to heritably maintain their transcriptional and functional fea-tures. FOXP3 expression maintains the TReg cell suppressive phenotype; when FOXP3 is experimentally ablated in mature TReg cells, the resulting cells are capable of producing effector cytokines and causing inflammation17.

FOXP3-dependent lineage commitment at least partly relies on the demethylation of a CpG island that is located in a regulatory element in CNS2 of the FOXP3 locus31,32. Deletion of CNS2 does not affect the initial acquisition of high levels of FOXP3 expres-sion, but it results in cell division-dependent loss of FOXP3 expression. FOXP3, with the assistance of the transcription factor runt-related transcription factor 1 (RUNX1), binds to CNS2 in the demethylated state to maintain FOXP3 expression32. Accordingly, some FOXP3-expressing cells, for example, those that have been generated in vitro in the presence of TGFβ or those that have been recently generated in vivo and that fail to demethylate the CpG island, lose FOXP3 expression despite the fact that it was ini-tially expressed at high levels3,19,33. These cells may represent uncommitted precursor cells that have yet to choose a lineage.

Glossary

Asymmetric T cell divisionA process by which two daughter cells can inherit different amounts of immune receptors and signalling components from a parent cell during T cell division. It has been suggested that this process occurs because of the polarity of the dividing cell that is associated with immunological synapse formation and that it could specify different fates to the progeny of an individual T cell.

Conserved non-coding sequence 2(CNS2). The element engaged in the positive auto-feedback loop that confers heritable maintenance of forkhead box P3 (FOXP3) expression once demethylated. Also known as the TSDR (TReg cell-specific demethylated region).

CpG islandA sequence of 0.5–2 kilobases that is rich in CpG dinucleotides. CpG islands are mostly located upstream of housekeeping genes and also upstream of some tissue-specific genes. They are constitutively non-methylated in all animal cell types.

Infectious toleranceThe ability of a tolerized population of T cells to induce tolerance in a new, naive population of T cells. Tolerance might be to the same antigens or to new antigens that are encountered in the same context (linked suppression). Newly tolerized T cells can, in turn, induce tolerance in other T cells.

NOD miceNon-obese diabetic (NOD) mice spontaneously develop a form of autoimmunity that closely resembles human type 1 diabetes.

P E R S P E C T I V E S

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 3

© 2013 Macmillan Publishers Limited. All rights reserved

Furthermore, other transcription factors have been shown to bind to CNS2 and other regulatory elements at the Foxp3 locus and may contribute to stable FOXP3 expression34; these transcription factors include nuclear factor of activated T cells (NFAT), cyclic AMP-responsive element-binding protein (CREB)–ATF, nuclear factor-κB (NF-κB) and ETS1 (which are downstream of TCR activa-tion), as well as STAT5 and SMADs (which are activated through IL-2 and TGFβ recep-tor signalling). Although the extent to which these factors and persistent IL-2R signalling stabilize FOXP3 expression is not yet clear, FOXP3 expression alone may be insufficient to maintain lineage commitment. Thus, the stability of the TReg cell lineage is probably acquired in a two-step manner: through the establishment of an epigenetic state, including the demethylation of CNS2, that is favourable for continued FOXP3 expression and through the subsequent stabilization of this state by FOXP3 autoregulation at the demethylated CNS2.

Cells that only transiently express FOXP3 for the aforementioned reasons or that fail to establish an epigenetic state that is favour-able for the binding of FOXP3 at target sites do not represent fully differentiated and functionally competent TReg cells; rather, they may represent an uncommitted intermediate cell state or a cell that has aborted TReg cell differentiation. The multistep process of TReg cell commitment affords uncommitted precursor cells the option of differentiating into alternative cell lineages as instructed by environmental cues. Once the stabilizing FOXP3 positive feedback regulatory pathway has been established, circumstances in vivo that disrupt this regulatory programme of lineage stability are probably rare but may exist in extreme inflammatory conditions or upon IL-2 deprivation13,14,35.

S.S. FOXP3+ TReg cells are functionally and phenotypically stable after many cell divisions in inflammatory sites13. This indicates that stability is maintained by particular types of constant cell-extrinsic stimuli (such as antigen, co-stimulation and cytokines) and/or by a cell-intrinsic mechanism that induces TReg cells to express TReg function-associated molecules.

