Lineage stability and phenotypic plasticity of Foxp3⁺ regulatory T cells.

Shohei Hori

Lineage stability and phenotypic plasticity of Foxp3+ regulatory T cells

Author’s address Shohei Hori1 1 Laboratory for Immune Homeostasis, RCAI, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan.

Summary: Regulatory T (Treg) cells expressing the transcription factor forkhead box protein 3 (Foxp3) constitute a unique T-cell lineage committed to suppressive functions. While their differentiation state is remarkably stable in the face of various perturbations from the extracellular environment, they are able to adapt to diverse and fluctuating tissue environments by changing their phenotype. The lineage stability and phenotypic plasticity of Treg cells thus ensure the robustness of self-tolerance and tissue homeostasis. Recent studies have suggested, however, that Treg cells may retain lineage plasticity, the ability to switch their cell fate to various effector T-cell types under certain circumstances such as inflammation, a notion that remains highly contentious. While accumulating evidence indicates that some Treg cells can downregulate Foxp3 expression and/or acquire effector T-helper celllike phenotypes, results from my laboratory have shown that Treg cells retain epigenetic memory of, and thus remain committed to, Foxp3 expression and suppressive functions despite such phenotypic plasticity. It has also become evident that Foxp3 can be promiscuously and transiently expressed in activated T cells. Here, I argue that the current controversy stems partly from the lack of the lineage specificity of Foxp3 expression and also from the confusion between phenotypic plasticity and lineage plasticity, and discuss implications of our findings in Treg cell fate determination and maintenance.

Correspondence to: Shohei Hori Laboratory for Immune Homeostasis, RCAI RIKEN Center for Integrative Medical Sciences 1-7-22 Suehiro-cho, Tsurumi, Yokohama Kanagawa 230-0045, Japan Tel.: +81 45 503 7069 Fax: +81 45 503 7068 e-mail: [email protected] Acknowledgements I sincerely thank Dr. Ruka Setoguchi for continuous encouragement as well as critical reading of the manuscript, and the members of my laboratory for discussion. This work was supported in part by Grants-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23390123 and 25118733) and by the Mitsubishi Foundation. The author has no conflicts of interest to declare.

This article is part of a series of reviews covering Regulatory Cells in Health and Disease appearing in Volume 259 of Immunological Reviews.

Immunological Reviews 2014 Vol. 259: 159–172 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Keywords: regulatory T cells, Foxp3, heterogeneity, cell fate determination, phenotypic plasticity, epigenetic memory

Introduction Protection of an organism from autoimmune diseases cannot be explained entirely by physical elimination or functional inactivation of autoreactive lymphocyte repertoires. Accumulating evidence indicates that immunological tolerance to body tissues may be rather dominant and rely on cellextrinsic regulation of pathogenic autoreactive lymphocytes by a subset of T lymphocytes called regulatory T (Treg) cells. Since the late 1980s, several laboratories have independently characterized CD4+ T-cell subsets capable of protecting self or foreign tissues from destructive immune responses using different disease or transplantation models and designated such tissue-protective or immune-suppressive cells as Treg cells (1–8). Those early studies culminated

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

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Hori Treg cell fate determination and maintenance

in the discovery of an unique Treg cell subset characterized by the expression of the transcription factor Forkhead box protein 3 (Foxp3) (9–12). The findings that deficiency of functional Treg cells caused by mutations in the Foxp3 gene or induced ablation of Foxp3+ T cells results in the development of a fatal autoimmune disease have provided compelling evidence that they are indeed indispensable for self-tolerance (10, 11, 13–15). It has also become evident that, in addition to restraining autoimmunity (16), Foxp3+ Treg cells have a potential to suppress apparently any forms of immune responses. They are capable of preventing collateral tissue damage triggered by immune responses against microbes or allergens (17–20), maintaining homeostasis with the commensal microbiota (21, 22), facilitating maternal tolerance to allogeneic fetus during pregnancy (23), promoting therapeutic tolerance toward transplanted organs (24), and sometimes helping tumor cells or certain pathogens to escape from immune surveillance (18, 25). Moreover, recent studies have also suggested that their functions go beyond regulation of immune responses and encompass regulation of tissue homeostasis in general (26). Because of their fundamental role in various immune and non-immune processes and because of their therapeutic potential, Foxp3+ Treg cells have gained tremendous interest. The findings that Foxp3+ Treg cells exert tissue-protective or suppressive functions under such diverse circumstances raise a question as to what mechanisms ensure the robustness of Treg cell functions in the face of various unpredictable perturbations from the extracellular environment. There is evidence that Foxp3+ Treg cells represent a stable cell lineage committed to suppressive functions and are thus distinct from conventional helper as well as cytotoxic T cells (27, 28). Yet, their phenotype is not rigidly fixed in that they are able to change gene expression in response to extrinsic cues (26, 28, 29). These features, namely lineage stability and phenotypic plasticity, are crucial for Treg cells to adapt to diverse and fluctuating tissue environments for protection of the integrity of body tissues. In recent years, however, increasing numbers of reports have suggested that, under certain circumstances such as inflammation and lymphopenia, Treg cells may switch their lineage fate to diverse effector helper T (Th) cell types by entirely changing their gene expression program, and proposed that such reprogrammed ‘exTreg’ cells promote inflammation and other immune responses (30–32). Because such lineage (or developmental) plasticity of Treg cells should greatly impinge on the robustness of selftolerance and tissue homeostasis, this emerging notion has

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provoked great controversies and remains highly contentious (33–35). In addition, these findings have also raised serious concerns about the validity and safety of ongoing clinical trials that utilize adoptive Treg cell transfer as a therapeutic strategy for graft-versus-host disease (GvHD) and autoimmune diseases (36, 37). Over the last several years, my laboratory has been addressing this issue of lineage stability and phenotypic plasticity of Treg cells. Here, I review the results of our own studies as well as of others, propose a model that could reconcile lineage stability with effector Th cell-like phenotypes of Treg cells, and discuss implications of those findings in understanding of the mechanisms responsible for Treg cell fate determination and maintenance. Treg cell differentiation and Foxp3 Early studies of thymic epithelium-induced transplantation tolerance to xeno- or allogeneic tissue grafts in birds and in mice (1) and of autoimmunity provoked in neonatally thymectomized mice or in thymectomized and c-irradiated rats (3, 7) provided the initial evidence for the existence of a thymus-derived T-cell subset that mediate dominant selftolerance. The former studies also suggested that at least some of autoreactive T cells are positively selected as Treg cells and ‘imprinted’ with tissue-protective functions in the thymus (2). The latter studies led to the discovery of a thymus-derived CD25+ CD4+ T-cell population capable of protecting animals from autoimmune diseases (38–41). These independent lines of work converged in later studies that showed that intrathymic differentiation of CD25+ CD4+ Treg cells requires high-affinity or high-avidity T-cell receptor (TCR) interactions with self-peptides/major histocompatibility complex class II molecules presented by thymic epithelium or dendritic cells (42–44), a notion that has gained further support from TCR repertoire analysis (45, 46). The findings that CD25+ CD4+ Treg cells are capable of maintaining their suppressive functions after rounds of cell division under various in vitro and in vivo conditions have strengthened the notion that Treg cells are stably committed to suppressive functions (47–50). In 2003, three groups reported that expression of the transcription factor Foxp3 faithfully identifies these naturally occurring Treg cells and confers a Treg cell-like phenotype on otherwise conventional CD4+ T cells (9–11). Moreover, loss-of-function mutations of the Foxp3 gene lead to defective development of functional CD25+ CD4+ Treg cells (10, 11). These findings collectively led to the notion that Foxp3+ Treg cells represent a stable cell lineage committed to suppressive © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014


