International Immunopharmacology 11 (2011) 576–588

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International Immunopharmacology

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All creatures great and small: regulatory T cells in mice, humans, dogs and otherdomestic animal species

O.A. Garden a,⁎, D. Pinheiro a, F. Cunningham b

a Regulatory T Cell Laboratory, Department of Veterinary Clinical Sciences, The Royal Veterinary College, Royal College Street, Camden Town, London, NW1 OTU, UKb Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, AL9 7TA, UK

Abbreviations: APC, antigen-presenting cell; CTLantigen-4; Tcon, conventional (i.e. non-regulatory) Tindoleamine dioxygenase; LFA-1, leukocyte function anadhesion molecule-1; LAG-3, lymphocyte activation gprotein-2; LAP, latency activation peptide; ThGC, germiauthors have favoured the term TFH — follicular B helprepresent the same subset is unclear); FasL, Fas ligand;co-stimulator; Gz, granzyme; DN, double negative (tumour necrosis factor-related apoptosis-inducing ligand5 or tumour necrosis factor receptor super family melymphocytic leukaemia/lymphoma 2 (Bcl-2)-interactinplasmacytoid dendritic cell; mTOR, molecular targimmunoglobulin-like transcript 3/4; ATP, adenosine tmonophosphate; COX-2, cyclo-oxygenase-2; PGE2, pros⁎ Corresponding author. Tel.: +44 20 7468 1222, +

7468 5204.E-mail address: ogarden@rvc.ac.uk (O.A. Garden).

1567-5769/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.intimp.2010.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 October 2010Accepted 1 November 2010Available online 18 November 2010

Keywords:Regulatory T cellFoxp3/FOXP3DogVeterinaryFunctional mechanismsImmunotherapy

Abnormalities of peripheral tolerance are thought to contribute to the pathogenesis of a number ofinflammatory, autoimmune and neoplastic diseases of both humans and animals. Furthermore, the inductionof allograft tolerance is the ‘holy grail’ of clinical transplantation. Of the various mechanisms underlyingperipheral tolerance, regulatory T cells (Tregs) have risen to particular prominence. Various Treg subsets havebeen characterised, including naturally occurring cells that develop along a regulatory lineage in the thymusand induced cells that arise in the periphery from conventional T cell precursors. The transcription factorForkhead box (Foxp3) serves a crucial role in stabilising the Treg transcriptome and is a faithful marker ofperipheral Tregs in the mouse, though its expression is somewhat more promiscuous in man. Regulatory Tcells display a wide spectrum of suppressive and cytotoxic mechanisms and may convert to specific T helpercell subsets in response to appropriate inflammatory cues. Although knowledge of Tregs in domestic animalspecies is still in its infancy, a growing body of literature is accumulating in the dog, cat, pig, cow, sheep andhorse. We highlight our own and other studies of Tregs in the dog, an important veterinary species and amodel for a number of human diseases. The ethos of ‘One Health, One Medicine’ is anticipated to accelerateefforts to close the knowledge gap between domestic animal and mainstream species in this field. We predictthat the prodigious pace of research into Tregs will continue unabated for years to come, fuelled by theexciting therapeutic potential of these cells.

A-4, cytotoxic T lymphocytecell; DC, dendritic cell; IDO,tigen-1; ICAM-1, intercellularene-3; FGL-2, fibrinogen-likenal centre helper T cell (moster T cell; whether these cellsICOS (CD278), inducible T celli.e. CD3+CD4−CD8−); TRAIL,; DR5 (CD262), death receptormber 10b; Bim, B cell chronicg mediator of cell death; pDC,et of rapamycin; ILT3/ILT4,riphosphate; AMP, adenosinetaglandin E2.44 7940 812188; fax: +44 20

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The rise, fall and enthusiastic resurrection of the suppressor T cell –latterly re-named the regulatory T cell (Treg) – represent definingevents in modern immunology [1–6]. Recovering from the doldrumsof the molecular era, Tregs have once again ascended into main-stream immunology following the seminal discovery of a population

of suppressive CD4+ T cells characterised by high constitutiveexpression of the IL-2Rα chain (CD25) [7–12]. Playing a cruciallyimportant role in peripheral tolerance, a number of interactingT cells with regulatory function are now known to exist, includingCD4+CD25+/high [13–16], T regulatory 1 (Tr1) [17–21], T helper 3(Th3) [22,23], CD4+CD25− lymphocyte-activation gene 3+ (LAG3+)[24,25], CD8+ [26–29], CD3+CD4−CD8− [30–36], γδ TCR+ [37–41]and natural killer T (NKT) [42–45] cells. The field of Treg biology isadvancing at an unprecedented rate, but new questions are beinggenerated as rapidly as new knowledge is being gained. Topical issuesthat are currently being researched include the heterogeneity,plasticity and stability of Tregs [46–54]; the interactions and relativeimportance of Tregs of various phenotypes [13,14,55]; the molecularmechanisms of regulation and their hierarchy [56–61]; and thetherapeutic potential of Tregs, with a view to increasing their numberand potency in the context of transplantation, autoimmune diseaseand allergy, and decreasing their number and potency in the contextof cancer and infectious disease [62–69]. Against this backdrop offrenetic activity in the murine and human systems is the increasingrecognition that there is a relative dearth of information on Tregs inveterinary species. Nevertheless, a number of studies on Tregs in bothcompanion and farm animals have been published in recent years,providing a platform from which to explore their involvement in a

577O.A. Garden et al. / International Immunopharmacology 11 (2011) 576–588

variety of diseases – many of which show striking similarities to theirhuman counterparts – and hence potential therapeutic utility. Thisreview summarises historical and current concepts in murine andhuman Treg biology, before considering what is known about Tregs inthe dog and other domestic animal species. We conclude by exploringthe therapeutic applications of Tregs and discussing unresolved issuesthat are likely to be the focus of vigorous research in this area in futureyears.