Regarding the cell-intrinsic mechanism, there is accumulating evidence to suggest that the epigenetic status of the TReg cell is crucial for the maintenance of its function and pheno type8,36,37. Among several epi-genetic mechanisms, DNA hypomethylation of specific genes is the most important for sustaining TReg cells as a cell lineage because

it is highly inheritable, relatively stable and linked to gene expression. A recent genome-wide study has shown that TReg cells retain a TReg-specific DNA hypomethylation pattern, especially in the genes encoding TReg function-associated or TReg-specific molecules such as CTLA4 and the zinc finger proteins Helios and Eos8. The induction of this TReg-type epi-genetic pattern is FOXP3 independent, and both TReg cell epigenetics and FOXP3 expres-sion depend on TCR stimulation. Developing TReg cells that have these epigenetic changes but lack expression of FOXP3 express low levels of CD25 and CTLA4; however, FOXP3 appears to sustain the high and constitutive expression of these molecules in TReg cells. Thus, FOXP3-expressing TReg cells with these TReg-type epigenetic changes are functionally and phenotypically stable. However, a small fraction of FOXP3+ T cells without stable epi-genetic modifications would be unstable and would lose FOXP3 expression8. By contrast, those T cells with these epigenetic changes but without expression of FOXP3 are primed to express FOXP3 following TCR stimula-tion and, once FOXP3 is expressed, would be driven to a stable TReg cell lineage3.

As seen in the process of TH cell differen-tiation from naive T cells after antigen exposure, TReg cells show adaptive properties by sensing environmental cues28,29. For exam-ple, like TH1 cells, FOXP3+ TReg cells that are recruited to a site of TH1-type inflammation express T-bet and CXC-chemokine receptor 3 (CXCR3), and they apparently produce interferon-γ (IFNγ)28; similarly, TReg cells expressing STAT3, retinoic acid receptor-related orphan receptor-γt (RORγt) and CC-chemokine receptor 6 (CCR6) as well as producing IL-17 accumulate at sites of TH17 cell-mediated inflammation29. Although the production of pro-inflammatory cytokines by TReg cells may appear to contradict their immunosuppressive function, this does not necessarily mean that TReg cells are plastic and have pro-inflammatory functions for the reasons discussed below.

First and most importantly, TReg cells do not exert suppressive activity until they receive strong antigenic stimulation, as illustrated by studies carried out in vitro38,39. Upon TCR stimulation, FOXP3 and other transcription factors, as well as co-repressor and co-activator molecules, assemble to repress or to activate a particular gene (for example, repression of Ifng in TReg cells) depending on the combination of factors assembled. In addition, the demon-stration of cytokine production at the level of mRNA in TReg cells that have been stimulated with the mitogens PMA (phorbol 12-myristate 13-acetate) and ionomycin, as has been shown

in many reports, does not necessarily mean that TReg cells actually secrete these cytokines in significant amounts. Thus, it is necessary to determine whether TCR and/or CD3 stimula-tion can induce the secretion of these pro-inflammatory cytokines by TReg cells and not that they merely induce the expression of the cytokine mRNA.

H.W. Antigen, in an appropriate context, is clearly a driver for the induction of TReg cells40. Historically, there are many exam-ples of regulation being destabilized when antigen is made unavailable41. However, if one wishes to understand how the context of antigen experience determines long-term stability, then more information is required.

First, information about markers of dif-ferentiation is necessary. Our best indicators of FOXP3+ T cells with a tractable level of commitment to regulation are currently FOXP3 expression together with demethyla-tion of some key genes — Foxp3 at intron 1 (the position of CNS2), tumour necrosis factor receptor superfamily member 18 (Tnfrsf18; the gene that encodes GITR) at exon 5, Ctla4 at exon 2 and Ikaros family zinc finger protein 4 (Ikzf4; the gene that encodes Eos) at intron 1 (REF. 8). Given that TReg cells lack many immune effector functions, the methylation status of the genes that mediate these functions may provide additional guid-ance. In due course we should expect other technologies, such as modern proteomic approaches, to offer further markers to guide us in determining the long-term stability of TReg cells.