functions and Foxp3 acts as their ‘master regulator’ or ‘lineage specification factor’. The discovery of Foxp3 has revolutionized the field and subsequent studies have started to uncover the molecular and cellular mechanisms of Treg cell differentiation and functions by using Foxp3 expression as a ‘specific’ molecular marker of Treg cells. Consistent with earlier studies, differentiation of Foxp3+ Treg cells was confirmed to take place primarily in the thymus at the CD4+ CD8 singlepositive stage concomitantly with positive and/or negative selection [thymus-generated Treg (tTreg cells)] (12). Additional Treg cells can also be generated in the periphery from naive CD4+ T cells under certain tolerogenic contexts in vivo [peripherally generated Treg (pTreg cells)] (20, 24). In addition, Foxp3+ T cells exhibiting some degree of suppressive functions can also be generated in vitro in the presence of transforming growth factor-b (TGF-b) costimulation (51). Many intrinsic as well as extrinsic factors that positively or negatively regulate Foxp3 expression have also been identified (28). Importantly, induction and maintenance of Foxp3 expression are two separable processes regulated by distinct cis-regulatory elements within the Foxp3 locus (52). In particular, one of the evolutionally conserved non-coding DNA sequence (CNS) elements, CNS2, was shown to be required for heritable maintenance of Foxp3 expression in dividing Treg cells (52). This CNS2 element is also called Treg cell-specific demethylation region (TSDR), because its CpG sites are completely demethylated in Treg cells (53, 54). Importantly, TGF-b-induced Foxp3+ T cells show no or only limited TSDR demethylation, which correlates well with their unstable Foxp3 expression and incomplete Treg cell phenotype (53, 55). In contrast, in vivo generated pTreg cells exhibit a largely demethylated TSDR (55, 56). Despite the essential role of Foxp3 in Treg cells, it has also become clear that Foxp3 alone is neither strictly necessary nor sufficient for differentiation of Treg cells. In humans, activated T cells transiently upregulate FOXP3 expression without acquiring a Treg cell phenotype (57– 61), although such ‘promiscuous’ Foxp3 expression was not seen in initial mouse studies (9–12). T cells that are transcribing the Foxp3 locus but unable to express functional Foxp3 protein lack suppressive functions but have some other features of Treg cells (62–64). A meta-analysis of the transcriptomes of Foxp3+ Treg cells and other Foxp3-expressing cells (e.g. TGF-b-induced Foxp3+ T cells and Foxp3-transduced T cells) has shown that Foxp3 accounts for only a fraction of the transcriptional land© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

scape of Treg cells (65). It is becoming apparent that, to establish the characteristic Treg cell phenotype, Foxp3 has to cooperate with other transcription factors (66, 67), cisregulatory elements (68), and epigenetic mechanisms (64). Recent studies have also shown that Foxp3+ Treg cells is not a homogeneous population but can change their migratory, functional and homeostatic properties in response to specific cues in the tissue or immune environment (26, 28, 29). Accumulating evidence indicates that such phenotypic plasticity of Treg cells is controlled by the same transcriptional machinery (such as T-bet, GATA-3, IRF4, Bcl6) that is employed by the very effector Th cells they are regulating. Thus, Treg cells express some phenotypic characteristics of effector Th cells, which endow them with the ability to adapt to diverse and fluctuating environments and to regulate various effector classes of immune responses. Lineage plasticity of Treg cells? Although earlier studies have suggested that Foxp3+ Treg cells represent a stable cell lineage committed to suppressive functions, its formal proof was missing. We therefore decided to evaluate stability of Treg cell phenotype and functions. With the aid of Foxp3-reporter mice, we sorted Foxp3+ T cells from peripheral lymphoid organs into high purity and adoptively transferred them into T cell-deficient (Rag1 / or Cd3e / ) mice (69). Four weeks after transfer, approximately 50% of the donor cells were found to be negative for Foxp3 expression in the spleen and lymph nodes. Spiking experiments demonstrated that this was caused by loss of Foxp3 expression but not by outgrowth of Foxp3 T cells that contaminated the sorted Foxp3+ donor cells. These Foxp3 T cells derived from Foxp3+ T cells (or so called exFoxp3 T cells) showed low expression of Treg cell signature molecules including CD25, GITR, and CTLA-4, produced significant amounts of effector cytokines (e.g. IFN-c, IL-2, and IL-17), and failed to inhibit proliferation of conventional T cells in vitro. Thus, at least some of Foxp3+ T cells can lose Foxp3 expression and acquire effector Th cell-like phenotypes. Similar results were also reported by others (70, 71). The accumulation and phenotype of exFoxp3 T cells was influenced by extrinsic signals derived from the extracellular environment. For instance, in CD3e-deficient recipients of Foxp3+ T cells, we found more extensive accumulation of exFoxp3 T cells in the Peyer’s patches than in the lymph nodes and spleen, many of which showed a follicular helper T (Tfh) cell phenotype (i.e.