2. Suppressor T cells — from opprobrium to reincarnation

An important new chapter in immunology opened with thediscovery of separate lineages of T and B cells, followed by therealisation that certain T cell subsets provide ‘help’ for antigen-specific antibody responses [70–73]. The suggestion that T cells areable to suppress and thus regulate these responses was only a shortstep away from the concept of help, yet it created a wave of protestwhen first introduced under the label ‘infectious tolerance’ in theground-breaking publications of Gershon and Kondo in the early1970s [74–76]. However, propelled by apparently convincing exper-imental evidence and the cogency of the argument for cellular controlof positive immune responses, the concept of suppressor T cellsrapidly gained traction in the mid to late 1970s [1–5]. A largeliterature accumulated, based on studies of suppressor T cellhybridomas performed in vitro. These immortalised cells wereclaimed both to secrete suppressor factors that bound antigen in theabsence of MHC molecules and to express the I–J determinant,thought to be encoded within the MHC between the ‘I–A’ (H2A) and‘I–E’ (H2E) loci — investigated using alloantisera raised between twoinbred murine strains that were MHC recombinants [77–81].Elaborate networks of suppressor cells were described, designatedTs1, Ts2 and Ts3, alongside contra-suppressor cells that were thoughtto mediate an anti-inhibitory function [82–86]. However, many ofthese original ideas were questioned with the advent of moleculargenetics in the early 1980s: in particular, molecular characterisationof the murine MHC class II region established that no I–J locus existed[87,88], and cloning of the TCR β chain genes demonstrated a lack ofre-arrangement in many of these ‘suppressor hybridomas’ [89,90].With astonishing rapidity, the concept of suppressor T cells lost favourand publications in this field were all but extinguished by the early1990s [91–93]. Nevertheless, transferable tolerance remained arobust phenomenon and some immunologists of the time nevercompletely lost faith in this concept [94–96].

Re-birth of the concept of T cell regulation in the mid 1990s camewith the seminal discovery of a population of peripheral CD4+CD25+

T cells in the mouse with regulatory properties in vitro and in vivo[7,8,10,11,97]. Similar cells were characterised in the rat [12,98] andhuman [99–104] and within very little time the field of suppressorT cell biology was once again alive and well, freshly re-branded underthe less emotive ‘Treg’ label. Such has been the reprisal of this area ofimmunology that most broad-based international immunologicalmeetings have a major session on Tregs; indeed, one or two largemeetings every year dedicated wholly to Tregs and their interactionswith Th cells are now commonplace, and Treg research activity riskseclipsing the continued investigation of the many alternativemechanisms of peripheral tolerance, which should not be forgotten[1].

3. Regulatory T cells — current concepts in mouse and man

Regulatory T cells may be broadly divided into naturally occurringsubsets (nTregs), which develop in the thymus along a regulatorylineage – for example, the canonical CD4+CD25+/high Treg; and induced –

or ‘adaptive’ – subsets,which arise in the periphery following activation ofconventional (non-regulatory) T cells (Tcons) in amicroenvironment richin regulatory cytokines, or following the interaction of Tcons with nTregs

in a process called infectious tolerance – for example, the Tr1, Th3 and‘induced (i) Treg’ cells [13,105–107]. Naturally occurring Tregs havetraditionally been thought to possess a broad repertoire of autoreactiveTCRs [97], supported by evidence in various murine models [108–113],though recent studies have questioned this postulate and suggest thatthere is considerable overlap between the repertoires of nTregs and Tcons[114], or even that the predominant cognate specificity of nTregs may benon-self antigens [115,116]. Regardless, nTregs clearlymake an importantcontribution to both peripheral tolerance of autoantigens[7,8,15,97,117,118] and the regulation of immune responses to non-selfantigens, such as those associated with pathogens [119–122] or themicrobiota of the gut [22,123,124]. Interestingly, despite their origin fromTcons and presumably predominantly non-self specificity, Tr1 cells – oneof thebest characterisedof the inducedTregs– also appear toplay a role inregulating immune responses to both self and non-self antigens in vivo[17,22]. The inter-relationships and relative importance of the variousTreg subsets remain to be fully characterised, but complementary roles ofCD4+CD25+ nTregs and Tr1 cells [125], and of CD4+CD25+ and CD8+

CD28- Tregs [126] – to name two examples – have been documented invivo.

