regulatory t cells and immune tolerance in the...

Regulatory T Cells and Immune Tolerancein the Intestine

Oliver J. Harrison1 and Fiona M. Powrie1,2

1Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom2Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, Experimental MedicineDivision, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom

Correspondence: [email protected]

A fundamental role of the mammalian immune system is to eradicate pathogens whileminimizing immunopathology. Instigating and maintaining immunological tolerancewithin the intestine represents a unique challenge to the mucosal immune system. RegulatoryT cells are critical for continued immune tolerance in the intestine through active control ofinnate and adaptive immune responses. Dynamic adaptation of regulatory T-cell populationsto the intestinal tissue microenvironment is key in this process. Here, we discuss special-ization of regulatory T-cell responses in the intestine, and howa breakdown in these processescan lead to chronic intestinal inflammation.

The mammalian host harbors a vast and di-verse commensal microbiota. The gastroin-

testinal tract is a site of preferential colonizationby commensal organisms, consisting of fun-gal, viral, and bacterial species. Initial microbialcolonization of the host occurs during birthand continues until a stable commensal micro-biota is established during childhood (Tannock2007). Colonization of the gastrointestinal tractis a vital triggering stimuli for maturation of themucosal immune system, and the presence of acommensal microbiota further benefits the hostby providing resistance to invading pathogensand metabolism of dietary components (Mac-pherson et al. 2005; Hooper et al. 2012). A dy-namic molecular dialogue between microbiotaand host ensures this colonization occurs as astate of mutualism, the breakdown of which canresult in chronic pathologies of the gastrointes-

tinal tract, such as inflammatory bowel diseases(IBD) (Kaser et al. 2010; Maloy and Powrie2011). Complex interactions between the mi-crobiota, mucosal immune system, and intesti-nal tissue cells provide multiple layers of regula-tion that control intestinal immunity. Here, wefocus on the role of regulatory T cells as keycomponents of intestinal homeostasis and dis-cuss how tissue-specific adaptations contributeto their function when patrolling this challeng-ing frontier.

THE GASTROINTESTINAL TRACT

The intestine constitutes a vast network ofnonlymphoid and secondary lymphoid tissues,and as such, is home to numerous populationsof leukocytes. Reflecting the unique challengeof maintaining intestinal immune tolerance,

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many of these cellular populations are found tobe enriched in, or exclusive to, the intestine. Inaddition to the array of hematopoietic cell pop-ulations critical for immune tolerance withinthe intestine, the intestinal mucosa is anatomi-cally specialized to promote homeostasis (Hilland Artis 2010; Harrison and Maloy 2011). Theintestinal epithelium consists of a single layer ofspecialized epithelial cell subsets derived frommultipotent and highly proliferative Lgr5þ stemcells located within the intestinal crypts. Al-though the cellular composition of the intesti-nal epithelium varies with anatomical location,both the colon and small intestine possess pop-ulations of absorptive and secretory cells, in-cluding enterocytes, colonocytes, goblet, endo-crine, and Paneth cells (Wright 2000; Simonsand Clevers 2011). Goblet-cell secretion ofmembrane tethered and soluble mucus com-ponents creates not only a viscous protectivebarrier, but a matrix loaded with secretory IgAand Paneth cell-derived antimicrobial peptides(AMP) to forge a layer impermeable to the ma-jority of intestinal bacteria (Hill and Artis 2010).Pattern-recognition receptors (PRR), includ-ing Toll-like receptors (TLR) and Nod-like re-ceptors (NLR), are germ-line-encoded sensorsof microbial and host-derived danger signals(Schroder and Tschopp 2010). Tonic PRR-signaling within intestinal epithelial cells (IECs)drives cell-intrinsic proliferation, survival, AMPproduction and fortification of intercellulartight junctions, limiting bacterial translocationto the lamina propria (Maloy and Powrie 2011).Furthermore, homeostatic PRR-signaling with-in the intestinal epithelium serves to regulatenot only localization but also composition ofthe microbiota. For example, deficiency in thecytosolic PRR, NLRP6, results in an altered in-testinal microbiota with elevated abundance ofthe bacterial phyla Bacteroidetes (Prevotella-ceae) and TM7 (Elinav et al. 2011). NLRP6-de-ficient mice have increased susceptibility to ex-perimental DSS (dextran sodium sulphate)colitis; elegant cross-fostering and cohousingexperiments revealed the colitogenic microbiotato be both vertically and horizontally transmis-sible. Thus, the intestinal epithelium uses mul-tiple pathways to influence the localization and

composition of the microbiota to promoteintestinal tolerance. Dysbiosis, an imbalance inthe composition of microbial communities,occurs in patients with IBD, although whetherthis is cause or a consequence of the inflamma-tory environment remains unclear (Kaser et al.2010).

ANTIGEN-PRESENTING CELLS PROMOTEINTESTINAL TOLERANCE

In line with important roles in host defense andtolerance, the intestine contains abundant andheterogeneous populations of myeloid antigen-presenting cells (APC). Subsets from distinctdevelopmental origins vary in anatomical local-ization, phenotype, and function (Coombes andPowrie 2008; Varol et al. 2010). Recent studies ofintestinal APC have used the differential expres-sion of CD103 (aE integrin) and CX3CR1 (re-ceptor for Fractalkine [CX3CL1]) to identifytwo major tissue resident populations, both ofwhich appear to contribute to intestinal toler-ance in distinct ways (Varol et al. 2010). Thus,CD103þ dendritic cells (DC) can take up in-testinal antigens and following CCR7-depen-dent migration to the mesenteric lymph node(MLN), initiate T-cell responses with an intes-tinal tropism through induction of intestinalhoming receptors CCR9 and a4b7 (Iwata et al.2004; Johansson-Lindbom et al. 2005; Jaenssonet al. 2008). Under homeostatic conditions,CD103þ DC preferentially promote regulatoryT-cell responses (Coombes et al. 2007; Sun et al.2007; Schulz et al. 2009). However, this functionis not hardwired, and as discussed below, is con-trolled by the intestinal microenvironment pro-viding a mechanism through which the balancebetween tolerance and immunity can be con-trolled (Laffont et al. 2010). In addition to effectson T-cell-mediated immunity, CD103þDC alsopromote T-cell-independent IgA class switchrecombination by intestinal B cells contribut-ing to host defense and intestinal barrier func-tion (Uematsu et al. 2008). In contrast, intestinalCX3CR1þAPC (consisting of dendritic cells andmacrophages) are thought to be nonmigratoryand poor primers of naı̈ve T cells (Schulz et al.2009), although this has recently been chal-

