foxp3+ regulatory t cells

FOXP3+ regulatory T cells:control of FOXP3 expression bypharmacological agentsNaganari Ohkura1,2, Masahide Hamaguchi1,2 and Shimon Sakaguchi1,2

1 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan2 WPI Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan

Review

Naturally arising CD4+CD25+ regulatory T cells (Tregs),which specifically express the forkhead family transcrip-tion factor FOXP3, are essential for the maintenance ofimmunological self-tolerance and immune homeostasis.Tregs can suppress the activation, proliferation and ef-fector function of other lymphocytes in physiologicaland pathological immune responses. Therefore, controlof the development, survival, and function of Tregs isinstrumental for effective control of immune responses.For example, cytokines such as interleukin-2 and trans-forming growth factor-b, monoclonal antibodies to theTreg-associated molecules such as interleukin-2 recep-tor a chain and cytotoxic T lymphocyte-associated 4, andpharmacological agents that alter signaling pathwaysfor Treg function, can augment or dampen the suppres-sive activity of Tregs. How these agents control thefunction of Tregs at the molecular level remains to beelucidated. However, it is envisaged that pharmacologi-cal control of the function and development of Tregs bytargeting FOXP3 or Treg-associated molecules will en-able better control of immune responses in variousclinical settings.

IntroductionNaturally arising CD4+CD25+ regulatory T cells (Tregs),which constitute 5–10% of peripheral CD4+ T cells innormal mice and healthy humans, are engaged in themaintenance of immunological self-tolerance and immunehomeostasis [1–3]. Natural Tregs specifically express theforkhead family transcription factor FOXP3 in addition tocharacteristic expression of cell-surface molecules such asinterleukin-2 receptor a chain (CD25) and cytotoxic Tlymphocyte-associated 4 (CTLA-4) [4–6]. Most Tregs areproduced by normal thymuses as functionally distinct andmature subpopulations of T cells. Depletion of CD4+CD25+

Tregs from the immune system leads to various autoim-mune diseases and provokes immune responses to com-mensal bacteria in the intestine, causing inflammatorybowel disease (IBD), and eliciting allergy [7]. By contrast,Treg depletion can evoke immune responses beneficial tothe host, for example, effective anti-tumor or anti-microbi-al immunity [8,9]. In addition, antigen-specific expansionof CD25+CD4+ Tregs is exploited not only to treat or

Corresponding author: Ohkura, N. ([email protected])

158 0165-6147/$ – see front matter � 2010 Elsevier Ltd. All rights reserved. doi:10

prevent autoimmune, immunopathological, or allergic dis-eases, but also to establish immunological tolerance toorgan grafts, prevent graft-versus-host disease after bonemarrow transplantation, and sustain feto–maternal toler-ance. Thus, naturally arising Foxp3+CD25+CD4+ Tregscould be key targets to control various physiological andpathological immune responses.

In addition to Foxp3+ Tregs (which are the major Tregpopulation in the immune system), there are Foxp3-non-expressing T cells with suppressive activity, includingthose secreting interleukin (IL)-10 (Tr1 cells) [10] or trans-forming growth factor-b (TGF-b) (Th3 cells) [11]. TheseFoxp3-non-expressing T cells could play significant partsin the maintenance of peripheral tolerance and immunesuppression. However, in this review, we shall focus onFoxp3+CD4+ Tregs because at present it is difficult toassess the possible contribution of non-Foxp3 Tregs toordinary immune responses and there is little evidencefor their roles in maintaining natural self-tolerance. Weshall first summarize the immunological characteristics ofFoxp3+ Tregs and the mechanisms by which they suppressimmune responses. We shall then discuss how the functionand development of Tregs can be controlled by pharmaco-logical agents for the benefit of the host.

Foxp3 as a ‘master regulator’ of the development ofTreg cellsThe Foxp3 gene was first identified as a defective gene inthe mouse strain Scurfy (an X-linked recessive mutantwith lethality in hemizygous males). Mutant animals ex-hibit hyperactivation of CD4+ T cells and overproduction ofproinflammatory cytokines [12]. In humans, mutations ofFOXP3 result in the deficiency or dysfunction ofCD4+CD25+ Tregs, and cause the disorder called ‘immunedysregulation, polyendocrinopathy, enteropathy, X-linkedsyndrome’ (IPEX syndrome). IPEX syndrome is character-ized by a combination of severe multi-organ autoimmunediseases, allergy, and IBD [13–15], which might be differ-ent from common forms of allergy and IBD. Foxp3 ispreferentially expressed in CD4+CD8-CD25+ thymocytesand peripheral CD4+CD25+ T cells in mice. Ectopic retro-viral transduction of the Foxp3 gene in CD25-CD4+ T cellscan convert them to CD25+CD4+ Treg-like cells that cansuppress proliferation of other T cells in vitro and inhibitthe development of autoimmune disease produced by Treg

.1016/j.tips.2010.12.004 Trends in Pharmacological Sciences, March 2011, Vol. 32, No. 3

mailto:[email protected]

http://dx.doi.org/10.1016/j.tips.2010.12.004

Review Trends in Pharmacological Sciences March 2011, Vol. 32, No. 3

depletion [4,5]. Foxp3 transduction in naı̈ve T cells upre-gulates the expression of CD25 and other Treg-associatedcell-surface molecules such as cytotoxic T lymphocyte-as-sociated 4 (CTLA-4) and glucocorticoid-induced TNFR-related gene (GITR), and also represses the productionof IL-2, interferon g (IFN-g), and IL-4. These functionaland phenotypic characteristics are very similar to thoseobserved in natural Tregs, indicating that Foxp3 is amaster regulator of the function and development of Tregs.However, in humans, FOXP3 can be expressed in conven-tional T cells upon antigenic stimulation without apparentsuppressive activity [16–20]. This suggests that there aredifferences between mice and humans in the mechanismsof the FOXP3 induction and FOXP3-dependent regulation.In addition, analyses of mouse strains in which Foxp3 wasreplaced with green fluorescent protein (GFP) showed thatexpression of Treg signatures was observed partially inFoxp3-GFP+ T cells [21,22], and that the overexpression ofFoxp3 was insufficient for inducing all of the Treg signa-ture genes [23,24]. Assuming that Foxp3 can confer sup-pressive activity to conventional T cells, determining thekeymechanism of suppression and how Foxp3 controls it isvery important.