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CXCR5high PD1high ICOShigh Bcl6+), efficiently induced germinal centers, and promoted IgA production in the intestine (72). The differentiation of exFoxp3 Tfh cells was not seen in other lymphoid tissues, indicating that environmental cues present in Peyer’s patches promote the accumulation of exFoxp3 T cells and their differentiation into Tfh cells. Others also reported increased accumulation of exFoxp3 T cells in the intestine of T-cell-deficient recipients of Foxp3+ T cells, particularly in the presence of inflammation (70, 71, 73). The accumulation of exFoxp3 T cells was also affected by the presence of conventional T cells; when Ly5.1 Foxp3+ T cells were cotransferred with Ly5.2 Foxp3 T cells into T-cell-deficient mice, more than 90% of the Ly5.1 donor cells remained Foxp3+ (69–71). Similarly, when Ly5.1 Foxp3+ T cells were transferred into sublethally irradiated Ly5.2 wildtype mice, which were lymphopenic but still harbored many radio-resistant host T cells, most of the donor cells remained Foxp3+ (author’s unpublished results). Because cytokines are key environmental cues that instruct Th cell differentiation, we activated highly purified Foxp3+ T cells with anti-CD3 and CD28 antibodies in the presence or absence of Th cell polarizing cytokines and assessed how cytokine signals affect the generation and phenotype of exFoxp3 T cells (69). Consistent with earlier reports (74, 75), we have also found that IL-6 costimulation resulted in the generation of IL-17+ Th17-like exFoxp3 cells. Similarly, IL-4 costimulation resulted in the generation of IL-4+ Th2like exFoxp3 cells. Neutralization of TGF-b also led to the generation of IL-2+ exFoxp3 T cells. Interestingly, IL-12 costimulation failed to affect Foxp3 expression but induced IFN-c+ Foxp3+ T cells (unpublished results), as reported by others (76–79). Other groups have also reported that, even in the absence of such deliberate polarization, some Foxp3+ T cells lose Foxp3 expression upon stimulation with prolonged TCR/CD28 signals (80) or with certain costimulatory signals (81, 82). Similar in vitro observations have also been made in human FOXP3+ T cells; generation of exFOXP3 T cells after repetitive TCR stimulation (83) and cytokine-driven conversion into a Th17 cell-like phenotype (84) were reported. To address whether some Foxp3+ T cells lose Foxp3 expression in vivo in normal non-lymphopenic mice, we have generated Foxp3GFPCre mice, in which a GFP-Cre fusion protein is expressed under the control of the endogenous Foxp3 locus and crossed them with ROSA26RFP Cre-reporter mice to identify GFP RFP+ exFoxp3 T cells in the normal T-cell repertoire (85). Similar genetic fate-mapping studies were

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also carried out by others who used Foxp3-GFPCre bacterial artificial chromosome (BAC) transgenic ROSA26YFP mice (86). Consistent with their findings, we found that approximately 10–20% of RFP+ CD4+ T cells are negative for GFP and Foxp3 expression. These GFP RFP+ exFoxp3 T cells resulted from Foxp3 downregulation in Foxp3+ T cells but not from leaky Foxp3 transcription that might take place in naive T cells or earlier progenitor cells. Furthermore, GFP RFP+ T cells showed low expression of Treg cellassociated surface markers, exhibited a CD44high effector or memory phenotype, and produced effector cytokines IFN-c, IL-2, IL-4, IL-17, and IL-21, indicating differentiation into diverse effector Th cell-like phenotypes. Bluestone and colleagues further examined whether autoreactive Foxp3+ T cells can become pathogenic effector Th cells upon loss of Foxp3 expression by introducing the diabetogenic BDC2.5 TCR transgene into their fate reporter mice (86). They found that, after in vitro activation and expansion with a nominal antigen, GFP RFP+ exFoxp3 T cells as well as GFP RFP conventional T cells transferred diabetes into T-cell-deficient recipient mice. Similar findings were also reported using an experimental autoimmune encephalomyelitis (EAE) model (87). On the basis of these findings, they have proposed that inflammation promotes Foxp3 downregulation in autoreactive Treg cells and their conversion into pathogenic effector Th cells. Under certain circumstances, some Foxp3+ T cells acquire effector Th cell-like features without losing Foxp3 expression, so exhibiting ‘hybrid’ phenotypes. When peripheral Foxp3+ T cells were stimulated in vitro in the presence of dendritic cells activated via a fungal recognition receptor, some expressed IL-17 and RORct together with Foxp3 (88). Such Foxp3+ RORct+ IL-17+ have been identified in vivo in humans (89, 90) and in mice, particularly in the intestine (91). Similarly, as described above, some Foxp3+ T cells acquire T-bet and IFN-c expression without losing Foxp3 expression, when stimulated under Th1 polarization conditions in vitro (76–79). In vivo evidence for the development of Foxp3+ T-bet+ IFN-c+ cells in inflammatory environments was reported in mice infected with Toxoplasma gondii (92) or neurotropic hepatitis virus (93) and in human patients suffering from multiple sclerosis (77) or type 1 diabetes (94). Other studies have also reported that, upon vaccination with protein antigens along with CpG-DNA adjuvants, some Foxp3+ T cells produce multiple cytokines and CD40L in an IL-6-dependent manner and help crosspriming of CD8+ T cells (95, 96). In most cases, however, those Foxp3+ T cells retain suppressive functions, leaving it © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014


unclear whether such hybrid-phenotypes reflect lineage plasticity of Treg cells (see below). These observations prompted some investigators to propose that Treg cells can switch their lineage fate to diverse effector T-cell types under certain conditions such as inflammation or lymphopenia and those reprogrammed exTreg cells promote inflammation or other immune responses (30, 31, 33). Alternatively, these findings may suggest that Treg cells represent a ‘metastable’ activation state rather than a distinct and stable cell lineage committed to suppressive functions. These emerging views have provoked great controversies particularly because they cannot be easily reconciled with the robustness of self-tolerance and tissue homeostasis. Because many Treg cells are positively selected based on their autoreactivity, their functional reprogramming to pathogenic effector Th cells could result in a catastrophic consequence to the host. In addition, these findings cannot be easily reconciled with a number of other findings that Treg cells are able to function under inflammatory conditions (17–19, 97). The controversies have been boosted further by other observations that dispute those plasticity experiments. One study has shown that, in an EAE model, myelin oligodendrocyte glycoprotein (MOG)-specific Foxp3+ and Foxp3 T cells display distinct TCR CDR3 sequences and are therefore derived from distinct clones, suggesting that there is no or only limited inter-conversion between the two populations during the autoimmune inflammation (98). Furthermore, Rudensky and colleagues (99) also conducted a genetic fatemapping study using a pulse-labeling approach and showed that Foxp3+ T cells display remarkably stable Foxp3 expression under steady state and various perturbed conditions in adult mice. They knocked-in cDNA encoding a GFPCre-ERT2 triple fusion protein into the endogenous Foxp3 locus and crossed the knockin mice with ROSA26YFP mice. When these mice were treated with tamoxifen to pulse label Foxp3+ T cells as adults and examined 2 weeks or 5 months later, less than 5% of YFP+ cells were found to be negative for Foxp3 expression, indicating that their Foxp3 expression is stable under the steady state. Furthermore, frequencies of the Foxp3 YFP+ T cells were not increased when the animals were challenged with various inflammatory stimuli or with a lymphopenic episode induced by sublethal irradiation. In addition, they also showed that double-sorted, highly purified Foxp3+ T cells do not convert to Foxp3 Th cells under autoimmune conditions in non-lymphopenic host mice. The only condition in which they observed appreciable downregulation of Foxp3 expression and © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