O.J. Harrison and F.M. Powrie

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lenged (Diehl et al. 2013). CX3CR1þ APC accu-mulate within the lamina propria following mi-crobial colonization where they promote intes-tinal tolerance through a variety of mechanisms(Niess and Adler 2010). CX3CR1þ APC lie ad-jacent to the epithelial barrier, and project trans-epithelial processes into the intestine to sampleluminal antigens (Rescigno et al. 2001; Niesset al. 2005). Highly phagocytic in nature, colonicmacrophages aid intestinal tolerance throughclearance of apoptotic cells and debris, whilealso acting to expand populations of regulatoryT cells (Murai et al. 2009; Hadis et al. 2011).Together the data indicate that lymphoid andtissue dwelling populations of APC collaborateto instigate and maintain a tolerogenic intestinalmicroenvironment.

Foxp3þ Treg IN INTESTINAL IMMUNETOLERANCE

The intestine contains a high frequency of reg-ulatory T-cell populations and early studiesrevealed their importance in preventing intesti-nal inflammation (Izcue et al. 2009). More re-cent studies have emphasized the importance ofFoxp3 (Forkhead box P3)-expressing popula-tions in particular. Foxp3 is a transcription fac-tor vital for the induction and maintenance of aregulatory T-cell phenotype (Josefowicz et al.2012a). Mutations in the X-linked gene encod-ing Foxp3 illustrate the essential role for Foxp3þ

Treg cells in immunological tolerance in bothmice and humans (Ziegler 2006). The majorityof patients with IPEX (immune dysregulationpolyendocrinopathy enteropathy X-linked) syn-drome bear loss-of-function mutations withinthe FOXP3 gene and suffer an early onset fatalautoimmunity (Chatila et al. 2000; Bennett et al.2001; Wildin et al. 2001). Although IPEX syn-drome affects many organs and tissues, the in-testine is most frequently affected, emphasizingthe key role of Foxp3þ Treg cells in establishingand maintaining tolerance within the intestine(Gambineri et al. 2003).

Induction of Foxp3 expression and acquisi-tion of the regulatory T-cell program occurs atboth thymic and extrathymic locations. Thy-mus-derived Foxp3þ Treg cells (tTreg) differen-

tiate in response to high-affinity TCR (T-cellantigen receptor) interactions with MHC (ma-jor histocompatibility complex) class II pre-sented self-antigens (Stritesky et al. 2012; Xingand Hogquist 2012). Extrathymic acquisition ofTreg function and Foxp3 expression is also de-pendent on TCR-signaling but also strongly in-fluenced by environmental factors (Bilate andLafaille 2012). Indeed, there is convincing evi-dence that the intestinal microenvironment is apreferential site of antigen-induced Treg (iTreg)development, most likely reflecting high im-mune challenge and the continuous need foractive immune suppression to maintain intesti-nal homeostasis.

Indeed, results in several mouse models ofcolitis have shown that disruption of regulato-ry T-cell networks can promote chronic intesti-nal inflammation in the presence of a triggeringintestinal microbiota (Izcue et al. 2009). We havefurther dissected control of colitis by regulatoryT cells using a T-cell transfer model. In this mod-el, adoptive transfer of naı̈ve CD4þ CD45RBHi

T cells to a lymphopenic host results in cachex-ia and severe intestinal inflammation (Powrieet al. 1993). Transfer of Foxp3þTreg, or original-ly, populations enriched in regulatory potential(CD45RBLo and then CD45RBLo CD25þ) aresufficient to prevent the onset of, or “cure” on-going, intestinal inflammation in this and othermodels (Powrie et al. 1993, 1994; Maloy et al.2003). Development of colitis is dependent onthe cytokine IL-23, which functions to promoteTh1 and Th17 cell-mediated pathogenic effec-tor responses as well as restrain iTreg differenti-ation in the intestine (Hue et al. 2006; Izcueet al. 2008; Ahern et al. 2010).

The presence of tTreg and iTreg popula-tions in the intestine raises the question of thefunctional roles of these populations (Fig. 1).This has been difficult to decipher, as there arefew markers that distinguish between these twopopulations. In vitro activation of mature naiveCD4þ T cells in the presence of TGF-b1 and IL-2 is now a commonly used method of Foxp3þ

iTreg generation. Although these cells showsome facets of Treg function, as assessed in vitro,the genetic composition of these cells is vastlydifferent to those generated in different tissue

Regulatory T Cells and IT in Intestine

Cite this article as Cold Spring Harb Perspect Biol 2013;5:a018341 3



contexts in vivo (Haribhai et al. 2009; Feuereret al. 2010). Helios, a putative tTreg marker andIkaros transcription factor family member, isencoded by Ikzf2, a gene enriched in the Tregcell signature (Hill et al. 2007; Thornton et al.2010). However, it is now apparent that a sub-stantial proportion of experimentally inducedFoxp3þ iTreg can express Helios, and as such,its use as a marker of Treg origin should beemployed with caution (Verhagen and Wraith2010; Gottschalk et al. 2012). In the absence ofinflammation, the use of neuropilin-1 (Nrp-1)as a selective marker of tTreg appears promising;mucosa-derived iTreg are Nrp-1lo comparedwith their tTreg Nrp-1Hi counterparts (Weisset al. 2012; Yadav et al. 2012). However, the ro-bustness of neuropilin-1 as a marker of tTregand its function in this context will require fur-ther experimental investigation.

