FOCUS | Review Article

FOCUS | Review Article https://doi.org/10.1038/s41590-018-0120-4

https://doi.org/10.1038/s41590-018-0120-4

Regulatory T cells in autoimmune disease

Margarita Dominguez-Villar1* and David A. Hafler1,2*

In recent years, the understanding of regulatory T cell (Treg cell) biology has expanded considerably. Key observations have
challenged the traditional definition of Treg cells and have provided insight into the underlying mechanisms responsible for the
development of autoimmune diseases, with new therapeutic strategies that improve disease outcome. This Review summarizes
the newer concepts of Treg cell instability, Treg cell plasticity and tissue-specific Treg cells, and their relationship to autoimmunity.
Those three main concepts have changed the understanding of Treg cell biology: how they interact with other immune and non-
immune cells; their functions in specific tissues; and the implications of this for the pathogenesis of autoimmune diseases.

M
ore than 20 years after their ‘re-discovery’, regulatory T cells
(Treg cells) have emerged as an important component in
understanding of the immune response to pathogens and
the mechanisms of peripheral tolerance that control the development
of allergies and autoimmune diseases. In mice and humans, Treg cells
are characterized by high expression of the cytokine IL-2 receptor
α​-chain (CD25) and expression of the transcription factor Foxp3,
which is required for their development, function and stability1–4. In
humans, as CD25 is also expressed by activated CD4+ T cells, absence
of the IL-7 receptor α​-chain (CD127) is used as a marker complemen-
tary to CD25 to more precisely identify human Treg cells5. Moreover,
numerous surface receptors have been described that are variably
specific for defined Treg cell subsets, indicative of the heterogeneity of
this population6. Treg cells can be broadly classified into two groups on
the basis of their developmental origin. Thymic Treg cells (tTreg cells),
also known as ‘natural Treg cells’, are generated in the thymus as a sepa-
rate lineage at the stage of CD4+ single-positive thymocytes and are
thought to show enrichment for T cell antigen receptors (TCRs) with
high affinity for self peptides7. Although the details of their suppres-
sive mechanisms are still not completely understood, and these mech-
anisms are most probably dependent on the microenvironment and
the target population to be controlled, in general these cells perform
their function both by cell-contact mechanisms that involve specific
cell-surface receptors and by the secretion of inhibitory cytokines such
as IL-10, TGF-β​and IL-357. Induced Treg cells (iTreg cells) develop from
conventional CD4+ T cells in the periphery after antigen encounter
and in the presence of specific factors such as TGF-β​ and IL-28. So
far there is (are) no definitive protein marker(s) that distinguish(es)
between these two Treg cell populations in vitro or in vivo, although
there are important differences in their epigenetic signatures, and par-
ticularly in the Foxp3 locus, that make iTreg cells intrinsically unstable
in inflammatory and/or stress conditions8,9.
From a historical perspective, the initial observations made in the
1970s that led to the definition of Treg cells in the 1990s are tightly
linked to autoimmune diseases. Seminal work by Nishizuka and
Sakakura back in 1969 demonstrated that thymectomy in healthy
neonatal mice led to inflammation and severe organ-specific auto-
immune pathology, which indicated the involvement of a thymus-
derived population in control of self-tolerance10. That observation
was further confirmed in adult rats that underwent thymectomy
and were subjected to sublethal irradiation11. Most notably, inocula-
tion with CD4+ T cells from healthy syngeneic mice inhibited dis-
ease in both systems, and once autoimmunity was established, CD4+
T cells isolated from these sick mice and adoptively transferred into
T cell–deficient mice were able to induce disease12. These experiments
demonstrated that T cells are able not only to serve as inducers of
autoimmune disease but also to inhibit such disease. That led to the
hypothesis that in the periphery of normal mice there are two popula-
tions of CD4+ T cells: one population potentially capable of inducing
autoimmunity, and a second population of ‘suppressor’ cells, different
from helper T cells, that would inhibit autoreactive T cells. Treg cell
research during those years floundered, in part due to the failure to find
specific cell markers that could define this population and the ambi-
guity observed in their suppressive mechanisms. The identification of
a population of CD4+ T cells responsible for controlling autoreactive
responses13, as well as the search for a cell-surface marker that would
define this population, indicated that a subset of CD25-expressing
CD4+ T cells present in the periphery of healthy mice was responsible
for the inhibition of autoimmunity. The transfer of cell suspensions
from the spleen of BALB/c mice depleted of CD25+ cells into athymic
nude mice induced autoimmunity that affected several organs, and
co-transfer of CD25+CD4+ T cells inhibited disease4. In 2001, human
Treg cells were identified in the thymus and peripheral blood of healthy
people as those CD4+ T cells with very high expression of CD25 and
phenotype and function very similar to that of their rodent counter-
part14,15. Although there is still no definitive marker for the isolation
of human Treg cells, these studies, together with the discovery of Foxp3
as the master transcription factor of Treg cells1,3,16, laid the groundwork
for the beginning of in-depth analysis of Treg cell biology in health and
disease (Box 1).
As for Treg cell function in human autoimmune disease, early
studies of patients revealed that in various autoimmune disor-
ders, there is a defect in either the number or the function of Treg
cells isolated from the peripheral blood, with those data being
supported by in vivo models of disease (a comprehensive list of
autoimmune diseases is in ref. 17). Although some of these early
data were misleading due to the confounding variable of the iden-
tification of human Treg cells based on the positive expression of
CD25 as the only marker for Treg cell identification, subsequent
work has clearly demonstrated that most autoimmune diseases,
such as type 1 diabetes18–20, multiple sclerosis21,22, systemic lupus
erythematosus23, myasthenia gravis24, rheumatoid arthritis25 and
others17, display defects in either the number or the function of
tTreg cells in peripheral blood.
Investigations during the past two decades have advanced
knowledge of the mechanisms that underlie the development of tTreg
cells in the thymus, as well as their gene-expression signature, their
role in controlling both immune and non-immune cell responses
and their effect on immune system–mediated diseases (reviewed in
refs 7,26), and, finally, the identity of the tissues in which they exert
functions beyond suppression27,28. Moreover, the traditional view
that Treg cells are a terminally differentiated population not capable

Department of Neurology, Yale School of Medicine, New Haven, CN, USA. 2Department of Immunobiology, Yale School of Medicine, New Haven, CN,
1