The transcription factor Forkhead box p3 (Foxp3) – designated inupper case letters (FOXP3) in humans – serves a crucial role instabilising the transcriptome of Tregs and is a faithful marker ofperipheral Tregs in the mouse [127–133]. Originally dubbed the‘master regulator’ of Tregs, Foxp3/FOXP3 is essential for themaintenance of functional competence of Tregs, as suggested by thesevere immunodeficiency diseases associated with its absence ordefective function in mice [134,135] and humans [136,137] – butwhether it is strictly necessary for Treg lineage commitment has beenquestioned in recent years [138–141]. Rather, a higher level ofregulation has been proposed, perhaps mediated by a network ofgenes [142]. As well as being expressed by nTregs, Foxp3 may also beinduced in murine Tcons by their stimulation in the presence ofexogenous TGF-β, yielding T cells – labelled ‘iTregs’ – with a basicphenotype (CD4+CD25+Foxp3+) that is identical to that of nTregs[143–147]; however, regulatory function following stimulation ofTcons in the presence of TGF-β has not been observed by all authors[142], while others have observed only transient regulatory functionassociated with unstable Foxp3 expression [148]. When successfullygenerated, iTregs have shown unique functional characteristics thatdistinguish them from nTregs [55,149–151]. The process of conver-sion of Tcons to iTregs is enhanced in the presence of retinoic acid,produced in vivo by specialised dendritic cells (DCs) in the gut [152–156]. Recent studies have identified an immediate Foxp3- precursor toFoxp3+ Tregs in the peripheral lymphoid organs of unmanipulatedmice: interestingly, TGF-β signalling played only a minor role in thegeneration and differentiation of these precursor cells [157]. HumanTcons, in contrast to those of the mouse, show upregulation of FOXP3with activation in the absence of exogenous TGF-β, the very lowconcentrations of this cytokine found in complete medium – coupledwith endogenous synthesis and activation – being sufficient to induceFOXP3 [158,159]; however, the induced FOXP3 expression is transientand the resulting CD4+CD25+FOXP3+ T cells lack or rapidly loseregulatory function in most [160–168] – but not all [169,170] –

studies. Indeed, FOXP3 expression is not so faithfully associated withregulatory function in humans as it is inmice: its ectopic expression inmurine Tcons readily confers regulatory properties on the cells [127–129], but forced over-expression of FOXP3 is required to conferregulatory properties on human Tcons, each of the two establishedsplice variants (FOXP3FL and FOXP3Δ2) behaving in a similar manner[171–175]. Similar functional data on the recently discovered thirdisoform of FOXP3 (FOXP3Δ7) [176] are currently lacking. A picture isthus emerging that robust regulatory function requires stable andhigh-level Foxp3/FOXP3 expression, established by epigenetic mod-ifications of the foxp3/FOXP3 locus that include demethylation[148,177,178] and acetylation [179–181]. Yet an auto-regulatory

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function of the transient upregulation of Foxp3/FOXP3 in Tcons,sufficient to curb their clonal expansion [182] or even to attain atransient Treg phenotype [183], cannot be discounted.

The full complexity of human CD4+FOXP3+ Tregs is only nowbeginning to come to light,with recent descriptions of naive/resting Tregs(CD25intermediateFOXP3lowCD127low/−CD45RA+HLA-DR−Ki67−), effector/activated Tregs (CD25highFOXP3highCD127lowCD45RA−HLA-DR−ICOS+/−

Ki67+) and terminal effector Tregs (CD25highFOXP3highCD127−CD45RA−

HLA-DR+ICOS+/−Ki67+); in addition, a non-suppressive CD4+

CD25intermediateFOXP3lowCD127lowCD45RA− population exists, whichis able to secrete IL-2, IFN-γ and IL-17 [14,50,184,185]. The contributionof CD4+FOXP3− T cells to the FOXP3+ Treg pool with activation andconversion in vivo remains to be defined [14,50], though evidencesuggests that this process does occur [186,187]. Foxp3 is not expressedby murine Tr1 cells [19], though the picture is less clear in the humansystem — initial studies suggesting an absence of constitutive FOXP3expression in this subset of Tregs [188], but more recent workapparently defining a population of FOXP3+ human Tr1 cells [189,190].

Mechanisms of suppression mediated by Tregs include thoseoperating in a contact-dependent or close range manner and thosedependent on soluble cytokines; both Tcons and antigen-presentingcells (APCs) – including macrophages and B cells – may be the targetof regulation (Table 1) [56–61], though Tregs are also thought toregulate NK cells, neutrophils and other innate immune cells [191–196]. However, not all Treg functions are suppressive or inhibitory innature: recent evidence suggests that they may act as helper cells forintestinal IgA responses [197] and systemic IgG responses followingintranasal immunization [198].

The potential plasticity of Tregs has recently been recognised, withreports of their conversion to a number of the Th subsets in appropriatepro-inflammatory microenvironments [46,47,49,52–54,199]. Indeed,the currentweight of evidence suggests that Treg Foxp3 expressionmaybe down-regulated or even turned off under the influence of sufficientlystrong environmental cues, despite the presence of epigenetic changesof the foxp3 locus thought to confer stability of expression. Foxp3 is thusthought to act as a rheostat of Treg function in vivo: high expressionendows the cells with full suppressive capability, while reducedexpression leads to preferential conversion to the Th2 lineage andloss of expression licenses Tregs to convert to any one of a number ofsubsets – including Th1, Th2, Th17 or T follicular B helper (Tfh) cells –under the influence of polarising cytokines [53,278]. The mechanismsunderlying such down-regulation of Foxp3 remain to be fullycharacterised, as does the biological function of Treg conversion invivo. Current thinking is that these ‘ex-Tregs’mayplay aneffector role inanti-pathogen or anti-cancer defence, acting in an innate-like mannerbefore the development of the adaptive immune response [49,199]. Inthis regard, a recent study revealed an unexpected benefit of nTregs inthe clearance of herpes simplex virus in a murine model [279],prompting the speculation that ex-Tregs were implicated in viralclearance [53,199]. Further support for the heterogeneity and plasticityof Tregs has come in the form of documentation of hybrid cells – withthe properties of both Tregs and Th1 or Th17 cells – in both the murine[280,281] and human [282–284] immune system, and bivalent(permissive and repressive) epigenetic marks associated with thetbx21, gata3 and rorcgenes inmurinenTregs [285], suggesting that thesecells are poised in readiness for re-programming to Th1, Th2 or Th17phenotypes if dictated by polarising influences within the microenvi-ronment [49]. Interestingly, recent data suggest that Tregs may expresstranscription factors usually associated with Th cells – T-bet [286] orIRF4 [287] – as an ‘adaptive’ response to the local microenvironment,thus generating ‘bespoke’ regulation of respective Th cells differentiat-ing within the same locale [288]. Nevertheless, not all studies reconcilewith the idea of Treg plasticity: recent work in a murine modeldemonstrated remarkable stability of Foxp3+ Tregs under physiologicaland inflammatory conditions in vivo, adding a counter-argument to thiscontroversial area of Treg biology [289].