The molecular basis of Foxp3 inductionFoxp3 induction in developing Tregs is controlled viavarious signaling pathways involving T cell receptors(TCRs), IL-2, signal transducers and activators of tran-scription (STAT), Smad, TGF-b, phosphatidylinositol3-kinase (PI3K)/Akt/mTOR, and Notch [25–28]. AfterTCR engagement with self-peptide/MHC ligands, Foxp3expression is initiated by the binding of several transcrip-tion factors to the promoter or enhancer regions of theFoxp3 gene. Within the 50 flanking region of the Foxp3transcription start site, there are several important tran-scription factor-binding sites, including those for AP-1 andNFAT, along with typical sites of eukaryotic promoters(including TATA and CAAT boxes). This region acts as thepromoter for Foxp3 transcription and is referred to as the‘proximal promoter region’, which is highly conserved overmany species. Recent studies have shown that these NFATand AP-1 binding sites positively regulate the transcrip-tional activation of the Foxp3 gene in response to TCRstimulation. NFAT and Smad3 cooperate to induce Foxp3gene expression, and NFAT maintains the activity of theenhancer and consequently Foxp3 gene expression [25].TCR-induced Foxp3 expression in Tregs is also controlledby sequence-specific binding of CREB/ATF. In addition, theimportance of epigenetics for the regulation of Foxp3 ex-pression has been shown in mice and humans [29,30]. Forexample, Foxp3 and other transcription factors bind to theconserved non-coding DNA sequence elements at theFoxp3 locus in a CpGDNA demethylation-dependent man-ner [31]. Taken together, these findings indicate thatseveral diverse intrinsic and extrinsic signals regulateFoxp3 expression. This suggests that FOXP3 expressioncould be controlled by pharmacological means.

Suppressive functions of Foxp3+CD25+CD4+ TregsFoxp3+ natural Tregs suppress the activation, prolifera-tion and effector function of other T cells. Many possible

mechanisms for Treg-mediated suppression have beenproposed [2,32]. The contribution of cell contact-dependentmechanisms is suggested by the in vitro inability of Tregsto suppress the proliferation of responder T cells if the twopopulations are separated by a semi-permeable membrane[33,34]. Cultured supernatants of antigen-stimulatedTregs also fail to exhibit suppressive activity. After cellcontact, antigen-activated Tregs might downregulate theexpression of CD80 and CD86 on antigen-presenting cells(APCs), particularly dendritic cells (DCs), and also stimu-late DCs to express the enzyme indoleamine 2,3-dioxygen-ase (IDO). IDO catabolizes the essential amino acidtryptophan to kynurenine, which is toxic to T cells[35,36]. These processes appear to be dependent uponthe CTLA-4 molecules that are constitutively expressedon the surface of Tregs. Tregs might also kill responder Tcells by a granzyme-dependent or perforin-dependentmechanism, or deliver a negative signal to responder Tcells to inhibit their proliferation [37–39]. In addition, non-production of IL-2 by Foxp3+ Tregs, together with theirconstitutive high expression of the high-affinity IL-2 re-ceptor (IL-2R), could enable them to efficiently absorb IL-2from the surroundings, thereby contributing to the hin-drance of the activation/expansion of responder T cells [40].Furthermore, Treg-mediated suppression might involvethe secretion of suppressive humoral factors such as IL-10, TGF-b, galectin-1, and IL-35 (which is a member of theIL-12 family and a heterodimer comprising Ebi3 and Il12a/p35) [2,41–43]. Overall, these findings indicate that multi-ple mechanisms (including cell-contact and factor-mediat-ed pathways) operate in Treg-mediated suppression.However, most of the suppressive mechanisms of Tregshave been established in mice. Further experiments arerequired to determine if these mechanisms share commonfeatures in mice and humans.

Control of the development and function of Tregs bysmall molecules (including cytokines)As discussed above, immune responses can be augmentedor dampened by controling the development, survival, andfunction of Foxp3+CD25+CD4+ Tregs. Indeed, cytokinessuch as IL-2 and TGF-b, and pharmacological agents suchas rapamycin and retinoic acid, can augment the suppres-sive activity of Foxp3+ Tregs and/or induce Foxp3+ Tregsfrom naı̈ve T cells (Figure 1).

TGF-b and IL-2

TGF-b has been shown to induce Foxp3 expression and aregulatory phenotype in peripheral T cells in mice. Forexample, Tregs can be generated by in vitro antigenicstimulation of naı̈ve T cells in the presence of TGF-band IL-2 [44]. Histone acetylation is induced in the en-hancer region of the Foxp3 gene by TGF-b treatmentcoupled with anti-CD3 and anti-CD28 stimulation in pri-mary CD4+ T cells. Moreover, TGF-b decreases methyla-tion of the CpG island in the Foxp3 first intron andincreases Foxp3 expression. A part of Th3 Tregs appearsto be TGF-b-induced Foxp3+ Tregs. TGF-b is also reportedto induce FOXP3 expression in human conventional Tcells. However, whether the resulting T-cell populationis endowed with suppressive activity is controversial.

159

[()TD$FIG]

Foxp3 gene

NFATSmad3

NFATRunx AP-1

c-Rel

Foxp3 Runx1 Cbf-β

ATG

-2a 1-1

IL-2R TGF-βR S1P1

RAR

JAK

STAT

SmadPI3K

Akt

ER

RAE2

TRENDS in Pharmacological Sciences

Figure 1. Possible pathways for inducing Foxp3 expression. The complex network of signaling pathways cooperatively regulates the expression of Foxp3. Tregs

constitutively express dozens of receptors, such as the IL-2 receptor (CD25), TGF-b receptor (TGF-bR) and S1P receptor (S1P1). TGF-b can induce Foxp3 expression and a

regulatory phenotype in peripheral T cells. Foxp3+ Tregs can be generated by antigenic stimulation of naı̈ve T cells in the presence of TGF-b and IL-2. The latter is

indispensable for the survival of Foxp3+ Tregs. Signaling from IL-2 via the IL-2 receptor also controls the expression of Foxp3 via STAT5. The vitamin-A metabolite retinoic

acid (RA) facilitates the differentiation of naı̈ve T cells to Tregs in the presence of TGF-b. Estrogen promotes the proliferation of Tregs. S1P1 delivers a signal for the thymic

generation, peripheral maintenance and suppressive activity of Tregs through the S1P1–PI3K–Akt pathway. Response elements for transcription factors within the Foxp3

gene are also shown [25,31]. S1P1, type-1 sphingosine 1-phosphate receptor; RA, retinoic acid; RAR, all-trans retinoic acid receptor; E2, estradiol; ER, estrogen receptor;

PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activator of transcription; JAK, Janus kinase;, NFAT, nuclear factor of activated T cells; Runx1, Runt-related

transcription factor 1; Cbf-b, core-binding factor, beta subunit; c-Rel, V-rel reticuloendotheliosis viral oncogene homolog.


Differences between humans and mice might be present inthe effect of TGF-b on Treg development [45].

IL-2 is indispensable for the survival of Foxp3+ Tregsbecause IL-2 deficiency substantially reduces the numberof Foxp3+ Tregs and produces fatal autoimmunity/inflam-mation (see below). Signaling from IL-2 via the IL-2 recep-tor also controls the expression of Foxp3 via STAT5.Administration of IL-2 has been shown to induce theproliferation of Tregs, for example, in cancer patients[46]. After IL-2 cessation, the number of Tregs droppedmore significantly in clinical responders than in non-responders. Furthermore, IL-2 treatment stimulates che-mokine receptor CXCR4 expression on Tregs, and enablesmigration of Treg cells towards the chemokine CXCL12 intumor microenvironments, and may therefore facilitateTreg accumulation in tumors. These observations indicatethat administration of IL-2 can alter the number andfunction of Foxp3+ Tregs. It is envisaged that combinationsof cytokines (including IL-2) and antigen stimulation canexpand natural Tregs in an antigen-specific fashion anddrive antigen-specific naı̈ve T cells to differentiate to Tregs.

Rapamycin

Rapamycin (Sirolimus) is an immunosuppressive drugcurrently used to prevent graft rejection in humans, andconsidered to be suitable for tolerance induction. Rapamy-cin inhibits the response to IL-2 and thereby blocks acti-

160

vation of T cells and B cells. Rapamycin binds the cytosolicprotein FK506-binding protein 12 (KFBP12), and then therapamycin–FKBP12 complex inhibits the mammaliantarget of rapamycin (mTOR) pathway by directly bindingmTOR complex 1 (mTORC1) [47,48] (Figure 2). In humans,rapamycin promotes in vitro expansion of natural Tregs viaselective inhibition of effector T-cell proliferation [49], anddoes not interfere with de novo induction of Tregs fromnaı̈ve CD4+ T cells [50]. In addition, in vivo administrationof rapamycin prevents type 1 diabetes in non-obese dia-betic (NOD) mice and re-establishes long-term self-toler-ance through the expansion of natural Tregs. Thesefindings indicate that rapamycin allows the expansion ofnatural FOXP3+ Tregs in humans and rodents. In addition,FOXP3+ Tregs isolated from patients with type 1 diabetesundergoing rapamycin treatment exhibited an increasedactivity to suppress proliferation of CD4+CD25- effector Tcells comparedwith before treatment [51].Moreover, in theabsence of mTOR, naı̈ve CD4+ T cells differentiate intoFoxp3+ Tregs under conditions that otherwise facilitateeffector T-cell differentiation [52]. Conversely, activation ofthe Akt-mTOR axis impairs the generation of Tregs in thethymus [53]. These results are consistent with the obser-vations that rapamycin can promote the generation ofTregs in vitro and in vivo [49,54,55]. The findings thatrapamycin has an in vivo direct effect on natural Tregfunction suggest that it can be used in immunosuppressive

[()TD$FIG]

Growth factorscytokine signals

PI3K

Akt

mTOR

FKBP12

Rapamycin

Rapamycin

p70S6K

Protein synthesis

p34cdc2

Cell cycle progression

TSC1

TSC2

Foxp3 ?

Treg activity

TRENDS in Pharmacological Sciences

Figure 2. Mechanisms of action of rapamycin. Rapamycin binds to FK506-binding protein (FKBP12). The complex then binds to the mammalian target of rapamycin

(mTOR). The rapamycin–FKBP12–mTOR complex inhibits several biochemical pathways required for cytokine signal transduction, cell-cycle progression, protein synthesis,

metabolism, ribosome biogenesis, and transcription. Rapamycin also affects the expression of Foxp3. p70S6K, 70-kDa S6 protein kinase; PI3K, phosphoinositide 3-kinase;

TSC1, hamartin; TSC2, tuberin; p34cdc2, cyclin-dependent kinase-1.


regimens for tolerance induction, and that the phosphoi-nositide 3-kinase (PI3K)-Akt-mTOR pathway may be apromising target for controling Treg generation.

The sphingosine 1-phosphate receptor agonist FTY720

(fingolimod)

As a novel immunosuppressant, the sphingosine 1-phos-phate receptor agonist FTY720 has been used to preventallograft rejection in organ transplantation. FTY720 canmarkedly prolong the survival of allografts by inducingapoptosis in antigen-reactive lymphocytes. It can alsoprevent T-cell migration to inflammation sites by bindingto the sphingosine-1 phosphate receptor 1 (S1P1), down-regulating its expression, and thereby leading to retentionof T cells in lymphoid organs. FTY720 was also recentlyshown to possess an additional effect that increases thefunctional activity of Tregs [56].

FTY720 can increase the number and enhance thefunctional activity of Foxp3+ Tregs in mixed lymphocytereaction (MLR). Such FTY720-treated cells can downregu-late the alloreactivity of lymphocytes. FTY720 treatmentprominently upregulates the expression of Foxp3, IL-10,TGF-b, and CTLA-4 in Tregs. Thus, FTY720 could be apromising new reagent for the treatment of autoimmuneand other immunological diseases.