USA. *e-mail: margarita.dominguez-villar@yale.edu; david.hafler@yale.edu

Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology 665

© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Review Article
|
Review Article FOCUS
https://doi.org/10.1038/s41590-018-0120-4
Box 1 | How are Treg cells studied in autoimmunity?
Studies of Treg cells in autoimmunity have been facilitated by the
existence of mouse models for many autoimmune diseases130.
While these models of autoimmune diseases allow fate-mapping
and gene-modification experiments that cannot be performed
in humans, they do not generally reflect the genetic architecture
that underlies human autoimmune diseases; that is, the models
often reflect the consequential aspects of disease pathogenesis
but not the underlying mechanisms that cause the activation of
autoreactive T cells.
Human Treg cell studies are hindered by the lack of a definitive
surface marker that uniquely isolates Treg cells from other
T cell populations. Thus, much of the early work assessing the
frequency of Treg cells in peripheral blood of patients needs to be
considered with caution, as Treg cells were defined solely by the
positive expression of CD25 and the results can be misleading,
as human conventional T cells can also express CD25 after
being activated. Moreover, human studies are limited to ex vivo
characterization or short in vitro experiments and depend on the
availability of samples from patients with autoimmunity.
Recent advances in high-throughput technologies that
require small numbers of cells will considerably affect research
into human Treg cell autoimmunity, allowing the analysis of rare
Treg cell populations from small tissue samples, etc. Currently
available technologies are able to determine the epigenetic and
transcriptional signature at the single-cell level. The combination
of these datasets and systems-biology approaches in the analysis
of serial samples obtained from people at different time points,
before and after therapeutic intervention and from different
anatomical locations is delivering an enormous amount of
information about the response of patients to treatment, disease
course and mechanistic insights.
of secreting pro-inflammatory cytokines and whose only function
is to suppress T cell responses has been challenged by recent data.
Specifically, Treg cells possess some degree of plasticity and instabil-
ity, although in-depth understanding of the molecular mechanisms
that drive these two states and the relationship between them in dis-
ease settings remains incomplete.
This Review will provide an update on recent discoveries in Treg
cell biology in the context of autoimmune disease. From our per-
spective, three main observations have changed the understand-
ing of what Treg cells are, how they function in peripheral lymphoid
organs and non-immune tissues, how they relate to other immune
and non-immune cells, and how their phenotype and function can
be modulated, with clear consequences for the development of
new therapeutic strategies for various autoimmune diseases. These
observations are as follows: 1) the instability of Treg cells and their
acquisition of an effector phenotype after losing Foxp3 expression
under inflammatory conditions; 2) the plasticity of the phenotype
of Treg cells, with their acquisition of effector-like properties while
maintaining Foxp3 expression; and 3) the discovery of tissue-spe-
cific Treg cells, which demonstrates again that Treg cells, like other
immune cells, are influenced by their environment. These obser-
vations have arisen at a time during which the development of
high-throughput genomic, epigenetic and proteomic technologies
is allowing the analysis of rare cell populations at a single-cell level,
which will undoubtedly improve knowledge of basic Treg cell biology
in general and of human Treg cell biology in particular.
Foxp3 as a master regulator of Treg cell phenotype and
function
The discovery of Foxp3 as the master regulator of Treg cell devel-
opment and function was critical for the understanding of Treg cell
666
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
NaTuRe Immunology
biology1–3. Inactivating mutations in Foxp3 result in the spontane-
ous development of severe autoimmunity with a scurfy phenotype
in mice29 and IPEX syndrome (‘immune dysregulation, polyendo-
crinopathy, enteropathy, X-linked’) in humans30,31. Foxp3 is neces-
sary for the development, maintenance and function of tTreg cells1–3,
although alone it is not sufficient for full recapitulation of the Treg
cell phenotype9,32. Beyond Foxp3, the second requirement necessary
for establishment of the Treg cell functional program is the genera-
tion of a specific epigenetic signature acquired during development
and finalized in the periphery9,33. Both have essential roles in main-
taining Treg cell function, and alteration of Foxp3 or epigenetic mod-
ifications in auto-inflammatory conditions are probably the cause
of the Treg cell instability and aberrant plasticity observed in several
autoimmune settings (Fig. 1). Due to the importance of Foxp3 in
Treg cell maintenance and the prevention of autoimmunity, regu-
lation of Foxp3 expression is a matter of active research. Foxp3 is
subjected to two main layers of regulation, transcriptional and post-
translational, both of which are responsive to positive and negative
regulation by factors in the tissue environment, including cytokines,
metabolic mediators and inflammatory factors.
Foxp3 expression is a tightly regulated process, and while the
mechanisms by which Foxp3 is transcriptionally regulated by tran-
scription factors during tTreg cell development and in mature Treg
cells have been clearly established (reviewed in ref. 34), data have
shown that in rodents, Foxp3 transcription is also controlled epige-
netically. The Foxp3 locus contains several conserved non-coding
sequences (CNSs) that are critical for the initiation and maintenance
of Foxp3 transcription35. Among the three CNSs described so far,
only CNS2 has been demonstrated to prevent autoimmunity. It is a
TCR-responsive enhancer with binding sites for Runx1–CBFβ​ tran-
scription-factor complexes that is important in maintaining Foxp3’s
stability. CNS2-deficient mice develop spontaneous autoimmunity,
which emphasizes the importance of CNS2 in the stability and func-
tion of Treg cells36,37. Furthermore, CNS2 contains a conserved CpG
island (TSDR) that is hypomethylated specifically in Treg cells; thus,
it is transcriptionally active in tTreg cells but is hypermethylated in
naive or effector T cells38,39. This region has been widely used to dis-
tinguish true tTreg cells from conventional T cells transiently upregu-
lating Foxp3 expression and iTreg cells38,39.
Three main post-translational modifications have been
described for Foxp3 protein: acetylation, phosphorylation and
ubiquitination. These modifications affect the stability and DNA-
binding capacity of Foxp3 and thus modulate Treg cell function
and the development of autoimmunity. The acetylation of spe-
cific lysine residues by lysine acetyltransferases globally stabilizes
Foxp3 expression and promotes Treg cell function by favoring the
binding of Foxp3 to its transcriptional targets40 and allowing it to
avoid proteasomal degradation40. Several acetylases and deacety-
lases, such as TIP6041 and p30040,42, have been shown to interact
with Foxp3 and control its acetylation. Other post-translational
processes include phosphorylation at serine and threonine resi-
dues by several kinases, including PIM-1, PIM-2 and CDK243–46,
and ubiquitination at lysine residues, which targets Foxp3 for
proteasomal degradation47. Inflammatory stimuli result in prote-
asome-dependent degradation of Foxp3 mediated by the ubiqui-
tinase Stub1, which binds to Foxp3 and promotes its Lys48-linked
ubiquitination48. Overexpression of Stub1 abolishes the suppres-
sive ability of Treg cells in vitro and in vivo and confers onto Treg
cells a phenotype with similarities to that of the TH1 subset of
helper T cells; this raises the question of whether the effector
T cell–like Treg cell phenotype in this setting is an intermediate
stage en route to instability and loss of Foxp3 protein expression.
In contrast, the deubiquitinating enzyme USP7 has high expres-
sion in Treg cells and is associated with Foxp3 in the nucleus,
where it regulates the turnover of Foxp3. Under inflammatory
conditions, this enzyme is downregulated, which facilitates the
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology
 FOCUS | Review
| Article
NaTuRe Immunology FOCUShttps://doi.org/10.1038/s41590-018-0120-4
Review Article

Health Autoimmunity

Treg cell instability (ex-Foxp3)

Foxp3 Global Observed in:

Transcriptional regulation protein epigenetic
Epigenetic regulation Environmental expression signature ‘ex-Foxp3’ cell
Post-translational modifications triggers NOD model
miRNAs Autoimmune EAE model
Others? inflammation Treg cell

Expression of pro-inflammatory cytokines

Downregulation of Foxp3
Pathogenic
Resolution of
Foxp3 Global inflammation
protein epigenetic
expression signature

Treg cell ? ?

Resolution of
inflammation Observed in:

TH1-like Treg cells

Treg cell plasticity Type 1 diabetes

Multiple sclerosis
HACs and HDACs
Autoimmune Foxp3 Global Sjögren syndrome
DNA methylation
inflammation protein epigenetic Autoimmune hepatitis
Histone modifications
expression signature Atherosclerosis
Others?
TH2-like Treg cells
Treg cell
Allergy and asthma
Systemic sclerosis

TH17-like Treg cells

Increased expression of helper T cell TFs
Expression of pro-inflammatory cytokines Psoriasis
Maintained Foxp3 expression Rheumatoid arthritis
Dysfunctional

Fig. 1 | The Treg cell functional program in health and autoimmunity. The Treg cell functional program depends on the following two main axes, both of
which are regulated by various factors (gray boxes) and are maintained under healthy conditions: Foxp3 expression, and a global epigenetic signature. In
genetically susceptible people, environmental triggers induce inflammation, and a proportion of Treg cells can lose Foxp3 expression and become unstable
(Treg cell instability; ex-Foxp3) or can maintain Foxp3 expression but alter their global epigenetic signature (Treg cell plasticity), secrete pro-inflammatory
cytokines and display reduced function. Treg cell instability leads to the so-called ‘ex-Foxp3 cells’, which have been observed in mouse models of diabetes
(NOD) and multiple sclerosis (EAE). Treg cell plasticity favors the acquisition of TH1-like, TH2-like or TH17-like properties by the Treg cells; each of these
outcomes have been observed in various autoimmune diseases in mice and humans. While the mechanistic connection between Treg cell instability and Treg
cell plasticity is not known, plastic Treg cells can become ‘normal’ Treg cells after resolution of inflammation. It remains to be determined whether this is also
the case for Treg cell instability. miRNA, microRNA; HACs, histone acetylases; HDACs, histone deacetylases; TFs, transcription factors.

degradation of Foxp340. Furthermore, conditional deletion of
USP7 in Treg cells leads to lethal autoimmunity with a decreased
number of Treg cells in the periphery that display an aberrant TH1-
like phenotype in vitro and in vivo49. Targeting USP7 decreases
recruitment of the acetyltransferase TIP60 to the CNS2 region
of Foxp3. TIP60 promotes acetylation-dependent dimerization
of Foxp350,51, and in the absence of TIP60, lethal autoimmunity
develops52. Other factors have also been shown to control Foxp3
expression, such as HIF-1α​, which is induced by stimulation with
IL-6 and the TCR and inhibits Foxp3 through ubiquitination53.
Both ubiquitination and acetylation target lysine residues, so
they might compete in the regulation of Foxp3 expression. In fact,
hyperacetylation of Foxp3 prevents its polyubiquitination and pro-
teasomal degradation, which increases its stability40. Such data sug-
gest that post-translational modifications of Foxp3 have a crucial
role in modulating the plasticity or instability of Treg cells, which
adds another layer of complexity to regulation of the Treg cell func-
tional program, with potential consequences for the development
of autoimmunity.
Foxp3 works in concert with other transcription factors and
proteins, forming multiprotein complexes that determine the tran-
scriptional signature and effector functions of Treg cells. Hundreds
of protein partners have been described, including the transcrip-
tion factors Gata-3, NFAT, Runx1, Eos and others54. Global analysis
of the Foxp3-interaction network suggests a model in which Foxp3
and its binding partners form multi-protein complexes that bind to
pre-existing DNA enhancers55 and regulate transcription positively
or negatively depending on the interacting proteins recruited56. As
mentioned above, Foxp3 also associates with proteins that mediate
epigenetic modifications, such as TIP60, Sirtuin 1 or HDAC741,57,
which alter the acetylation state of partner loci and Foxp3 itself,
with consequences for the binding of transcription factors and his-
tone modifications. In this context, disruption of Foxp3’s interac-
tions with specific proteins diminishes Treg cell function and leads
to autoimmune responses due to increased polyubiquitination of
Foxp358,59. Other evidence supporting the proposal of the impor-
tance of Foxp3-interacting partners in Treg cell function includes the
observation that different FOXP3 mutations result in a wide range

Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology 667

© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Review Article
|
Review Article FOCUS
https://doi.org/10.1038/s41590-018-0120-4
of IPEX disease severity, reflective of the relative importance of the
residues affected in the integrity of the Foxp3 protein and the pro-
tein partners that form the DNA-binding complexes. For example,
the most common mutation in IPEX, p.A384T, which disrupts the
sequence specificity of Foxp3’s DNA binding and alters Foxp3’s
interactions with specific target genes31, inhibits Treg cell function but
preserves the ability of Treg cells to repress inflammatory cytokine
production, due in part to a specific inhibition of Foxp3’s interaction
with TIP6060. Furthermore, experiments with mice have shown that
this mutation specifically in Treg cells perturbs the binding of Foxp3
to specific target genes, including Batf, which is partly responsible
for the induction of a unique pattern of tissue-restricted inflamma-
tion in certain non-lymphoid tissues due to defective function of
these Treg cells61. Additionally, a Foxp3 reporter mouse that expresses
a fusion of Foxp3 with enhanced green fluorescent protein (EGFP)
at its amino terminus (EGFP-Foxp3), which disrupts the interac-
tion of Foxp3 with many cofactors, including TIP60, p300 and Eos,
does not develop apparent autoimmunity, but its Treg cells display
alterations in function in vivo, with autoimmune-prone mice of the
non-obese diabetic (NOD) strain developing diabetes faster than
their wild-type counterparts do58. Interestingly, these same Treg cells
are potent suppressors of antibody-mediated arthritis due to ‘pref-
erential’ interaction of EGFP-Foxp3 with the transcription factor
IRF462. These findings not only demonstrate that certain cofactors
are crucial for Treg cell–mediated function but also suggest that Treg
cells might be ‘tuned’ to control particular types of inflammation
by modulating the constituents of Foxp3 protein complexes under
specific environmental conditions. Furthermore, we are tempted
to speculate that the variety of functions that Treg cells perform in
different tissue environments could be accompanied by the forma-
tion of specific multi-protein complexes with tissue-specific pro-
teins that would act cooperatively with Foxp3 in performing Treg cell
functions in specific environments.
Treg cell instability
Both the expression of Foxp3 and its stability have crucial roles
in the maintenance of Treg cell function63. Thus, conditional dele-
tion of a Foxp3 allele in mature Treg cells results in effector T cells
that are capable of causing inflammatory tissue lesions64. Although
the instability of Foxp3 in iTreg cells has been widely observed and
is intrinsic to their developmental origin8,9, tTreg cells have been
investigated to determine how the instability of Foxp3 expression
under basal or inflammatory conditions in specific tissues affects
the development and resolution of autoimmunity65–67. Loss of Foxp3
expression by tTreg cells has been observed in vitro68,69, in the adop-
tive transfer of cells into lymphopenic hosts70, in infectious settings71
and in graft-versus-host disease72. A fate-mapping mouse model
in which a yellow fluorescent protein reporter marks all cells that
at any time expressed Foxp3 in both homeostatic conditions and
autoimmune inflammatory conditions has been generated. In this
model, a small proportion of apparently stable Treg cells lost Foxp3
expression and acquired an effector-memory phenotype with dif-
ferent levels of secretion of the pro-inflammatory cytokines IFN-γ​
and IL-1767. These ‘ex-Foxp3 cells’ were able to induce autoimmu-
nity in an adoptive transfer model on the NOD background and
consisted of a mixed population, on the basis of their level of TSDR
demethylation, which would suggest that not all ex-Foxp3 cells in
this model were once de facto tTreg cells. Subsequent data have sug-
gested that this fate-mapping mouse model was detecting a propor-
tion of Foxp3+ tTreg cells that were either transiently upregulating
Foxp3 or were not fully committed to the tTreg cell lineage73. Further
data in support of the proposal of tTreg cell instability have shown
that Foxp3 expression is lost in tTreg cells specific for an epitope of
myelin oligodendrocyte glycoprotein (MOG; amino acids 38–49)
during the development of experimental autoimmune encephalitis
(EAE), with an increased frequency of ex-Foxp3 cells in the central
668
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
NaTuRe Immunology
nervous system at the preclinical and peak stages of EAE that
decreases during EAE resolution. These ex-Foxp3 Treg cells express
IFN-γ​and are able to transfer EAE74. It remains to be determined
whether the decrease in ex-Foxp3 cells during disease resolution is
due to re-acquisition of Foxp3 expression by ex-Foxp3 cells.
Another genetic fate-mapping mouse model has provided
evidence that the majority of mature tTreg cells in the spleen and
lymph nodes are relatively stable under homeostatic conditions66.
This model, based on inducible labeling of Foxp3+ cells upon treat-
ment with tamoxifen, marks all those cells that expressed Foxp3
at the moment of tamoxifen administration, and, in contrast to
models with continuous labeling67, prevents the detection of cells
transiently expressing Foxp3. Although Foxp3 expression is stable
under homeostatic conditions, depriving cells of growth factor or
blocking the IL-2 receptor, which induces autoimmunity75, results
in substantially decreased Foxp3 expression per cell in mature tTreg
cells, and a small population loses Foxp3 expression completely.
However, they do not produce pro-inflammatory cytokines66, sug-
gestive of some degree of instability under specific environmental
settings. This apparent discrepancy might arise from the different
fate-mapping mouse models used and the type of labeling of Foxp3
cells, which could lead to the labeling of uncommitted Treg cells67
or the absence of labeling of ex-Foxp3 cells that appeared before
tamoxifen administration66. The discordance in results could also
depend on the inflammatory stimuli used to assess Foxp3’s stabil-
ity. Regardless, in both fate-mapping models, there is a small pop-
ulation of tTreg cells that lose Foxp3 expression, and those Treg cells
that remain ‘stable’ display diminished Foxp3 expression at the
single-cell level66. As decreased levels of Foxp3 in Treg cells isolated
from inflammatory sites have been observed in mouse models of
autoimmunity63,76 and in patients with autoimmune diseases24,77–81,
further work with these models is warranted in order to identify
the mechanisms and consequences of long-term decreases in Treg
cell Foxp3 expression. Further data have confirmed the obser-
vation that most mature tTreg cells are stable under steady-state
conditions, in a new fate-mapping mouse model in which Foxp3
lacks CNS1, but these cells become unstable when stimulated in
vitro and in vivo in a model of EAE, losing Foxp3 expression and
acquiring TH1 cell– and TFH cell–like features73. While epigenetic
changes such as re-methylation of the CNS2 region could account
for the loss of Foxp3 expression in these settings67,73, the molecu-
lar mechanisms responsible for the decrease in Foxp3 protein
and the potential contribution of post-translational modifica-
tions of Foxp3 protein on the generation of ex-Foxp3 cells remain
to be explored.
Finally, deletion of specific Foxp3 partners can also precipitate the
appearance of ex-Foxp3 cells. For example, Treg cell–specific deletion
of the chaperone GP96 on the NOD background leads to lethal auto-
immunity due to defective suppressive abilities of Treg cells in models
of diabetes and colitis. In this system, Treg cells progressively lose
Foxp3 expression and gain IFN-γ​secretion, although they maintain
their specific TSDR-demethylation pattern59. Mice with Treg cell–
specific deletion of the transcription factor Helios develop systemic
autoimmune pathology characterized by increased germinal-center
formation, lymphocytic infiltration into non-lymphoid organs and
glomerulonephritis82. Although Helios does not form protein com-
plexes with Foxp3 or bind to the Foxp3 locus82, Helios-deficient Treg
cells have increased expression of IFN-γ​and IL-17 and are unstable,
with decreased expression of Foxp3 and a tendency to completely
lose Foxp3 expression82,83. Treg cells deficient in another transcrip-
tion factor, Eos, exhibit increased expression of IL-2 and IFN-γ​
along with reduced suppressive ability, while forced overexpression
of Eos in Treg cells prevents Treg cell instability, even in inflamma-
tory environments. EosloFoxp3+ tTreg cells are detectable in vivo and
have regulatory function, with the ability to acquire helper T cell–
like effector characteristics while maintaining Foxp3 expression;
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology

NaTuRe Immunology
however, such cells exhibit specific changes in their global DNA-
methylation pattern84.
Treg cell plasticity
Plasticity is a property inherent to most if not all immune cells,
which allows them to adapt their phenotype and function to the
changing environment and extracellular ‘danger’ signals. Thus,
it is not surprising that Treg cells possess some degree of plasticity.
Treg cells have the ability to acquire features specific to the type of
immune response they control, which is driven mostly by ‘mas-
ter’ transcription factors and is regulated by environmental sig-
nals. Thus, Treg cells acquire expression of the transcription factor
T-bet to restrain type 1 inflammation during infection85,86, and they
utilize the transcription factors IRF4 and STAT3 to inhibit TH2
cell responses87 and TH17 cell responses88, respectively. While this
modality of plasticity seems to be advantageous for the host and
beneficial to the outcome of the immune response, aberrant plastic-
ity of Treg cells is also observed in several autoimmune diseases, with
Treg cells expressing pro-inflammatory cytokines, acquiring helper
T cell–like phenotypes and displaying diminished function in most
cases but maintaining Foxp3 expression21,83,89–95. Paradoxically, these
helper T cell–like Treg cells utilize the same transcription factors used
by Treg cells to inhibit specific types of immune responses. Therefore,
the secretion of IFN-γ​by TH1-like Treg cells requires T-bet expres-
sion21,91–93, while IL-6-driven TH17-like Treg cells require STAT3 for
the secretion of IL-1772, and IL-4-driven TH2-like Treg cells upregu-
late IRF4 and Gata-395,96. In most cases, helper T cell–like Treg cells
have a demethylated TSDR in the Foxp3 locus even though they
share effector features, which suggests that their phenotype might
be reversible21,90,93. They display alterations in their epigenetic sig-
nature characteristic of those of Treg cells9,32,34,97, which might be the
underlying mechanism that allows the secretion of pro-inflamma-
tory cytokines. Current efforts are focused on understanding the
signaling pathways that drive this plasticity in specific autoimmune
diseases, to harness this flexibility to the treatment of human dis-
ease92 and the role of Treg cell plasticity in autoimmune disease–
related tissues.
TH1-like Treg cells
Perhaps the best-characterized tTreg cell plasticity event is the acqui-
sition of TH1 cell–like features. In mouse models and patients with
autoimmune diseases, such as type 1 diabetes93, multiple sclero-
sis21,91, autoimmune hepatitis98 and Sjogren syndrome99, there is
an increased frequency of IFN-γ​+Foxp3+ tTreg cells in the periph-
ery that display lower suppressive ability than that of Treg cells from
healthy age-matched subjects. TH1-like Treg cells upregulate the tran-
scription factor T-bet and other TH1 cell markers, such as CCR5 and
CXCR3. Furthermore, in the apolipoprotein E–deficient (Apoe–/–)
mouse model of atherosclerosis, which shares various pathogenic
similarities with autoimmune disorders, there is accumulation of
IFN-γ​-producing TH1-like Treg cells in the aorta that display altered
suppressive ability in vitro and in vivo90. Studies of an in vitro model
in which TH1-like Treg cells are generated through the use of IL-12
have demonstrated that TH1-like Treg cells possess an activated PI(3)
K and Akt kinase pathway and the transcription factor FoxO, which
is partly responsible for their secretion of IFN-γ​and decreased sup-
pressive capacity91. Interestingly, Treg cells isolated from patients
with relapsing–remitting multiple sclerosis also display an activated
PI(3)K–Akt–FoxO pathway ex vivo, and their suppressive ability is
corrected by blockade of the PI(3)K pathway94. In vivo, activation of
PI(3)K–Akt by Treg cell–specific deletion of the PI(3)K phosphatase
PTEN elicits a type 1 autoimmune disorder, with Treg cells downreg-
ulating their expression of CD25 and Foxp3 and displaying reduced
functionality100,101. Moreover, FoxO itself has been linked to the reg-
ulation of Treg cell plasticity. Mice with Treg cell–specific deletion of
FoxO succumb to a lethal autoimmunity similar to that observed in
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
FOCUS | Review
| Article
FOCUShttps://doi.org/10.1038/s41590-018-0120-4
Review Article
scurfy mice94, with Treg cells displaying a TH1-like Treg cell phenotype
and being unable to prevent disease in a colitis model. IFN-γ​ seems
to be involved in the defective function of Treg cells, as Foxo1–/–Ifng–/–
mice partially recover from the wasting syndrome associated with
this model94.
Transcriptomic analysis of IFN-γ​+ TH1-like Treg cells at the popu-
lation level91 or single-cell level90 has demonstrated that they exhibit
lower expression of genes encoding immunosuppressive molecules
than that of Treg cells and have altered expression of costimulatory
molecules, migratory properties and specific signaling pathways. It
is unknown whether TH1-like Treg cells have a role in disease patho-
genesis and/or protection in specific tissues. TH1-like Treg cells have
been observed among MOG-specific Treg cells infiltrating the cen-
tral nervous system of mice during EAE development. These Treg
cells are not able to suppress the infiltration of the central nervous
system by MOG-specific effector T cells or prevent disease onset;
they secrete IFN-γ​at the onset and peak of disease but at a reduced
frequency during the recovery phase102. In contrast, T-bet+Foxp3+
cells in pancreatic tissue seem to be protective in a mouse model of
type 1 diabetes103. Finally, while most work on TH1-like Treg cells has
suggested that IL-12 and/or type 1 cytokines induce the phenotype,
the fact that PI(3)K–Akt, a major pathway that integrates diverse
environmental signals into cell function, is involved in their genera-
tion suggests that other environmental cues might have the ability
to induce Treg cell plasticity, as it has been observed, for example,
with increased concentrations of dietary salt or NaCl104.
TH17-like Treg cells
A small proportion of human peripheral Treg cells produce IL-17 in
healthy people and upregulate the gene encoding the transcription
factor RORγ​t (RORC) (TH17-like Treg cells) ex vivo105 while conserv-
ing their suppressive ability105,106. Given the well-established devel-
opmental relationship between Treg cells and TH17 cells, it remains
to be determined whether TH17-like Treg cells are a transient stage in
the de-differentiation of tTreg cells into TH17 cells, as has been sug-
gested69,107. In support of that proposal, the conversion of Foxp3+ Treg
cells into TH17 cells has a crucial role in the pathogenesis of autoim-
mune arthritis in a collagen-induced arthritis mouse model. This
conversion is driven by IL-1β​108 and IL-6, and Foxp3+IL-17+ Treg cells
are observed in the synovium of subjects with active rheumatoid
arthritis109. Moreover, the conversion of Treg cells into TH17 cells
has also been reported in the CD18hypo PL/J mouse model of
psoriasis108, and TH17-like Treg cells have been observed in skin tissue
of psoriatic patients89.
Perhaps the tissue in which TH17-like Treg cells have been best
identified is the gastrointestinal tract. The lamina propria seems
to be enriched for iTreg cells with high expression of the TH17 cell–
defining transcription factor RORγ​t. These TH17-like Treg cells seem
to have a beneficial function, as their absence exacerbates pathogen-
esis in several models of mucosal autoimmunity110,111. Furthermore,
studies have suggested that Foxp3+RORγ​t+ Treg cells control glomer-
ulonephritis112, and a lack of TH17-like Treg cells results in increased
mortality and organ pathology associated with systemic lupus ery-
thematosus113. In addition to the importance of IL-1β​and IL-6 in
promoting the secretion of IL-17 by Treg cells114,115, other environ-
mental factors, including indoleamine 2,3-dioxygenase116, ligation
of the pattern-recognition receptor TLR2117 and certain infec-
tions118, have been shown to modulate the conversion of TH17-like
Treg cells either indirectly or directly.
TH2-like Treg cells
Patients with systemic sclerosis display an increased frequency
of TH2-like Treg cells in the skin but not in the peripheral blood,
characterized by the secretion of IL-4 and IL-13 and upregulation
of Gata-3 and IRF-4. Peripheral Treg cells from these patients have
high expression of ST2, the receptor for the alarmin IL-33. The