4. Regulatory T cells in the dog — what do we know?

The concept of peripheral tolerance in the dog is not new: indeed,Weiden and colleagues postulated that a suppressor T cell populationwas responsible for preventing graft-versus-host disease in stablecanine radiation chimaeras challenged by the infusion of donorlymphocytes [290]. Furthermore, circulating suppressor cells werereported in German shepherd dogs with progressive myelopathy in1980: these cells were radioresistant, present within both the nylonwool-adherent and non-adherent populations of leukocytes, andinhibited by indomethacin – suggesting that their activitywasmediatedby prostaglandins [291]. These early findings were surprisinglyinsightful given the general nascence of immunological knowledge atthe time, pre-dating by several decades much more recent studies thathave described monocytes and induced Treg cells in humans withsuppressive activity mediated by PGE2 [168,267–270]. Further workcharacterised a short-lived suppressor population – thought to beneutrophils – present in the peripheral blood of healthy dogs,mediatingits effect in an indomethacin-independent manner by the release of asoluble factor [292]. These findings are harder to reconcile with thecurrent literature, though recent work has suggested that neutrophilsmay indeed serve a regulatory role in some circumstances [293–295].

A study performed in the early 1990s described the inhibitoryactivity of spontaneous and induced suppressor lymphocyte popula-tions – assumed to be T cells – in peripheral blood of dogs with atopicdermatitis [296]. Further studies documented the ability of theazaspirane drug SK&F 105685 to elicit suppressor cell activity in thespleen and bone marrow of healthy beagle dogs, putatively by theinduction of ‘suppressive T cells’ [297,298]. The lack of globalimmunosuppression elicited by this drug suggested that it heldpromise as a future therapy for autoimmune disease [297]. The roleof immunosuppressive cytokines was also recognised in this earlywork – for example, levels of TGF-β mRNA were assessed in canineatopic skin by semi-quantitative reverse transcriptase PCR [299] – butfurther substantive advances in thefield of canine peripheral tolerancewere not made until more than a decade later, when we and othersnoted the ability of the anti-mouse/rat Foxp3 mAb FJK-16s to stain apopulation of CD4+ T cells in the peripheral blood and lymph nodes ofdogs [300,301]. We observed that FJK-16s stained far fewer CD8+ Tcells and deduced that a peripheral population of CD4−CD8− (doublenegative) FJK-16s+ T cells existed [300]. We speculated that the CD4+

FJK-16s+ cells were Tregs, though initially had no direct evidence fortheir regulatory function. Stimulation of both peripheral blood andlymph node cells with ConA yielded impressive increases in both theproportion and absolute numbers of CD4+ and CD8+ FJK-16s+ T cells:interestingly, in our hands this increase in expression of FJK-16sstaining did not require exogenous TGF-β – recapitulating similarfindings in human but notmurine peripheral lymphoid cells – but bothTGF-β and IL-2 supplements were required in the studies performedby Biller and colleagues [301], perhaps reflecting differences inexperimental design.

While these early studies provided indirect evidence of Tregs in thedog on the basis of assumed cross-reactivity of the FJK-16s mAb, thespecificity of this antibody for canine FOXP3 was subsequentlyconfirmed by examining cell lines over-expressing the canine FOXP3gene [302]. Cross-reactivity of the anti-human CD25 mAb ACT-1 withthe canine protein was also confirmed using the same strategy [302],supporting earlier work showing positive staining of activated canine TcellswithACT-1 [303]. Subsequent studies have verified cross-reactivityof ACT-1 for canine CD25 [304], but have suggested that the affinity ofthis antibody for the canine protein is low [305]; indeed, Abrams andcolleagues have developed a novel, higher-affinity, canine-specific anti-CD25 mAb (P4A10), which is now commercially available [305].

A number of recent studies have examined the changes inproportions of CD4+FOXP3+ T cells occurring in canine cancer,following exploratory work performed by Biller and colleagues on a

Table 1A summary of regulatory T cell effector mechanisms.

Key molecule(s) Expression pattern Targetcell(s)

Mechanism(s) References

Modulation of APC functionCTLA-4 (CD152) High constitutive expression

on murine nTregs and humanCD45RA-FOXP3high effector Tregs

APC Down-regulates or prevents up-regulation of CD80 and CD86 by APCs, thus inhibitingtheir co-stimulatory function.

[200]

T cell Mediates ‘outside-in’ signalling by interaction with CD80 and CD86 expressed byactivated Tcons, inhibiting their expansion and effector function.

[201]

Also required to condition DCs to express IDO (see below)CTLA-4:B7 (CD80/CD86) interactions thought to represent a ‘core’ Tregmechanism.

[59]

LFA-1 (CD11a/CD18or αLβ2)

Murine nTregs and humanCD4+CD127-CD25high Tregs

APC Treg LFA-1 interacts with ICAM-1 expressed by DCs, preventing up-regulation ofCD80 and CD86 by the DCs; thought to be first part of a two-stepcontact-dependent mechanism – initial LFA-1–dependent formation of Tregaggregates on immature DCs, then LFA-1 and CTLA-4–dependent down-modulationof CD80 and CD86.