appearance of exFoxp3 T cells was IL-2 neutralization, but the resulting exFoxp3 T cells did not show effector Th celllike phenotypes. These observations are apparently contradictory with other plasticity experiments, but the root of the apparent conflicts was not clear. The ‘heterogeneity model’: not all Foxp3+ T cells are committed to Treg cell fate To reconcile those apparently contradictory observations, we have proposed that lineage heterogeneity of Foxp3+ T cells, rather than lineage plasticity of Treg cells, accounts for the observed conversion of Foxp3+ T cells to exFoxp3 effector Th cells. This ‘heterogeneity model’ postulated that many Foxp3+ T cells are committed to the Treg cell fate but some others remain uncommitted and retain the options to adopt alternative effector Th cell fates (100). According to this model, the remarkable accumulation of exFoxp3 Th cells under lymphopenic or inflammatory environments is explained by conversion and selection (by preferential proliferation, survival, immigration, and/or retention) of the minor uncommitted population, rather than by induced lineage reprogramming of committed Treg cells. This model is based on our observations that CD25low Foxp3+ T cells preferentially give rise to exFoxp3 effector Th cells when transferred into T-cell-deficient mice or stimulated in vitro in the presence of IL-4, IL-6, or antiTGF-b antibodies (69). In contrast, the vast majority of CD25high Foxp3+ T cells are resistant to conversion into exFoxp3 Th cells under those conditions. The heterogeneity model remained unproven, however, because the nature of such uncommitted Foxp3+ T cells as well as the origin of exFoxp3 effector T cells was unknown. In addition, the CD25 expression is not a ‘clean’, although useful, marker to separate the stable and unstable populations, because CD25 expression on Foxp3+ Treg cells is known to be regulated dynamically depending on local IL-2 availability and proliferation status of the cells (12, 47, 48, 101, 102). Moreover, CD25low Foxp3+ T cells are still heterogeneous and contain many stable cells and CD25high Foxp3+ T cells still contain some, although much fewer, unstable cells (69). This has also left open a possibility that committed Treg cells may still retain lineage plasticity. Regarding the nature of uncommitted Foxp3+ T cells, we initially hypothesized that the uncommitted state represents developmental intermediates that are on the way to differentiate into committed Treg cells but still retain options to adopt alternative effector Th cell differentiation pathways (100). Another non-exclusive possibility was that it reflects

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an alternative mode of Foxp3 expression that is independent of Treg cell differentiation, which would correspond to promiscuous and transient FOXP3 expression observed in human activated T cells. We preferred the former possibility because previous studies including our own showed that mouse conventional T cells do not show such promiscuous Foxp3 expression (9–11). We have realized, however, that this is no longer the case. When naive CD4+ T cells were activated with anti-CD3 and CD28 antibodies in vitro, Foxp3 expression was readily induced in 10–20% of them even in the absence of TGF-b signals (85). Much like their human counterpart, those mouse activation-induced Foxp3+ T cells were not Treg cells, because they showed a gene expression profile distinct from Treg cells (except for being Foxp3+) but similar to activated Foxp3 T cells, produced IL-2 as abundantly as activated Foxp3 T cells, and lacked suppressive activity. Moreover, they exhibited a fully methylated TSDR and readily lost Foxp3 expression upon restimulation in vitro. We have found that previous studies failed to detect this activation-induced Foxp3 expression because na€ıve CD4+ T cells were activated with anti-CD3 for over 2 days or in the presence of CD44high effector-memory cells or antigen-presenting cells, which secrete cytokines (e.g. IFN-c, IL-4, IL-6, and IL-21). Such prolonged TCR signals or cytokine signals prevented na€ıve T cells to upregulate Foxp3 expression. To determine whether the normal T-cell repertoire harbors such transient Foxp3+ non-Treg cells and whether exFoxp3 effector Th cells generated in T-cell-deficient or inflammatory environments are derived from them, we performed adoptive transfer experiments to identify peripherally induced Foxp3+ T cells in normal lymphoreplete mice (85). Ly5.1 Foxp3 CD4+ T cells were first transferred into Ly5.2 congenic host mice. After a period of 2–8 weeks, total peripheral Foxp3+ T cells containing the donor cells that had induced Foxp3 expression during the residence in lymphoreplete mice were transferred into Rag1 / mice or activated in vitro in the presence of IL-4, IL-6, or anti-TGF-b antibodies to drive differentiation into exFoxp3 effector Th cells. When compared to the host Foxp3+ T cells, the peripherally induced Foxp3+ T cells readily lost Foxp3 expression and those exFoxp3 T cells underwent preferential population expansion. Moreover, the frequencies of the Ly5.1 donor-derived exFoxp3 T cells inversely correlated with the extent of their population expansion and with the duration of their residence in the lymphoreplete host mice, indicating that, at the population level, peripherally induced Foxp3+ T cells acquire more stable Foxp3 expression over

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time primarily by losing the potential to expand in lymphopenic mice. In contrast, thymus-derived Foxp3+ T cells exhibited markedly stable Foxp3 expression after 2 or 8 weeks of residence in lymphoreplete host mice and did not acquire effector Th cell-phenotypes under those conditions. It was of note, however, that Foxp3+ thymocytes gave rise to some exFoxp3 T cells when directly transferred into Rag1 / mice or stimulated in vitro. These results indicate that some of ‘newly developed’ Foxp3+ T cells, particularly those generated in the periphery, exhibit transient Foxp3 expression, whereas ‘resident’ Foxp3+ T cells, particularly those generated in the thymus, exhibit markedly stable Foxp3 expression. Because environmental cues positively or negatively affect the accumulation and phenotype of exFoxp3 Th cells, we addressed how they influence the behavior of peripherally induced Foxp3+ T cells and thymus-derived Foxp3+ T cells using this adoptive transfer approach. When Peyer’s patches of the CD3e-deficient recipients of Foxp3+ T cells were analyzed, we found that exFoxp3 T cells, including Tfh cells, are derived from peripherally induced Foxp3+ T cells but not from thymus-derived resident Foxp3+ T cells. Significantly, those exFoxp3 T cells derived from peripherally induced Foxp3+ T cells underwent more extensive population expansion in Peyer’s patches than in lymph nodes and spleen (author’s unpublished results). Conversely, when Foxp3+ T cells were cotransferred with Foxp3 T cells into Rag1 / mice, we found that Foxp3 T cells inhibited the extensive population expansion of the exFoxp3 T cells derived from peripherally induced Foxp3+ T cells but promoted the population expansion of Foxp3+ T cells, thereby resulting in the apparent maintenance of Foxp3 expression in the Foxp3+ donor T cells (85). Thus, these findings fully support the heterogeneity model in that extrinsic cues present in Peyer’s patch environment or T cell-deficient environment promote the accumulation of exFoxp3 Th cells by driving conversion and preferential population expansion of a minor population of peripherally and recently induced Foxp3+ T cells but not by inducing conversion of thymusderived resident Foxp3+ Treg cells. These results, while demonstrating that some of newly developed Foxp3+ T cells exhibit transient Foxp3 expression and preferentially give rise to exFoxp3 effector Th cells, do not clarify whether such newly developed transient Foxp3expressing cells are developmental intermediates on the way to differentiate into stable Treg cells or they are activated conventional T cells exhibiting promiscuous and transient Foxp3 expression independently of Treg cell differentiation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014


