f o C u s o n To l e r A n C e i n T r A n s p l A n TAT i o n

An update on regulatory T cells in transplant

tolerance and rejection
Xian Chang Li and Laurence A. Turka
Abstract | Several types of T cells with immunosuppressive properties have been identified, but FOXP3+
regulatory T (TREG) cells have emerged as a dominant cell type; they are critically involved in the induction
and maintenance of immune tolerance. Manipulation of this cell type for the induction of transplant
tolerance including renal transplant tolerance has attracted considerable attention. Studies in this area have
demonstrated unexpected complexities, and attempts to translate TREG cells towards clinical utility have met
with unanticipated difficulties. In this Review, a broad overview is provided on recent progress in the study
of TREG cells, focusing on challenges, opportunities, and emerging approaches in exploiting T REG cells for the
induction of transplant tolerance.
Li, X. C. & Turka, L. A. Nat. Rev. Nephrol. 6, 577–583 (2010); published online 3 August 2010; doi:10.1038/nrneph.2010.101

Introduction
one of the major goals in transplantation is the induction
of tolerance, that is, indefinite allograft survival without
the need for life-long immunosuppression. over the
past 10 years, the approach of the scientific community
towards achieving this goal has evolved from one that
focused on inhibition of t-effector cells to one that also
involves promotion of regulatory t (treG) cells.1 the
current paradigm is that the balance of alloantigen-
reactive treG cells and t-effector cells ultimately deter-
mines whether the graft is accepted or rejected, and that
promoting the development and maintenance of regula-
tory responses represents a promising approach for the
induction of transplantation tolerance.
although the existence of treG cells is indisputable,
harnessing them for therapeutic purposes has not
been straightforward. For example, the local micro-
environment in which treG cells reside can have
considerable influence on their functional status. Certain
inflammatory cytokines can either disarm treG cells
or render t-effector cells resistant to suppression. 2
additionally, treG cells are extremely heterogeneous
and consist of many different subsets. moreover, some of
these subsets exhibit considerable plasticity in that they
are neither terminally differentiated nor hardwired for
suppression;3 their regulatory programs can be readily
turned off or reprogrammed.4 as a consequence, former
treG cells can, under some circumstances, such as in an
inflammatory milieu, acquire the phenotype and func-
tional capabilities of t-effector cells and mediate tissue
damage. Clearly, these complexities present unexpected
Competing interests
L. A. Turka declares associations with the following companies:
Biogen IDEC, Bristol–Myers Squibb, GlaxoSmithKline and
Novartis. See the article online for full details of the
relationships. X. C. Li declares no competing interests.
challenges in targeting treG cells for the induction of
transplant tolerance.
although several subsets of t cells have been identi-
fied to date that have immune regulatory properties, in
this review focus will be placed specifically on FoXP3+
treG cells. this FoXP3+ regulatory cell type is certainly
the most studied and best understood, and the existence
of an autoimmune syndrome called immune dysregula-
tion, polyendocrinopathy, enteropathy, X-linked (iPeX)
syndrome in humans who lack a functional FOXP3 gene
is a clear evidence of its role in peripheral tolerance and
immune homeostasis.5 in this review we provide an
overview of treG cells and tolerance induction in various
transplant models including kidney transplantation. the
reason why discussion does not solely focus on kidney
transplantation is because progress and issues in other
models are applicable to renal transplantation and vice
versa. this review highlights recent progress in the
study of FoXP3+ treG cells, and summarizes emerging
approaches for their manipulation in the induction of
transplant tolerance.
T cells with regulatory properties
although multiple types of immune regulatory t cells
exist, they are often broadly divided into two categories—
FoXP3– and FoXP3+ regulatory cells. FoXP3– regulatory
cells include CD4+il-10-producing tr1 cells,6 a subset
The Transplant
of CD8+ t cells, and CD3+CD4–CD8– double-negative Institute, Beth Israel
t cells.7 these cells suppress t-cell activation through Deaconess Medical
Center, Harvard
a variety of different mechanisms and have been shown Medical School,
to contribute to transplant tolerance in selected animal 330 Brookline Avenue,
models. in addition, natural killer t cells that are also CLS‑605, Boston,
MA 02215, USA
FoXP3– can have potent immune suppressive activities (X. C. li, l. A. Turka).
in some transplant models.8 although the FoXP3 protein
Correspondence to:
can be found in both CD4+ and CD8+ t cells, the former X. C. Li
(that is, CD4+FoXP3+ cells, referred to as treG cells) xli@bidmc.harvard.edu

