Regulatory T-cells in systemic lupus erythematosus and rheumatoid arthritis
Konstantia-Maria Chavele, Michael R. Ehrenstein
Centre of Rheumatology, The Rayne Building, University College London, 5 University Street, London WC1E 6JF, UK
Received 24 June 2011
Accepted 28 July 2011
Available online 4 August 2011
Edited by Richard Williams, Alexander
Flügel, and Wilhelm Just
Regulatory T-cells (Tregs)
Regulatory T-cells (Tregs) are the guardians of peripheral tolerance acting to prevent autoimmune
diseases such as systemic lupus erythomatosus (SLE) and rheumatoid arthritis (RA). Defects in Tregs
have been reported in these two diseases despite signiﬁcant differences in their clinical phenotype
and pathogenesis. In both diseases the potency of Treg fails to keep pace with the activation of effec-
tor cells and are unable to resist the ensuing inﬂammation. This review will discuss the phenotypic,
numeric, and functional abnormalities in Tregs and their role in patients and murine models of SLE
2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Autoimmune diseases such as systemic lupus erythomatosus
(SLE) and rheumatoid arthritis (RA) arise due to a failure of immu-
nological self-tolerance. Despite central mechanisms of tolerance,
some T-cells recognizing self-antigens are released into the periph-
ery. One of the mechanisms employed to eliminate or control these
potentially damaging cells is regulatory T-cells (Tregs). The impor-
tance of Tregs is underscored by the overwhelming inﬂammation
and autoimmunity that results in their absence. This review will
highlight the role of Tregs in two autoimmune rheumatic diseases,
SLE and RA.
A number of immune regulatory cells have been described but
this review will focus on CD4
Tregs, which can be divided into nat-
urally occurring and adaptive . In mouse, CD4 Tregs constitute
around 5% of the peripheral CD4
lymphocyte population 
whereas in humans only 1–2% . Human Tregs were originally
characterized by Sakaguchi et al. as CD4
T-cells that constitutively
express the receptor of a chain of IL-2 (CD25) . Unlike the
mouse, several human studies have suggested that only those
T-cells expressing the highest levels of CD25 (CD25
in vitro suppressing activity. Although CD25 was the ﬁrst Treg
marker to be identiﬁed, it is also expressed on activated CD4
cells. CD127 (the
chain of the IL-7 receptor) has more recently
been used to distinguish human Tregs from activated CD25
cells. Tregs are considered as CD127
whereas activated CD25
T-cells are CD127
[5,6]. Foxp3 is a critical transcription factor
for the development and function of Tregs and is vital to their phe-
notypic identiﬁcation [7–9]. In mice, mutation  or depletion
 of the Foxp3 gene resulted in fatal autoimmune lymphoprolif-
erative disease whereas in human, several mutations of Foxp3 gene
have been linked to a disease called immune dysregulation, poly-
endocrinopathy, enteropathy X-linked syndrome (IPEX) [12,13].
Although Foxp3 is essential for Treg function, as with CD25, CD4
T-cells can upregulate the expression of Foxp3 upon activation
[14,15]. These activated Foxp3
T-cells are capable of producing
IL-2 in vitro indicating a lack of Treg function. This ﬁnding could
be relevant to diseases such as RA and SLE, which are characterized
by marked T cell activation.
Apart from the naturally occurring thymicaly derived Tregs,
stimulation of peripheral CD4
T-cells with anti-CD3 and anti-
CD28 in the presence of IL-2 and TGF-b can induce Foxp3 expression
[15,16]. These induced or adaptive Tregs are CD4
Thus the peripheral CD4
Treg population is a mixture
of both natural and induced Tregs. Recently, Helios has been
identiﬁed as a transcription factor that is expressed by natural, but
not induced, Tregs. Thornton et al. showed that all CD4
mocytes were Helios
whereas Helios expression in peripheral Tregs
was restricted to approximately 70% of CD4
in both human
and mice. Induced Tregs, either in vitro with anti-CD3 and anti-
CD28 antibody in the presence of IL-2 and TGF-b, or in vivo by expo-
sure to antigen orally, do not express Helios suggesting that Helios is
a thymic-derived Treg maker . Apart from distinguishing differ-
ent populations of Tregs, Helios is also important in their function.