We therefore undertook a different approach to identify and characterize newly developed peripheral Foxp3+ T cells exhibiting unstable Foxp3 expression in normal mice by taking advantage of Foxp3GFPCre.ROSA26RFP mice (85). Because it takes some time for the ROSA26 locus to undergo Cremediated recombination and for RFP protein to accumulate in cells after induction of Cre activity [estimated to take approximately 4 days in ES cells (103)], this system allows us to distinguish GFP+ RFPlow newly developed Foxp3+ T cells, which have recently initiated Foxp3 transcription, and GFP+ RFPhigh Foxp3+ T cells, which have continued to express Foxp3 for some time. By using this molecular timer, we were able to show that peripheral GFP+ RFPlow T cells exhibit more unstable Foxp3 expression than GFP+ RFPhigh T cells. Importantly, GFP+ RFPlow cells were found to be phenotypically and functionally heterogeneous; CD25low GFP+ RFPlow cells showed low expression of Treg cell signature molecules including Foxp3, GITR, and OX40, little suppressive activity in vitro, fully methylated TSDR, and unstable Foxp3 expression. In contrast, CD25high GFP+ RFPlow cells already expressed Treg cell phenotypic markers and potent suppressive activity. Nonetheless, they showed incomplete TSDR demethylation and slightly but significantly less stable Foxp3 expression than CD25high GFP+ RFPhigh cells, indicating that a few unstable Foxp3+ cells contained in the CD25high subset are also newly developed Foxp3+ T cells. GFP+ RFPhigh cells exhibited potent suppressive activity, stable Foxp3 expression, and a fully demethylated TSDR, irrespective of their CD25 expression. Notably, CD25low GFP+ RFPhigh cells contained more effector- or memory-phenotype (e.g. CD44high CD62Llow CCR7low) cells than CD25high GFP+ RFPhigh cells, a finding consistent with previous observations that Treg cells downregulate CD25 expression upon in vivo activation and proliferation (12, 47, 48). Although newly developed CD25high Foxp3+ T cells contained some unstable cells, they exhibited largely stable Foxp3 expression and suppressive activity and thus most of them are already committed to stable Foxp3 expression and suppressive functions. These results support the hypothesis that the ‘uncommitted’ Foxp3+ T cells that give rise to exFoxp3 effector Th cells are activated non-Treg cells exhibiting promiscuous and transient Foxp3 expression, enriched in newly developed CD25low Foxp3+ T cells, rather than developmental intermediates of Treg cells, enriched in newly developed CD25high Foxp3+ T cells. To validate our hypothesis further, we have recently performed a high-throughput TCR repertoire analysis using © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

Foxp3GFPCre.ROSA26RFP.TCRCa+/ mice expressing a fixed single TCRb chain, and compared Va2 CDR3 sequences among GFP RFP CD44low (naive) cells, GFP RFP CD44high (effector- or memory-phenotype) cells, GFP RFP+ (exFoxp3) cells, and GFP+ (Foxp3+) cells. The results showed that the TCR repertoire of exFoxp3 T cells is most similar to GFP RFP CD44high effector- or memory-phenotype cells (author’s unpublished results). These data provide further evidence that the majority of exFoxp3 T cells are derived from activated T cells that transiently expressed Foxp3 in the course of differentiation into effector Th cells. Our results provide evidence for the heterogeneity model by demonstrating that only a minor population of nonregulatory Foxp3+ T cells gives rise to exFoxp3 effector Th cells in response to lymphopenia or inflammatory cytokine signals, whereas Foxp3+ T cells exhibiting suppressive functions do not, irrespectively of their thymic or peripheral origins. Although originating from a minor population, those non-Treg-derived exFoxp3 effector Th cells are able to accumulate by selective and extensive population expansion under those conditions. ‘Latent’ Treg cells: epigenetic memory of Treg cell fate If all exFoxp3 T cells originate from activated conventional T cells that have transiently upregulated Foxp3, their TSDR should be completely methylated. This was not the case, however, as exFoxp3 T cells isolated from Foxp3GFPCre.ROSA26RFP mice showed a partially demethylated TSDR at the population level (85). When activated in vitro with anti-CD3 and CD28 antibodies in the presence of IL-2, approximately 30% of GFP RFP+ CD4+ T cells re-acquired Foxp3 expression. Likewise, when adoptively transferred into Rag1 / mice, approximately 10% of them re-acquired Foxp3 expression when examined 2 weeks later (author’s unpublished results). Those that became Foxp3+ showed nearly complete TSDR demethylation and were fully suppressive, whereas those that remained Foxp3 showed fully methylated TSDR and lacked suppressive activity. ExFoxp3 T cells generated in lymphopenic mice also behaved similarly (69, 85). This Foxp3 re-induction was robust in that it did not depend on TGF-b signals and was not inhibited by IL-4 or IL-6 signals, in contrast to de novo Foxp3 induction from naive T cells. These results suggest that the exFoxp3 T-cell population is also heterogeneous and consists not only of effector Th cells derived from activated non-Treg cells but also of Treg cells that have lost Foxp3 expression but retain epigenetic memory of Foxp3 expression and suppressive functions. We therefore designated the latter exFoxp3 Tcell population as ‘latent’ Treg cells.