Despite these caveats, sophisticated geneticmodels have provided strong support for col-laboration between iTreg and tTreg populationsin control of intestinal inflammation. Thus,efficient prevention of T-cell transfer colitis bytTreg cells requires deviation toward an iTregphenotype by the colitogenic transferred T cells(Haribhai et al. 2009). A nonredundant rolefor iTreg in maintenance of intestinal toleranceunder homeostatic conditions has been shownthrough a series of elegant genetic manipu-lations of the Foxp3 locus. Within the Foxp3locus, conserved noncoding DNA sequences(CNS) promote size, stability, and compositionof the Foxp3þ Treg population (Zheng et al.2010). In vivo, CNS3 deficiency leads to drasti-

cally decreased thymic output of Foxp3þ tTreg,and CNS2-dependent mechanisms appear re-sponsible for heritable maintenance of Foxp3expression in proliferating Treg. Foxp3 CNS1contains a TGF-b/NFAT response element, andsynergistic binding of Smad3 and NFATat CNS1are suggested to be required for Foxp3 induc-tion (Tone et al. 2008). In contrast, CNS1-defi-cient mice have unaffected tTreg populations,but defective induction of Foxp3þ iTreg, partic-ularly within the GALT (gut-associated lym-phoid tissue). This highly selective blockadein extrathymic Foxp3þ Treg induction did notlead to fatal autoimmune disorders, nor exacer-bated Th1 or Th17 responses in the intestine, asmay be expected if there is loss of intestinalimmune tolerance. Instead, complete loss of in-tronic Foxp3 CNS1 manifested as pronouncedTh2 responses, primarily at mucosal sites (Jo-sefowicz et al. 2012b). In line with this, agedCNS1-deficient mice develop B-cell-dependentintestinal inflammation with antibodies to foodand intestinal antigens, elevated IgE and IgA,and accumulation of Gata3þ CD4þ T cells pro-ducing IL-4, IL-5, and IL-13. These data areconsistent with earlier studies showing a keyrole for iTreg cell development in oral tolerancetoward nominal antigens and control of allergicresponses, and illustrate the importance of im-mune deviation toward a Foxp3þ Treg programfor mucosal tolerance (Curotto de Lafaille et al.2008). The reason for the deviation toward Th2-mediated pathology in the absence of iTreg mayreflect the prevailing environmental conditions,such as, for example, the lack of particular mi-

CNS1-independent

Oral tolerance, Fetal tolerance

Self and foreign antigen specificity

Complementary or overlapping functions?

Stability and longevity of iTreg?

Inflammatory context of induction?

Imprinting and heritability of tissue adaptation?

Peripherally induced, CNS1-dependent

Foxp3Enriched self-antigen specificity

Foreign antigen responsiveness of tTreg?

Organ and tissue localization?

Shared or distinct suppressor mechanisms?

Tissue adaptation linked to suppressor mechanisms?

CTLA-4

TCR CD25GITR

Thymic origin, Central tolerance

tTreg iTregNeuropilin-1

Figure 1. Properties of Foxp3þ Treg from thymic and extrathymic origins.





crobial stimuli required to elicit strongly in-flammatory Th1/Th17 responses. Indeed, itwill be of interest to test whether infectionwith known colitogenic bacteria elicits exacer-bated inflammation in CNS1-deficient mice.Alternatively, it is possible that deficiency iniTreg is not sufficient to drive a pathogenicTh1/Th17 response to the microbiota and thatthere are multiple layers of regulation, includingtTreg cells and IgA among others, that contrib-ute to this important function (Feng et al. 2010).

Together, the above results suggest impor-tant roles for both tTreg and iTreg populationsin maintaining intestinal homeostasis. Whetherthere is shared responsibility or specific func-tional roles of these developmentally distinctpopulations is not clear. One obvious possibil-ity is that tTreg and iTreg populations contrib-ute different antigen specificities, increasing thefrequency of potentially responsive Treg popu-lations that can be activated. It is known thatFoxp3þ Treg cells use a broad ab-TCR reper-toire distinct from that of Foxp32 effector Tcells (Hsieh et al. 2006; Pacholczyk et al. 2006).Recent studies indicate that colonic Foxp3þTreguse a TCR repertoire distinct from Foxp3þ Tregfound in systemic tissues, suggesting that thelocal antigenic milieu shapes the colonic Foxp3þ

Treg pool (Lathrop et al. 2011). As may beexpected, microbial specificities are increasedwithin colonic Foxp3þ Treg populations, andtransgenic mice bearing a TCR repertoire de-rived from colonic Foxp3þ Treg could not selectthymic Foxp3þ Treg, suggesting they are specificfor exogenous antigens. Furthermore, when Tcells bearing these TCR were genetically prevent-ed from adopting a Treg cell fate, they developedcolitogenic potential. These data are compatiblewith the notion that the intestinal microenviron-ment supports local differentiation of microbe-reactive iTreg as a means of preventing devel-opment of inflammatory effector responses.This local adaptation to the intestinal micro-environment provides one layer of control thatmay be complemented by tTreg cells, some ofwhich may also be reactive with microbial an-tigens. Indeed, it would be interesting to deter-mine whether thymectomized germ-free micealso show accumulation of Foxp3þ Treg on col-

onization. Further understanding of the specif-icity of intestinal Foxp3 expressing cell popula-tions is required to fully understand the role ofthese cells in antigen-specific tolerance induc-tion.