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Key points
■ Regulatory T (TREG) cells are indispensable in transplant tolerance
■ TREG cells are heterogeneous and are not inherently stable; their suppressive
programs can be turned off or they may be reprogrammed to become T‑effector
cells
■ Some effector cell types, such as memory T cells, can evade TREG‑cell‑mediated
suppression
■ Mechanisms that regulate the in vivo induction and stability of TREG cells are
poorly defined
■ The mechanism by which FOXP3+ TREG cells interact with other types of
regulatory cells in transplant tolerance is unknown
■ Potential complications of immunodeficiency exist from nonspecific expansion
of TREG cells
FOXP3+ TREG cells
FOXP3, CD25, GITR,
CTLA-4, IL-10, CD39,
OX40, galectin-9
nTREG cells iTREG cells
■ Thymus-derived ■ Generated in the periphery
■ Selected by autoantigens ■ Derived from T-effector cells
■ Possess an autoreactive TCR repertoire ■ Induction requires TCR, CD28 signaling,
■ Cross-reactive to alloantigens and certain cytokines (e.g. TGF-β, IL-2)
■ Suppress autoimmunity ■ Positively and negatively regulated by many
■ Contact-dependent fashion in suppression other pathways
Figure 1 | Key features and differences between nTREG cells and iTREG cells. Both
subsets express the transcription factor FOXP3 and a variety of other cell surface
factors, but they are strikingly different in origin, induction, TCR repertoire and in
antigen specificity. Abbreviations: iTREG cell, induced regulatory T cell; nTREG cell,
natural regulatory T cell; TREG cell, regulatory T cell; TCR, T‑cell receptor.
have emerged as the dominant focus of research and
application in both preclinical and clinical studies.9
CD4+foXp3+ Treg cells
the discovery that the transcription factor FoXP3 was
both necessary and sufficient for treG-cell function in an
entire class of CD4+ t cells, firmly re-established the study
of immune regulation in general, and sharply focused
the spotlight on these cells in particular.10 Phenotypically,
these treG cells express several cell surface markers such
as CD25, Ctla-4, Gitr (glucocorticoid-induced tumor
necrosis factor receptor), CD134, CD103, CD39 and
CD73, many of which are also expressed by activated
CD4+ t-effector cells, and are therefore not treG-cell
specific. under basal conditions, treG cells account for
approximately 5–10% of the total CD4+ t cells in the
periphery, and can expand via lymphopenia-driven or
antigen-driven proliferation in vivo.11
even as defined by universal FoXP3 expression,
treG cells are not a uniform population, but are broadly
subdivided into those that developed in the thymus
(so-called natural (n)treG cells) and those that were
converted from t-effector cells in the periphery (termed
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© 2010 Macmillan Publishers Limited. All rights reserved
adaptive treG cells, or more commonly, induced (i)treG
cells) (Figure 1).12 ntreG cells and itreG cells share some
common features in terms of phenotype and function.
For example, both types of cells express FoXP3 and both
potently suppress the proliferation of t-effector cells.12
additionally, t-cell receptor (tCr) signaling is required
for the induction of both ntreG cells and itreG cells, as
targeted mutations that disrupt proximal tCr signal-
ing events often result in a complete absence of thymic
Foxp3+ t cells.13 similarly, suppression of tCr signal-
ing by calcineurin inhibitors blocks the conversion of
t-effector cells to itreG cells.14 Finally, cytokines such as
tGF-β and il-2 are critical to intrathymic development
of ntreG cells15 and are also mandatory for the induc-
tion of itreG cells in the periphery.16 this observation
is consistent with the finding that the FOXP3 promoter
region contains binding sites for smaD proteins and
stat5, key molecules involved in tGF-β and il-2
signaling, respectively.
Key features of nTreg and iTreg cells
ntreG cells and itreG cells also have striking differ-
ences (Figure 1). ntreG cells are selected and matured
in the thymus by autoantigens, before being exported to
the periphery. thus, the tCr repertoire of these cells is
primarily autoreactive.17 By contrast, as itreG cells are
converted from conventional t-effector cells in the
periphery, they supposedly have a similar tCr repertoire
to that of the t-effector cells from which they are derived.
therefore, although itreG cells are initially selected by
autoantigens in the thymus, as are all t cells, they may be
considered to be more skewed toward foreign antigens.
