Lineage stability and phenotypic plasticity of Foxp3 + regulatory T cells

Shohei Hori Lineage stability and phenotypicplasticity of Foxp3+ regulatory Tcells

Author’s address

Shohei Hori1

1Laboratory for Immune Homeostasis, RCAI, RIKEN Center

for Integrative Medical Sciences, Kanagawa, Japan.

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-in-

Aid 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.

Summary: Regulatory T (Treg) cells expressing the transcription factorforkhead box protein 3 (Foxp3) constitute a unique T-cell lineagecommitted to suppressive functions. While their differentiation state isremarkably stable in the face of various perturbations from the extra-cellular environment, they are able to adapt to diverse and fluctuatingtissue environments by changing their phenotype. The lineage stabilityand phenotypic plasticity of Treg cells thus ensure the robustness ofself-tolerance and tissue homeostasis. Recent studies have suggested,however, that Treg cells may retain lineage plasticity, the ability toswitch their cell fate to various effector T-cell types under certain cir-cumstances such as inflammation, a notion that remains highly conten-tious. While accumulating evidence indicates that some Treg cells candownregulate Foxp3 expression and/or acquire effector T-helper cell-like phenotypes, results from my laboratory have shown that Treg cellsretain epigenetic memory of, and thus remain committed to, Foxp3expression and suppressive functions despite such phenotypic plasticity.It has also become evident that Foxp3 can be promiscuously and tran-siently expressed in activated T cells. Here, I argue that the currentcontroversy stems partly from the lack of the lineage specificity ofFoxp3 expression and also from the confusion between phenotypicplasticity and lineage plasticity, and discuss implications of our findingsin Treg cell fate determination and maintenance.

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

Introduction

Protection of an organism from autoimmune diseases cannot

be explained entirely by physical elimination or functional

inactivation of autoreactive lymphocyte repertoires. Accumu-

lating evidence indicates that immunological tolerance to

body tissues may be rather dominant and rely on cell-

extrinsic regulation of pathogenic autoreactive lymphocytes

by a subset of T lymphocytes called regulatory T (Treg)

cells. Since the late 1980s, several laboratories have indepen-

dently characterized CD4+ T-cell subsets capable of protect-

ing self or foreign tissues from destructive immune

responses using different disease or transplantation models

and designated such tissue-protective or immune-suppres-

sive cells as Treg cells (1–8). Those early studies culminated

Immunological Reviews 2014

Vol. 259: 159–172

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© 2014 John Wiley & Sons A/S. Published by John Wiley & SonsLtdImmunological Reviews0105-2896

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdImmunological Reviews 259/2014 159

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 develop-

ment 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 collat-

eral tissue damage triggered by immune responses against

microbes or allergens (17–20), maintaining homeostasis

with the commensal microbiota (21, 22), facilitating mater-

nal tolerance to allogeneic fetus during pregnancy (23),

promoting therapeutic tolerance toward transplanted organs

(24), and sometimes helping tumor cells or certain patho-

gens to escape from immune surveillance (18, 25). More-

over, 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 robust-

ness of Treg cell functions in the face of various unpredict-

able perturbations from the extracellular environment. There

is evidence that Foxp3+ Treg cells represent a stable cell

lineage committed to suppressive functions and are thus dis-

tinct 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 pro-

posed 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 self-

tolerance and tissue homeostasis, this emerging notion has

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 autoim-

mune 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 rec-

oncile 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 self-

tolerance. 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 recep-

tor (TCR) interactions with self-peptides/major histocom-

patibility 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 conven-

tional 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 find-

ings 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 Ltd160 Immunological Reviews 259/2014

Hori � Treg cell fate determination and maintenance

functions and Foxp3 acts as their ‘master regulator’ or ‘line-

age 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’ molecu-

lar marker of Treg cells. Consistent with earlier studies, dif-

ferentiation of Foxp3+ Treg cells was confirmed to take

place primarily in the thymus at the CD4+ CD8� single-

positive stage concomitantly with positive and/or negative

selection [thymus-generated Treg (tTreg cells)] (12). Addi-

tional 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 suppres-

sive 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 posi-

tively or negatively regulate Foxp3 expression have also

been identified (28). Importantly, induction and mainte-

nance of Foxp3 expression are two separable processes regu-

lated 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 func-

tional 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-

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), cis-

regulatory elements (68), and epigenetic mechanisms

(64).

Recent studies have also shown that Foxp3+ Treg cells is

not a homogeneous population but can change their migra-

tory, 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 transcrip-

tional 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 regu-

late 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.