TOLEROGENIC MODULES USEDBY INTESTINAL Foxp3þ Treg

Although a source of early debate, it is nowwidely accepted that Foxp3þ Treg cells use mul-tiple regulatory mechanisms to control hostimmune responses. Thus, production of regu-latory cytokines (TGF-b, IL-10, and IL-35),modulation of APC function (LAG-3, CTLA-4, Granzyme/Perforin), and alteration of cel-lular metabolism (CD25, CD39/CD73, IDO in-duction in DC), have all been implicated in Tregcell function in distinct inflammatory settings(Vignali et al. 2008; Shevach 2009). We focusbriefly on IL-10 and TGF-b as key cytokinepathways necessary for immune tolerance with-in the intestine.

IL-10, a regulatory cytokine, is produced bymultiple leukocyte populations and protects thehost by limiting inflammatory responses to mi-crobial challenge (Saraiva and O’Garra 2010).IL-10 signals via a cytokine receptor complexconsisting of IL-10Ra and IL-10Rb subunits,promoting STAT3 activation via Jak1 and Tyk2(Glocker et al. 2011). In contrast with the fatalconsequences of Foxp3 deficiency, genetic ab-lation of IL-10 or IL-10R leads to colitis in thepresence of a triggering microbiota (Kuhn et al.1993; Davidson et al. 1996; Sellon et al. 1998).Mutations in genes encoding IL-10 or IL-10Rsubunits are found in patients with very severeearly-onset IBD (eo-IBD) and are associatedwith immune hyperactivation within the intes-tine (Glocker et al. 2009). Many of these patientsare unresponsive to immunosuppressive thera-pies and require bone marrow transplantation.Recent studies suggest up to 15% of eo-IBDcases are attributable to mutations in the IL-10/IL-10R axis, clearly illustrating the impor-tant role of this pathway in intestinal homeosta-sis (Kotlarz et al. 2012). Single nucleotide poly-morphisms in IL10 have also been associatedwith susceptibility to adult ulcerative colitis





and Crohn’s disease, although in this setting thegenetic contribution to disease is modest(Franke et al. 2008, 2010; Jostins et al. 2012).

IL-10 production is enriched within muco-sal tissues and CD4þ T cells at these sites are anabundant source. Furthermore, both Foxp3þ

and Foxp32 populations contribute to the pro-duction of IL-10 within the gastrointestinaltract. However, strikingly there is compartmen-talization of this response, with high levels of IL-10 production in the colon primarily restrictedto Foxp3þ cells and the reverse in the small in-testine where Foxp32 cells represent the majorsource (Uhlig et al. 2006; Maynard et al. 2007).Functional studies support the expression dataas the adoptive transfer of IL-102/2 CD4þ Tcells to lymphopenic hosts results in severe in-testinal inflammation even in the face of hostderived IL-10 production, and development ofcolitis ensues following specific deletion of IL-10 from CD4þ T cells (Davidson et al. 1996;Asseman et al. 1999; Roers et al. 2004). Earlystudies showed that IL-10-deficient Treg cellscould prevent, but not cure, colitis in a T-celltransfer model (Asseman et al. 1999; Uhlig et al.2006). In a lymphocyte-replete model, deletionof IL-10 from Foxp3þ Treg also leads to intes-tinal inflammation, albeit with less severitythan deletion in total CD4þ T cells, implicatingCD4þ Foxp32 cells as a functional source of IL-10 that contributes to intestinal homeostasis(Rubtsov et al. 2008).

HOW DOES IL-10 ACT TO PROMOTEINTESTINAL TOLERANCE?

The IL-10 receptor complex is expressed on in-nate and adaptive immune cell populationswithin the intestine and acts to control intes-tinal CD4þ T cells through direct and indi-rect mechanisms. Myeloid-specific ablation ofSTAT3 leads to spontaneous colitis driven bydysregulated CD4þ T-cell responses. In this set-ting, defective IL-10R signaling in colonic mac-rophages results in elevated levels of costimu-latory molecules CD80/86 and hyperactivationof colitogenic CD4þ T cells (Takeda et al. 1999;Kayama et al. 2012). Direct IL-10R signalinginto CD4þT cells also serves to limit colitogenic

responses within the intestine. Intestinal Th17and Foxp3þ Treg express IL-10Ra, and CD4-specific expression of a dominant-negative IL-10Ra, results in selective expansion of Th17and IFN-g/IL-17A coproducing populationsduring intestinal inflammation (Huber et al.2011). Under these circumstances, IL-10 pro-duction from both Foxp3þ and Foxp32 CD4þ

T cells is required to control colitis.In addition to effects on effector T cells,

there is also accumulating evidence that IL-10R signaling into Foxp3þ cells is importantfor their function. Thus, IL-10Rb2/2 Foxp3þ

Treg cells fail to protect mice from intestinal in-flammation following naı̈ve T-cell transfer(Murai et al. 2009). In that study, IL-10Rb2/2

Treg cells lost Foxp3 expression and regulatoryfunction following transfer into a lymphopenia-induced disease setting. In a genetic model, spe-cific deletion of IL-10Ra in Foxp3þ Treg cellsresulted in spontaneous intestinal inflammationcharacterized by selective dysregulation of Th17cells (Chaudhry et al. 2011). However, in thatand other lymphocyte replete settings, Foxp3þ

Treg cells show remarkable stability and are notobserved to lose expression of Foxp3 (Rubtsovet al. 2010; Chaudhry et al. 2011). Whether theloss of Foxp3 expression in lymphopenia-drivencolitis represents a failure to maintain the Tregprogram or is a specific consequence of the celltransfer system requires further study, especiallygiven the interest in Treg cell therapy in the clin-ical setting. Finally, it should not be overlookedthat many other cell types produce IL-10, in-cluding B cells and macrophages, and there isevidence that this plays a functional role in in-testinal homeostasis (Mizoguchi et al. 2002;Murai et al. 2009; Hadis et al. 2011).