this difference in tCr specificity is important in trans-
plantation where tolerance is often defined by donor
antigen specificity.18 moreover, many other factors and
pathways facilitate the induction of itreG cells, includ-
ing retinoic acid,19 leukemia inhibitory factor (liF),20
Ctla-4, and PD-1–PD-l1 interaction.21 equally impor-
tant is the finding that induction of itreG cells can be
inhibited by several mechanisms, including by certain
cytokines (for example, il-4, il-6, iFn-γ), by oX40 and
t cell-ig mucin molecule-1 (tim-1) costimulation or by
sustained akt–mtor activation.4,22,23 intriguingly, these
pathways seem to have little effect on the intrathymic
development of ntreG cells. in addition, knockout of
the gene that encodes the membrane-associated guany-
late kinase Carma‑1 results in selective deficiency of
ntreG cells in the thymus, although CD4 + Foxp3 –
t-effector cells in the periphery can be readily converted
to Foxp3+ itreG cells by tGF-β,13 suggesting key differ-
ences in the development of ntreG cells and itreG cells.
interestingly, itreG cells are particularly unstable and tend
to lose FoXP3 expression more easily than ntreG cells,
which is probably related to epigenetic differences in
the FOXP3 gene in both subsets.24 such differences may
be important in the selective manipulation of ntreG cells
and itreG cells by therapeutic means.
with few exceptions, conversion of t-effector cells to
itreG cells in vivo may be surprisingly rare.25 Despite the
fact that t-effector cells are capable of becoming itreG
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 f o C u s o n To l e r A n C e i n T r A n s p l A n TAT i o n
cells under certain defined conditions,16 in vivo studies
showing consistent and reproducible induction of itreG
cells, either in naïve hosts or under tolerizing conditions,
are few and far between. tolerizing protocols are hoped
to induce prominent itreG cells that confer donor spe-
cific tolerance in transplant models, but clear-cut evi-
dence supporting this claim in tolerant recipients, is still
lacking. in fact, a lack of information about the in vivo
conditions that promote the conversion of t-effector cells
to treG cells in transplant recipients indicates that more
studies are warranted in this area. in this regard, it should
be noted that the relative contribution of ntreG cells and
itreG cells to the peripheral pool of treG cells and the
relevance of each subset to transplant tolerance are not
known. whether ntreG cells and itreG cells are syner-
gistic in promoting tolerance or are merely redundant
is not yet clear; however this important issue requires
further investigation.
Mechanisms of TREG-cell action
Both innate and adaptive immune cells are targets
of treG-cell-mediated suppression, and treG cells are
known to employ a variety of mechanisms to mediate
these effects. treG cells directly suppress many functions
of CD4+ and CD8+ t cells, ranging from their prolifera-
tion to their differentiation into t-helper (tH)-1, tH2, and
tH17 subsets. in some cases, treG cells induce apoptosis of
responding t cells.26 treG cells also suppress the activation
of B cells, thereby inhibiting humoral immune responses.
other prominent targets of treG cells include dendritic
cells, macrophages, natural killer cells, mast cells, and
osteoblasts.26 treG cells are also involved in tissue repair
and in the resolution of tissue inflammation,27 suggesting
a potential role for treG cells in the regulation of non-
immune cells. in fact, treG cells can inhibit the develop-
ment of transplant vasculopathy,28 a complex process that
involves many immune and nonimmune cells, supporting
a broad role for treG cells in tissue remodeling.
the mechanism by which treG cells regulate such
diverse cell types both inside and outside the immune
system is an area of considerable interest. treG cells prob-
ably employ several different mechanisms to suppress
pathogenic t-effector cells. In vitro assays have demon-
strated that activation of treG cells via tCr stimulation
is required to mediate suppression of t-effector cells and
that this suppression requires strict cell–cell contact.26
under some conditions, treG cells have been shown to
deprive t-effector cells of survival and growth factors or
to directly kill activated t cells via granzyme-dependent
mechanisms. Furthermore, treG cells express CD39 and
CD73, the ectoenzymes that break down the extracellular
atP into adenosine.29 this process has been shown to
turn an atP-rich inflammatory milieu to one that is