Retinoic acids

The vitamin-A metabolite retinoic acid (RA) facilitates thedifferentiation of naı̈ve T cells to Foxp3+ Tregs in thepresence of TGF-b [57–59]. RA can promote TGF-b-depen-dent generation of Foxp3+ Tregs but decrease the TGF-b-and IL-6-dependent generation of inflammatory Th17 cells

in mice [60]. RA-induced Foxp3+ T cells can efficientlysuppress target cells and, thus, have a regulatory functiontypical of Foxp3+ Tregs. RA induces histone acetylation atthe Foxp3 gene promoter and expression of Foxp3 proteinin CD4+ T cells. The induction of RA-induced Foxp3+ T cellsis mediated by the nuclear retinoic acid receptor a (RARa)and involves T-cell activation driven by mucosal DCs andCD28 co-stimulation. A unique cellular feature of theseTregs is their capacity to ‘home’ to the gut, particularly tothe lamina propria of the small intestine [59]. Thus, RA is apositive regulatory factor for the generation of gut-homingFoxp3+ Tregs. Whether RA can be exploited to induce oraltolerance to treat immunological diseases such as autoim-mune disease and allergy has yet to be evaluated.

Aromatase inhibitors

Aromatase is the key enzyme responsible for estrogenbiosynthesis. It has been shown in mice and humans thatestrogen can promote immune tolerance by expandingTregs [61,62]. Aromatase inhibitors have been consideredto be potent therapeutic agents for estrogen-dependentbreast cancer [63]. In addition, tumor infiltration by Tregsis associatedwith increased relapse and shorter survival inpatients with breast cancer [64]. Therefore, blocking estro-gen receptor-a signaling could be effective for breast cancersby (at least in part) abrogating Tregs. In a Phase II random-ized controlled trial of the aromatase inhibitor letrozoletogetherwith orwithout cyclophosphamide in patientswithbreast cancer, a significant reduction in the number of Tregswas observed in primary tumors after treatment with theinhibitor but not by the addition of cyclophosphamide [65].These reports suggest that aromatase inhibitors have

161


significant immunomodulatory roles that affect Tregproliferation.

Probiotics

The beneficial effects of probiotics have been described inmany diseases. A mixture of probiotics that upregulateCD4+Foxp3+ Tregs was recently reported [66]. Adminis-tration of the probiotic mixture induced T-cell and B-cellhyporesponsiveness and downregulated Th1, Th2, andTh17 cytokine production without apoptosis in these cells.It also induced generation of Tregs from the CD4+CD25-

population and increased the suppressor activity of naturalTregs. Administration of probiotics has been shown to havetherapeutic effects in experimental IBD, atopic dermatitis,and rheumatoid arthritis. How they exhibit these immu-nomodulatory effects needs to be investigated further.

Control of Foxp3+ Tregs by targeting Treg-associatedmoleculesFoxp3 directly or indirectly controls the expression of�700genes and directly binds to �10% of them. Among Foxp3-controlled genes, deficiency of IL-2-related genes (i.e. IL-2,IL-2 receptor a-chain [CD25], and b-chain [CD122]) orCTLA-4 (CD152) produces severe autoimmune/inflamma-tory disease similar to that observed in Foxp3-deficient ormutant mice. IL-2 and its signaling via the IL-2 receptorare mainly required for Treg survival, and a CTLA-4-dependent mechanism of suppression might constitute acore mechanism of suppression in vivo [67]. Modulation ofother molecules such as GITR, OX40, and folate receptor-4(FR4) expressed on the cell surface of Foxp3+ Tregs canalter Treg function by the use of blocking or agonisticmonoclonal antibody (mAb). Furthermore, those molecules(e.g. CD25 and FR4) expressed on Tregs at much higherlevels than non-Tregs cells in the activated or resting statecan be suitable targets to deplete Tregs using cytotoxicmAbs.

CTLA-4

CTLA-4 delivers inhibitory signals to activated T cells andserves as a negative regulator of immunity. T-cell activa-tion requires two signals: TCR activation and co-stimula-tion via the interaction of CD28 on T cells and B7 (Cd80/CD86) on antigen-presenting cells such as dendritic cells.CTLA-4, another ligand for B7, is upregulated after acti-vation, and delivers an inhibitory signal, leading to sup-pression of T-cell proliferation [68]. Importantly, CTLA-4 isconstitutively expressed by Foxp3+ Tregs in rodents andalso in terminally differentiated FOXP3highCD25highCD4+

T cells in humans. For the past decade, the role of CTLA-4in Treg function has been controversial. Several findingssupport the notion that CTLA-4 is essential for Tregfunction. First, blockade of CTLA-4 expressed by naturalTregs (not responder T cells) abrogated in vivo and in vitroTreg suppression activity in studies in which Tregs fromCTLA-4-intact mice were co-cultured with CTLA-4-defi-cient responder T cells in vitro or co-transferred to SCIDmice [69]. Second, Foxp3 together with other transcriptionfactors upregulates the expression of CTLA-4 by binding tothe promoter region of theCTLA-4 gene [70]. This indicatesthat Foxp3 might sustain high expression of CTLA-4 in

162

Foxp3+ Tregs. Most importantly, a recent study with Treg-specific CTLA-4-deficient mice clearly showed that micesuccumbed to lymphoproliferation with splenomegaly andvarious autoimmune diseases, and developed hyperpro-duction of IgE, as seen in Foxp3 deficiency [67]. Treg-specific CTLA-4 deficiency also augmented tumor immuni-ty. Yet, exclusive blockade of the CTLA-4 signal inFoxp3+CD25+CD4+ Tregs or non-Treg T cells in mice indi-cates that CTLA-4 is required for CD25+CD4+ Tregs andactivated effector T cells, and that CTLA-4 blockade aug-ments tumor inhibition by attenuating Treg suppressionand augmenting effector T-cell activity. Clinical trialsusing anti-CTLA-4 blocking antibody demonstrated en-hanced anti-tumor immune responses that occasionallyaccompanied autoimmunity (most commonly IBD) [71].It remains to be determined in humans how CTLA-4blockade augments tumor immunity (i.e. if it attenuatesTreg function, enhances effector T cell activity, or both)[72].