Second, understanding the heterogene-ity of TReg cells is important. In terms of the pre-existing heterogeneity in the TReg cell population, it would be helpful to establish epigenetic and functional parameters of TReg cell clones that have been isolated from both mice and humans, and to correlate these parameters with the functional status of the cell. A recent report clearly demonstrated that single-cell isolates of human natural TReg cells exhibit significant heterogeneity, which is not visible in bulk cultures26. Following various forms of inflammatory stimuli it would be helpful to understand what features of the TReg cell, when ‘called to action’, enable it to selectively repress the initiating inflammation, but that may be inappropriate in other inflammatory settings (for example, comparing TH1-, TH2- and TH17-based inflammatory events).

Third, it is important to understand the decision-making processes that are involved in the expansion of the TReg cell population. What has been missing in the

P E R S P E C T I V E S

4 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/immunol

© 2013 Macmillan Publishers Limited. All rights reserved

analyses of TReg cell ‘plasticity’, especially in the case of pTReg cells, has been the assess-ment of outcomes in sequential divisions of daughter cells that are derived from individual TReg cells. Recent work with other lymphocyte populations indicates that there are a range of factors that can determine whether divisions progress symmetrically or asymmetrically (asymmetric T cell division)42; asymmetric division provides the poten-tial for the diversification of cell fates and for daughter TReg cells to be diluted in an expanding population. Modern flow cytometry imaging approaches should allow a better understanding of how daughter cells progress under diverse stimulatory conditions and should provide guidelines for how to steer division to give rise to more homogeneous or heterogeneous progeny, as might be desired.

Is there a difference in the stability of mouse and human TReg cells?

S.S. Recent studies have shown that FOXP3+ T cells in mice and humans are heterogeneous in their function, phenotype and epigenetic status. When consider-ing the argument for ‘TReg cell plasticity’, this heterogeneity needs to be taken into consideration because plasticity could be a property of only a particular subpopula-tion of TReg cells rather than being a general property of all FOXP3+ TReg cells. This is especially the case in humans20.

FOXP3+ T cells in humans can be divided into three populations: FOXP3lowCD45RAhiCD25low TReg cells (designated as resting or naive TReg cells); FOXP3hiCD45RAlowCD25hi TReg cells (known as effector or activated TReg cells); and FOXP3lowCD45RAlowCD25low non-TReg cells, which are not suppressive and are capable of producing pro-inflammatory cytokines43. The TReg-type epigenetic status, including DNA hypomethylation of the CNS2 region in FOXP3, is also different among these populations of cells: naive and effector TReg cells are more demethylated than non-TReg cells. Upon TCR stimula-tion, naive TReg cells upregulate FOXP3 expression, proliferate and differentiate into effector TReg cells, which are highly proliferative and terminally differentiated, and the majority of effector TReg cells die by apoptosis after exhibiting strong suppres-sive activity. Therefore, there is a possibility that the production of pro-inflammatory cytokines (which does not necessarily mean cytokine secretion) by FOXP3+ T cells can be attributed to the presence

of non-TReg cells in the population sample. It needs to be determined in humans whether the observations that have been ascribed to TReg cell plasticity could be, at least partly, due to the heterogeneity of FOXP3+ T cells, and how each population of FOXP3+ T cells differentiate to or from other FOXP3+ or FOXP3− T cells.

The heterogeneity of FOXP3+ T cells is less clear in mice than in humans. The use of cell-fate reporter mice has revealed the presence of phenotypically unstable — plas-tic — FOXP3+ T cells14. FOXP3+ T cells with a full TReg-type epigenetic pattern are highly stable in terms of function and phenotype, whereas FOXP3+ T cells that have not yet established this epigenetic pattern are unstable (some of them differentiate into full TReg cells with a complete TReg-type epi-genetic pattern, whereas others lose FOXP3 expression and revert to naive T cells)8. In addition, using cell-fate reporter mice, one can detect a small population of cells that has once expressed FOXP3 and has then lost its expression3. These ex-TReg cells appear to retain the TReg-type epigenetic pattern and can easily be reconverted to FOXP3+ T cells. In addition, as discussed above, the conversion of FOXP3+ T cells into FOXP3− T cells can be prevented by IL-2 secretion by other T cells or by the converted T cells themselves2. It is currently unclear whether such ex-FOXP3-expressing T cells constitute a significant fraction of T cells in the physiological state.