Helios binds the Foxp3 promoter and partially regulates its expres-
sion. Indeed in vitro inhibition of Helios by siRNA oligonucleotides
results in down-regulation of Foxp3 . Additionally, natural Tregs
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FEBS Letters 585 (2011) 3603–3610
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have a stable Foxp3 gene expression, which is controlled by epige-
netic mechanisms. Human CD4
Tregs display a demethy-
lated FOXP3 promoter in contrast to CD4
T cells, where
FOXP3 is partially methylated. Furthermore, stimulated CD4
T cells transiently express FOXP3 but remained partially methyl-
ated, suggesting promoter methylation as a mechanism for regula-
tion of stable FOXP3 expression and Treg commitment . Based
on Foxp3 expression, Tregs can also be divided in different popula-
tions. Miyara et al. deﬁned these populations as naïve or resting
) and activated Tregs (CD25
) both of which are suppressive in vitro. There is
also a non-regulatory population that is CD25
. Several additional markers of Tregs have been identiﬁed
including cytotoxic T lymphocyte-associated protein 4 (CTLA-4),
glucocorticoid-induced TNF-receptor (GITR), lymphocyte activated
gene-3 (LAG-3), neutropilin-1 (Nrp1), CD62L
,CD39 and CD72
but once again they are not exclusive to Tregs and integrating these
molecules into a clear picture of Treg function remains elusive.
2. Properties and function of Tregs
Regardless of the origin of Tregs (natural or adaptive Tregs),
these cells are considered suppressor cells and able to play a piv-
otal role in the maintenance of immune tolerance. Their impor-
tance in the development of autoimmune diseases was
recognised by Sakaguchi and colleagues who were the ﬁrst to show
that transfer of CD4
T-cells depleted of CD25
T-cells, by speciﬁc
monoclonal antibodies against CD25, into BALB/c athymic nude
mice caused spontaneous development of T-cell dependent
autoimmune diseases (such as thyroiditis, gastritis, insulitis, sialo-
adenitis, adrenalitis, oophoritis, glomerulonephritis (GN), and pol-
yarthritis). When these mice were reconstituted by CD4
cells within a limited period after CD4
T-cell transfer, the
autoimmune disease development was successfully prevented .
Most of our knowledge of the function of human Tregs comes
from in vitro experiments. In vitro, Tregs upon antigen stimulation
are anergic and characterized by low proliferation rate and low
secretion of IL-2. Tregs can be expanded in vitro in the presence
of anti-CD3 and IL-2 or when simulated with anti-CD3/CD28. In
vivo, these cells have a high proliferation rate . Tregs after
in vitro activation can suppress proliferation and cytokine produc-
tion of CD4
T-cells [3,21,22]. Tregs can also suppress mono-
cytes, macrophages  B-cells  and dendritic cells . Tregs
exert their suppressive function through different mechanisms,
which includes secretion of inhibitory cytokines and cytotoxic fac-
tors or by metabolic disruption, or by modulating antigen present-
ing cell (APC) maturation and function.
One mechanism by which Tregs exert suppressive function is
via the secretion of inhibitory cytokines such as IL-10, TGF-b 
and IL-35  as well as cytotoxic factors including perforin and
granzymes. Deletion of IL-10 speciﬁcally in Treg leads to inﬂamma-
tion at mucosal surfaces but does not trigger systemic autoimmu-
nity . IL-10 affects the differentiation of dendritic cells and
inhibits the production of IL-12 thus impairing the ability of den-
dritic cells to promote T-cell activation and Th1 differentiation
. Tregs express TGF-b on their surface which is increased upon
in vitro stimulation with anti-CD3, in the presence of monocytes its
membrane expression is increased. Blockade of Treg TGF-b expres-
sion with neutralizing antibodies , results in disruption of Treg
suppression suggesting that TGF-b is an important mediator of
Treg suppression activity. IL-35 is another inhibitory cytokine se-
creted by Tregs and required for their suppressive activity. It is a
heterodimer of Epstein-Barr virus-induced gene 3 (Ebi3) and p35
). Both genes are highly expressed on mouse Foxp3
[27,30] but not by resting and activated T-effector cells. In contrast
the heterodimer cytokine IL-35 is constitutively expressed by Treg
but not T-effector cells. Mouse Tregs deﬁcient in Ebi3 and p35 had
signiﬁcant reduced suppression activity in vitro, and fail to control
homeostatic proliferation and to cure inﬂammatory bowel disease
in vivo. In addition, ectopic expression of IL-35 confers regulatory
activity on naive T cells, whereas recombinant IL-35 suppresses
T-cell proliferation .