TRANSFORMING GROWTH FACTOR(TGF)-b

The transforming growth factor (TGF)-b fam-ily of cytokines have developmental and physi-ological roles throughout the mammalian life-span. Encoded by individual genes, the TGF-bfamily consists of three isoforms, TGF-b1, TGF-b2, and TGF-b3, which signal via the TGF-bR complex (TGF-bRI and TGF-bRII). TGF-





bR activation triggers intrinsic kinase activityand phosphorylation of downstream signalingcomponents, of which transcription factorsSmad2/3 are an example (Derynck and Zhang2003). Inhibitory Smads, for example, Smad7,act to negatively regulate TGF-bR signaling,making this a finely tuned system. Furthercontrol occurs at the posttranscriptional levelas TGF-b is secreted to the cell surface in a la-tent complex, requiring further posttranslation-al modification to become biologically active.Activation of the latent TGF-b1 complex re-quires enzymatic cleavage of latency-associatedpeptides, or integrin-mediated conformationalchange. The dominant role for TGF-b1 in im-munological tolerance is highlighted by the T-cell-dependent, early onset, fatal autoimmunedisease of TGF-b12/2 mice that occurs withinweeks of birth (Shull et al. 1992; Diebold et al.1995; Letterio et al. 1996; Kobayashi et al. 1999).Similar to Foxp3 deficiency, complete ablationof TGF-b1 production, or TGF-bR expressionin T cells, leads to catastrophic autoinflamma-tory disease early after birth. In contrast withcomplete loss of this pathway, quantitative re-ductions in mice with impaired but not abol-ished TGF-b regulatory pathways leads to intes-tinal inflammation, indicating the intestine isparticularly sensitive to TGF-b signaling. Ge-netic perturbation of Smad signaling, throughloss of Smad3/4, or forced expression of Smad7results in intestinal inflammation, implicatingactive TGF-b in intestinal tolerance. Likewise,inability to activate latent TGF-b in either T cellor myeloid cells results in loss of intestinal ho-meostasis. Mice with APC-specific ablation ofintegrin subunits, av or b8, or T-cell-specificdeletion of convertase furin are prone to devel-opment of intestinal inflammation (Lacy-Hul-bert et al. 2007; Travis et al. 2007; Yang et al.2007; Pesu et al. 2008). In addition to the im-portance of T-cell-produced TGF-b1, it is alsocrucial that T cells can respond to this cytokine(Li et al. 2006). Naı̈ve T cells unable to respondto TGF-b cannot be controlled by Treg cells(Fahlen et al. 2005); however, whether TGF-bmediates suppression by inhibition of prolifer-ation, acquisition of colitogenic potential, orgeneration of iTreg cell populations warrants

further investigation. Furthermore, the distinctsignaling pathways initiated by different TGF-bisoforms remains to be examined in this andmultiple other contexts.

Foxp3þ Treg-CELL SPECIALIZATIONIN THE INTESTINE

An emerging concept in T-cell biology is that ofadaptation to the tissue environment and con-text-dependent specialization of the adaptiveimmune response is well documented (Littmanand Rudensky 2010). Differentiation of CD4þTcells to distinct effector populations is depen-dent on appropriate TCR signaling, costimula-tion, and surrounding cytokine milieu. Expres-sion of lineage-specifying transcription factorsT-bet, Gata3, and ROR(g)t is required for Th1,Th2, and Th17 responses, respectively (Zhengand Flavell 1997; Szabo et al. 2000; Ivanov et al.2006). However, expression of these differenti-ation programs is not mutually exclusive and itis increasingly appreciated that interplay be-tween these factors contributes to the fate andflexibility of effector T-cell responses (Oestreichand Weinmann 2012). As has been described foreffector T-cell populations, it is now evident thatFoxp3þTreg can acquire expression of transcrip-tion factor modules that allow context-depen-dent immune suppression (Barnes and Powrie2009). For example, in response to IFN-g or IL-27, Foxp3þ Treg cells up-regulate expression ofthe Th1 cell transcription factor, T-bet (Kochet al. 2009; Hall et al. 2012). T-bet expressionin Foxp3þ Treg promotes CXCR3 expressionand migration to Th1-inflamed sites facilitatingcontrol of the Th1 response (Koch et al. 2009).Equally, the reliance of Th17 cells on STAT3-de-pendent cytokines for their differentiation ismirrored by the STAT3-dependency of Foxp3þ

Treg-mediated control. In that case, targeted de-letion of STAT3 (or IL-10Ra, which signals viaSTAT3) in Foxp3þ Treg lead to severe intestinalinflammation dominated by accumulation ofTh17 cells (Chaudhry et al. 2009, 2011).

In contrast with the Th1-context-dependentexpression of T-bet in Foxp3þTreg, the majorityof peripheral Foxp3þ Treg express transcriptionfactors IRF4 and/or Gata3, suggesting these





transcription factors may play more general rolesin Treg cell function (Zheng et al. 2009; Wanget al. 2011; Wohlfert et al. 2011; Rudra et al.2012). Indeed, the specific loss of IRF4 inFoxp3þ Treg results in an autoimmune syn-drome characterized by mucosal Th2 responses,similar in reported phenotype to mice deficientin Foxp3 CNS1 (Zheng et al. 2009). IRF4 seemsto be an important pioneer transcription factorinvolved in multiple T-cell effector phenotypesincluding Th2, Th17, and Follicular Helper T-cell (TFH) differentiation (Lohoff et al. 2002; Re-ngarajan et al. 2002; Huberet al. 2008; Bollig et al.2012; Glasmacher et al. 2012). Based on thesefindings, IRF4 may play multiple roles in Foxp3þ

Treg cells that promote specific modules of sup-pression. In support of this, Blimp-1 is a regula-torof IL-10 production and its expression in Tregis dependent on IRF4 (Cretney et al. 2011). Fur-thermore, IRF4-deficient Foxp3þ Treg cells areunable to control Th1 and Th17 responses in aT-cell transfer model of colitis (Zheng et al.2009). Further understanding of the variousmolecular mechanisms through which IRF4 ex-pression in Foxp3þTreg cells contributes to mu-cosal immune tolerance is required.