Taken together, these findings in humans and mice indicate that, although some FOXP3+ T cells might lose FOXP3 expres-sion, this might not be a general property of FOXP3+ TReg cells; indeed, these ex-TReg cells may re-express FOXP3 if they retain the TReg-type epigenetic status. These findings also suggest that the conversion of FOXP3– TReg cells to FOXP3+ TReg cells might not be frequent in the physiological state because conventional T cells secrete IL-2, which prevents this process and sustains the expression of FOXP3 by TReg cells.

H.W. Within this framework of uncertainty regarding TReg cell stability and plasticity, there have also been concerns that human TReg cells are more unstable than mouse TReg cells — the cells from which we have gained much of our optimism for therapeutic manipulation. This concern partly derives from our inability to purify human TReg cells owing to poorly defined combinations of surface markers; in mice, the availability of markers that can be expressed under the control of the Foxp3 locus guarantees pure

starting populations. Given other confound-ing factors, such as the uncertainty of what determines a TReg cell that is committed to regulation or the uncertainty of the cellular microenvironment that delivers the final ‘stable’ functional regulators, it seems premature to make, let alone worry about, such comparisons.

A.Y.R. and R.E.N. The cell-intrinsic mecha-nisms that confer stability of FOXP3 expres-sion in mouse and human TReg cells are likely to be similar. The characterized regulatory DNA elements at the FOXP3 locus, includ-ing CNS2, are well conserved between mice and humans. FOXP3-expressing T cells in mice and, more prominently, in humans are heterogeneous in terms of expression levels of FOXP3, CD25, CTLA4 and other factors that are associated with TReg cell activation or function.

As in mice, human TReg cells are defined by a suppressive phenotype and by sus-tained high FOXP3 expression. Human FOXP3-expressing T cells can be divided into three categories based on their expres-sion of FOXP3, CD25 and CD45RA. FOXP3lowCD25medCD45RA+ T cells and FOXP3hiCD25hiCD45RA− T cells appear to represent competent TReg cells with potent suppressive capacities; these cells are characterized by CNS2 demethylation and stable expression of FOXP3 in vivo. By contrast, FOXP3lowCD25medCD45RA− T cells are not suppressive, have extensive methylation at the CNS2 and are predict-ably prone to proliferation-dependent loss of FOXP3 expression43. Thus, as in mice, FOXP3 expression alone is insufficient to identify human TReg cells. Activation of human CD4+ T cells in vitro can result in transient unstable expression of FOXP3 and, while this phenomenon has also been observed in mice, it is more pronounced in humans20. We suggest that in humans, as in mice, the committed, CNS2- demethylated, suppressive TReg cells represent a stable population that serves to mitigate inflammation-induced tissue injury in a variety of settings.

D.A.A.V. This is an important question but I feel we do not have enough information to provide a definitive answer. Given that TReg cell stability is often determined by loss of expression of FOXP3, the expression of FOXP3 by activated human T cells serves to further complicate this issue. My general view is that the mechanisms that maintain TReg cell stability and that may drive their instability are likely to be similar in humans

P E R S P E C T I V E S

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 5

© 2013 Macmillan Publishers Limited. All rights reserved

and mice. However, there may be important differences in the extent to which pathways that maintain stability are required in differ-ent disease settings. This may have signifi-cant clinical implications. This important question will be easier to address when we have a full appreciation of all the pathways that maintain and impinge on TReg cell stability.

What are the functional implications of TReg cell stability or plasticity?

H.W. Why does the research community seem preoccupied with understanding the plasticity of TReg cells rather than other CD4+ T cell subsets? There are very good reasons for this that are related to under-standing immunological diseases and their treatment. One reason is that TReg cells with self-reactive TCRs might, if destabi-lized, cause autoimmune disease23. Other reasons relate to the need to enhance a patient’s own TReg cells either within the patient themselves or by expanding the cells ex vivo and injecting them back into the patient in order to minimize the use of immunosuppressive drugs44. Such strategies would benefit from an understanding of how TReg cells could be operationally stabi-lized. Finally, there is a growing optimism in cancer immunotherapy that if one could destabilize immune regulation then a more effective mode of tumour immunization would be possible.