[202-204]

A direct effect on Treg suppressive function independent of APCs has also beendescribed.

[205]

LAG-3 (CD223) Activated murine nTregs APC Binds to MHC class II molecules, mediating an inhibitory signal that suppresses DCmaturation and stimulatory function.

[206-207]

Neuropilin-1 Murine nTregs APC Promotes prolonged interactions of Tregs with immature DCs, thus giving them anadvantage over naive Th cells in modulating function of DCs.

[208-209]

FGL-2 Murine B cells, macrophagesand DCs

APC Binds to FcγRIIB, inhibiting maturation and inducing apoptosis of APCs (e.g. bonemarrow-derived DCs).

[210-211]

Inhibitory cytokinesTGF-β Controversial: resting and activated

murine nTregs (surface TGFβ+);activated murine nTregs and humanCD4+CD25high Tregs (surface LAP+ only);murine Th3 cells; human ThGC cells

T cell Controversial: most studies suggest that cytokines are not required for nTregfunction in vitro; however, several support their involvementin vivo.

[185, 203,212-219]

TGF-β may be released as a soluble cytokine or tethered to the membrane,where it suppresses Tcons in a contact-dependent manner, inhibitingproliferation and pro-inflammatory cytokine production. Surface TGF-β alsoconverts Tcons into iTregs (infectious tolerance) and may bedelivered to

APC? DCs, thus inhibiting their function. [47]Early studies suggest the existence of a Th3 subset dependent on soluble TGF-β,though whether they represent a separate lineage is questionable. HumanCD4+CD57+FOXP3- ThGC cells mediate suppression by TGF-β, IL-10 and Fas/FasL(see below).

[220]

IL-10 Activated murine nTregs and humanCD4+ICOS+FOXP3+ Tregs; murineand human Tr1 cells; Th1, Th2, Th9and Th17 cells; human ThGC cells

APC Down-regulates both co-stimulatory molecules and synthesis of pro-inflammatorycytokines, thus inhibiting APC function.

[17, 185,220-224]

T cell Directly inhibits IL-2 and TNF-α synthesis by CD4+ T cells.Tregs also stimulate IL-10 synthesis by APCs and their subsequent B7-H4 expression,rendering the APCs immunosuppressive.

[225]

Synthesis of IL-10 by Th cells is thought to serve an auto-regulatory (negative feedback)function.

[20, 226]

IL-35 Murine nTregs T cell Inhibits Tcon proliferation [227-228]Induces IL-10 synthesis by CD4+CD25-CD39+ T cells. [229]

APC? [Human Tregs do not synthesise IL-35, though alternative sources (e.g. trophoblast cellsat the feto-maternal interface) may be important.]

CytolysisGranzyme A or B ±perforin

Murine nTregs; human CD4+CD25high

Tregs and Tr1 cellsT cell Mediates cytolysis of Tcons.

Various expression patterns and mechanisms have been suggested:• human Tr1 cells: GzB + perforin [230]• human CD4+CD25high Tregs: GzA + perforin [231]• murine nTregs: GzB, perforin-independent [232-233]• murine Tregs: GzB + perforin [234]

APC Cytolysis of APCs in some in vitro studies. [231-232]Fas (CD95);FasL (CD178)

Murine nTregs and DN Tregs; humanCD4+CD25highFOXP3+ and ThGC cells

T cell Mediates cytolysis of Tcons.Various expression patterns have been observed:• human CD4+CD25+CCR4+ Tregs: FasL [235]• human CD4+CD25highFOXP3+ Tregs: Fas and FasL [236]• human CD4+CD57+FOXP3- ThGC cells: FasL [220]• murine DN Tregs: FasL [35-36]

APC Mediates cytolysis of APCs.Various expression patterns have been observed:• human CD4+CD25+FOXP3+ Tregs: FasL [237]• murine nTregs: FasL [238]

TRAIL Murine nTregs T cell Mediates cytolysis of Tcons by interaction with DR5. [239]IL-2 deprivation Murine and human nTregs (CD25) T cell Compete for IL-2, depriving Tcons and leading to Bim-mediated apoptosis (cytolysis);

also induces nTreg synthesis of IL-10[224, 240-241]

Controversial: some studies suggest IL-2 not required for function, butimportant for self-renewal and ‘metabolic fitness’; or that Tregs disengage upstream Tcon IL-2R signalling from downstream cell cycleprogression

[242–244]

(continued on next page)

579O.A. Garden et al. / International Immunopharmacology 11 (2011) 576–588

Table 1 (continued)

Key molecule(s) Expression pattern Targetcell(s)

Mechanism(s) References

Immunosuppressive metabolitesIDO → kynurenines Tolerogenic DCs (e.g. IDO+ pDCs,

CD8α+CD19+ DCs andmonocyte-derived DCs)

T cell nTregs induce DC expression of IDO by CTLA4-B7–dependent signalling, requiring IFN-γproduction by the DCs in some but not all studies.Suggested mechanisms:• tryptophan deprivation elicits stress response and mTOR inhibition g [245-247]• production of kynurenines induces apoptosis and cell cycle arrest• tryptophan deprivation and kynurenine synthesis lead to generation of iTregs,involving DC expression of ILT3 and ILT4 in some studies

[248-250]

• IDO+ DCs activate and expand nTreg numbers [251-253]CD39, CD73 Murine nTregs and human

CD4+CD25highFOXP3+ Tregs;human Tr1 cells

T cell Ectonucleotidases that sequentially catabolise ATP into AMP and then adenosine, whichacts on A2A receptors expressed by Tcons:• suppresses Tcon proliferation and cytokine synthesis [254-261]• induces anergy and generation of iTregs, involving LAG-3 [262]

cAMP Murine nTregs T cell Inhibits Tcon proliferation and IL-2 synthesis; enters Tcons from activated nTregs viagap junctions that form between the contiguous cells. cAMP also rapidly down-regulates CD80/CD86 expression by DCs, while up-regulating inhibitory B7-H3.