Cytolysis through secretion of cytotoxic factors like perforin
and granzyme B is another mechanism used by Tregs to mediate
their suppressive activity. Perforin is a cytotoxic protein that
polymerizes in target-cell membranes to form transmembrane
pores. Granzymes (A and B) is a serine protease, which activates
apoptosis once in the cytoplasm of the target cell. Human adap-
tive Treg cells preferentially express granzyme B whereas acti-
vated human CD4
natural Treg cells express granzyme A
but very little granzyme B. Furthermore, both Treg subtypes dis-
play perforin-dependent cytotoxicity against autologous target
cells suggesting that the perforin/granzyme pathway is one of
the mechanisms that Treg cells harness to control immune re-
Recently, ‘metabolic disruption’ has been described as a further
mechanism of T-effector cells suppression by Tregs and is thought
to involve cytokine deprivation leading to T-effector cell apoptosis.
There is a debate as to whether the high levels of CD25 expression
in Tregs results in local consumption of IL-2 causing IL-2 depriva-
tion of T-effector cells and their apoptosis. Pandiyan et al. showed
in a mouse model of inﬂammatory disease that cytokine depriva-
tion-induced apoptosis is a prominent mechanism by which Tregs
inhibit T-effector cell responses  whereas in humans IL-2
depletion alone was not required by Tregs to suppress T-effector
cells . Tregs can also directly suppress T-effectors by transfer-
ring the inhibitory second messenger cyclin adenosine monophos-
phate (cAMP) through gap junctions to T-effector cells . Finally,
Tregs express ectoenzymes like CD39 and CD73 that generate per-
icellular adenosine, which suppressed T-effector cell function
through activation of adenoisine receptor 2A (A2aR) [35–37].
Adenosine binds to A2aR leading to the induction of Treg cells
through inhibition of IL-6 expression while promoting TGF-b secre-
Tregs can also exert their suppressive function by directly
affecting dendritic cells (DC). One of the molecules involved in
this mechanism is CTLA-4 which is constantly expressed by
Tregs but also by CD4
T-cells upon activation. CTLA-4, like
CD28, binds to CD80/CD86 co-stimulatory molecules expressed
by antigen presenting cells and sends a negative signal that
causes inhibition of T-cell activation. Blockade of Treg CTLA-4 re-
sults in reduced T-effector cell suppression mediated by DC .
Recently, Qureshi et al. revealed the mechanism of CTLA-4 ac-
tion. CTLA-4 can capture CD80 and CD86 from opposing cells
by a process of trans-endocytosis. After removal, these costimu-
latory ligands are degraded inside CTLA-4 expressing cells,
resulting in impaired costimulation via CD28 . Another way
that Tregs control APCs is by upregulating expression of indole-
amine 2,3-dioxygenase (IDO) which catalyzes tryptophan degra-
dation and kynurenine generation which is associated with T-cell
hyporesponsiveness and apoptosis. IDO is up-regulated by proin-
ﬂammatory cytokines in an attempt to modulate immune
responsiveness and therefore represents a negative regulatory
pathway. Additionally, CTLA-4 signaling through CD80/CD86 in
APC may also up-regulate IDO . IDO expression by DC results
in inhibition of T-effector cell function, including proliferation
and clonal expansion, and reduced survival. Moreover, IDO-
expressing DC may favor the emergence of CD4
Tregs by the expansion/conversion from naive CD4
T cells [42,43].
3604 K.-M. Chavele, M.R. Ehrenstein / FEBS Letters 585 (2011) 3603–3610
It appears that Treg have multiple mechanisms at their disposal
to suppress immune responses which vary according to the envi-
ronmental and inﬂammatory context.