669
Review Article
|
Review Article FOCUS
https://doi.org/10.1038/s41590-018-0120-4
skin shows enrichment for this cytokine, which suggests that it
might have a role in the reprogramming of Treg cells into a TH2-like
phenotype95. TH2-like Treg cells have also been observed in mutant
(Il4raF709) mice susceptible to allergy and in Treg cells from the
peripheral blood of food-allergic patients. TH2-like Treg cells secrete
IL-4 and/or IL-13 and upregulate the transcription factors IRF4 and
Gata-396. Moreover, mice with Treg cell–restricted deletion of the
ubiquitin ligase Itch show autoimmune features, and Treg cells are
not able to control type 2 inflammation. This defect is associated
with the acquisition of a TH2-like phenotype by the Treg cells and
with increased expression of Gata-3, activation of STAT6 and secre-
tion of IL-4119.
Many questions remain to be answered about Treg cell plasticity.
Does Treg cell plasticity reflect initial heterogeneity of the Treg cell
population, with only a specific subset of Treg cells being able to
acquire effector-like properties65? Do helper T cell–like Treg cells rep-
resent a transient stage on the path to becoming ex-Foxp3 cells? Can
Treg cell plasticity be harnessed for the design of new therapeutic
strategies aimed at modulating Treg cell function in diverse disease
settings? What is the role of helper T cell–like Treg cells in autoim-
mune tissues during disease development and progression?
Tissue-resident Treg cells
One of the main recent advances in the Treg cell field has been the
discovery that Treg cells populate specific tissues in the body during
physiological and stress conditions. The characterization of these
populations and their mechanisms of action will have important
implications for understanding the development, maintenance and
resolution of autoimmunity in specific organs. Tissue-resident Treg
cells actively perform non-immunological functions and work at
maintaining tissue homeostasis and wound repair, roles that could
be important for tissue homeostasis in autoimmunity settings.
Studies have begun to define the specific phenotype of tissue-resi-
dent Treg cells in the muscles, skin, lungs, gastrointestinal tract, liver
and adipose tissue, among other locations (reviewed in ref. 28). In
most cases, these Treg cells seem to be of thymic origin120–122, with
considerable oligoclonality of their TCR repertoire, which indicates
that particular antigens in the tissue might be responsible for the
accumulation of Treg cells in specific niches120,122,123. They are char-
acterized by the expression of tissue-specific transcription factors
and mediators that drive the function of other cells in that tissue, in
support of the notion that the microenvironment exerts important
effects on the phenotype of Treg cells in a given location. The molec-
ular mechanisms by which tissue-resident Treg cells acquire their
tissue-specific program have only begun to be explored. Epigenetic
analysis of mouse Treg cells isolated from various tissues and com-
pared with Treg cells from the lymph nodes has demonstrated that
tissue Treg cells undergo extensive epigenetic reprograming that is
globally tissue specific, but that there is a common tissue Treg cell
population characterized by expression of the activation marker
KLRG1 and ST2124.
The best-studied tissue-specific Treg cell population is the one
that is resident in visceral adipose tissue (VAT). VAT Treg cells serve
important roles in the defense against associated metabolic disor-
ders. In mice, lean fat tissue is populated by T cells with a TH2 cell
phenotype and shows enrichment for Treg cells that maintain the
predominance of resident anti-inflammatory macrophages. Treg
cells accumulate over time and acquire a TH2-like phenotype, with
expression of Gata-3, BATF, IRF4, CCR4 and IL-10121, as well as of
PPARγ​, a transcription factor that controls adipocyte differentiation
and mediates the accumulation, phenotype and function of VAT Treg
cells121,125. Treg cell–specific deletion of PPARγ​results in the specific
loss of VAT Treg cells, which demonstrates the important role of this
transcription factor in the development and/or maintenance of
VAT Treg cells121. The frequency of VAT Treg cells diminishes with age
in obese mice, which suggests that they have a role in modulating
670
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
NaTuRe Immunology
Box 2 | Treg cell–specific therapies for autoimmune diseases
One of the many therapeutic strategies being investigated in the
autoimmunity field is the restoration of peripheral tolerance, and
one avenue that has been explored is the development of Treg cell–
based therapeutic strategies. Interesting results have been ob-
tained with immunotherapies based on the adoptive transfer of
polyclonally expanded Treg cells into patients with autoimmune
diseases such as type 1 diabetes, cutaneous lupus erythematosus
or Crohn’s disease131–134. Furthermore, with the development of
technologies to engineer epitope-specific TCRs, such as chimeric
antigen receptors135, future approaches will potentially achieve
an optimal number of Treg cells with TCRs specific for disease-
relevant antigens for adoptive transfer into patients with auto-
immunity in those cases in which the autoantigens are known.
Although it is still too early to know the long-term consequences
of these therapies, the first results are encouraging and hold
promise for the adoption of these technologies for other autoim-
mune diseases.
Another potential therapeutic approach to the restoration of
peripheral tolerance is the targeting of specific molecules and/or
pathways that are dysfunctional in the existing pool of Treg cells
in patients. For this approach, it will be important to determine
the physiological relevance and molecular mechanisms driving
the plasticity and instability of Treg cells in the patients, as well as
to improve knowledge of how the dysfunction of tissue-specific
Treg cells affect the development of certain autoimmune diseases.
In this context, there are many questions still unresolved (as
discussed in the main text and Box 3) that will undoubtedly
improve the design of Treg cell–specific therapeutic options for
patients with autoimmune disease.
obesity-associated fat-tissue inflammation123. Given that obesity is
an established risk factor in autoimmune disease, it will be impor-
tant to define the features and functions of VAT Treg cells in the
development of autoimmunity.
Similarly, lung and muscle Treg cells express amphiregulin, the
ligand for epidermal growth factor receptor, to promote tissue
repair. Muscle Treg cells accumulate in acutely injured skeletal mus-
cle in mouse models of muscle injury and muscular dystrophy, in
which they control muscle inflammation after injury and promote
tissue repair by acting on immune and non-immune cells27,120. Data
in a zebrafish model further support the proposal of a dual role
for Treg cells in the immunoregulation and tissue regeneration
observed in mice. In zebrafish, Treg cell–like cells126 rapidly migrate
to damaged organs in models of spinal-cord, heart and retina regen-
eration, and their function is dependent on the secretion of organ-
specific regenerative factors. Treg cell–specific ablation inhibits
organ regeneration127.
Treg cells populate healthy skin and help maintain homeostasis.
In healthy people and rodents, skin Treg cells possess an effector–
memory phenotype, with expression of Treg cell–specific surface
markers and greater proliferative capacity ex vivo than that of their
blood counterparts. They express IL-17 and IL-10 and display a
demethylated TSDR, which would suggest that they are tTreg cells122.
Interestingly, Treg cells reside in close apposition to hair follicles,
where they contribute to the activation of hair-follicle stem cells by
promoting their proliferation and differentiation through the Notch
signaling pathway. Ablation of Treg cells completely abolishes the
ability of the hair follicle to regenerate, which indicates that Treg cells
are a key component of the skin stem-cell niche128.
The maintenance of local tissue homeostasis is of particu-
lar importance in the intestinal mucosa, where the immune sys-
tem must be able to effectively discriminate between pathogens
and dietary factors or commensal flora. Accordingly, deregulated
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology

NaTuRe Immunology
Box 3 | Unanswered questions
• What markers can be used to distinguish tTreg cells from iTreg
cells?
• What are the molecular mechanisms and environmental
triggers responsible for the generation of ex-Foxp3 cells in
autoimmunity?
• What are the details of the molecular mechanisms and envi-
ronmental cues that drive the development of TH1-, TH22-
and TH17-like Treg cell phenotypes in ‘plastic’ Treg cells in
specific autoimmune settings?
• What is the contribution of the Treg cell epigenetic signature
to Treg cell instability and plasticity in autoimmunity?
• What are the epigenetic and phenotypic signatures of human
Treg cells in specific tissues, and how do these signatures con-
tribute to disease pathology?
• Does Treg cell plasticity reflect the initial heterogeneity of the
Treg cell population, with only a specific subset of Treg cells
being able to acquire effector-like properties?
• What is the role of Treg cell plasticity and instability in auto-
immune tissues during the development, progression and
resolution of disease?
• What are the mechanisms that Treg cells utilize to maintain
tissue integrity, and what is the involvement of tissue-specific
Treg cells in tissue damage in autoimmunity?
immune responses to luminal flora in genetically susceptible peo-
ple are generally recognized as key factors in the pathogenesis of
inflammatory bowel disease. While the role of commensal flora in
inducing Treg cells has been well characterized, the dynamics and
characterization of Treg cells in the mucosal tissue has only begun
to be appreciated. Thus, the lamina propria seems to show enrich-
ment for Treg cells with high expression of RORγ​t110,129, and although
most of these are not tTreg cells, they seem to have a positive function
in maintaining tissue homeostasis, and their absence exacerbates
pathogenesis in several models of mucosal autoimmunity.
How do Treg cells maintain tissue integrity, and what is the
involvement of tissue-specific Treg cells in autoimmune disease?
One could speculate that defects in specific tissue-resident Treg cells
would be involved in the development, maintenance or resolution
of autoimmune disorders that affect that specific tissue; for example,
is there a specific role for muscle-resident Treg cells in the pathology
of myasthenia gravis, or for VAT Treg cells in autoimmune disorders
in which obesity is an important risk factor, or for skin Treg cells in
psoriasis? Finally, while most research performed so far has studied
mice, and although there are intrinsic difficulties with translating
these observations to humans, it will be crucial to determine the
phenotype and function of tissue-specific Treg cells in healthy people
and in patients with autoimmune diseases to understand the role
of tissue-resident Treg cells in the development and maintenance of
human autoimmunity.
Conclusions
Intense research in the Treg cell field has improved knowledge of
their biology and has provided evidence of the existence of instabil-
ity and plasticity within the Treg cell compartment. Accumulating
data in recent years have shown the presence of ex-Foxp3 cells
and helper T cell–like Treg cells in various autoimmune patholo-
gies. Current efforts are focused on determining the molecular
mechanisms responsible for the induction of each of these func-
tional states in the periphery and autoimmune organs, as well as the
environmental triggers that drive them. The ultimate goal of these
studies is to harness the mechanisms that generate the instability
and plasticity of Treg cells to modulate Treg cell function not only in
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
FOCUS | Review
| Article
FOCUShttps://doi.org/10.1038/s41590-018-0120-4
Review Article
autoimmune pathologies but also in other diseases such as cancer.
As for tissue-resident Treg cells, although research has begun to elu-
cidate the mechanisms and factors that control their function in tis-
sues, many questions remain to be answered, mostly those related
to the mechanisms that tissue Treg cells employ to maintain tissue
integrity in autoimmune diseases and the characterization of tissue-
resident Treg cells in humans (Box 2). Answers to these questions
will undoubtedly improve the design of Treg cell–specific therapeutic
options for patients with autoimmune diseases.
Received: 4 December 2017; Accepted: 29 January 2018;
Published online: 20 June 2018
References
1. Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Foxp3 programs the
development and function of CD4+CD25+ regulatory T cells. Nat. Immunol.
4, 330–336 (2003).
2. Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell
development by the transcription factor Foxp3. Science 299,
1057–1061 (2003).
3. Khattri, R., Cox, T., Yasayko, S. A. & Ramsdell, F. An essential role for
Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4,
337–342 (2003).
4. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. & Toda, M. Immunologic
self-tolerance maintained by activated T cells expressing IL-2 receptor
alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance
causes various autoimmune diseases. J. Immunol. 155, 1151–1164 (1995).
5. Liu, W. et al. CD127 expression inversely correlates with FoxP3 and
suppressive function of human CD4+ T reg cells. J. Exp. Med. 203,
1701–1711 (2006).
6. Sakaguchi, S., Miyara, M., Costantino, C. M. & Hafler, D. A. FOXP3+
regulatory T cells in the human immune system. Nat. Rev. Immunol. 10,
490–500 (2010).
7. Josefowicz, S. Z., Lu, L. F. & Rudensky, A. Y. Regulatory T cells:
mechanisms of differentiation and function. Annu. Rev. Immunol. 30,
531–564 (2012).
8. Kanamori, M., Nakatsukasa, H., Okada, M., Lu, Q. & Yoshimura, A.
Induced regulatory T cells: their development, stability, and applications.
Trends Immunol. 37, 803–811 (2016).
9. Ohkura, N. et al. T cell receptor stimulation-induced epigenetic changes
and Foxp3 expression are independent and complementary events required
for Treg cell development. Immunity 37, 785–799 (2012).
10. Nishizuka, Y. & Sakakura, T. Thymus and reproduction: sex-linked
dysgenesia of the gonad after neonatal thymectomy in mice. Science 166,
753–755 (1969).
11. Penhale, W. J., Farmer, A., McKenna, R. P. & Irvine, W. J. Spontaneous
thyroiditis in thymectomized and irradiated Wistar rats. Clin. Exp.
Immunol. 15, 225–236 (1973).
12. Sakaguchi, S., Takahashi, T. & Nishizuka, Y. Study on cellular events in
postthymectomy autoimmune oophoritis in mice. I. Requirement of Lyt-1
effector cells for oocytes damage after adoptive transfer. J. Exp. Med. 156,
1565–1576 (1982).
13. Sakaguchi, S., Fukuma, K., Kuribayashi, K. & Masuda, T. Organ-specific
autoimmune diseases induced in mice by elimination of T cell subset. I.
Evidence for the active participation of T cells in natural self-tolerance;
deficit of a T cell subset as a possible cause of autoimmune disease.
J. Exp. Med. 161, 72–87 (1985).
14. Baecher-Allan, C., Brown, J. A., Freeman, G. J. & Hafler, D. A.
CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167,
1245–1253 (2001).
15. Stephens, L. A., Mottet, C., Mason, D. & Powrie, F. Human CD4+CD25+
thymocytes and peripheral T cells have immune suppressive activity in
vitro. Eur. J. Immunol. 31, 1247–1254 (2001).
16. Gavin, M. A. et al. Foxp3-dependent programme of regulatory T-cell
differentiation. Nature 445, 771–775 (2007).
17. Miyara, M. et al. Human FoxP3+ regulatory T cells in systemic autoimmune
diseases. Autoimmun. Rev. 10, 744–755 (2011).
18. Brusko, T. M., Wasserfall, C. H., Clare-Salzler, M. J., Schatz, D. A. &
Atkinson, M. A. Functional defects and the influence of age on the
frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes 54,
1407–1414 (2005).
19. Haseda, F., Imagawa, A., Murase-Mishiba, Y., Terasaki, J. & Hanafusa, T.
CD4+ CD45RA− FoxP3high activated regulatory T cells are functionally
impaired and related to residual insulin-secreting capacity in patients with
type 1 diabetes. Clin. Exp. Immunol. 173, 207–216 (2013).
20. Lindley, S. et al. Defective suppressor function in CD4+CD25+ T-cells from
patients with type 1 diabetes. Diabetes 54, 92–99 (2005).
671
Review Article
|
Review Article FOCUS
https://doi.org/10.1038/s41590-018-0120-4
21. Dominguez-Villar, M., Baecher-Allan, C. M. & Hafler, D. A. Identification
of T helper type 1-like, Foxp3+​regulatory T cells in human autoimmune
disease. Nat. Med 17, 673–675 (2011).
22. Viglietta, V., Baecher-Allan, C., Weiner, H. L. & Hafler, D. A. Loss of
functional suppression by CD4+CD25+ regulatory T cells in patients with
multiple sclerosis. J. Exp. Med. 199, 971–979 (2004).
23. Bonelli, M. et al. Quantitative and qualitative deficiencies of regulatory
T cells in patients with systemic lupus erythematosus (SLE). Int. Immunol.
20, 861–868 (2008).
24. Thiruppathi, M. et al. Functional defect in regulatory T cells in myasthenia
gravis. Ann. NY Acad. Sci. 1274, 68–76 (2012).
25. van Roon, J. A., Hartgring, S. A., van der Wurff-Jacobs, K. M., Bijlsma, J.
W. & Lafeber, F. P. Numbers of CD25+Foxp3+​T cells that lack the IL-7