[263-265]APC [266]

COX-2→PGE2 Human CD4+CD25highFOXP3+ iTregs;human LPS-activated monocytes

T cell Expression of COX-2' release of PGE2, which suppresses Tcon proliferation and synthesisof pro-inflammatory cytokines via action on EP2 and EP4 receptors and cAMP synthesis.PGE2 also mediates autocrine and paracrine induction of FOXP3 in Tregs and Tconsrespectively.

[261, 267-270]

The concept of a prostaglandin-producing ‘suppressor cell’ is not new. [271]

MiscellaneousGalectins 1, 3, 9and 10

Murine nTregs and human CD4+CD25high

TregsT cell Possible functions: cell cycle arrest and cytolysis of Tcons (e.g. galectins 1, 9 and 10);

intrinsic anti-apoptotic and pro-anergic properties (e.g. galectins 3 and 10); Treginduction (e.g. galectin 9)Various expression patterns have been observed:• murine nTregs: galectin 3 and 9 [272-275]• human CD4+CD25high Tregs: galectins 1 and 10 [276-277]

Notes: In all studies, murine nTregs were identified on the basis of the CD4+CD25+(Foxp3+) phenotype: the contribution of iTregs with the same phenotype would have beendifficult to exclude in most studies, though they would have represented only a low proportion of total peripheral CD4+CD25+ T cells in naive animals; similarly, unless statedotherwise human Tregs were assumed to be naturally occurring, but we have noted the specific phenotype applied in each study owing to their more variable definition.

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small number of animals with oral melanoma (n=4), osteosarcoma(n=3),mast cell tumour (n=2) and soft tissue sarcoma (n=1) [301].When compared to healthy control animals, the proportions of CD4+

FOXP3+ cells in dogswith a variety of neoplasms– including round celltumours, carcinomas and sarcomas – were greater in both the bloodand, when assessed, tumour-draining lymph nodes [301,306]; whenindividual tumour types were analysed, those animals with carcino-mas had significantly higher proportions of these cells than the controldogs [306]. Similarly, neoplastic dogs showed an increase in the ratioof peripheral CD4+FOXP3+: CD8+ T cells; in particular, lymphomapatients demonstrated significantly higher ratios than control animals[306]. The presence of metastasis further increased the proportions ofperipheral blood CD4+FOXP3+ T cells in dogs with a range of tumours[307]. Further work performed by the same group demonstratedincreased proportions of CD4+FOXP3+ T cells in the peripheral bloodand tumour beds of canine oral malignant melanoma patients [308];furthermore, the proportion of peripheral blood CD4+FOXP3+ T cellsshowed a positive correlation with tumour stage and negativecorrelations with the proportions of Th1 and cytotoxic T cells inthese animals [309]. However, not all studies of canine tumours haveyielded such a clear message: for example, Rissetto and colleaguesfound no difference in the proportions of CD4+FOXP3+ cells in theperipheral blood or lymph nodes – either regional or distant – in dogswith osteosarcoma [304], yet Biller and colleagues documentedincreased proportions of these cells in the blood, but not lymphnodes, of dogs with the same type of tumour, with an associateddecrease in both the proportion of CD8+ T cells and the ratio of CD8+:CD4+FOXP3+ cells [310]; indeed, these authors also demonstratedthat CD8+: CD4+FOXP3+ ratios lower than the mean value in thepatient group were associated with significantly shorter survivaltimes, raising the intriguing possibility that the Tregs in these dogswere implicated in the immune evasion of the cancer [310]. Studiesinterrogating CD4+FOXP3+/CD25+ T cells or FOXP3 expression inother areas of caninemedicine have been scant. Keppel and colleagues

provided evidence for an increase in the proportionof peripheral bloodCD4+FOXP3+ T cells and concentration of serum IL-10 in dogs withatopic dermatitis receiving allergen-specific immunotherapy, associ-ated with a decrease in serum IgE concentration [311]; while Veenhofand colleagues demonstrated increased FOXP3 expression in bothlesional and non-lesional skin of dogs with cutaneous adverse foodreactions [312], and de Lima and others showed diminished propor-tions of CD4+CD25+ T cells and increased concentrations ofsupernatant IFN-γ in cultures of PBMCs from dogs immunized with avaccine against Leishmania spp [313].