3. Tregs in systemic lupus erythromatosus
SLE is a systemic autoimmune disease targeting multiple or-
gans including the skin, joints, kidneys, and central nervous
system. SLE is characterized by abnormalities in both the B-
and T-cell compartments associated with loss of tolerance fol-
lowed by activation and expansion of autoreactive lymphocytes,
the production of inﬂammatory cytokines and abundant produc-
tion of a wide array of potentially pathogenic autoantibodies. In
SLE, autoantibodies are directed against intranuclear nucleic
acids, proteins and nucleoprotein complexes. Thus autoantibod-
ies in conjunction with loss of tolerance play an important role
in the pathogenesis and clinical manifestation of disease. In both
murine and human SLE, the mechanisms of central tolerance ap-
pears to be unaffected implying that there might be a break-
down in the peripheral tolerance. Given that Tregs are involved
in controlling peripheral tolerance even subtle defects could con-
tribute to the development of disease. The uncontrolled activa-
tion of B- and T-cells and pathogenesis of SLE may be partly
attributed to defects in Tregs.
3.1. Tregs in murine models of SLE
The role of Tregs in lupus-prone mice has been extensively
studied. The ﬁrst evidence that Tregs have protective effects came
form Treg depletion studies. Hayashi et al. showed that the admin-
istration of anti-mouse CD25
T-cell monoclonal antibody (PC61.5)
in autoimmune-prone female NZB NZW F1 (B/WF1) mice, 3 days
after birth, induced the development of nephritis with an increase
in IgG2a antinuclear antibody, elevated IL-6 and IFN-
creased TGF-b production, . New Zealand Mixed 2328
(NZM2328) mice spontaneously develop lupus-like disease with
high circulating levels of autoantibodies to double stranded DNA
and fatal glomerulenephritis. Three-day thymectomy, resulted in
an accelerated double stranded DNA autoantibody response and
early onset of severe proliferative glomerulenephritis with exten-
sive mesangial immune complexes. Transfer of CD25
6-week old asymptomatic donors effectively suppressed autoanti-
body production and the development of autoimmune diseases,
with the exception of proliferative lupus nephritis and sialoadeni-
tis . Scalapino et al. nephritis showed that in (New Zealand
Black New Zealand White) F (1) (B/W) lupus-prone mice, adop-
tive transfer of puriﬁed and ex vivo expanded (with IL-2 and TGF-
thymic-derived Tregs reduced the inci-
dence of renal disease, or slowed the progression of renal disease
when administered after development of proteinuria .
In many of these murine lupus-prone mice strains, such as
(New Zealand Black New Zealand White) F1 (BWF1) and (SWR
Xx New Zealand Black) F1 (SNF1) that spontaneously develop
lupus-like disease the number CD4
Tregs is signiﬁcantly
lower compared to other non-autoimmune mouse strains [46–
48]. In addition, NZM2410 mice that are congenic for the Sle1 locus
have reduced numbers of Foxp3
Tregs which correlates with auto-
antibody production and lupus like features . Some of the data
on Treg function in lupus murine models are conﬂicting. Lupus
prone mouse strains have hyperactive B-  and T-cells  with
a lower threshold of activation. Several groups have found that
Tregs isolated from lupus-prone mice and cultured in vitro re-
tained their suppressive activities [46,48] whereas other groups
have reported the opposite [52,53]. In addition, T cells from
MLR/lpr mice appear resistant to Treg suppression . Tregs are
therefore not able to control T-cell activation and proinﬂammatory
cytokine production leading to chronic inﬂammation. MLR/lpr
mice also have an altered Treg phenotype (CD62L
with a profound reduction in Dicer expression and an altered
microRNA proﬁle compared to non-autoimmune strains of mice
. Overall, these observations suggest that both the quantity
and quality of Tregs are important in the development of lupus-like
These ﬁndings also illustrate the important concept that in
diseases such as SLE, Treg function needs to keep pace with
autoreactive T cells causing inﬂammation and autoimmunity.
Thus if therapies are directed at Treg, restoration of Treg func-
tion to healthy levels may not be sufﬁcient. In order to regain
control of ongoing inﬂammation, Treg with increased potency
are required. The murine model of chronic graft versus host
(cGVHD) disease is characterized by persistent lymphoid hyper-
plasia and SLE-like disease with splenomegaly, B-cell expansion,
autoantibodies and severe immune complex glemerulonephritis.