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The majority of exFoxp3 T cells that reacquired Foxp3 expression were Helioshigh, suggesting that many of latent Treg cells are derived from tTreg cells (85). Consistent with this possibility, others have previously shown that there is some overlap in TCR repertoire between exFoxp3 T cells and Foxp3+ thymocytes (86). In our own analyses, we also found some CDR3 sequences shared between the two populations (unpublished results). Latent Treg cells present in the exFoxp3 T-cell population likely account for this overlap. Many issues remain to be addressed concerning the biology and physiology of latent Treg cells. One important issue is the extrinsic cues that drive Foxp3+ Treg cells to become latent Treg cells. Because Foxp3 re-induction was dependent on TCR/CD28 stimulation (85) and inhibited partly by antiIL-2 antibodies (author’s unpublished results), it appears that limited availability of TCR ligands and/or IL-2 may be such extrinsic cues that lead to reversible Foxp3 downregulation in Treg cells. Consistent with this possibility, it has been shown that IL-2 neutralization promotes (99), whereas provision of IL-2 signals prevents (70, 87), Foxp3 downregulation and appearance of exFoxp3 T cells. Other important issues include their functions and cell fates, particularly under inflammatory conditions. Although it is theoretically possible that latent Treg cells may switch their cell fate into Th cells under inflammatory conditions, this is unlikely because the Foxp3 re-induction is a robust process that takes place even in the presence of inflammatory cytokine signals. Nevertheless, it remains possible that latent Treg cells may transiently exhibit some effector Th cell activities before Foxp3 is re-induced. To address these issues, it will be important to find a marker that allows us to prospectively isolate and characterize latent Treg cells. Although there are many questions that need to be addressed in future studies, the existence of latent Treg cells that retain epigenetic memory of Foxp3 expression and suppressive functions (and hence Treg cell fate) provides further evidence that Treg cells represent a stable cell lineage committed to suppressive functions independently of continuous Foxp3 expression. These findings also suggest that the demethylated TSDR ensures stability of Treg cell fate, rather than stability of Foxp3 expression per se, and hence represents a specific signature of the committed Treg cell lineage. Mechanistically, the demethylated TSDR ensures epigenetic memory of Foxp3 expression probably by maintaining the Foxp3 locus accessible to the transcription factors (including CREB/ATF, Ets-1, and likely NF-jB as well) that bind to the TSDR and trans-activate Foxp3 tran-

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scription in a demethylation-dependent manner (104, 105). A recent study has shown that genomic regions within some other Treg signature genes including Il2ra, Ctla4, Tnfrsf18 (encoding GITR), and Ikzf4 (encoding Eos) are also demethylated preferentially in Treg cells (64). Although functions of these demethylated regions in transcriptional regulation remain unknown, those epigenetic modifications may also contribute to the lineage stability of Treg cells. A revised ‘heterogeneity model’: reconciling lineage stability with effector Th cell-like phenotypes of Treg cells Although our results provide evidence for the heterogeneity model, its initial form postulated the lineage heterogeneity of Foxp3+ T cells only and did not take into account the presence of latent Treg cells among exFoxp3 T cells (100). Therefore, the model needs to be revised to incorporate the lineage heterogeneity of exFoxp3 T cells as well. After the publication of our results (85), some studies continue to suggest that committed Treg cells can be reprogrammed to effector Th cells under certain inflammatory conditions. Thus, the issue of lineage stability versus lineage plasticity of Treg cells still remains contentious (35). Some of the recent studies did not rule out the possibility that exFoxp3 or Foxp3+ effector Th phenotype cells are derived from recently activated T cells exhibiting promiscuous and transient Foxp3 expression. For instance, some studies used CD25high Foxp3+ CD4+ T cells as ‘bona fide Treg’ cells and showed that some of them gave rise to exFoxp3 Th cells, but as discussed above, these cells still contain a few uncommitted cells, particularly recently developed CD25high Foxp3+ T cells, which might have expanded under the inflammatory conditions used in those studies (87, 106). More importantly, in light of the revised heterogeneity model, even if committed Treg cells can downregulate Foxp3 expression and/or acquire effector Th cell-like phenotypes (such as the potential to produce pro-inflammatory cytokines), such phenotypes do not necessarily indicate lineage plasticity or even functional plasticity of Treg cells, as I discuss here. The first issue that needs to be discussed is whether loss of Foxp3 expression in Treg cells indicates their effector Th cell functions in vivo. Recent studies have shown that, in response to certain inflammatory signals, at least some of bona fide Treg cells downregulate Foxp3 expression. Two recent papers have shown that, in response to LPS, © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014


heat-shock, or pro-inflammatory cytokines such as IL-6, Foxp3 protein is polyubiquitinated and subjected to proteasome-dependent degradation, at least partly due to upregulation of the E3 ubiquitin ligase Stub1 (107) and due to downregulation of the deubiquitinase USP7 (108). Consistent with these results, we also noted previously that Foxp3 expression levels in CD25high Foxp3+ T cells are uniformly downregulated when activated in the presence of IL-6, although few cells completely lost Foxp3 expression (69). Another recent paper has shown that, upon immunization of Foxp3-GFPCre BAC transgenic ROSA26RFP mice with a MOG peptide in Complete Freund’s adjuvant, frequencies of exFoxp3 T cells are increased in some MOG-specific, but not polyclonal, RFP+ CD4+ T cells infiltrating into the central nervous system during the induction and peak phases of EAE (87). Approximately 10% of MOG-specific exFoxp3 T cells, while few MOG-specific Foxp3+ T cells, were IFN-c+. DNA methylation analysis revealed that the TSDR of those antigen-specific exFoxp3 T cells is partially demethylated (more demethylated than GFP RFP T cells and polyclonal exFoxp3 T cells, but less demethylated than antigen-specific as well as polyclonal Foxp3+ T cells), although the extent of the TSDR demethylation in MOG-specific exFoxp3 T cells showed considerable individual variations. These findings suggest that exFoxp3 T cells are derived from both Foxp3+ Treg cells exhibiting demethylated TSDR and uncommitted Foxp3+ T cells exhibiting methylated TSDR. These studies, however, do not provide any evidence that those Tregderived exFoxp3 T cells exhibit effector Th cell functions in vivo and thus have switched their cell fate. Because Foxp3 is required for suppressive functions of Treg cells, it is conceivable that suppressive functions are abrogated, but this does not indicate that those Treg-derived exFoxp3 cells function as effector Th cells in vivo. In the EAE study, the authors showed that exFoxp3 T cells transferred EAE into T cell-deficient mice, but their heterogeneous origins make it unclear which population, Treg or non-Treg (or both), is the pathogenic one (87). In addition, it should be pointed out that exFoxp3 T cells needed to be activated and expanded for a long period of time in vitro before the adoptive transfer. Because Treg cells (including latent Treg cells) proliferate poorly as compared to non-Treg cells in vitro, this raises a possibility that non-Treg-derived exFoxp3 T cells underwent selective population expansion and diluted Tregderived exFoxp3 T cells during the in vitro culture. More importantly, in light of our findings that Treg cells retain epigenetic memory of Foxp3 expression and suppressive functions, it is likely that inflammation-induced © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