We and others have recently provided direct evidence of theregulatory function of canine CD4+CD25highFOXP3high T cells. Devel-oping a novel canine-specific anti-CD25 mAb, Abrams and colleaguesshowed that immunomagnetically selected CD4+CD25+ T cells wereable to inhibit the proliferation of responder T cells in one-way mixedleukocyte reactions; indeed, these cells showed suppressive potentialat ratios of Treg:responder cell of 1:20, underlining their potencyin this assay system [305]. Using the cross-reactive anti-CD25 mAbACT-1, we have demonstrated that CD4+ T cells with the highest CD25expression were enriched for FOXP3 and stained positively with thenewly developed anti-murine/human Helios mAb 22F6 [314],suggesting that they expressed Helios, an Ikaros transcription factorfamily member that has recently been associated with nTregs in bothmice [315,316] and humans [317]. Following activation of CD4+ Tcells derived from either peripheral blood or lymph nodes with ConAand subsequent withdrawal of the stimulus, we were able to demon-strate the emergence of two populations of T cells defined on the basisof FOXP3 and IFN-γ expression [314]. Thus, we observed CD4+

FOXP3highIFN-γ− T cells, which we speculated were activated Tregs,and CD4+FOXP3intermediateIFN-γ−/+ T cells, which we speculatedcomprised a more heterogeneous population of predominantlyactivated Tcons [314]. We used flow-assisted cell sorting (FACS™) toisolate CD4+CD25high T cells, demonstrating their regulatory functionin classical suppression assays in which fresh CD4+ T cells were added

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as a responder population and cell proliferation was measured by theincorporation of tritiated thymidine [314]. Current studies are focusedon elucidating themechanisms of suppression of these Tregs and theirimplication in a number of canine diseases, important both because ofthe veterinary significance of this species and its use as a large animalmodel of various human diseases [318–323].

5. Regulatory T cells in other domestic animal species — anexpanding panoply

The existence of Tregs has also been established in a number ofdomestic animal species other than the dog. ‘Suppressor cells’ were firstdescribed in the cat in the early 1980s [324–326], but it was not until thepivotal work of Wayne and Mary Tompkins and colleagues that CD4+

CD25+ Tregs were characterised in detail, in the context of thepathogenesis of feline immunodeficiency virus (FIV) infection [327–333]. These cells expressed FOXP3 [331]; mediated contact-dependentsuppression of proliferation and IL-2 synthesis in vitro [327]; werepresent in FIV-positive cats in a pre-activated state, providing a cellular‘sanctuary’ for productive FIV infection [328–330,333]; and could berecruited from the Tcon pool by stimulation with Con-A and TGF-β,depending on TGF-β/TGF-β-RII signalling for their regulatory function[332].More recentworkhasdescribed thedepletionof Tregs in vivo [334]and its impact on anti-viral responses mounted in cats chronicallyinfectedwith FIV [335]. Tregs have also been described in the pig, initiallyin the context of fetal tolerance [336] and renal and cardiac allotrans-plantation [337–341]; more recently, CD4+CD25high Tregs [342] andporcine FOXP3 [343,344] havebeen specifically characterised, and furtherstudies have described the induction of CD4+CD25+FOXP3+ [345] andTh3 cells [346] by porcine reproductive and respiratory syndrome virus.Retinoic acid was implicated in the augmentation of a diverse Th1, Th2and Treg response in pigs infected with Ascaris suum [347] andpreliminary studies have provided evidence for the induction of Tregsby dendritic cells treated with the immunosuppressive drug mycophe-nolic acid [348]. A growing body of knowledge of intestinal mucosalimmunology exists in the pig and studies of Tregs in this context are likelyto be forthcoming in future years [349]. In addition to Tregs, other cellswith a regulatory function in the pig have included alveolarmacrophages,which are able to suppress the proliferation of T cells [350].

Early reports of bovine ‘suppressor cells’ described a population of Tcells capable of suppressing the proliferation of autologous respondercells by the release of a soluble factor [351]; these cells, generated by theactivation of lymphocytes with ConA, did not require cell: cell contactand resembled human ConA-induced suppressor T cells documented inthe preceding few years [352,353]. Whether these cells were bona fideTregs or activated Tcons, which in some assay systems are capable ofmediating non-specific suppression [354,355], remains unclear. Anumber of subsequent studies presented indirect evidence for popula-tions of CD4+, CD8+ and γδ T cells with regulatory activity [355–361],but T cells expressing FOXP3 were first documented in 2009: thus, Seoand colleagues developed novel mAbs against bovine FOXP3, demon-strating that its expression was limited to CD4+CD25+ T cells [362],while Gerner and colleagues documented cross-reactivity of the anti-mouse/rat Foxp3 mAb FJK-16s, showing that the majority of FOXP3+ Tcells were CD4+ but that minor populations of CD8β+ and γδ+ FOXP3+

lymphocytes could also be identified [363]. However, the regulatoryproperties of the CD4+CD25highFOXP3+ population were called intoquestion by work performed by Hoek and colleagues, who suggestedthat these cells were neither anergic nor suppressive; rather, WC1.1+

and WC1.2+ γδ T cells, and CD14+ monocytes, showed regulatoryproperties in their experiments, supportedby theobservation that thesecells transcribed IL-10 [364].

Ovine ConA-induced ‘suppressor cells’ were described in 1985[365]. Unlike the bovine suppressor cells generated in a similarmanner, the ovine cells appeared not to mediate their regulatoryfunction in vitro by means of a soluble factor, but by the depletion of

IL-2 [365]. Interestingly, these findings reconcile with recent workdescribing IL-2 deprivation by murine and human nTregs[224,240,241]. Subsequent ovine studies presented indirect evidencefor both CD4+ and CD8+ Tregs [366,367], as well as endometrialsuppressor cells that were unlikely to be conventional T, B or NK cells[368,369]. Ovine FOXP3 has recently been sequenced, showing highhomology with FOXP3 from other species [370]. Immunohistochem-ical studies have revealed infiltration of FOXP3+ T cells into the skin ofsheep infested with Psoroptes ovis [370] and recent work hasdocumented the kinetics of IFN-γ and IL-10 synthesis by activatedovine peripheral blood mononuclear cells [371].