Beyond its essential role in embryogenesis,within the hematopoietic system, the trans-cription factor Gata3 is required for thymic T-cell development and mature CD4þ T-cell-dif-ferentiation to a Th2 lineage (Zheng and Flavell1997). In addition, this lineage-specifying fac-tor promotes immune tolerance through tran-scriptional enhancer activity at Foxp3 CNS2 tomaintain Foxp3 expression in regulatory T cells(Wang et al. 2011). Genetic ablation of Gata3 inFoxp3þ Treg results in a systemic autoimmunesyndrome, dermatitis, and intestinal inflamma-tion (dependent on experimental system) dueto reduced expression of Foxp3 mRNA and pro-tein on a per-cell basis and associated decreasein regulatory capability (Wang et al. 2011;Wohlfert et al. 2011; Rudra et al. 2012). Along-side decreased Foxp3 expression, Foxp3þ Treg-specific ablation of Gata3 results in uncon-trolled intestinal Th2 responses, with increasedfrequencies of Gata3þ, IL-4, 5, and 13 produc-ing populations in the intestinal lamina propria(Rudra et al. 2012) (Fig. 2A).

Although it is clear that Foxp3 and Gata3cooperate to promote intestinal tolerance, theprecise mechanisms are not known. Foxp3 me-diates its effects at a molecular level throughmultimeric protein complexes, and directly reg-ulates the expression of many of its interactingpartners, including Gata3 (Rudra et al. 2012).Cooperation between Foxp3 and Gata3 pro-motes the Treg transcriptional program andsubset stability. In the case of CNS1-deficientmice, it is possible that reduced Foxp3 expres-sion during iTreg induction leads to sole bind-ing of Gata3 at classically Foxp3/Gata3 co-bound sites, resulting in an aberrant Gata3transcriptional response that leads to mucosalTh2 pathologies. As such, the mucosal Th2 re-sponse observed in CNS1-deficient mice mayrepresent a cell intrinsic failure of effector T cellsto divert their fate to a Foxp3þ iTreg phenotype,rather than specific requirement for Foxp3þ

iTreg cells to control aberrant Th2 responses tothe local antigenic milieu (Fig. 2B).

It is important to note that expression levelsof effector T-cell transcription factors by Foxp3þ

Treg cells are low relative to their Foxp32 coun-terparts, suggesting that Foxp3þ Treg cells mayuse this strategy to adopt some, but not all as-pects of distinct effector programs. For example,the acquisition of homing receptors, CXCR3in the example of T-bet, without productionof proinflammatory cytokine IFN-g, rendersFoxp3þ Treg cells able to migrate, but not con-tribute, to Th1-driven pathologies. In the in-stance of T-betþ Foxp3þ Treg, incomplete com-mitment to an effector phenotype is regulated bysuppression of IL-12Rb2 expression requiredfor STAT4-mediated acquisition of effectorfunction (Koch et al. 2012). The mechanismsby which IFN-g/STAT1 signals in effector Tcells, but not Foxp3þ Treg, lead to IL-12Rb2expression remain to be elucidated. As yet,whether the role of effector T-cell-associatedtranscription factors in Foxp3þ cells is similarfor tTreg and iTreg populations remains to beexamined. Interestingly, Gata3 has been shownto inhibit the induction of Foxp3 in TGF-b-induced iTreg (Mantel et al. 2007), in com-parison to the requirement for maintenance ofFoxp3 in mature Treg.





Adaptation of Foxp3þ Treg to the tissue mi-croenvironment and the ability to use multipleregulatory mechanisms is key to instigating andmaintaining active immunological tolerance.Distinct antigen specificities, enrichment inregulatory cytokine production and utilizationof effector transcription factor networks show

that adaptation of Foxp3þ Treg to the tissuemicroenvironment is critical in maintainingintestinal immune tolerance. The role of thehost immune system in continuous toleranceto the microbiota is evident; yet, the commensalmicrobiota itself is intimately involved in thedynamic molecular dialogue required for im-

WT

A

B

Normal Foxp3 expressionFunctional Gata3 regulatory moduleTh2 immune tolerance

Mucosal immune toleranceRegulated Th2 response

Absent Gata3 regulatory moduleImpaired Foxp3 expression

Foxp3+

Gata3+

Foxp3+

Treg cell

Foxp3+

iTreg

NaïveT cell WT

Th2 deviation fromiTreg precursor

Dysregulated Th2 responseIntestinal inflammation

Defective iTreggeneration

CNS1–

Gata3-deficient

Dysregulated Th2 responseIntestinal inflammation

Figure 2. Foxp3þ Treg specialization on microenvironment adaptation maintains mucosal immune tolerance.(A) Gata3-dependent mechanisms promote Foxp3 expression and regulation of mucosal Th2 responses. Wheth-er these mechanisms are distinct is not known. (B) CNS1 deficiency and loss of Foxp3þ iTreg-mediatedregulation of mucosal Th2 responses may occur through cell-extrinsic (absolute requirement for Foxp3þ iTregin control of Th2 responses) or cell-intrinsic (naı̈ve T-cell precursors to Foxp3þ iTreg deviate to Th2 phenotypein absence of CNS1) mechanisms.





mune tolerance at both mucosal and peripheralsites.

MICROBIOTA AND DIETARY METABOLITESAFFECT INTESTINAL CD4þ T-CELL SUBSETS

Colonization of the host by the commensal mi-crobiota and the presence of multiple dietarycomponents is key to proper mucosal immunecell function (Fig. 3). Indeed, individual bacte-rial species promote differentiation of distinctintestinal T-cell populations. Reared in sterilesurrounds, and on a diet free of live microbes,germ-free (GF) mice provide a useful experi-mental tool in understanding the relationshipbetween host immune system and commensalmicrobiota. Controlled colonization of germ-

free mice allows analysis of the role for individ-ual or multiple bacterial species in mutualisticinteractions with the host immune system.