If we reflect on how immunological homeostasis is maintained, and what might need to be done to reset a dysfunctional immune system, it may well be that thera-peutically we only need to modestly tip the balance between regulatory and effector immune function to restore a long-term ‘ceasefire’. This might be achieved by encouraging more death of effector T cells than of TReg cells; by the selective expansion of regulatory populations; or by guiding more ‘would-be’ TReg cells to a commit-ted functional status. Recent studies have described a real potential for epigenetic therapeutics45,46 to enhance commitment to the TReg cell lineage. Once the overall stability threshold of the population of TReg cells has been raised to a certain level, this state might be sustained via intrinsic mechanisms of infectious tolerance44, thereby limiting the duration of treatments and the risks of unwanted side effects. In short, for therapeutic applications we might only need to think about stability and plastic-ity in quantitative terms rather than as an all-or-nothing dilemma.

S.S. The main task of FOXP3+ tTReg cells is to maintain immune self-tolerance and homeostasis. Functional stability is crucial for the ability of FOXP3+ tTReg cells to sup-press autoimmune disease because they are more self-reactive than conventional T cells with respect to their TCR repertoire and are therefore more hazardous when they are converted to FOXP3− non-TReg cells. The functional stability of TReg cells is also neces-sary to control a variety of inflammatory responses because some of the pro-inflam-matory cytokines, such as IL-6, may tem-porarily antagonize the TReg cell suppressive function. Therefore, it is not their plasticity but their capacity to adaptively differentiate (while sustaining FOXP3 expression) that is crucial for TReg cell function. In addition, they can acquire a migratory capacity to specific inflammatory sites, as well as addi-tional suppressive activity — for example, to secrete IL-10 or to produce granzyme B and perforin depending on the type, tissue site or chronicity of the inflammatory response47. It remains to be determined whether TReg cell functional instability, for example, due to anomalies in TReg cell epigenetics, contributes to any immunological diseases in humans.

D.A.A.V. The functional and translational implications of TReg cell stability and plastic-ity could be substantial. First, TReg cells have been shown to represent a major barrier to effective antitumour immunity as well as to sterilizing immunity to chronic viral infections10,48. Identifying the pathways that maintain TReg cell stability in inflammatory sites could present potentially important new therapeutic targets to undermine intratumoural or antiviral TReg cells and to enhance disease clearance. Second, TReg cells may be unstable or may not function effectively in autoimmune or inflammatory diseases. It is conceivable that the pathways that maintain stability are not optimally used in certain disease settings, which could lead to TReg cell insufficiency. One could potentially enhance pathways that maintain TReg cell stability to limit or to cure disease. Third, clinical trials using in vitro generated or expanded TReg cells are in progress. Once the pathways that maintain or undermine TReg cell stability in vivo are known, these cells could be manipulated during their in vitro preparation and/or fol-lowing transfer into the patient to optimize their clinical efficacy. Likewise, an in-depth understanding of the pathways that mediate TReg cell plasticity could also be used to limit or to enhance TReg cell activity in different disease settings.

A.Y.R. and R.E.N. TReg cell stability is a pre-requisite for their ability to limit transient, and to prevent lasting, tissue damage in response to various types of inflammation or injury. A loss of TReg cell function in a particular environment can have potentially devastating consequences. Different tissue and inflammatory environments can impose adaptive changes on TReg cells, modify-ing their functional properties, through the induced expression of transcriptional regulators and of homing and growth factor receptors. Although the stability of FOXP3 expression, which is imprinted as the result of a positive FOXP3 auto-feedback loop, is essential for immune homeostasis, it remains unknown whether particular ‘states’ of TReg cells that are induced in response to specific tissue or inflammatory cues are reversible (that is, plastic) or lasting. The stability of bona fide TReg cells implies that they have robust potential for cell-based therapies. Nevertheless, unstable FOXP3-expressing non-TReg cells (as described above) rep-resent a potential complication of these approaches and need to be taken into account. A mechanistic understanding of stable FOXP3 expression will facilitate the use of techniques to manipulate, and assays to validate, stable FOXP3 expression, which should facilitate efficacious use of TReg cells for immunotherapy.

Shimon Sakaguchi is at the World Premier International Immunology Frontier Research Center,

Osaka University, Suita 565–0871, Japan and at the Department of Experimental Pathology, Institute for

Frontier Medical Sciences, Kyoto University, Kyoto 606–8507, Japan.