immunosuppressive, as adenosine inhibits the activa-
tion of dendritic cells and macrophages, which in turn
prevents t-cell priming. treG cells also express Ctla-4
on their surface, which can directly engage the periph-
eral membrane protein B7 on antigen-presenting cells
(aPCs) to inhibit aPC activation via different mech-
anisms.30 in addition, treG cells can produce copious
nature reviews | nephrology
© 2010 Macmillan Publishers Limited. All rights reserved
amounts of immune suppressive cytokines, including
tGF-β, il-10, and il-35, which are known to inhibit a
wide spectrum of cellular activities.31 tGF-β exerts broad
antiproliferative and anti-inflammatory effects. il-10
strongly inhibits activation of macrophages and dendritic
cells, and il-35 is a key mediator of treG-cell-induced
immunosuppression.32 Despite advances in our under-
standing of treG-cell function, the processes by which
this catalog of in vitro mechanisms contributes to in vivo
immunosuppression by treG cells is still not known.
Harnessing TREG cells for tolerance
Despite what many immunologists believe to be the tre-
mendous potential of treG cells for inducing transplant
tolerance, translating this strategy into reality has not
been straightforward. many challenging issues face this
area that require further study.
nTreg cells versus iTreg cells
when compared in a variety of assays in vitro and in vivo,
ntreG cells and itreG cells show comparable potency in
suppressing t-effector cells.4 However, as noted above,
the relative importance of ntreG cells and itreG cells in
transplant models remains unknown. whether both
subsets are required or whether one subset predomi-
nates the other in the induction of transplant tolerance
is not clear. a subset of ntreG cells can be alloreactive,
presumably owing to their cross-reactivity to allo-
antigens. in fact, in several transplant models, global
depletion of ntreG cells prevents tolerance induction to
allografts.9 the exact proportion and identity of ntreG
cells that are alloreactive is unclear, however, as no
specific markers exist to distinguish alloreactive ntreG
cells from nonalloreactive ones. ideally, therapies that
promote treG-cell-mediated tolerance should selec-
tively and specifically activate and expand alloreactive
ntreG cells. targeting ntreG cells en-mass in transplant
models, though beneficial, may also induce unwanted
effects owing to a stimulation of nonalloreactive treG
cells that could potentially render patients susceptible to
immunodeficient complications.
on the other hand, itreG cells are derived from
t-effector cells and should have the same antigen speci-
ficity as alloreactive t-effector cells. thus, itreG cells
may have a particularly important role in donor-specific
tolerance. in fact, studies that have tried to expand itreG
cells ex vivo and then adoptively transfer them to induce
tolerance to allografts in vivo have considerable attrac-
tion, as in selected models itreG cells can be key players
in tolerance induction.33 However, the paucity of itreG
cells in vivo, even under tolerizing conditions in trans-
plant models, raises concerns. one issue is that itreG
cells must compete with ntreG cells in vivo for space,
resources, and survival, and in this setting, itreG cells
may have a competitive disadvantage. another concern
is that the induction of itreG cells can be diverted, leading
to the formation of pathogenic tH17 cells in the presence
of inflammatory cytokines.34 whether blocking tissue
inflammation can enhance induction of itreG cells in vivo
remains to be studied.
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FOXP3, IL-2
miRNA
Epigenetics
Immune suppression
Cytopathic effects
Unstable TREG cells
Committed TREG cells
Figure 2 | Lineage plasticity of TREG cells in the periphery. Multiple pathways and
mechanisms are involved in regulating the peripheral pool of TREG cells, including
miRNA, epigenetic mechanisms, and pathways that involve FOXP3 and IL‑2. The
TREG cell pool consists of committed TREG cells that are relatively stable and
dedicated to suppression and unstable TREG cells. Unstable TREG cells can lose
FOXP3 and acquire T‑effector cell functions. Abbreviations: miRNA, microRNA;
TREG cell, regulatory T cell.
lineage plasticity of Treg cells
as alluded to above, although treG cells were initially
believed to be highly specialized and fully committed to
suppression of t-effector cells at all times, in fact their
lineage and suppressive programs exhibit unexpected
plasticity.3 For example, ntreG cells can lose Foxp3 expres-