Work on equine Tregs is still in a relatively nascent state. Followingthe generation of novel mAbs against equine IL-10, Wagner andcolleagues defined a population of CD4+IL-10+IFN-γ+ cells that werethought to represent Tr1 cells [372]. Further indirect evidence for therole of immunosuppressive cytokines was generated by cultures ofperipheral blood mononuclear cells derived from Icelandic horses,which suggested that heavy helminth burdens imposed a suppressiveeffect on IL-4 production by inducing IL-10 and TGF-β synthesis [373].Further work by the same group revealed that the immune responseof young foals is Th1-biased, but that regulatory IL-10 production by Tcells is developmentally mature within the first three months of life[374]. Recent work has documented the expression of FOXP3 byequine CD4+ T cells using cross-reactive anti-human FOXP3(PCH101) and anti-murine Foxp3 (FJK-16s) mAbs (A.M. de Mestre,personal communication). Other species in which regulatory cellshave been described include the guinea pig [375–378], baboon[379,380], macaque [381–383] and chimpanzee [384]. Furthermore,a recent study described the generation of IL-10+ T cells from chickenCD4+CD25− T cells by culturing them with anti-CD3/CD28-coatedbeads, IL-2 and TGF-β[385], while the anti-human FOXP3 mAb 236A/E7 yielded positive immunohistochemical staining of the mesentericlymph nodes of harbour seals and a walrus [386]. The gene encodingFOXP3 in the zebrafish was recently characterised [387], promptingspeculation that Tregs may also exist in teleost fish.

6. Gazing into the crystal ball — future perspectives

Recent years have seen an increasing interest in the potentialtherapeutic applications of Tregs in a variety of clinical contexts[14,62,63,68]. For example, allogeneic Tregs have been infused intohaemopoietic stem cell transplant recipients in the treatment of graft-versus-host disease [388,389]. Treatments targeting Tregs in experi-mentalmodels and human patients have adopted a number of differentstrategies, either with the aim of augmenting regulation in the contextof inflammation, autoimmune disease and allotransplantation, or ofattenuating regulation in the context of cancer [62–69]. Such strategieshave included (i) activation and expansion of nTreg numbers in vivo,either in an antigen-specific fashion by the administration of peptides inlow, repeated subcutaneous – or in some cases, sublingual or oral –doses, or in a polyclonal fashion by the administration of mAbs, smallmolecules or cytokines [62,67,390]; (ii) in vitro activation andexpansion of nTregs – or the induction of iTregs from Tcons – inpreparation for their adoptive transfer into thepatient [62,390,391]; and(iii) reductionof Tregnumbers and, or functional competence in tumourbeds and their draining lymph nodes [392–395]. Before such immuno-therapeutic strategies can beuniversally embraced, protocols to activateand expand specific subsets of Tregswithout effects on Tcons need to bebetter defined, as do conditions fostering the phenotypic stability ofTregs in vivo inorder to avoid their conversion intopathogenic effector Tcells. The inter-relationships of the human FOXP3high effector Tregsubsets, the role of iTregs in the human immune system, the plasticityand true extent of inter-conversion of the various Th and Treg subsets invivo, and the question of whether Tregs – in particular, human Tregs –mount memory and recall responses all need to be clarified. Thehierarchy and spectrum of Treg suppressive mechanisms in vivo must

582 O.A. Garden et al. / International Immunopharmacology 11 (2011) 576–588

be elucidated and more focused methods to eliminate Tregs in cancermust be designed to mitigate collateral autoimmunity. Proof-of-principle experiments have demonstrated the feasibility of ‘designer’Tregs, genetically engineeredbyTCRgene transfer toensure appropriateantigen specificity [396–397]; however, these approaches must befurther evaluated to assess their safety and efficacy in disease settings.Current understanding of Treg biology in domestic animal species isgenerally still in its infancy, lagging behind knowledge in both themouse andman. This gapmust be closed as new reagents are developedfor use in veterinary immunological studies and the concept of ‘OneHealth, One Medicine’ is increasingly embraced for the benefit of bothhuman and animal well-being [399,400]. The evolutionary aspects ofTreg biology also present a rich area for future research.

In conclusion, we predict that the exponential growth of Tregresearch following the pioneering work of the original proponents ofthis field and the more recent luminaries who resurrected it from thedoldrums of the early 1990s looks set to continue, fuelledby the excitingtherapeutic potential of these cells: much has been learned, but evenmore remains to be discovered in the years ahead. Only time will tellwhether Treg immunotherapy ultimately makes it into mainstreammedical practice, but of one thing we can be sure: in one form oranother, Tregs are undeniably here to stay! Per aspera ad astra!

7. Acknowledgements and author contributions

OAG gratefully acknowledges funding in his laboratory for work oncanine regulatory T cells from the Biotechnology and Biological SciencesResearch Council (BBSRC), Novartis Animal Health, the EuropeanCollege of Veterinary Internal Medicine and the American College ofVeterinary Emergency and Critical Care. Special thanks are due to DrsIan Thompson, LinaWilliamson, John E. Peel andMatthew Jones – all ofNovartis Animal Health – for insightful discussions throughout theduration of this work. OAGwould also like to thank the many students,both undergraduate and postgraduate, who have passed through hislaboratory and have been a constant source of inspiration – eachcontributing to thiswork in his or her own valuableway. OAGwrote thereview andwas the principal investigator for the caninework describedin thismanuscript; DPwas a PhDstudent inOAG's laboratory at the timeof writing of this review and performed the majority of experiments oncanine Tregs; and FC contributed to the design of experiments, madeinsightful comments on this manuscript andwas a co-supervisor for DPthroughout the duration of her PhD.

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