receptor are increased intra-articularly and have impaired suppressive
function in RA patients. Rheumatology (Oxford) 49, 2084–2089 (2010).
26. Ohkura, N., Kitagawa, Y. & Sakaguchi, S. Development and maintenance of
regulatory T cells. Immunity 38, 414–423 (2013).
27. Arpaia, N. et al. A Distinct function of regulatory T cells in tissue
protection. Cell 162, 1078–1089 (2015).
28. Panduro, M., Benoist, C. & Mathis, D. Tissue Tregs. Annu. Rev. Immunol.
34, 609–633 (2016).
29. Brunkow, M. E. et al. Disruption of a new forkhead/winged-helix protein,
scurfin, results in the fatal lymphoproliferative disorder of the scurfy
mouse. Nat. Genet. 27, 68–73 (2001).
30. Bennett, C. L. et al. The immune dysregulation, polyendocrinopathy,
enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.
Nat. Genet. 27, 20–21 (2001).
31. Wildin, R. S. et al. X-linked neonatal diabetes mellitus, enteropathy and
endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat.
Genet. 27, 18–20 (2001).
32. Hill, J. A. et al. Foxp3 transcription-factor-dependent and -independent
regulation of the regulatory T cell transcriptional signature. Immunity 27,
786–800 (2007).
33. Morikawa, H. & Sakaguchi, S. Genetic and epigenetic basis of Treg cell
development and function: from a FoxP3-centered view to an epigenome-
defined view of natural Treg cells. Immunol. Rev. 259, 192–205 (2014).
34. Huehn, J. & Beyer, M. Epigenetic and transcriptional control of Foxp3+
regulatory T cells. Semin. Immunol. 27, 10–18 (2015).
35. Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3
gene in regulatory T-cell fate. Nature 463, 808–812 (2010).
36. Feng, Y. et al. Control of the inheritance of regulatory T cell identity by a
cis element in the Foxp3 locus. Cell 158, 749–763 (2014).
37. Li, X., Liang, Y., LeBlanc, M., Benner, C. & Zheng, Y. Function of
a Foxp3 cis-element in protecting regulatory T cell identity. Cell 158,
734–748 (2014).
38. Baron, U. et al. DNA demethylation in the human FOXP3 locus
discriminates regulatory T cells from activated FOXP3(+​) conventional
T cells. Eur. J. Immunol. 37, 2378–2389 (2007).
39. Floess, S. et al. Epigenetic control of the foxp3 locus in regulatory T cells.
PLoS Biol. 5, e38 (2007).
40. van Loosdregt, J. et al. Regulation of Treg functionality by acetylation-
mediated Foxp3 protein stabilization. Blood 115, 965–974 (2010).
41. Li, B. et al. FOXP3 interactions with histone acetyltransferase and class II
histone deacetylases are required for repression. Proc. Natl Acad. Sci. USA
104, 4571–4576 (2007).
42. Beier, U. H., Akimova, T., Liu, Y., Wang, L. & Hancock, W. W. Histone/
protein deacetylases control Foxp3 expression and the heat shock response
of T-regulatory cells. Curr. Opin. Immunol. 23, 670–678 (2011).
43. Deng, G. et al. Pim-2 kinase influences regulatory T cell function and
stability by mediating Foxp3 protein N-terminal phosphorylation.
J. Biol. Chem. 290, 20211–20220 (2015).
44. Li, Z. et al. PIM1 kinase phosphorylates the human transcription factor
FOXP3 at serine 422 to negatively regulate its activity under inflammation.
J. Biol. Chem. 289, 26872–26881 (2014).
45. Morawski, P. A., Mehra, P., Chen, C., Bhatti, T. & Wells, A. D. Foxp3
protein stability is regulated by cyclin-dependent kinase 2. J. Biol. Chem.
288, 24494–24502 (2013).
46. Nie, H. et al. Phosphorylation of FOXP3 controls regulatory T cell
function and is inhibited by TNF-α​in rheumatoid arthritis. Nat. Med 19,
322–328 (2013).
47. Barbi, J., Pardoll, D. M. & Pan, F. Ubiquitin-dependent regulation of Foxp3
and Treg function. Immunol. Rev. 266, 27–45 (2015).
48. Chen, Z. et al. The ubiquitin ligase Stub1 negatively modulates regulatory
T cell suppressive activity by promoting degradation of the transcription
factor Foxp3. Immunity 39, 272–285 (2013).
49. Wang, L. et al. Ubiquitin-specific protease-7 inhibition impairs Tip60-
dependent Foxp3+ T-regulatory cell function and promotes antitumor
immunity. EBioMedicine 13, 99–112 (2016).
672 Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
NaTuRe Immunology
50. Song, X. et al. Structural and biological features of FOXP3 dimerization
relevant to regulatory T cell function. Cell Reports 1, 665–675 (2012).
51. Xiao, Y. et al. Dynamic interactions between TIP60 and p300 regulate
FOXP3 function through a structural switch defined by a single lysine on
TIP60. Cell Rep. 7, 1471–1480 (2014).
52. van Loosdregt, J. et al. Stabilization of the transcription factor Foxp3 by the
deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity 39,
259–271 (2013).
53. Dang, E. V. et al. Control of TH17/Treg balance by hypoxia-inducible factor
1. Cell 146, 772–784 (2011).
54. Rudra, D. et al. Transcription factor Foxp3 and its protein partners form
a complex regulatory network. Nat. Immunol. 13, 1010–1019 (2012).
55. Samstein, R. M. et al. Foxp3 exploits a pre-existent enhancer landscape for
regulatory T cell lineage specification. Cell 151, 153–166 (2012).
56. Kwon, H. K., Chen, H. M., Mathis, D. & Benoist, C. Different molecular
complexes that mediate transcriptional induction and repression by FoxP3.
Nat. Immunol. 18, 1238–1248 (2017).
57. Tao, R. et al. Deacetylase inhibition promotes the generation and function
of regulatory T cells. Nat. Med. 13, 1299–1307 (2007).
58. Bettini, M. L. et al. Loss of epigenetic modification driven by the Foxp3
transcription factor leads to regulatory T cell insufficiency. Immunity 36,
717–730 (2012).
59. Zhang, Y. et al. GP96 is a GARP chaperone and controls regulatory T cell
functions. J. Clin. Invest. 125, 859–869 (2015).
60. Bin Dhuban, K. et al. Suppression by human FOXP3+ regulatory T cells
requires FOXP3-TIP60 interactions. Sci. Immunol. 2, eaai9297 (2017).
61. Hayatsu, N. et al. Analyses of a mutant Foxp3 allele reveal BATF as a
critical transcription factor in the differentiation and accumulation of tissue
regulatory T cells. Immunity 47, 268–283 e269 (2017).
62. Darce, J. et al. An N-terminal mutation of the Foxp3 transcription factor
alleviates arthritis but exacerbates diabetes. Immunity 36, 731–741 (2012).
63. Wan, Y. Y. & Flavell, R. A. Regulatory T-cell functions are subverted and
converted owing to attenuated Foxp3 expression. Nature 445,
766–770 (2007).
64. Williams, L. M. & Rudensky, A. Y. Maintenance of the Foxp3-dependent
developmental program in mature regulatory T cells requires continued
expression of Foxp3. Nat. Immunol. 8, 277–284 (2007).
65. Komatsu, N. et al. Heterogeneity of natural Foxp3+ T cells: a committed
regulatory T-cell lineage and an uncommitted minor population retaining
plasticity. Proc. Natl Acad. Sci. USA 106, 1903–1908 (2009).
66. Rubtsov, Y. P. et al. Stability of the regulatory T cell lineage in vivo. Science
329, 1667–1671 (2010).
67. Zhou, X. et al. Instability of the transcription factor Foxp3 leads to the
generation of pathogenic memory T cells in vivo. Nat. Immunol. 10,
1000–1007 (2009).
68. Hoffmann, P. et al. Loss of FOXP3 expression in natural human
CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.
Eur. J. Immunol. 39, 1088–1097 (2009).
69. Koenen, H. J. et al. Human CD25highFoxp3pos regulatory T cells differentiate
into IL-17-producing cells. Blood 112, 2340–2352 (2008).
70. Duarte, J. H., Zelenay, S., Bergman, M. L., Martins, A. C. & Demengeot, J.
Natural Treg cells spontaneously differentiate into pathogenic helper cells in
lymphopenic conditions. Eur. J. Immunol. 39, 948–955 (2009).
71. Oldenhove, G. et al. Decrease of Foxp3+​Treg cell number and acquisition
of effector cell phenotype during lethal infection. Immunity 31,
772–786 (2009).
72. Laurence, A. et al. STAT3 transcription factor promotes instability of nTreg
cells and limits generation of iTreg cells during acute murine graft-versus-
host disease. Immunity 37, 209–222 (2012).
73. Zhang, Z. et al. Activation and functional specialization of regulatory
T cells lead to the generation of Foxp3 instability. J. Immunol. 198,
2612–2625 (2017).
74. Bailey-Bucktrout, S. L. et al. Self-antigen-driven activation induces
instability of regulatory T cells during an inflammatory autoimmune
response. Immunity 39, 949–962 (2013).
75. Setoguchi, R., Hori, S., Takahashi, T. & Sakaguchi, S. Homeostatic
maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by
interleukin (IL)-2 and induction of autoimmune disease by IL-2
neutralization. J. Exp. Med. 201, 723–735 (2005).
76. Tang, Q. et al. Central role of defective interleukin-2 production in the
triggering of islet autoimmune destruction. Immunity 28,
687–697 (2008).
77. Balandina, A., Lécart, S., Dartevelle, P., Saoudi, A. & Berrih-Aknin, S.
Functional defect of regulatory CD4+CD25+ T cells in the thymus of
patients with autoimmune myasthenia gravis. Blood 105, 735–741 (2005).
78. Huan, J. et al. Decreased FOXP3 levels in multiple sclerosis patients.
J. Neurosci. Res. 81, 45–52 (2005).
79. Long, S. A. et al. Defects in IL-2R signaling contribute to diminished
maintenance of FOXP3 expression in CD4+CD25+ regulatory T-cells of type 1
diabetic subjects. Diabetes 59, 407–415 (2010).