sion, with a resultant loss of immunosuppressive function,
and a potential acquisition of effector function, rendering
them capable of causing tissue destruction.35 another sur-
prise is that expression of Foxp3, though instrumental to
the development of treG cells, is not an on-and-off switch
to treG-cell functionality. in fact, differences in levels of
Foxp3 expression also profoundly influence their sup-
pressor programs, and reduced Foxp3 expression tends
to induce impaired treG-cell functions.36
a number of studies exist demonstrating functional
instability of ntreG cells. stimulation of highly purified
ntreG cells in vitro in the presence of il-6 resulted in the
loss of Foxp3 and such treG cells then became il-17-
producing t-effector cells.37 moreover, genetic muta-
tions that disrupt the micro (mi)rna pathways cause
treG cells to lose their suppressive function despite main-
tenance of Foxp3 expression.38 in humans, CD4+CD25low
cells that express lower levels of FoXP3 than normal
CD4+CD25+ cells can readily lose FoXP3 expression
and acquire il-17-producing ability.39 in 2009, Bluestone
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© 2010 Macmillan Publishers Limited. All rights reserved
and colleagues used an elegant lineage tracking tech-
nique to demonstrate that a notable fraction of ntreG
cells fail to maintain Foxp3 expression; such ‘ex-treG cells’
produced substantial amounts of iFn-γ and mediated
the development of autoimmune diabetes.35 the treG-cell
pool therefore seems to contain a population of com-
mitted treG cells and a small fraction of uncommitted
treG cells. such uncommitted treG cells seem to easily
lose FoXP3 expression and become t-effector cells
(Figure 2). whether the a priori identification of uncom-
mitted treG cells is possible, is not known, and although
demethylation of the FOXP3 locus may prove to be a
useful marker to identify uncommitted treG cells, its
assessment in live cells is difficult.24 although all of the
factors involved in the regulation of ntreG-cell stability
are probably not known, certain inflammatory cytokines
(for example, il-1, il-6, tnF) and costimulatory mol-
ecules (for example, oX40, tim-1) seem to be critically
involved.40 whether treG cells can be stabilized in vivo by
blocking these pathways remains to be determined.
Further concerns are held with regard to the instabil-
ity of itreG cells, as a genome-wide analysis of itreG cells
and ntreG cells identified many epigenetic differences
critical to treG-cell stability.24 For example, CpG islands,
histone methylation and acetylation as well as mirna
status are major mechanisms that contribute to the stabil-
ity of FoXP3+ treG cells,41 whereas itreG cells exhibit an
epigenetic signature similar to that of t-effector cells
as opposed to that of treG cells.24 this variation may
be one of the key reasons for the instability of itreG
cells, and, therefore, the paucity of itreG cells in vivo
in transplant recipients, even under tolerizing condi-
tions. Determining whether such mechanisms can be
targeted to stabilize itreG-cell populations once they are
induced in transplant models to promote tolerance is of
critical importance.
presentation of target alloantigen to Treg cells
a key issue unique to transplantation is the presence
of donor and host aPCs in transplant recipients; they
process and present alloantigens in strikingly differ-
ent fashions. 42 Donor aPCs use the direct pathway
whereas host aPCs use the indirect pathway to stimulate
t-effector cells and treG cells. this difference in antigen
presentation may have considerable influence on the
induction, homeostasis, and function of treG cells.43
whether donor aPCs and host aPCs are equally potent
in the induction of itreG cells or in the expansion of ntreG
cells remains to be defined, nor is it clear whether donor
and host aPCs differentially affect ntreG cells and itreG
cells. in some models, treG cells that induce transplant
tolerance demonstrate indirect antigen specificity, 44
implying that host aPCs that present allopeptides indi-
rectly may be more important than those that present
allopeptides directly in this process. aPCs may be key
targets of treG-cell-mediated suppression in vivo, yet it
is not known if treG cells are equally effective in suppres-
sion of donor and host aPCs, nor whether ntreG cells and
itreG cells recognize the same or different alloantigens in
the induction of tolerance in transplant models.
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 f o C u s o n To l e r A n C e i n T r A n s p l A n TAT i o n