Segmented filamentous bacteria (SFB) areGram-positive members of the Clostridiales ge-nus able to penetrate the small intestinal mucuslayer and interact intimately with the epitheli-um to colonize the host. Colonization withSFB results in accumulation of effector T cellswithin the small intestine, primarily Th17, andto a lesser extent, Th1 cells (Gaboriau-Routhiauet al. 2009; Ivanov et al. 2009). The mechanismsby which SFB promotes effector T-cell polari-zation and accumulation within the intestineremain largely unknown. Whether direct in-teractions between SFB and the intestinal epi-thelium, host immune cells, or production of

Clostridia

IEC

APCTGF-β1?

IL-10TGF-β1

RATGF-β1

IL-17AIFN-γ

EffectorT cellSTAT3-activating cytokines

Systemic inflammationEAE + arthritis

Systemic immunityOral tolerance

Foxp3+ Treg

Helicobacterhepaticus Microbiota

?

? ?

Segmented filamentous bacteria

Figure 3. Commensal microbiota and dietary metabolites influence intestinal T-cell adaptation to direct mu-cosal and peripheral immune responses. The intestinal mucosa is constantly exposed to a diverse array of foreignantigens, including dietary antigens, metabolites, and components of the commensal microbiota. The presenceof distinct microbial species promotes specialized host immune responses through differentiation of appropriateeffector and regulatory T-cell populations. Segmented filamentous bacteria promote effector T-cell accumula-tion, particularly of Th17 cells, within the intestine. Accumulation of Foxp3þ regulatory T-cell populations andinduction of IL-10 production arise in response to colonization of germ-free mice with a restricted flora(Clostridiales IV and XIVa, or Altered Schaedler Flora), or conventionally housed mice with Helicobacter hep-aticus. In both cases, the mechanism of induction and antigen-specificity of the response require furtherinvestigation. Antigen-presenting cell production of soluble mediators heavily influences the balance betweeneffector and regulatory T-cell responses. Production of STAT3 activating cytokines (IL-6, IL-23, IL-21) favorseffector T-cell differentiation, whereas retinoic acid (RA) and TGF-b1 production modulate the balance be-tween effector and regulatory responses, depending on the inflammatory microenvironment and associatedcytokine production.





unique metabolic modulators is required forinduction of effector T cells is not understood.Equally, whether the immune response to SFBcolonization is antigen-specific remains to beestablished. Identifying the genetic attributesof SFB that drive this highly immunostimula-tory response will be crucial to understandinghow distinct bacterial species modulate the mu-cosal immune system.

Regulatory T-cell populations are also af-fected by microbial colonization, which canlead to accumulation of intestinal Foxp3þ Tregpopulations and induction of IL-10 production(Honda and Littman 2012). For example, Bac-teroides fragilis is a human commensal able toinduce IL-10 production from intestinal T cellsand limit Th17 responses during intestinal in-flammation (Round and Mazmanian 2010).Colonization of germ-free mice with a cocktailof 46 spore-forming murine Clostridia strainspromotes accumulation of colonic Foxp3þ

Treg (Atarashi et al. 2011). The nature of theexact strains, microbial attributes, and associat-ed host molecular responses to this complexgnotobiotic mixture are as yet unknown. How-ever, accumulation of Foxp3þ Treg and induc-tion of IL-10 production are associated with in-creased colonic levels of active TGF-b1 in thesegnotobiotic mice. As discussed earlier, althoughthe reliability of Helios expression to determinetTreg from iTreg is still controversial, Clostri-dum-colonized gnotobiotic mice showed a shiftfrom Heliosþ Foxp3þ to Helios2 Foxp3þ Tregfollowing colonization, indicative of iTreg gen-eration. The antigen-specificity of Clostridia-induced colonic Foxp3þ Treg was not assessed,but in the context of TCR repertoire of colonicFoxp3þ Treg, it is possible to hypothesize thatthese iTreg would bear TCR-specificity for Clos-tridia-derived epitopes. Furthermore, analysisof TCR repertoire in this setting may aid iden-tification of individual Clostridia strains re-sponsible for Foxp3þ Treg accumulation and/or IL-10 induction. In addition, colonizationwith a benign altered Schaedler flora (ASF) pro-moted accumulation of colonic Helios2 Foxp3þ

Treg that served to promote intestinal toleranceduring experimental DSS colitis (Geuking etal. 2011). Similarly, infection with the mouse

pathogen Helicobacter hepaticus induces an IL-10-producing Treg response, which when miss-ing leads to bacteria-driven chronic intestinalinflammation (Kullberg et al. 2002). Together,these data suggest that induction of a Treg/IL-10-producing response may be an impor-tant part of host-commensal mutualism allow-ing persistence of microbes in the absence of adamaging inflammatory response. It is clearlyof interest to further understand the molecu-lar pathways and metabolites that control mi-crobe-induced Treg function, as these may pro-vide novel immune modulators and underliethe poorly characterized properties of probioticbacteria. Clostridia are fermenting bacteria andit is notable that fermentation products such asshort-chain fatty acids promote gut health(Maslowski et al. 2009). Whether iTreg induc-tion and IL-10 production following bacterialcolonization requires constant microbial expo-sure remains to be examined and the use ofreversible colonization systems will be a usefultool to examine this question.