downregulation of Foxp3 expression in Treg cells is transient and reversible. Consistent with this possibility, Foxp3 expression in MOG-specific RFP+ CD4+ T cells was restored during the resolution phase of EAE (87). Similarly, downregulation of Foxp3 expression and abrogation of suppressive functions in Treg cells stimulated with a TLR2 agonist were also shown to be transient and reversible (109). In addition, human T-cell clones derived from CD25high Treg cells show downregulation of Foxp3 expression after repetitive TCR stimulation, but these cells do not show effector Th cell-like phenotypes and this Foxp3 downregulation appears to be transient and reversible (83, 110). Thus, these recent data do not provide unequivocal evidence that inflammationinduced or repetitive TCR stimulation-induced Foxp3 downregulation in Treg cells leads to their conversion into effector Th cells. The second issue is whether acquisition by Treg cells of effector Th cell-like phenotypes such as expression of proinflammatory cytokines (e.g. IFN-c and IL-17) and CD40L is indicative of their lineage plasticity. As discussed earlier, Treg cells can produce these molecules under certain conditions, but these cells do retain suppressive functions in most cases. More importantly, some reports suggested that IFN-c produced from Treg cells is rather required for their suppressive functions (111, 112). For instance, in a model of lethal GvHD, in which adoptive transfer of bone marrow donor-type Foxp3+ Treg cells has been shown to protect the hosts from the disease (113), many donor Foxp3+ T cells as well as exFoxp3 T cells produced IFN-c (112). Strikingly, Foxp3+ T cells isolated from IFN-c-deficient mice failed to protect the host mice from the lethal GvHD, indicating that IFN-c production from Foxp3+ and/or exFoxp3 T cells is critical for their in vivo suppressive functions (112). Little is known about in vivo functions of IL-17 produced from Foxp3+ T cells. Considering the recent findings that even Th17 can be sometimes suppressive under certain inflammatory conditions (114, 115), however, it is reasonable to suggest that IL-17 production from Treg cells does not necessarily indicate their pro-inflammatory functions. Thus, in order to claim that Treg cells can be functionally reprogrammed, it is not sufficient to show that they express effector cytokines but is necessary to demonstrate that they do show effector Th cell functions in vivo. However, few studies actually examined helper functions of Treg cells in vivo. Although Sharma et al. showed that Foxp3+, but not Foxp3 , T cells function as Th cells that promote crosspriming of CD8+ T cells upon vaccination with protein antigens along with CpG-DNA adjuvants (95, 96), these

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results contradict with other findings that Treg cell-depletion in combination with CpG-based vaccination greatly enhanced anti-tumor or anti-bacterial CD8+ T-cell responses (116, 117). Although further studies are necessary to settle these two issues, I argue that inflammation-induced loss of Foxp3 expression and/or acquisition of effector Th cell-like phenotypes reflect phenotypic plasticity, but not lineage plasticity, of Treg cells. From a teleological point of view, it has been proposed that functional reprogramming of Treg cells into effector Th cells is necessary for immune responses against infectious agents and tumor cells to be initiated (30–32). A recent study showed, however, that Treg cell depletion induced the activation and expansion of low-avidity CD8+ T-cell clones but prevented the activation of high-avidity ones, thereby impairing memory responses to pathogens (118). Thus, suppressive functions of Treg cells do not necessarily impede protective immune responses against pathogens but may be rather required for appropriate coordination of protective immunity as well as for prevention of collateral tissue damage (118, 119).

Implications of the revised heterogeneity model in Treg cell fate determination and maintenance The revised heterogeneity model has also implications with respect to the differentiation pathways of Treg cells and the mechanisms of Treg cell fate determination and maintenance (Fig. 1). Thymic and peripheral Treg cell differentiation has been suggested to take place through a two-step process; TCR signals induces upregulation of CD25 (IL-2Ra chain), rendering these CD25+ Foxp3 thymocytes receptive to subsequent IL-2 signals that induce Foxp3 expression and differentiation into CD25high Foxp3+ Treg cells (120–123). Our findings demonstrated that newly developed peripheral Foxp3+ T cells are heterogeneous and consist of committed Treg cells enriched in CD25high cells and of non-Treg cells enriched in CD25low cells. We have recently found that newly developed Foxp3+ thymocytes are also heterogeneous; CD25high cells exhibit largely stable Foxp3 expression and suppressive activity, whereas CD25low cells exhibit more unstable Foxp3 expression, little suppressive activity, and are

Fig. 1. Treg cell fate determination and maintenance as viewed from the ‘revised heterogeneity model’. During thymic or peripheral Treg cell differentiation, uncommitted precursor cells adopt either Treg cell or conventional T (Tconv) cell fates upon activation through TCR/CD28, interleukin 2 (IL-2), and other signals. The commitment to the Treg cell fate is made probably before Foxp3 induction at the Foxp3 CD25+ Treg precursor stage and executed by the transcription factor network elicited by extrinsic signals from the extracellular environment. The same signals also induce expression of Treg cell signature genes (including Foxp3, CD25, and others) and epigenetic modifications at some cisregulatory elements (including DNA demethylation of the Foxp3 Treg cell-specific demethylation region (TSDR) and some other regions). Foxp3 is incorporated into the pre-existing transcription factor network and the resulting ‘Foxp3 interactome’ establishes the characteristic Treg-cell phenotype in cooperation with the remodeled cis-regulatory elements. The individual components of the network (indicated by x, y), however, may change during Treg cell differentiation. The Foxp3 complexes bind to the demethylated TSDR and auto-regulate Foxp3 transcription (red arrow). Treg cells further undergo phenotypic changes (including CD25 downregulation) in response to extrinsic cues and may also downregulate Foxp3 expression under certain circumstances (such as inflammation or limited availability of IL-2). These ‘latent’ Treg cells remain committed to the Treg cell fate because they retain the Treg cell-specific epigenetic mechanisms which ‘memorize’ Foxp3 expression and suppressive functions. On the other hand, when activated thymocytes or T cells express Foxp3 without engagement of the transcription factor network that controls Treg cell lineage commitment, Foxp3 expression alone cannot establish the characteristic Treg-cell phenotype. As a result, the activated Foxp3+ T cells readily lose Foxp3 expression, adopt the alternative Tconv cell fate and differentiate into effector Th cells. At early phases of the fate decision process, Treg and Tconv cells may still retain options to adopt the alternative lineage fate, before the epigenetic mechanisms fully establish the differentiated cellular states (dashed line arrows). The signals that direct this cell fate decision process as well as the identity of the transcription factor network that controls Treg cell lineage commitment remain to be elucidated.