Induction Tolerance
TREG pool

nTREG cells

nTREG cells
Alloantigen specific
Stable and long lasting
Naïve T cells

Reduction in number
of T-effector cells

T-effector cells
iTREG cells

Time
Figure 3 | Evolution of TREG cells in transplant tolerance. Induction of donor‑specific TREG cells is critically important in
tolerance, and both nTREG cells that cross‑react to alloantigens and iTREG cells that are derived from alloreactive T‑effector
cells may contribute to the alloreactive TREG‑cell pool. The longevity and alloantigen specificity of such TREG cells is essential
to tolerance maintenance. Abbreviations: iTREG cell, induced regulatory T cell; nTREG cell, natural regulatory T cell; TREG cell,
regulatory T cell.

Treg cells within and outside of lymphoid tissue
in addition to being present in lymphoid tissues, treG
cells are also found in large numbers at extralymphoid
sites, although the function of extra-lymphoid treG
cells is only beginning to be studied. treG cells at non-
lymphoid sites are not dormant cells. For example, treG
cells in adipose tissue have been shown to regulate met-
abolic processes;45 such treG cells are different from those
in the spleen. interestingly, treG cells in fat from obese
mice are altered in both phenotype and function.45 in the
transplant setting, treG cells can be found residing in
tolerant grafts,46 but the relative importance of treG cells
in the grafts versus those in the lymphoid tissues is not
known. whether they are the same type of cells present at
different sites or they are inherently different is not clear.
it is also not apparent if they are functionally equivalent.
in most models of immune responses, priming of t cells
for rejection takes place in the draining lymph nodes, and
treG cells at these sites may be particularly important for
the suppression of t-effector cells. treG cells may then
migrate to grafts where they have a key role in the control
of intragraft inflammation, which perpetuates the induc-
tion of new treG cells in situ. Graft acceptance has been
suggested to require the presence of intragraft treG cells.
a 2009 study by Bromberg and colleagues demonstrated a
sequential migration of treG cells from blood to the trans-
planted grafts and then to the draining lymph nodes in
order to achieve tolerance,47 which suggests that the role
of treG cells in transplant settings may be very dynamic.
longevity and homeostasis of Treg cells in vivo
t cells, regardless of their phenotypes and functions, have
a finite lifespan. even for memory t cells that are thought
to be long-lived, there is, in fact, ongoing slow but sub-
stantial attrition in the periphery.48 little is known about
the lifespan of alloreactive treG cells, but they are unlikely
to defy the factors that govern the life and death of t cells.
one potential mechanism that could overcome this
limitation is ‘infectious tolerance’ where treG cells per-
petuate tolerance by recruiting a new cohort of t cells to
become alloreactive treG cells to constantly replenish
the treG-cell pool, so that tolerance can be continuously
enforced in vivo.49 in selected models, this mechanism
is clearly potent in the maintenance of a tolerant state.50
whether the newly recruited treG cells are itreG cells,
(that is, recruited from naïve t-effector cells) or are
derived from the ntreG-cell pool, or a combination of
both, is not known (Figure 3). Clearly, more studies in
this area are undoubtedly needed.
resistance to Treg-cell-mediated suppression
ample exceptions exist where competent treG cells fail
to restrain t-effector cells. For example, treG cells often
completely fail to inhibit the responses of memory
t cells.51 Furthermore, t-effector cells activated under
inflammatory conditions are highly resistant to treG-cell-
mediated immunosuppression,52 suggesting that target
cells can overturn the suppressive function of treG cells.
t-effector cells can, therefore, ‘fight back’ to evade treG
cells. in addition, certain cells of the innate immune
system, for example, natural killer cells, can kill treG
cells upon activation.53 under such conditions, trans-
plant tolerance is difficult to establish, and alternative
approaches that target cells that are resistant to treG-cell-
mediated suppression or that restore their sensitivity to
treG cells are important in the induction of tolerance.