CD25

CD25 is not only a keymolecule for Treg function but is alsoa reliable cell-surface marker for natural Foxp3+ Tregs.Reduction of Foxp3+CD25+CD4+ Tregs can augment tumoror microbial immunity in experimental animals [73]. Inhumans, administration of the CD25-directed immuno-toxin RFT5-SMPT-dgA to patients with metastatic mela-noma induced a transient (but robust) reduction in thenumber of CD25high CD4+ T cells in vivo [74]. The reductionin Foxp3+ CD4+ T cell number was less complete withselective persistence of a stable number of CD25low

Foxp3+CD4+ T cells in vivo. Although significant anti-tumor responses were not observed with the treatment,it is not clear if thorough eradication of Tregs is required toelicit effective anti-tumor immunity. Alternatively, thetreatment might deplete not only CD25+ Tregs but alsoCD25+-activated effector T cells capable of attacking tumorcells. Similarly, depletion of human Tregs by denileukindiftitox (an immunotoxin-conjugated IL-2) is undergoingclinical trials for patients with advanced carcinoembryonicantigen (CEA)-expressing malignancies [75].

GITR

GITR is a co-stimulatory molecule expressed at differentlevels in resting CD4+ and CD8+ T cells, and is upregulatedby T-cell activation [76]. Themolecule is also constitutivelyexpressed on CD25+CD4+Tregs at high levels, and activa-tion of GITR signaling with agonistic anti-GITR mAb orGITR ligand can inhibit the suppressive activity ofCD25+CD4+ Tregs. A recent study in GITR knockout miceshowed that a reversal of suppression by GITR signalingwas attributed to the co-stimulatory activity of GITR onresponder CD4+CD25- T cells, which made them resistantto CD25+CD4+ Treg suppression. Notably, stimulation ofGITR with agonistic an anti-GITR mAb or GITR ligandsincreased tumor-specific CD4+ and CD8+ T-cell responses[77,78]. Furthermore, anti-GITR treatment was more ef-fective if it was initiated after a tumor had grown to acertain size compared with anti-GITR treatment before orimmediately after tumor inoculation [79]. This indicatesthat anti-GITR stimulation enhances the activity and

Table 1. Summary of current Treg-targeting immunotherapy

A. Small molecules including cytokines

Targets Small molecules

and cytokines

Systems Subjects Outcome PMID

TGF-b Recombinant TGF-b Mouse/in vivo Increasing Tregs 14676299

TGF-b blockade Mouse/in vivo Decreasing Tregs 15937545

IL-2 Recombinant IL-2 Human/Phase II Ovarian cancer Advantage in cancer

immunotherapy

17671219

Recombinant

IL-2 + CTLA4 blockade

Human/Phase II Advanced melanoma No advantage 16283570

mTOR Rapamycin (Sirolimus) Human/Observational

study

Type 1Diabetes

Mellitus

Up regulating

Suppressive

functions of Tregs

18559659

Human/Observational

study

Renal transplantation Up regulating

Suppressive

functions of Tregs

17097943

S1P1 FTY720 (Fingolimod) Mouse/in vivo Increasing Tregs 19659769

19692647

20702533

Estrogen Aromatase inhibitor

(Letrozole)

Human/Phase II Breast Cancer Advantage in cancer

immunotherapy

19064988

Retinoic

acid receptor

Retinoic acids Mouse/in vivo Increasing Tregs 19006694

17620363

19204112

Probiotics Mouse/in vivo Increasing Tregs 20080669

B. Monoclonal antibodies targeting Treg-associated molecules

Targets Antibodies Systems Subjects Outcome PMID

CTLA-4 Tremelimumab Human/Phase II Advanced colorectal cancer No advantage 20498386

Human/Phase II Advanced gastric and

esophageal adenocarcinoma

No advantage 20179239

Human/Phase II Advanced melanoma Advantage in cancer

immunotherapy

20086001

19139427

CD25 Daclizumab Human/Phase II Multiple sclerosis Advantage in disease control 19364932

20067954

15161974

19364933

Daclizumab Human/Observational

study

Type 1 Diabetes Mellitus Inconsistent in literature 17709711

19808924

20067954

Daclizumab Human/Observational

study

Advanced breast cancer Advantage in cancer

immunotherapy

19769742

Denileukin diftitox Human/Phase I Cancer vaccine Advantage in cancer

immunotherapy

17315189

LMB-2 Human/Observational

study

Advanced melanoma Advantage in cancer

immunotherapy

17878392

RFT5-SMPT-dgA Human/Observational

study

Advanced melanoma Advantage in cancer

immunotherapy

18481388

GITR Anti GITR monoclonal

antibody (DTA1)

Mouse/in vivo Decreasing Tregs 16868552

OX40 Agonist anti OX40

monoclonal

antibody (OX86)


FR4 Anti monoclonal

FR4 antibody



expansion of antigen-primed effector T cells (rather thantheir generation or antigen priming). Also, if GITR stimu-lation is combined with tumor antigen stimulation, theinduction of tumor-antigen-specific effector T cells is aug-mented and the induced tumor-antigen-specific T cells arerefractory to suppression by CD25+CD4+ Tregs [80]. Thus,agonistic anti-GITR mAbs are a promising candidate forimmunotherapy of cancer and microbial diseases.

OX40

OX40 is a co-stimulatory molecule of the tumor necrosisfactor (TNF) receptor family. It is expressed transiently on

activated T cells and constitutively on CD25+CD4+ Tregs.Studies have shown that activation of OX40 signaling withagonistic anti-OX40 mAb could inhibit the suppressiveactivity of CD25+CD4+ Tregs [81,82]. Furthermore,intra-tumoral injection of anti-OX40 mAb induced stronginhibition of tumor growth [82]. Using OX40-deficientCD25+CD4+ Tregs and CD25-CD4+ effector T cells, itwas shown that the OX40 signal could alter the functionsof CD25+CD4+ Tregs and effector T cells [82]. This sug-gested that agonistic anti-OX40 mediated the anti-tumoreffect by attenuating suppression by CD25+CD4+ Tregsand activating effector T-cell function.