Zhang et al. showed that administration of short-term low-dose
anti-CD3 antibody treatment induced signiﬁcant remission of
proteinuria, production of autoantibodies and renal immune
complex deposition in lupus nephritic mice. These improvements
are associated with up-regulation of renal Foxp3 and IL-10
mRNA expression compared to those treated with control IgG
suggesting that anti-CD3 therapy may induce IL-10 secreting
Tregs that eventually suppress T-effector function and
ameliorate lupus nephritis .
3.2. Tregs in human SLE
Considerable efforts have been made to delineate the role of
Tregs in the development of SLE in patients. Most of the initial
studies have focused on phenotypic characterization of circulating
Tregs in SLE patients and comparing them with healthy individuals
as well as other patients with autoimmune diseases. Unfortu-
nately, due to limitation in Treg speciﬁc markers, this type of inves-
tigation has been challenging to interpret and the results obtained
by different investigators are contradictory. Early reports revealed
that in patients with SLE the number of circulating CD4
Tregs were decreased [55–58]. These studies were open to doubt
since identiﬁcation of Tregs relied on CD25 which can be expressed
by activated CD4
T-cells. In more recent studies, investigators
analyzed Tregs as CD4
and still found a decrease percent-
age in SLE patients [59–62]. A number of groups have described
inversely correlate with disease activity. Active
SLE patients also appear to have decreased levels of CD4
Tregs compared to inactive SLE patients and healthy individuals
. In addition, different types of treatments used on SLE patients
such as corticosteroid therapy , therapeutic plasmapheresis
 and B-cell depletion with Rituximab [65,66] increase the cir-
culating percentage of CD4
Tregs. In contrast to most
studies, three different groups reported similar levels of circulating
Tregs in SLE patients and healthy individuals
The phenotypic characterization of circulating Tregs in SLE
patients has also been analyzed based on expression of intracel-
lular Foxp3 transcription factor. The results from these studies
did little to resolve the contradictory results from studies based
on CD25 expression. Three reports have shown a decrease of
Tregs in SLE patients compared to healthy individu-
als [61,70,71] and another three revealed normal levels of
Tregs [67,68,72]. Studies that showed a de-
crease in CD4
Tregs in SLE patients found an inverse cor-
relation with disease activity [61,70] or showed no correlation
. There are also reports showing an increase of circulating
Tregs that correlates with disease activity
K.-M. Chavele, M.R. Ehrenstein / FEBS Letters 585 (2011) 3603–3610
[59,73–76]. Recently, Bonelli et al. showed that SLE patients have
increased numbers of CD4
Tregs rather than activated T-cells.
In vitro functional analysis on these cells revealed that they
are able to suppress T-cell proliferation but not IFN-
. Zhao et al. reported that the proportion of blood
T-cells of the SLE patients was not signiﬁ-
cantly different from that of the healthy individuals. However,
the proportions of CD4
T-cells and CD4
T-cells of SLE patients were signiﬁcantly lower than those of
the healthy individuals .
Trying to quantify circulating Tregs in patients with SLE may
be inﬂuenced by the deposition of these cells in lymph nodes
and inﬂamed tissues. Unfortunately, there is limited information
about Tregs inﬁltration and deposition in lymph nodes and in-
ﬂamed organs such as the skin and kidney in SLE patients. Miy-
ara et al. reported a decreased number of Foxp
Tregs in lymph
nodes from patients with SLE and reduced levels of Foxp3 mRNA
isolated from SLE patients with active nephritis . Another
study done in patients with cutaneous SLE, also found a de-
creased number of Foxp3
Tregs in skin specimens obtained de-
spite the fact that these patients had a normal number of
Apart from the quantitative analysis of Tregs in SLE patients,
several studies were also focused on the qualitative and functional
analysis of Tregs in SLE patients. These studies also show diverse
results. Several groups have reported abnormalities in the suppres-
sive function of CD4
whereas other groups did not conﬁrm such alteration [57,75,79–
81]. In most studies, the suppressive defects of SLE patients were
attributed to Tregs, whereas others have indicated an increased
resistance to suppression by T-responder cells. Venigalla et al.