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Differences between humans and mice
many key differences exist in treG cells between humans
and mice. one of the outstanding features is that in
humans, FoXP3 is a t-cell activation molecule rather
than an exclusive marker of treG cells, as stimulation of
highly purified human t-effector cells is associated with
transient expression of FoXP3, and t cells that temporar-
ily acquire FoXP3 do not always show suppressive func-
tions.54 evidence exists that human CD4+ treG cells and
non-treG cells can be segregated by differential expression
of CD25 and CD127 (the alpha chain of il-7 receptor)
where functional treG cells are confined exclusively to
the CD25+CD127– fraction.55,56 studies from sakaguchi’s
research group have demonstrated additional strik-
ing differences between human and mouse treG cells.57
Based on levels of FoXP3 expression and various CD45
isoforms, they have identified at least three different
human treG-cell subsets. in essence, resting treG cells are
CD45ra+FoXP3low, but they readily acquire a CD45ra–
FoXP3 high phenotype upon activation; both subsets
are potent suppressor cells when deliberately tested.
interestingly, the activated CD45ra–FoXP3high treG cells
are extremely sensitive to apoptotic cell death. However,
the CD45ro+FoXP3low subset does not have suppressive
activities, but instead produces proinflammatory cyto-
kines, and therefore exhibits effector functions.57 Clearly,
these unique features should be carefully considered in
targeting human treG cells in the transplant setting. also,
different isoforms of FoXP3 exist in humans but not in
mice,58 suggesting that FoXP3 and the FoXP3-mediated
programs may be regulated differently in humans and in
mice. these differences inject a key note of caution
in extrapolating strategies and successes from mouse
models into approaches in the clinic.
Emerging approaches
two broad approaches exist in the use of treG cells
to promote transplant tolerance. the first approach is to
first expand treG cells in vitro and then apply expanded
treG cells as a cell therapy in vivo. the advantage of this
approach is that antigen-specific treG cells can be created
in vitro using donor antigens. this approach, however,
relies on the successful cultivation of treG cells in vitro
and stability of expanded treG cells in vivo following
adoptive cell transfer. as discussed above, both can be
technically challenging. nonetheless, this approach
undoubtedly holds great promise in harnessing treG
cells for therapeutic purposes. the second approach
is to selectively and specifically stimulate treG cells
in vivo, by taking advantage of fundamental differences
between the biology of treG cells and t-effector cells.
For example, treG cells constitutively express the high
affinity il-2 receptor, and therefore have a competitive
advantage in the utilization of il-2.59 in an islet trans-
plant model in the mouse, an il-2–ig fusion protein or
an il-2–anti-il-2 complex that has a long half-life in vivo
can promote robust expansion of treG cells, which are
permissive for islet allograft tolerance.60,61 although in
theory, il-2 might also stimulate t-effector cells, studies
of il-2 in humans suggest a preferential ability to expand
treG cells. this effect may be even more prominent when
il-2 is combined with mtor inhibitors such as rapa-
mycin. 61 in addition, other approaches that include
inhibition of innate immune cells, modification of epi-
genetic mechanisms, and prevention of memory t-cell
activation are also being actively explored and likely to
be important facets of promoting treG-cell-mediated
tolerance in transplantation.
Conclusions
treG cells are a key cell type in the induction and main-
tenance of immune tolerance, thus modulation of treG
cells could provide new strategies in creating trans-
plant tolerance. many issues and challenges still need
to be addressed in this area, however. as such barriers
continue to be identified and overcome, therapeutic
approaches harnessing the power of treG cells may even-
tually become a key element of a multifaceted strategy in
the induction of transplant tolerance.
Review criteria
The information discussed in this Review is based on
the experience of the authors and literature accumulated
over their years working in this field. No specific database
searches were performed.

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Acknowledgments
X. C. Li and L. A. Turka are supported by the NIH, USA.
Author contributions
X. C. Li researched data for the article and
contributed to the discussion of content, writing the
article, and reviewing/editing the manuscript before
submission. L. A. Turka contributed to the discussion
of content, writing the article, and reviewing/editing
the manuscript before submission.
volume 6 | oCtoBer 2010 | 583