163


FR4

In rodents, Foxp3+ Tregs express a higher level of FR4 thannaı̈ve T cells and, upon TCR stimulation, Foxp3+ Tregsupregulate the expression to a much higher level thanFoxp3- T cells [83], therefore enabling distinction betweenactivated Tregs and activated effector T cells. Thus, anti-FR4 depleting mAb is highly useful in provoking tumorimmunity by predominantly depleting activated Tregswhile preserving tumor-reactive effector T cells. If thehuman counterpart of mouse FR4 has similar anti-tumoractivity needs to be studied.

Future directionIn this review, we discussed various agents that can controlthe function and development of Tregs, including monoclo-nal antibodies against Treg-associated molecules andsmall molecules (including cytokines) affecting the gener-ation and suppressive activity of Tregs (Table 1). Giventhat many Tregs infiltrate into tumors, natural Tregs mayhamper the potential immune responsiveness of cancerpatients to tumors. Treg depletion might elicit autoimmu-nity in genetically susceptible individuals, but it can pro-voke and enhance effective tumor immunity [84,85]. Thus,targeting Tregs is a promising approach for cancer immu-notherapy (for example, local depletion of Tregs in tumorsand attenuation of Treg function at the time of therapeuticvaccination with tumor antigen). Moreover, strategies forexpanding antigen-specific natural Tregs may help to in-duce transplantation tolerance, suppress graft rejectionand inhibit graft-versus-host disease after bone-marrowtransplantation. In the presence of Tregs that activelymaintain graft tolerance, naı̈ve T cells newly recruitedto the graft site could differentiate into graft-specific Tregs(infectious tolerance) [86]. The same principle could beapplied to the treatment of autoimmune disease, as wellas allergy and other inflammatory diseases.

Discovery and development of small molecules and bio-logics (such as monoclonal antibodies) that modulate thefunction and/or development of Tregs are envisaged. ThePI3K–Akt–mTOR axis can be a good target of small mole-cules because it has a central role in controling the expres-sion of FOXP3 (and thus Treg development) by integratingenvironmental cues such as cytokines, TCR stimulations,and growth factors. Screening of pharmacological agentsthat control the activity of the axis may be a promisingapproach for developing novel therapies for immune dis-eases and cancers via regulation of Treg function. Monoclo-nal antibodies targetingTreg-associatedmoleculesmayalsobe able to control Treg function. Although it is not certain ifstrictly Treg-specific molecules other than Foxp3 exist, it islikely that some monoclonal antibodies specific for Treg-associated cell-surfacemolecules can differentially alter thefunctions of Treg and effector T cells, thereby augmenting ordamping immune responses. Further elucidation of thecellular and molecular basis underlying the developmentand function of Tregswill help to develop novel therapies forimmunological diseases and cancers.

Concluding remarksOver the last decade, considerable progress has been madein our understanding of Treg immunobiology. In particu-

164

lar, the identification of FOXP3 as a key regulator of thefunction and development of natural Tregs has provided aclue to elucidating the molecular mechanisms underpin-ning such development and function. Given that the bal-ance between Tregs and effector T cells is critical for thecontrol of immune responses, pharmacological treatmentsthat aim to modulate the balance can be a key therapeuticstrategy for the treatment and prevention of various im-munological diseases and for the control of physiologicalimmune responses against cancer, microbes, and organtransplants.

Conflicts of InterestThe authors have no conflicts of interest.

AcknowledgementsThis work was supported by grants-in-aids from the Ministry ofEducation, Sports and Culture and the Ministry of Human Welfare ofJapan.

References1 Sakaguchi, S. et al. (2007) Regulatory T cells—a brief history and

perspective. Eur. J. Immunol. 37 (Suppl. 1), S116–S1232 Shevach, E.M. (2009)Mechanisms of foxp3+ T regulatory cell-mediated

suppression. Immunity 30, 636–6453 Sakaguchi, S. et al. (2010) FOXP3+ regulatory T cells in the human

immune system. Nat. Rev. Immunol. 10, 490–5004 Hori, S. et al. (2003) Control of regulatory T cell development by the

transcription factor Foxp3. Science 299, 1057–10615 Fontenot, J.D. et al. (2003) Foxp3 programs the development and

function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–3366 Khattri, R. et al. (2003) An essential role for Scurfin in CD4+CD25+ T

regulatory cells. Nat. Immunol. 4, 337–3427 Singh, B. et al. (2001) Control of intestinal inflammation by regulatory

T cells. Immunol. Rev. 182, 190–2008 Wang, H.Y. andWang, R.F. (2007) Regulatory T cells and cancer.Curr.

Opin. Immunol. 19, 217–2239 Belkaid, Y. and Rouse, B.T. (2005) Natural regulatory T cells in

infectious disease. Nat. Immunol. 6, 353–36010 Wu, K. et al. (2007) IL-10-producing type 1 regulatory T cells and

allergy. Cell Mol. Immunol. 4, 269–27511 Mills, K.H. andMcGuirk, P. (2004) Antigen-specific regulatory T cells–

their induction and role in infection. Semin. Immunol. 16, 107–11712 Brunkow, M.E. et al. (2001) Disruption of a new forkhead/winged-helix

protein, scurfin, results in the fatal lymphoproliferative disorder of thescurfy mouse. Nat. Genet. 27, 68–73

13 Bennett, C.L. et al. (2001) The immune dysregulation,polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) iscaused by mutations of FOXP3. Nat. Genet. 27, 20–21

14 Gambineri, E. et al. (2008) Clinical andmolecular profile of a new seriesof patients with immune dysregulation, polyendocrinopathy,enteropathy, X-linked syndrome: inconsistent correlation betweenforkhead box protein 3 expression and disease severity. J. AllergyClin. Immunol. 122, 1105–1112 e1

15 Ochs, H.D. et al. (2005) FOXP3 acts as a rheostat of the immuneresponse. Immunol. Rev. 203, 156–164

16 Allan, S.E. et al. (2005) The role of 2 FOXP3 isoforms in the generationof human CD4+ Tregs. J. Clin. Invest. 115, 3276–3284

17 Allan, S.E. et al. (2007) Activation-induced FOXP3 in human T effectorcells does not suppress proliferation or cytokine production. Int.Immunol. 19, 345–354