showed that CD4
T-responder cells isolated from patients
with active SLE were signiﬁcantly less sensitive than those from
patients with inactive SLE to the suppressive function of autolo-
gous or normal donor CD4
a signiﬁcant inverse correlation was observed between regulatory
T cell suppressor function and the level of lupus disease activity
. Consistent with this ﬁnding, other groups have reported im-
paired Treg function when cultured with autologous T-effector
cells but not when T-effector cells come from healthy individuals
suggesting a possible resistance of lupus T effector cells to Treg
Despite the plethora of studies focused on Tregs and SLE, their
role in disease initiation and development remains elusive. A num-
ber of reasons are likely to account for this lack of clarity. The
markers that deﬁne Treg such as CD25 and Foxp3 can also be in-
duced at least transiently upon T-cell activation [1,14,82,83]. One
of the basic features of SLE is persistent T cell activation. For this
reason, studies based on phenotypic characterization of circulating
Tregs must be regarded with caution. In addition, SLE is a disease
with a very heterogeneous patient population and their pathogen-
esis is unlikely to be uniform. Disease activity and different thera-
pies could also alter Treg numbers or function. For example active
disease was positively correlated with decreased numbers and
suppressive function of Tregs whereas patients with inactive dis-
ease showed no difference to healthy individuals . Addition-
ally, patients treated with corticosteroid therapy ,
therapeutic plasmapheresis  and B-cell depletion with Ritux-
imab [65,66] have increased numbers of Tregs in their circulation.
Discrepancies in ﬁndings related to Treg function could be due to
different methods used to isolate Tregs from blood (e.g. Treg isola-
tion with magnetic beads versus FACS sorting), the different cell
markers used to isolate Tregs, the different in vitro stimuli used
to activate T-effector cells as well as the presence or absence of
accessory cells (e.g. monocytes) in the in vitro assays.
4. Tregs in rheumatoid arthritis
Rheumatoid arthritis (RA) is characterized by swelling of the
synovium and damage of the cartilage around the joints leading
ﬁnally to the joint destruction. Several factors play a role in dis-
ease development. The role of TNF-
and IL-6 as key mediators
of RA has been proven and blokade of these cytokines has been
successfully introduced as therapies in RA patients. The role of
Tregs has been demonstrated in both murine models of autoim-
mune arthritis and patients with RA. On the whole, Treg func-
tion has been reported to be impaired whereas the number of
circulating Tregs varies depending on the study. Recently inves-
tigations into Tregs focus on the balance between Tregs and
other proinﬂammatory T-cell populations such as Th17 cells
4.1. Tregs in murine models of rheumatoid arthritis
Collagen-induced arthritis (CIA) is the most common murine
model used to investigate the role of Tregs in disease and is in-
duced after immunization of mice with bovine type II collagen
emulsiﬁed in complete Freund’s adjuvant [86,87]. Morgan et al.
Tregs in these arthritic mice by administra-
tion of a monoclonal antibody speciﬁc for CD25. As a result,
these anti-CD25 treated mice had worse disease than controls.
Disease exacerbation was accompanied by higher antibody titers
against collagen, and in vitro tests showed increased prolifera-
tion of collagen-speciﬁc T cells. When CD4
administered back into CD25
depleted mice, at the time point
of immunization, disease severity was reduced . Tregs slo-
wed disease progression although there was no difference in T-
and B-cell responses. Adoptively transferred CD4
were located in the synovial tissue of affected joints soon after
transfer indicating that regulation may occur locally in the joint
. Transfer of Foxp3-transduced CD4
T-cells also ameliorated
CIA although the time point of transfer is critical for modulating
disease severity. CD4
Tregs were able to reduce the
severity of CIA when transferred prior to collagen immunization.
When transferred 20 days after collagen immunization, a higher
number of CD4
Tregs was needed suggesting increased
resistance to suppression whereas if transferred even later after
the booster immunization they had no effect on disease modula-
tion. Apart from the time point of Treg transfer, the route of
administration is also important. Tregs administered systemically
rather than directly into the inﬂamed joint were more effective
in modifying disease .