CXCR5high PD1high ICOShigh Bcl6+), efficiently induced ger-

minal centers, and promoted IgA production in the intes-

tine (72). The differentiation of exFoxp3 Tfh cells was not

seen in other lymphoid tissues, indicating that environmen-

tal cues present in Peyer’s patches promote the accumula-

tion of exFoxp3 T cells and their differentiation into Tfh

cells. Others also reported increased accumulation of ex-

Foxp3 T cells in the intestine of T-cell-deficient recipients

of Foxp3+ T cells, particularly in the presence of inflamma-

tion (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). Simi-

larly, when Ly5.1 Foxp3+ T cells were transferred into sub-

lethally 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+ Th2-

like 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 costimu-

latory 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 pro-

tein 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

also carried out by others who used Foxp3-GFPCre bacterial

artificial chromosome (BAC) transgenic ROSA26YFP mice

(86). Consistent with their findings, we found that approxi-

mately 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 cell-

associated 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 auto-

reactive 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 expres-

sion, 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 condi-

tions in vitro (76–79). In vivo evidence for the development

of Foxp3+ T-bet+ IFN-c+ cells in inflammatory environ-

ments 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 cross-

priming of CD8+ T cells (95, 96). In most cases, however,

those Foxp3+ T cells retain suppressive functions, leaving it



unclear whether such hybrid-phenotypes reflect lineage

plasticity of Treg cells (see below).

These observations prompted some investigators to pro-

pose that Treg cells can switch their lineage fate to diverse

effector T-cell types under certain conditions such as inflam-

mation 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 rec-

onciled with the robustness of self-tolerance and tissue

homeostasis. Because many Treg cells are positively selected

based on their autoreactivity, their functional reprogram-

ming to pathogenic effector Th cells could result in a cata-

strophic 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 con-

ditions (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 oligoden-

drocyte 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 fate-

mapping study using a pulse-labeling approach and showed

that Foxp3+ T cells display remarkably stable Foxp3 expres-

sion under steady state and various perturbed conditions in

adult mice. They knocked-in cDNA encoding a GFP-

Cre-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 ani-

mals were challenged with various inflammatory stimuli or

with a lymphopenic episode induced by sublethal irradia-

tion. 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

appearance of exFoxp3 T cells was IL-2 neutralization, but

the resulting exFoxp3 T cells did not show effector Th cell-

like phenotypes. These observations are apparently contra-

dictory 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 pro-

liferation, 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 anti-

TGF-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 prolif-

eration 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 differen-

tiate 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



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 suppres-

sive 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 per-

formed adoptive transfer experiments to identify peripher-

ally 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

time primarily by losing the potential to expand in lymp-

hopenic 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 condi-

tions. 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 ana-

lyzed, 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. Signifi-

cantly, those exFoxp3 T cells derived from peripherally

induced Foxp3+ T cells underwent more extensive popula-

tion 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 pro-

moted 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 pres-

ent in Peyer’s patch environment or T cell-deficient environ-

ment 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 thymus-

derived 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 Foxp3-

expressing 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.



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 Cre-

mediated 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 signifi-

cantly 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 suppres-

sive activity, stable Foxp3 expression, and a fully demethy-

lated 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 con-

tained 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 exhib-

iting 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

Foxp3GFPCre.ROSA26RFP.TCRCa+/� mice expressing a fixed sin-

gle 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-pheno-

type 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 non-

regulatory Foxp3+ T cells gives rise to exFoxp3 effector Th

cells in response to lymphopenia or inflammatory cytokine

signals, whereas Foxp3+ T cells exhibiting suppressive func-

tions 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, how-

ever, 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. Like-

wise, when adoptively transferred into Rag1�/� mice, approx-

imately 10% of them re-acquired Foxp3 expression when

examined 2 weeks later (author’s unpublished results). Those

that became Foxp3+ showed nearly complete TSDR demethy-

lation 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 ex-

Foxp3 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 suppres-

sive functions. We therefore designated the latter exFoxp3 T-

cell population as ‘latent’ Treg cells.