18 Pillai, V. et al. (2007) Transient regulatory T-cells: a state attained byall activated human T-cells. Clin. Immunol. 123, 18–29

19 Tran, D.Q. et al. (2007) Induction of FOXP3 expression in naive humanCD4+FOXP3 T cells by T-cell receptor stimulation is transforminggrowth factor-beta dependent but does not confer a regulatoryphenotype. Blood 110, 2983–2990

20 Passerini, L. et al. (2008) STAT5-signaling cytokines regulate theexpression of FOXP3 in CD4+CD25+ regulatory T cells andCD4+CD25- effector T cells. Int. Immunol. 20, 421–431


21 Gavin, M.A. et al. (2007) Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445, 771–775

22 Lin, W. et al. (2007) Regulatory T cell development in the absence offunctional Foxp3. Nat. Immunol. 8, 359–368

23 Hill, J.A. et al. (2007) Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptionalsignature. Immunity 27, 786–800

24 Sugimoto, N. et al. (2006) Foxp3-dependent and -independentmolecules specific for CD25+CD4+ natural regulatory T cellsrevealed by DNA microarray analysis. Int. Immunol. 18, 1197–

120925 Tone, Y. et al. (2008) Smad3 and NFAT cooperate to induce Foxp3

expression through its enhancer. Nat. Immunol. 9, 194–20226 Samon, J.B. et al. (2008) Notch1 and TGFbeta1 cooperatively regulate

Foxp3 expression and themaintenance of peripheral regulatory T cells.Blood 112, 1813–1821

27 Kim, J.K. et al. (2009) Impact of the TCR signal on regulatory T cellhomeostasis, function, and trafficking. PLoS One 4, e6580

28 Sakaguchi, S. et al. (2008) Regulatory T cells and immune tolerance.Cell 133, 775–787

29 Baron, U. et al. (2007) DNA demethylation in the human FOXP3 locusdiscriminates regulatory T cells from activated FOXP3(+) conventionalT cells. Eur. J. Immunol. 37, 2378–2389

30 Floess, S. et al. (2007) Epigenetic control of the foxp3 locus in regulatoryT cells. PLoS Biol. 5, e38

31 Zheng, Y. et al. (2010) Role of conserved non-coding DNA elements inthe Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812

32 Sakaguchi, S. et al. (2009) Regulatory T cells: how do they suppressimmune responses? Int. Immunol. 21, 1105–1111

33 Takahashi, T. et al. (1998) Immunologic self-tolerance maintained byCD25+CD4+ naturally anergic and suppressive T cells: induction ofautoimmune disease by breaking their anergic/suppressive state. Int.Immunol. 10, 1969–1980

34 Thornton, A.M. and Shevach, E.M. (1998) CD4+CD25+immunoregulatory T cells suppress polyclonal T cell activation invitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296

35 Oderup, C. et al. (2006) Cytotoxic T lymphocyte antigen-4-dependentdown-modulation of costimulatorymolecules on dendritic cells in CD4+CD25+ regulatory T-cell-mediated suppression. Immunology 118, 240–

24936 Grohmann, U. et al. (2002) CTLA-4-Ig regulates tryptophan catabolism

in vivo. Nat. Immunol. 3, 1097–110137 Gondek, D.C. et al. (2005) Cutting edge: contact-mediated suppression

by CD4+CD25+ regulatory cells involves a granzyme B-dependent,perforin-independent mechanism. J. Immunol. 174, 1783–1786

38 Cao, X. et al. (2007) Granzyme B and perforin are important forregulatory T cell-mediated suppression of tumor clearance.Immunity 27, 635–646

39 Bopp, T. et al. (2007) Cyclic adenosine monophosphate is a keycomponent of regulatory T cell-mediated suppression. J. Exp. Med.204, 1303–1310

40 Pandiyan, P. et al. (2007) CD4+CD25+Foxp3+ regulatory T cells inducecytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat.Immunol. 8, 1353–1362

41 Tang, Q. and Bluestone, J.A. (2008) The Foxp3+ regulatory T cell: ajack of all trades, master of regulation. Nat. Immunol. 9, 239–244

42 Vignali, D.A. et al. (2008) How regulatory T cells work. Nat. Rev.Immunol. 8, 523–532

43 Collison, L.W. et al. (2007) The inhibitory cytokine IL-35 contributes toregulatory T-cell function. Nature 450, 566–569

44 Curotto de Lafaille, M.A. and Lafaille, J.J. (2009) Natural and adaptivefoxp3+ regulatory T cells: more of the same or a division of labor?Immunity 30, 626–635

45 Ziegler, S.F. (2007) FOXP3: not just for regulatory T cells anymore.Eur. J. Immunol. 37, 21–23

46 Wei, S. et al. (2007) Interleukin-2 administration alters theCD4+FOXP3+ T-cell pool and tumor trafficking in patients withovarian carcinoma. Cancer Res. 67, 7487–7494

47 Zhou, H. et al. (2010) Updates of mTOR inhibitors. Anticancer AgentsMed. Chem. 10, 571–581

48 Powell, J.D. and Delgoffe, G.M. (2010) The mammalian target ofrapamycin: linking T cell differentiation, function, and metabolism.Immunity 33, 301–311

49 Battaglia, M. et al. (2006) Rapamycin promotes expansion of functionalCD4+CD25+FOXP3+ regulatory T cells of both healthy subjects andtype 1 diabetic patients. J. Immunol. 177, 8338–8347

50 Valmori, D. et al. (2006) Rapamycin-mediated enrichment of T cellswith regulatory activity in stimulated CD4+ T cell cultures is not due tothe selective expansion of naturally occurring regulatory T cells but tothe induction of regulatory functions in conventional CD4+ T cells. J.Immunol. 177, 944–949

51 Monti, P. et al. (2008) Rapamycin monotherapy in patients with type 1diabetesmodifies CD4+CD25+FOXP3+ regulatory T-cells.Diabetes 57,2341–2347

52 Delgoffe, G.M. et al. (2009) The mTOR kinase differentially regulateseffector and regulatory T cell lineage commitment. Immunity 30, 832–

84453 Haxhinasto, S. et al. (2008) The AKT-mTOR axis regulates de novo

differentiation of CD4+Foxp3+ cells. J. Exp. Med. 205, 565–57454 Kang, J. et al. (2008) De novo induction of antigen-specific