Dario A. A. Vignali is at the Department of Immunology, St. Jude Children’s Research Hospital,

Memphis 38105–3678, Tennessee, USA.

Alexander Y. Rudensky and Rachel E. Niec are at the Immunology Program and Howard Hughes Medical

Institute at Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

Herman Waldmann is at the Sir William Dunn School of Pathology, University of Oxford, South Parks Road,

Oxford OX1 3RE, UK.

Correspondence to S.S., D.A.A.V., A.Y.R. and H.W. e-mails: rudenska@mskcc.org;

shimon@ifrec.osaka-u.ac.jp; vignali.lab@stjude.org; Herman.Waldmann@path.ox.ac.uk

doi:10.1038/nri3464 Published online 17 May 2013

1. Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. Cell 133, 775–787 (2008).

2. Duarte, J. H., Zelenay, S., Bergman, M. L., Martins, A. C. & Demengeot, J. Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions. Eur. J. Immunol. 39, 948–955 (2009).

3. Miyao, T. et al. Plasticity of Foxp3+ T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity. 36, 262–275 (2012).

P E R S P E C T I V E S

6 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/immunol

© 2013 Macmillan Publishers Limited. All rights reserved

4. Vignali, D. A. A., Collison, L. W. & Workman, C. J. How regulatory T cells work. Nature Rev. Immunol. 8, 523–532 (2008).

5. Benoist, C. & Mathis, D. Treg cells, life history, and diversity. Cold Spring Harb. Perspect. Biol. 4, a007021 (2012).

6. Rudensky, A. Y. Regulatory T cells and Foxp3. Immunol. Rev. 241, 260–268 (2011).

7. Fu, W. et al. A multiply redundant genetic switch ‘locks in’ the transcriptional signature of regulatory T cells. Nature Immunol. 13, 972–980 (2012).

8. Ohkura, N. et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 37, 785–799 (2012).

9. Sakaguchi, S. Regulatory T cells: history and perspective. Methods Mol. Biol. 707, 3–17 (2011).

10. Workman, C. J., Szymczak-Workman, A. L., Collison, L. W., Pillai, M. R. & Vignali, D. A. The development and function of regulatory T cells. Cell. Mol. Life Sci. 66, 2603–2622 (2009).

11. Collison, L. W., Pillai, M. R., Chaturvedi, V. & Vignali, D. A. Regulatory T cell suppression is potentiated by target T cells in a cell contact, IL-35- and IL-10-dependent manner. J. Immunol. 182, 6121–6128 (2009).

12. Abbas, A. K. et al. Regulatory T cells: recommendations to simplify the nomenclature. Nature Immunol. 14, 307–308 (2013).

13. Rubtsov, Y. P. et al. Stability of the regulatory T cell lineage in vivo. Science 329, 1667–1671 (2010).

14. Zhou, X. et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nature Immunol. 10, 1000–1007 (2009).

15. Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).

16. Fontenot, J. D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329–341 (2005).

17. Williams, L. M. & Rudensky, A. Y. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nature Immunol. 8, 277–284 (2007).

18. Samstein, R. M. et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 151, 153–166 (2012).

19. Hori, S. Stability of regulatory T-cell lineage. Adv. Immunol. 112, 1–24 (2011).

20. Sakaguchi, S., Miyara, M., Costantino, C. M. & Hafler, D. A. FOXP3+ regulatory T cells in the human immune system. Nature Rev. Immunol. 10, 490–500 (2010).

21. Tian, L., Humblet-Baron, S. & Liston, A. Immune tolerance: are regulatory T cell subsets needed to explain suppression of autoimmunity? BioEssays 34, 569–575 (2012).

22. Nie, H. et al. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nature Med. 19, 322–328 (2013).

23. Zhou, X., Bailey-Bucktrout, S., Jeker, L. T. & Bluestone, J. A. Plasticity of CD4+ FoxP3+ T cells. Curr. Opin. Immunol. 21, 281–285 (2009).

24. Lal, G. et al. Distinct inflammatory signals have physiologically divergent effects on epigenetic regulation of Foxp3 expression and Treg function. Am. J. Transplant. 11, 203–214 (2011).

25. Yurchenko, E. et al. Inflammation-driven reprogramming of CD4+ Foxp3+ regulatory T cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo. PLoS ONE 7, e35572 (2012).