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 biol-

ogy 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 anti-

IL-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 downregu-

lation 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 downre-

gulation 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) pro-

vides further evidence that Treg cells represent a stable cell

lineage committed to suppressive functions independently

of continuous Foxp3 expression. These findings also sug-

gest 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 transcrip-

tion factors (including CREB/ATF, Ets-1, and likely NF-jB

as well) that bind to the TSDR and trans-activate Foxp3 tran-

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 tran-

scriptional 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 heterogene-

ity model, its initial form postulated the lineage heteroge-

neity 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 incor-

porate 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 repro-

grammed 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 heterogene-

ity model, even if committed Treg cells can downregulate

Foxp3 expression and/or acquire effector Th cell-like phe-

notypes (such as the potential to produce pro-inflammatory

cytokines), such phenotypes do not necessarily indicate line-

age 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,



heat-shock, or pro-inflammatory cytokines such as IL-6,

Foxp3 protein is polyubiquitinated and subjected to protea-

some-dependent degradation, at least partly due to upregu-

lation of the E3 ubiquitin ligase Stub1 (107) and due to

downregulation of the deubiquitinase USP7 (108). Consis-

tent 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 cen-

tral 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 Treg-

derived 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 con-

ceivable 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 adop-

tive 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 Treg-

derived 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 suppres-

sive functions, it is likely that inflammation-induced

downregulation of Foxp3 expression in Treg cells is tran-

sient 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, downre-

gulation 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 inflammation-

induced 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 pro-

inflammatory 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 condi-

tions, 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 sup-

pressive 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 inflamma-

tory conditions (114, 115), however, it is reasonable to

suggest that IL-17 production from Treg cells does not nec-

essarily indicate their pro-inflammatory functions. Thus, in

order to claim that Treg cells can be functionally repro-

grammed, 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 cross-

priming of CD8+ T cells upon vaccination with protein

antigens along with CpG-DNA adjuvants (95, 96), these



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 pheno-

types 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 nec-

essarily impede protective immune responses against patho-

gens but may be rather required for appropriate

coordination of protective immunity as well as for preven-

tion 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), ren-

dering these CD25+ Foxp3� thymocytes receptive to subse-

quent 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 heteroge-

neous; 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 Tregcell 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 samesignals also induce expression of Treg cell signature genes (including Foxp3, CD25, and others) and epigenetic modifications at some cis-regulatory elements (including DNA demethylation of the Foxp3 Treg cell-specific demethylation region (TSDR) and some other regions). Foxp3 isincorporated into the pre-existing transcription factor network and the resulting ‘Foxp3 interactome’ establishes the characteristic Treg-cellphenotype 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 (redarrow). Treg cells further undergo phenotypic changes (including CD25 downregulation) in response to extrinsic cues and may alsodownregulate Foxp3 expression under certain circumstances (such as inflammation or limited availability of IL-2). These ‘latent’ Treg cells remaincommitted to the Treg cell fate because they retain the Treg cell-specific epigenetic mechanisms which ‘memorize’ Foxp3 expression andsuppressive functions. On the other hand, when activated thymocytes or T cells express Foxp3 without engagement of the transcription factornetwork 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 earlyphases of the fate decision process, Treg and Tconv cells may still retain options to adopt the alternative lineage fate, before the epigeneticmechanisms fully establish the differentiated cellular states (dashed line arrows). The signals that direct this cell fate decision process as well asthe identity of the transcription factor network that controls Treg cell lineage commitment remain to be elucidated.



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 uncom-

mitted 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+ thymo-

cytes 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 contin-

ued to express Foxp3 when stimulated even under condi-

tions 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+ thy-

mocytes and T cells raises important questions as to what

are the extrinsic signals that controls this fate decision pro-

cess and what intrinsic mechanisms translate these signals

into distinct cell fates. Considering the importance of auto-

reactivity 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 pos-

sible 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 impor-

tant factors is epigenetic mechanisms, particularly DNA

demethylation of the Foxp3 TSDR and other Treg cell-associ-

ated gene loci (54, 64). Our results indicate that newly

developed CD25high Foxp3+ T cells showed only partial

TSDR demethylation but exhibited largely stable Foxp3

expression and suppressive functions. A recent study by

Huehn and colleagues (125) also showed that most imma-

ture CD24high Foxp3+ thymocytes displayed largely methy-

lated 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 main-

tains stability of Treg cell fate but not as the cell fate deter-

mining 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 cofac-

tors, 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) pro-

posed 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 reg-

ulatory components but rather by a self-perpetuating prop-

erty 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 instru-

mental in elucidation of the mechanisms responsible for

Treg cell fate determination and maintenance.

The existence of latent Treg cells in the normal T-cell rep-

ertoire 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 popula-

tion 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 re-

induction 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 het-

erogeneity 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



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 line-

age 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 het-

erogeneity model and facilitate our understanding of the

mechanisms of Treg cell fate determination and mainte-

nance.

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Lineage stability and phenotypic plasticity of Foxp3 + regulatory T cells