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prone to activation-induced cell death (author’s unpublished results). These results indicate that the decision for precursor cells to adopt the Treg cell fate is made as soon as, or even before Foxp3 is induced, probably at the CD25+ Foxp3 precursor stage. On the other hand, a recent study has suggested that CD25low Foxp3+ thymocytes also represent Treg cell precursors that differentiate into CD25high Foxp3+ cells in response to IL-2 signals (124). It is currently unclear, however, whether these data indicate that uncommitted CD25low Foxp3+ thymocytes make the decision to adopt the Treg cell fate after Foxp3 induction in response to IL-2 signals. It is also possible that CD25low Foxp3+ thymocytes contain some committed Treg cells that upregulate CD25 in response to IL-2 signals. Because some of newly developed CD25low Foxp3+ thymocytes and T cells continued to express Foxp3 when stimulated even under conditions that drive effector Th cell differentiation (i.e. in the presence of IL-4, IL-6, or anti-TGF-b antibodies) (author’s unpublished results), we think that the latter possibility is more likely. However, it is currently difficult to dissect these two possibilities, because of the lack of methods to trace fates of individual cells. The existence of two alternative cell fates (i.e. Treg cell fate and exFoxp3 conventional T-cell fate) in Foxp3+ thymocytes and T cells raises important questions as to what are the extrinsic signals that controls this fate decision process and what intrinsic mechanisms translate these signals into distinct cell fates. Considering the importance of autoreactivity for tTreg-cell differentiation, it is very likely that TCR signals play a key role. However, TCR signals are also required for activation-induced promiscuous and transient Foxp3 expression (85), suggesting that differences in the quantity and/or quality of TCR signals may be translated into these distinct cell fates. In addition to TCR signals, the observed association of CD25 expression with stability of Foxp3 expression would suggest a role for IL-2 signals, but again IL-2 signals are also required for promiscuous and transient Foxp3 expression (85). Because CD25 expression depends on TCR signals, this observed association may be a consequence of differential TCR signals. It is obviously possible that there may be other signals that control Treg cell fate determination, which are yet to be identified. Regarding the intracellular mechanisms that control Treg cell fate determination and maintenance, one of the important factors is epigenetic mechanisms, particularly DNA demethylation of the Foxp3 TSDR and other Treg cell-associated gene loci (54, 64). Our results indicate that newly developed CD25high Foxp3+ T cells showed only partial © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 259/2014

TSDR demethylation but exhibited largely stable Foxp3 expression and suppressive functions. A recent study by Huehn and colleagues (125) also showed that most immature CD24high Foxp3+ thymocytes displayed largely methylated TSDR yet already exhibited stable Foxp3 expression. These results indicate that TSDR demethylation is initiated only after commitment to the Treg cell fate has taken place and thus TSDR demethylation acts as a safeguard that maintains stability of Treg cell fate but not as the cell fate determining factor in the initial commitment process. Although the intracellular mechanisms that commit precursor cells to the Treg cell fate remain elusive, two recent studies have provided some insights by showing that Foxp3, its cofactors, and genes encoding them form a molecular circuitry with multiple and redundant feedback loops (66, 67). On the basis of these findings, Benoist and colleagues (66) proposed that such a molecular network may function as a genetic switch that ‘locks-in’ the characteristic Treg cell transcriptional signature. These findings suggest a view that the Treg cell fate is not determined solely by individual regulatory components but rather by a self-perpetuating property of the transcriptional network as a whole (66, 126). Such a network perspective of cellular differentiation has been emerging as an important paradigm particularly in the field of stem cell biology (127–129), and should be instrumental in elucidation of the mechanisms responsible for Treg cell fate determination and maintenance. The existence of latent Treg cells in the normal T-cell repertoire has implications with respect to the origin of pTreg cells. Although it has been assumed that any Foxp3+ T cells that are derived from peripheral Foxp3 T cells are pTreg cells, generated de novo in the periphery, this may not be always the case because the starting Foxp3 T-cell population may contain latent Treg cells, some of which may be derived from tTreg cells. In future studies, it is therefore important to distinguish de novo Foxp3 induction from reinduction when addressing many questions concerning pTreg cells. Conclusions and future perspectives The issue of lineage stability versus lineage plasticity of Treg cells still remains contentious. In an attempt to resolve the ongoing controversy, I have herein proposed a revised heterogeneity model, which considers the lineage heterogeneity of Foxp3+ T cells and exFoxp3 T cells. Our findings indicate that Foxp3 expression does not segregate entirely with Treg cell fate because some Foxp3+ T cells are not committed to Treg cell fate and some exFoxp3 T cells retain epigenetic

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memory of, and thus remain committed to, Treg cell fate. I argue that the current controversy stems in part from the lack of the lineage specificity of Foxp3 expression and also from the confusion between phenotypic plasticity and lineage plasticity. By distinguishing these two notions, the revised heterogeneity model would provide a coherent framework that reconciles lineage stability with effector Th cell-like phenotypes of Treg cells. This model still remains hypothetical, however, because we have so far dealt with cell populations and been unable to monitor functions and fates of individual Foxp3+ and exFoxp3 T cells over time in

vivo. Practically, the boundary between phenotypic plasticity and lineage plasticity therefore remains obscure. To define the boundary more clearly, it is important to understand what commitment to Treg cell fate really means at the molecular as well as system levels. The revised heterogeneity model might also provide a framework for future studies into this direction. The recent technical advances in single cell biology will help proving or disproving the revised heterogeneity model and facilitate our understanding of the mechanisms of Treg cell fate determination and maintenance.

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RORγt(+)Foxp3(+) Cells are an Independent Bifunctional Regulatory T Cell Lineage and Mediate Crescentic GN.

Histone methylation mediates plasticity of human FOXP3(+) regulatory T cells by modulating signature gene expressions.

Epigenetic and transcriptional control of Foxp3+ regulatory T cells.

FOXP3+ regulatory T cells and their functional regulation.

Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells.

Transcriptome profiling of human FoxP3+ regulatory T cells.

Tr1-Like T Cells - An Enigmatic Regulatory T Cell Lineage.

Development of thymic Foxp3(+) regulatory T cells: TGF-β matters.

IL-17+Foxp3+ T cells: an intermediate differentiation stage between Th17 cells and regulatory T cells.

Adipose tissue macrophages induce PPARγ-high FOXP3(+) regulatory T cells.

Hepatocytes induce Foxp3⁺ regulatory T cells by Notch signaling.

Foxp3+ regulatory T cells promote lung epithelial proliferation.

Unstable FoxP3+ T regulatory cells in NZW mice.

Harnessing FOXP3+ regulatory T cells for transplantation tolerance.

SOCS1 and regulation of regulatory T cells plasticity.

Regulatory mechanisms link phenotypic plasticity to evolvability.

γδ T cells restrain extrathymic development of Foxp3+-inducible regulatory T cells via IFN-γ.

Regulatory T cells, especially ICOS+ FOXP3+ regulatory T cells, are increased in the hepatocellular carcinoma microenvironment and predict reduced survival.

The balance of intestinal Foxp3+ regulatory T cells and Th17 cells and its biological significance.

Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells.

Induction of antigen specific CD4(+)CD25(+)Foxp3(+)T regulatory cells from naïve natural thymic derived T regulatory cells.

UXT is a novel regulatory factor of regulatory T cells associated with Foxp3.

Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations.

Antigen-Specific Development of Mucosal Foxp3+RORγt+ T Cells from Regulatory T Cell Precursors.