Antigen-induced arthritis (AIA) is another model of murine
rheumatoid arthritis and is induced by pre-immunization of
mice with methylated bovine serum albumin in complete Fre-
und’s adjuvant followed 21 days later by an intra-articular injec-
tion of the knee joint with methylated bovine serum albumin
diluted in saline . In AIA, depletion of CD25
in immunized animals before arthritis induction led to an exac-
erbation of arthritis as indicated by knee swelling and histolog-
ical scores and increased cellular and humoral immune
responses to the inducing antigen. Transfer of CD4
into immunized mice at the time of induction of antigen-in-
duced arthritis decreased the severity of disease but was not
able to cure established arthritis. As in the CIA models, trans-
cells accumulated in the inﬂamed joint .
Regardless of the murine model of disease, Treg depletion and
adoptive transfer experiments have shown that Tregs are able
to modify disease severity and immune responses. However,
the exact mechanism by which Treg modulate disease remain
3606 K.-M. Chavele, M.R. Ehrenstein / FEBS Letters 585 (2011) 3603–3610
Manipulation of Tregs either directly or indirectly has also been
proven successful in treating arthritis in murine models of disease.
Wright et al. showed that adoptive transfer of antigen speciﬁc
Tregs (generated by retroviral T-cell receptor gene transfer into
Tregs) in mice with AIA resulted in reduction
of Th17 cells and a signiﬁcant decrease in arthritic bone destruc-
tion . Recently, an indirect way of targeting Tregs in the AIA
model of arthritis by using tolerogenic DC loaded with antigen
has been described. Administration of DC transfected with an
ERK activator and OVA in mice with AIA, maintains DC in an imma-
ture state. As a result, DC present antigen at suboptimal levels,
leading to inhibition of CD8 T-cell expansion and secretion of
TGF-b that directs antigen speciﬁc Treg differentiation and prolifer-
ation rather than expansion of CD4
T-effector cells. As a result
arthritis is signiﬁcantly inhibited through antigen speciﬁc Tregs
. Finally, administration of anti-CD3 monoclonal antibody in
the CIA murine model of arthritis has proven to be effective in
treating disease severity through expansion of naturally occurring
Tregs and the generation of CD8
4.2. Tregs in human rheumatoid arthritis
As with SLE, many human RA studies focused on quantifying
Treg numbers in the peripheral blood, but also at the site of inﬂam-
mation. In RA patients the number of Tregs present in synovial
ﬂuid is higher than that in the peripheral blood. Tregs accumulated
in inﬂamed joints express high levels of surface and intracellular
CTLA-4, GITR, OX-40, and Foxp3 . Data regarding the number
of Tregs in the circulation of RA patients compared to healthy indi-
viduals are inconclusive and contradictory. Some studies showed a
decrease of circulating Treg in RA patients [97–99] whereas others
showed no difference in circulating Treg numbers compared to
healthy individuals [96,100–102] and some indicated an increase
in circulating Treg numbers [103,104].
Studying Treg function in patients with RA has been informa-
tive. Although Tregs present in the synovial ﬂuid of patients with
RA have an enhanced capacity to suppress both T-cell proliferation
and cytokine production (TNF-
) disease is still able to
progress. T-responder cells present in the synovial ﬂuid were less
susceptible to suppression compared with circulating T-responder
cells . This data is consistent with the observation that
strongly activated CD4
T-cells are resistant to Treg suppression
. Our group has reported that CD4
are able to suppress proliferation of T-effectors but were unable
to suppress proinﬂammatory cytokine secretion from activated
T-cells and monocytes . This deﬁciency of RA Tregs is associ-
ated with defective expression and function of CTLA-4, a key mol-
ecule linked to their suppressive function. Expression of CTLA-4 is
reduced in RA Tregs compared to healthy Tregs which correlates
with reduced function. Artiﬁcial induction of CTLA-4 expression
on RA Treg in vitro restored their suppressive capacity. Further-
more, CTLA-4 blockade impaired healthy Treg suppression of T-cell
production, but not T-cell proliferation. These data suggest
that Tregs control T-cell proliferation and cytokine production
through different mechanisms . Furthermore, CTLA-4 gene
polymorphisms have also been correlated with autoimmune dis-
eases like RA  and SLE .