26. d’Hennezel, E., Yurchenko, E., Sgouroudis, E., Hay, V. & Piccirillo, C. A. Single-cell analysis of the human T regulatory population uncovers functional heterogeneity and instability within FOXP3+ cells. J. Immunol. 186, 6788–6797 (2011).

27. Pasare, C. & Medzhitov, R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299, 1033–1036 (2003).

28. Koch, M. A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nature Immunol. 10, 595–602 (2009).

29. Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 326, 986–991 (2009).

30. Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control TH2 responses. Nature 458, 351–356 (2009).

31. Polansky, J. K. et al. DNA methylation controls Foxp3 gene expression. Eur. J. Immunol. 38, 1654–1663 (2008).

32. Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).

33. Josefowicz, S. Z. et al. Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 482, 395–399 (2012).

34. Josefowicz, S. Z., Lu, L.-F. & Rudensky, A. Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).

35. Oldenhove, G. et al. Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31, 772–786 (2009).

36. Floess, S. et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 5, e38 (2007).

37. Wei, G. et al. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 30, 155–167 (2009).

38. Takahashi, T. et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10,1969–1980 (1998).

39. Thornton, A. M. & Shevach, E. M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296 (1998).

40. Daniel, C., Wennhold, K., Kim, H. J. & von Boehmer, H. Enhancement of antigen-specific Treg vaccination in vivo. Proc. Natl Acad. Sci. USA 107, 16246–16251 (2010).

41. Cobbold, S. P., Adams, E., Marshall, S. E., Davies, J. D. & Waldmann, H. Mechanisms of peripheral tolerance and suppression induced by monoclonal antibodies to CD4 and CD8. Immunol. Rev. 149, 5–33 (1996).

42. Chang, J. T. et al. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315, 1687–1691 (2007).

43. Miyara, M. et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 30, 899–911 (2009).

44. Kendal, A. R. et al. Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance. J. Exp. Med. 208, 2043–2053 (2011).

45. Beier, U. H. et al. Histone deacetylases 6 and 9 and sirtuin-1 control Foxp3+ regulatory T cell function through shared and isoform-specific mechanisms. Sci. Signal. 5, ra45 (2012).

46. Zhang, H., Xiao, Y., Zhu, Z., Li, B. & Greene, M. I. Immune regulation by histone deacetylases: a focus on the alteration of FOXP3 activity. Immunol. Cell Biol. 90, 95–100 (2012).

47. Wing, J. B. & Sakaguchi, S. Multiple Treg suppressive modules and their adaptability. Front. Immunol. 3, 178 (2012).

48. Wang, H. Y. & Wang, R. F. Regulatory T cells and cancer. Curr. Opin. Immunol. 19, 217–223 (2007).

AcknowledgementsS.S. acknowledges the daily discussions he had with his col-leagues that helped him to write his comments for this article. D.A.A.V. apologises to those investigators who contributed important observations related to the questions he covered that he could not discuss or quote owing to space limitations. D.A.A.V. is supported by the US National Institutes of Health (NIH) (grants AI091977, AI039480, AI052199, DK089125), American Asthma Foundation (grant10-0128), National Cancer Institute Comprehensive Cancer Center grant (CA21765) and ALSAC.A.Y.R. is supported by an NIH grant (R37 AI034206) and is an investigator at the Howard Hughes Medical Center. R.E.N. is supported by an NIH Medical Scientist Training Program grant (GM07739) and a National Institute of Neurological Disorders and Stroke grant (1F31NS073203-01).H.W. wishes to acknowledge the help of his colleagues D. Howie, R. Hilbrands and S. Cobbold in writing his comments for this article.

Competing interests statementThe authors declare competing financial interests. See Web version for details.

FURTHER INFORMATIONShimon Sakaguchi’s homepage: www.ifrec.osaka-u.ac.jpDario A. A. Vignali’s homepage: http://www.stjude.org/vignaliAlexander Y. Rudensky’s homepage: http://www.mskcc.org/research/lab/alexander-rudenskyHerman Waldmann’s homepage: http://www.path.ox.ac.uk/dirsci/immunology/waldmann

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

P E R S P E C T I V E S

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 7

© 2013 Macmillan Publishers Limited. All rights reserved