CD4+CD25+Foxp3+ regulatory T cells in vivo following systemicantigen administration accompanied by blockade of mTOR. J.Leukoc. Biol. 83, 1230–1239

55 Kopf, H. et al. (2007) Rapamycin inhibits differentiation of Th17 cellsand promotes generation of FoxP3+ T regulatory cells. Int.Immunopharmacol. 7, 1819–1824

56 Zhou, P.J. et al. (2009) Immunomodulatory drug FTY720 inducesregulatory CD4(+)CD25(+) T cells in vitro. Clin. Exp. Immunol. 157,40–47

57 Mucida, D. et al. (2007) Reciprocal TH17 and regulatory T celldifferentiation mediated by retinoic acid. Science 317, 256–260

58 Sun, C.M. et al. (2007) Small intestine lamina propria dendritic cellspromote de novo generation of Foxp3 T reg cells via retinoic acid. J.Exp. Med. 204, 1775–1785

59 Benson, M.J. et al. (2007) All-trans retinoic acid mediates enhanced Treg cell growth, differentiation, and gut homing in the face of highlevels of co-stimulation. J. Exp. Med. 204, 1765–1774

60 Kang, S.G. et al. (2007) Vitamin A metabolites induce gut-homingFoxP3+ regulatory T cells. J. Immunol. 179, 3724–3733

61 Polanczyk, M.J. et al. (2006) Estrogen-mediated immunomodulationinvolves reduced activation of effector T cells, potentiation of Treg cells,and enhanced expression of the PD-1 costimulatory pathway. J.Neurosci. Res. 84, 370–378

62 Prieto, G.A. and Rosenstein, Y. (2006) Oestradiol potentiates thesuppressive function of human CD4 CD25 regulatory T cells bypromoting their proliferation. Immunology 118, 58–65

63 Jiao, J. et al. (2010) Recent advancement in nonsteroidal aromataseinhibitors for treatment of estrogen-dependent breast cancer. Curr.Med. Chem. 17, 3476–3487

64 Bates, G.J. et al. (2006) Quantification of regulatory T cells enables theidentification of high-risk breast cancer patients and those at risk oflate relapse. J. Clin. Oncol. 24, 5373–5380

65 Generali, D. et al. (2009) Immunomodulation of FOXP3+ regulatory Tcells by the aromatase inhibitor letrozole in breast cancer patients.Clin. Cancer Res. 15, 1046–1051

66 Kwon, H.K. et al. (2010) Generation of regulatory dendritic cells andCD4+Foxp3+ T cells by probiotics administration suppresses immunedisorders. Proc. Natl. Acad. Sci. U.S.A. 107, 2159–2164

67 Wing, K. et al. (2008) CTLA-4 control over Foxp3+ regulatory T cellfunction. Science 322, 271–275

68 Greenfield, E.A. et al. (1998) CD28/B7 costimulation: a review. Crit.Rev. Immunol. 18, 389–418

69 Takahashi, T. et al. (2000) Immunologic self-tolerance maintained byCD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic Tlymphocyte-associated antigen 4. J. Exp. Med. 192, 303–310

70 Wu, Y. et al. (2006) FOXP3 controls regulatory T cell function throughcooperation with NFAT. Cell 126, 375–387

71 Hodi, F.S. (2010) Overcoming immunological tolerance to melanoma:Targeting CTLA-4. Asia Pac. J. Clin. Oncol. 6 (Suppl. 1), S16–S23

72 Comin-Anduix, B. et al. (2008) Detailed analysis of immunologic effectsof the cytotoxic T lymphocyte-associated antigen 4-blockingmonoclonal antibody tremelimumab in peripheral blood of patientswith melanoma. J. Transl. Med. 6, 22–36

73 Shimizu, J. et al. (1999) Induction of tumor immunity by removingCD25+CD4+ T cells: a common basis between tumor immunity andautoimmunity. J. Immunol. 163, 5211–5218

165


74 Powell, D.J., Jr et al. (2008) Partial reduction of human FOXP3+ CD4 Tcells in vivo after CD25-directed recombinant immunotoxinadministration. J. Immunother. 31, 189–198

75 Morse, M.A. et al. (2008) Depletion of human regulatory T cellsspecifically enhances antigen-specific immune responses to cancervaccines. Blood 112, 610–618

76 Shevach, E.M. and Stephens, G.L. (2006) The GITR-GITRLinteraction: co-stimulation or contrasuppression of regulatoryactivity? Nat. Rev. Immunol. 6, 613–618

77 Cohen, A.D. et al. (2006) Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity. Cancer Res. 66,4904–4912

78 Ramirez-Montagut, T. et al. (2006) Glucocorticoid-induced TNFreceptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhancesantitumor immunity. J. Immunol. 176, 6434–6442

79 Ko, K. et al. (2005) Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+regulatory T cells. J. Exp. Med. 202, 885–891

166

80 Nishikawa, H. et al. (2008) Regulatory T cell-resistant CD8+ T cellsinduced by glucocorticoid-induced tumor necrosis factor receptorsignaling. Cancer Res. 68, 5948–5954

81 Valzasina, B. et al. (2005) Triggering of OX40 (CD134) on CD4(+)CD25+T cells blocks their inhibitory activity: a novel regulatory role for OX40and its comparison with GITR. Blood 105, 2845–2851

82 Piconese, S. et al. (2008) OX40 triggering blocks suppression byregulatory T cells and facilitates tumor rejection. J. Exp. Med. 205,825–839

83 Yamaguchi, T. et al. (2007) Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 27,145–159

84 Danke, N.A. et al. (2004) Autoreactive T cells in healthy individuals. J.Immunol. 172, 5967–5972

85 Nishikawa, H. et al. (2005) CD4+ CD25+ regulatory T cells control theinduction of antigen-specific CD4+ helper T cell responses in cancerpatients. Blood 106, 1008–1011

86 Waldmann, H. et al. (2006) Infectious tolerance and the long-termacceptance of transplanted tissue. Immunol. Rev. 212, 301–313

foxp3+ regulatory t cells

Documents