NaTuRe Immunology
80. Zhang, B., Zhang, X., Tang, F., Zhu, L. & Liu, Y. Reduction of forkhead box
P3 levels in CD4+CD25high T cells in patients with new-onset systemic lupus
erythematosus. Clin. Exp. Immunol. 153, 182–187 (2008).
81. Moes, N. et al. Reduced expression of FOXP3 and regulatory T-cell
function in severe forms of early-onset autoimmune enteropathy.
Gastroenterology 139, 770–778 (2010).
82. Kim, H. J. et al. Stable inhibitory activity of regulatory T cells requires the
transcription factor Helios. Science 350, 334–339 (2015).
83. Nakagawa, H. et al. Instability of Helios-deficient Tregs is associated with
conversion to a T-effector phenotype and enhanced antitumor immunity.
Proc. Natl Acad. Sci. USA 113, 6248–6253 (2016).
84. Sharma, M. D. et al. An inherently bifunctional subset of Foxp3+ T helper
cells is controlled by the transcription factor eos. Immunity 38,
998–1012 (2013).
85. Koch, M. A. et al. The transcription factor T-bet controls regulatory T cell
homeostasis and function during type 1 inflammation. Nat. Immunol. 10,
595–602 (2009).
86. Levine, A. G. et al. Stability and function of regulatory T cells expressing
the transcription factor T-bet. Nature 546, 421–425 (2017).
87. Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription
factor IRF4 to control TH2 responses. Nature 458, 351–356 (2009).
88. Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a
Stat3-dependent manner. Science 326, 986–991 (2009).
89. Bovenschen, H. J. et al. Foxp3+​regulatory T cells of psoriasis patients
easily differentiate into IL-17A-producing cells and are found in lesional
skin. J. Invest. Dermatol. 131, 1853–1860 (2011).
90. Butcher, M. J. et al. Atherosclerosis-driven Treg plasticity results in
formation of a dysfunctional subset of plastic IFNγ​+ Th1/Tregs. Circ. Res.
119, 1190–1203 (2016).
91. Kitz, A. et al. AKT isoforms modulate Th1-like Treg generation and
function in human autoimmune disease. EMBO Rep. 17, 1169–1183 (2016).
92. Kitz, A. & Dominguez-Villar, M. Molecular mechanisms underlying
Th1-like Treg generation and function. Cell. Mol. Life Sci. 74,
4059–4075 (2017).
93. McClymont, S. A. et al. Plasticity of human regulatory T cells in healthy
subjects and patients with type 1 diabetes. J. Immunol. 186,
3918–3926 (2011).
94. Ouyang, W. et al. Novel Foxo1-dependent transcriptional programs control
Treg cell function. Nature 491, 554–559 (2012).
95. MacDonald, K. G. et al. Regulatory T cells produce profibrotic cytokines in
the skin of patients with systemic sclerosis. J. Allergy Clin. Immunol. 135,
e9 946–e949 (2015).
96. Noval Rivas, M. et al. Regulatory T cell reprogramming toward a
Th2-cell-like lineage impairs oral tolerance and promotes food allergy.
Immunity 42, 512–523 (2015).
97. Wei, G. et al. Global mapping of H3K4me3 and H3K27me3 reveals
specificity and plasticity in lineage fate determination of differentiating
CD4+ T cells. Immunity 30, 155–167 (2009).
98. Arterbery, A. S. et al. Production of proinflammatory cytokines by
monocytes in liver-transplanted recipients with de novo autoimmune
hepatitis is enhanced and induces TH1-like regulatory T cells. J. Immunol.
196, 4040–4051 (2016).
99. Yamada, A. et al. Impaired expansion of regulatory T cells in a neonatal
thymectomy-induced autoimmune mouse model. Am. J. Pathol. 185,
2886–2897 (2015).
100. Huynh, A. et al. Control of PI(3) kinase in Treg cells maintains homeostasis
and lineage stability. Nat. Immunol. 16, 188–196 (2015).
101. Shrestha, S. et al. Treg cells require the phosphatase PTEN to restrain TH1
and TFH cell responses. Nat. Immunol. 16, 178–187 (2015).
102. Korn, T. et al. Myelin-specific regulatory T cells accumulate in the CNS but
fail to control autoimmune inflammation. Nat. Med 13, 423–431 (2007).
103. Tan, T. G., Mathis, D. & Benoist, C. Singular role for T-BET+CXCR3+
regulatory T cells in protection from autoimmune diabetes. Proc. Natl
Acad. Sci. USA 113, 14103–14108 (2016).
104. Hernandez, A. L. et al. Sodium chloride inhibits the suppressive function of
FOXP3+ regulatory T cells. J. Clin. Invest 125, 4212–4222 (2015).
105. Ayyoub, M. et al. Human memory FOXP3+ Tregs secrete IL-17 ex vivo and
constitutively express the TH17 lineage-specific transcription factor RORγ​t.
Proc. Natl Acad. Sci. USA 106, 8635–8640 (2009).
106. Beriou, G. et al. IL-17-producing human peripheral regulatory T cells retain
suppressive function. Blood 113, 4240–4249 (2009).
107. Radhakrishnan, S. et al. Reprogrammed FoxP3+ T regulatory cells become
IL-17+ antigen-specific autoimmune effectors in vitro and in vivo.
J. Immunol. 181, 3137–3147 (2008).
108. Singh, K. et al. Reduced CD18 levels drive regulatory T cell conversion into
Th17 cells in the CD18hypo PL/J mouse model of psoriasis. J. Immunol.
190, 2544–2553 (2013).
109. Komatsu, N. et al. Pathogenic conversion of Foxp3+ T cells into TH17 cells
in autoimmune arthritis. Nat. Med. 20, 62–68 (2014).
Nature Immunology | VOL 19 | JULY 2018 | 665–673 | www.nature.com/natureimmunology
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
FOCUS | Review
| Article
FOCUShttps://doi.org/10.1038/s41590-018-0120-4
Review Article
110. Sefik, E. et al. Mucosal immunology. Individual intestinal symbionts
induce a distinct population of RORγ​+ regulatory T cells. Science 349,
993–997 (2015).
111. Yang, B. H. et al. Foxp3+ T cells expressing RORγ​t represent a stable
regulatory T-cell effector lineage with enhanced suppressive capacity during
intestinal inflammation. Mucosal Immunol 9, 444–457 (2016).
112. Kluger, M. A. et al. Stat3 programs Th17-specific regulatory T cells to
control GN. J. Am. Soc. Nephrol. 25, 1291–1302 (2014).
113. Kluger, M. A. et al. Treg17 cells are programmed by Stat3 to suppress Th17
responses in systemic lupus. Kidney Int. 89, 158–166 (2016).
114. Kitani, A. & Xu, L. Regulatory T cells and the induction of IL-17.
Mucosal Immunol 1, S43–S46 (2008).
115. Xu, L., Kitani, A., Fuss, I. & Strober, W. Cutting edge: regulatory T cells
induce CD4+CD25–Foxp3– T cells or are self-induced to become Th17 cells
in the absence of exogenous TGF-β​. J. Immunol. 178, 6725–6729 (2007).
116. Sharma, M. D. et al. Indoleamine 2,3-dioxygenase controls conversion of
Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes. Blood 113,
6102–6111 (2009).
117. Nyirenda, M. H. et al. TLR2 stimulation drives human naive and effector
regulatory T cells into a Th17-like phenotype with reduced suppressive
function. J. Immunol. 187, 2278–2290 (2011).
118. Smith, A. A. et al. Characterization of Th17-like Tregs during late stages of
infection with B. pertussis in mice. Possible immunomodulation by type I
interferon. J. Immunol. 196, 196.8 (2016).
119. Jin, H. S., Park, Y., Elly, C. & Liu, Y. C. Itch expression by Treg cells
controls Th2 inflammatory responses. J. Clin. Invest. 123, 4923–4934 (2013).
120. Burzyn, D. et al. A special population of regulatory T cells potentiates
muscle repair. Cell 155, 1282–1295 (2013).
121. Cipolletta, D. et al. PPAR-γ​is a major driver of the accumulation and
phenotype of adipose tissue Treg cells. Nature 486, 549–553 (2012).
122. Sanchez Rodriguez, R. et al. Memory regulatory T cells reside in human
skin. J. Clin. Invest. 124, 1027–1036 (2014).
123. Feuerer, M. et al. Lean, but not obese, fat is enriched for a unique
population of regulatory T cells that affect metabolic parameters. Nat. Med.
15, 930–939 (2009).
124. Delacher, M. et al. Genome-wide DNA-methylation landscape defines
specialization of regulatory T cells in tissues. Nat. Immunol. 18,
1160–1172 (2017).
125. Hamaguchi, M. & Sakaguchi, S. Regulatory T cells expressing PPAR-γ​
control inflammation in obesity. Cell Metab. 16, 4–6 (2012).
126. Kasheta, M. et al. Identification and characterization of Treg-like cells in
zebrafish. J. Exp. Med. 214, 3519–3530 (2017).
127. Hui, S. P. et al. Zebrafish regulatory T cells mediate organ-specific
regenerative programs. Dev. Cell 43, 659–672 (2017).
128. Ali, N. et al. Regulatory T cells in skin facilitate epithelial stem cell
differentiation. Cell 169, 1119–1129 (2017).
129. Ohnmacht, C. et al. Mucosal immunology. The microbiota regulates type 2
immunity through RORγ​t+ T cells. Science 349, 989–993 (2015).
130. Yu, X., Huang, Q. & Petersen, F. History and milestones of mouse models
of autoimmune diseases. Curr. Pharm. Des. 21, 2308–2319 (2015).
131. Bluestone, J. A. et al. Type 1 diabetes immunotherapy using polyclonal
regulatory T cells. Sci. Transl. Med. 7, 315ra189 (2015).
132. Desreumaux, P. et al. Safety and efficacy of antigen-specific regulatory T-cell
therapy for patients with refractory Crohn's disease. Gastroenterology 143,
1207–1217 (2012).
133. Marek-Trzonkowska, N. et al. Therapy of type 1 diabetes with
CD4+CD25highCD127– regulatory T cells prolongs survival of pancreatic
islets - results of one year follow-up. Clin. Immunol. 153,
23–30 (2014).
134. Marek-Trzonkowska, N. et al. Administration of CD4+CD25highCD127–
regulatory T cells preserves β​-cell function in type 1 diabetes in children.
Diabetes Care 35, 1817–1820 (2012).
135. Dawson, N. A. J. & Levings, M. K. Antigen-specific regulatory T cells: are
police CARs the answer? Transl. Res. 187, 53–58 (2017).
Acknowledgements
We thank members of the Hafler and Dominguez-Villar laboratories for critical reading
of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence should be addressed to M.D. or D.A.H.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
673