Retinoic acid (RA) is a metabolite of diet-derived vitamin A, a nutrient essential for mul-tiple physiological processes including an arrayof immunological functions (Hall et al. 2011b).Within the GALT, RA promotes both regulatoryand effector immune responses through im-printing of intestinal homing receptors a4b7and CCR9 on activated T cells, and acts as acofactor in the de novo generation of Foxp3þ

iTreg by CD103þDC (Coombes et al. 2007; Sunet al. 2007). Induction of Foxp3 expressionby CD103þ DC is dependent on TGF-b1 andis enhanced by RA, accordingly CD103þ DChighly express mRNA for aldh1a1 and aldh1a2,the retinal metabolizing enzymes required forRA synthesis (Coombes et al. 2007). In vivocorroboration of the role for RA in iTreg in-duction stems from the observation that micereared on a vitamin-A deficient diet are defec-tive in iTreg generation on oral antigen expo-sure (Hall et al. 2011a). As such, during in-testinal homeostasis, RA promotes regulatoryresponses to bolster immunological toleranceto dietary antigens. However, recent studiesshow context-dependent roles for this metabo-lite in intestinal immune tolerance. Thus, RA





acts to promote effector responses in settings ofintestinal inflammation. Oral infection withToxoplasma gondii is controlled by a strong in-testinal Th1 response, which is impaired in vi-tamin-A deficient mice (Suzuki et al. 1988; Hallet al. 2011a). Furthermore, normal tolerance todietary antigens is disturbed in patients withCeliac disease, whereby aberrant immune re-sponses to gluten, a component of wheat, resultin intestinal inflammation. Contrary to its rec-ognized role in oral tolerance, RA, in combina-tion with IL-15, a cytokine highly abundant inthe mucosa of patients with Celiac disease, pro-motes proinflammatory cytokine productionfrom intestinal APC to drive intestinal inflam-mation (DePaolo et al. 2011). As such, RA actsas a proinflammatory coadjuvant in settings ofintestinal stress, while promoting intestinal tol-erance during homeostasis.

THE INTESTINAL MICROBIOTA AFFECTSSYSTEMIC IMMUNITY

The effect of the commensal microbiota on dis-tinct lymphocyte subsets modulates immunefunction and tolerance beyond the intestinalmicroenvironment. Many models of autoim-munity or chronic inflammation are attenu-ated in the absence of a commensal microbiota(Chervonsky 2010). Although Th17 cells arestrongly associated with immunopathology inmultiple models of autoimmunity, colonizationwith SFB does not result in immunopathologyand instead aids in protection from intestinalinfection with murine pathogen Citrobacter ro-dentium (Ivanov et al. 2009). However, coloni-zation with SFB is required for inflammation inmodels of intestinal, joint, and central nervoussystem pathologies (Stepankova et al. 2007;Wu et al. 2010; Lee et al. 2011). Therefore, SFBcolonization can promote tissue pathology inexperimental settings predisposed to chronicinflammation, either in the absence of Treg (co-litis) or in autoimmune prone strains (arthritisand experimental autoimmune encephalomy-elitis [EAE]). However, whether SFB can beclassed as a true commensal species is conten-tious, given its intimate interaction with the in-testinal epithelium. The regulatory response to

SFB remains ill-defined. Although clearly highlyimmunostimulatory, SFB is not essential for tis-sue inflammation. Transgenic SJL/J mice thatexpress a myelin peptide-specific TCR developrelapsing-remitting EAE under conventional,but not GF conditions, indicating a requirementfor microbial colonization as a triggering fac-tor for disease pathogenesis (Berer et al. 2011).The colonization of GF mice with commensalmicrobiota, but not monocolonization withSFB, results in the acquisition of relapsing-re-mitting disease, highlighting the requirementfor microbiota-derived signals as priming stim-uli to trigger tissue pathologies in settings al-ready predisposed to chronic inflammation. Inthe context of intestinal inflammation, the ex-plosion in knowledge and techniques used indetailing the intestinal microbiota will be fun-damental to understanding whether dysbiosisrepresents a triggering factor or is a consequenceof inflammation, particularly given the high fre-quency of IBD-susceptibility SNPs within thehealthy population.

CONCLUSION

The mucosal immune system promotes intes-tinal tolerance by controlling localization andcomposition of the commensal microbiota.Equally, the commensal microbiota is a funda-mental stimulus for priming and activation ofboth mucosal and peripheral immune respons-es. However, dysregulated immune responsesto the commensal microbiota have profoundeffects on human health. The dynamic inter-actions between microbiota and the immunesystem represent the key to targeted future ther-apies in chronic intestinal inflammation. Touse natural tolerogenic mechanisms occurringunder homeostatic microbial-mammalian dia-logue, we require not only greater understand-ing of how the immune system regulates themicrobiota, but equally how the distinct micro-bial communities influence host immunity.

ACKNOWLEDGMENTS

The authors are grateful for support from theWellcome Trust (PhD Studentship to O.J.H.,





Investigator Award to F.M.P.) and for helpfuldiscussions from past and present members ofthe Powrie and Maloy research groups.

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2013; doi: 10.1101/cshperspect.a018341Cold Spring Harb Perspect Biol Oliver J. Harrison and Fiona M. Powrie Regulatory T Cells and Immune Tolerance in the Intestine

Subject Collection Immune Tolerance

IntestineRegulatory T Cells and Immune Tolerance in the

Oliver J. Harrison and Fiona M. PowrieIntestineRegulatory T Cells and Immune Tolerance in the

Oliver J. Harrison and Fiona M. Powrie

Immunological ToleranceDendritic Cells: Arbiters of Immunity and

Kanako L. Lewis and Boris Reizis

Microbiota and AutoimmunityAlexander V. Chervonsky

to Restore Tolerance in Autoimmune DiseasesCurrent and Future Immunomodulation Strategies

Jeffrey A. Bluestone and Hélène Bour-Jordan

Treg Cells, Life History, and DiversityChristophe Benoist and Diane Mathis

T-Cell Tolerance: Central and PeripheralYan Xing and Kristin A. Hogquist Commensalism Gone Awry?

Frustrated−−Infectious (Non)tolerance

Jesse C. Nussbaum and Richard M. LocksleyCentral B-Cell Tolerance: Where Selection Begins

Roberta Pelanda and Raul M. TorresHistorical Overview of Immunological Tolerance

Ronald H. Schwartz

DiseaseThe Immunogenetic Architecture of Autoimmune

An Goris and Adrian ListonSelf-Control?Natural Killer Cell Tolerance: Control by Self or

Baptiste N. Jaeger and Eric Vivier

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