In RA, Tregs especially those present in the synovial ﬂuid are
also inﬂuenced by the cytokine proﬁle. TNF-
, IL-6, IL-15, and IL-
1 present in the inﬂamed joint act to increase the number of
inﬁltrating Tregs in the inﬂamed joint but at the same time im-
pair their function. For example IL-6 secreted by DC after TLR
stimulation induces T-responder cell resistance to Treg mediated
suppression . TNF-
and IL-7 secreted by activated mono-
cytes in the inﬂamed joint have a direct effect on CD4
Tregs by abrogating their suppressive activity . In vitro
addition of TNF-
at high concentrations in Treg suppression as-
says inhibit the suppressor function of Tregs by down regulating
Foxp3 expression .
Different types of RA treatment can also affect the function of
Tregs. Our group has demonstrated that after the resolution of
inﬂammation by administration of anti-TNF therapy (anti-TNF
antibody inﬂiximab) the Treg function that was originally im-
paired appeared to be restored . Patients treated with inf-
liximab had two different populations of Tregs. Natural Tregs
characterized by CD62 Ligand expression (CD62L
) and induced
Tregs that were CD62L
. In patients who responded to anti-
TNF therapy, the CD62L
Tregs were still unable to suppress
production by T-responder cells whereas in-
Tregs from the same patients suppressed TNF-
production by T-responder cells through the produc-
tion of IL-10 and TGF-b rather than CTLA-4 . Although
anti-TNF treatment works for most patients, after withdrawal
of treatment relapse usually ensues raising the possibility that
to induce long lasting remission natural Tregs need to be func-
tionally restored. Some of these ﬁndings have recently been con-
ﬁrmed using a mouse model of arthritis. Regulatory T cells with
a reduced expression of CD62L are increased in a TNF driven ar-
thritic mouse model treated with anti-TNF .
Treg stability in inﬂammatory conditions like RA and their rela-
tionship with inﬂammatory Th17 cells has been studied in the last
few years. In an inﬂammatory condition like RA, it is quite possible
that Tregs in the presence of the different proinﬂammatory cyto-
kines will become unstable and convert to pathogenic T-cells. Treg
secreting Th17 have been described though their function is not
necessarily impaired. Ayyoub et al. showed that circulating mem-
ory Tregs secrete IL-17 ex vivo . In addition, memory Tregs
when cultured in vitro with IL-1b and IL-6 secrete IL-17 whereas
IL-17 secretion was prevented in the presence of TGF-b suggesting
that inﬂammatory conditions impair Tregs cell function and pro-
mote IL-17 production . The mechanisms affecting Treg sta-
bility under inﬂammatory conditions are linked to regulation and
epigenetic modiﬁcation of the Foxp3 gene. Complete demethyla-
tion of CpG residues in the proximal promoter of Foxp3 gene is re-
quired for stable Foxp3 expression. IL-6 and possibly other
proinﬂammatory cytokines like TNF-
and IL-1b promote reme-
thylation of CpG residues in the proximal promoter of Foxp3 gene
resulting in downregulation of Foxp3 expression by Tregs
Tregs play a pivotal role in controlling autoimmune responses
and inﬂammation. Subtle changes in Treg number, function or
phenotype could lead to the development of autoimmune dis-
eases such as SLE and RA, but also may be a consequence of
the inﬂammatory environment (Fig. 1). In comparison to data
from murine models of disease, clear conclusions as to the nat-
ure of Treg defects in patients with SLE and RA have yet to be
realized due to the many contradictory reports. The discovery
of new speciﬁc Treg markers will help to resolve these issues
but disease heterogeneity and the number of therapies available
also contributes to some of the conﬂicting ﬁndings. Accurate pa-
tient classiﬁcation and analysis of subgroups of patients will be
important in future studies. In addition, the importance of Treg
at different phases of disease may vary. Evidence from both dis-
eases suggest that the potency of T-effectors and the suppressive
effects of Treg need to be carefully balanced. Further research is
needed in human diseases to determine how to increase the
suppressive power of Tregs, perhaps only for a short period, in
order to restore immunological tolerance.
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autoimmunity failure or Tregs may reﬂect inadequate number of Tregs, defective Treg function and phenotype or resistance of the T-effector cell population to suppression by
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