Review

Structure and function of lymphoid tissues

Lymphoid microenvironments andinnate lymphoid cells in the gutClaire Pearson, Holm H. Uhlig and Fiona Powrie

Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, John Radcliffe Hospital,

University of Oxford, Oxford OX3 9DU, UK

Gut-associated lymphoid tissue (GALT) is a sensor re-gion for luminal content and plays an important role inlymphoid maturation, activation and differentiation. Itcomprises isolated and aggregated lymphoid follicles,cryptopatches (CPs) and tertiary lymphoid tissue. Innatelymphoid cells (ILCs) play a central role within GALT.Prenatal GALT development is dependent on ILC lym-phoid-inducer function. Postnatally, these cells rapidlyrespond to commensal and pathogenic intestinal bacte-ria, parasites and food components by polarized cyto-kine production [such as interleukin (IL)-22, IL-17 or IL-13]and further contribute to GALT formation and function.Here, we discuss how ILCs shape lymphoid intestinalmicroenvironments and act as amplifier cells for innateand adaptive immune responses.

Innate lymphoid cells (ILCs) as regulators of the gutmicroenvironmentCoexistence of vertebrates with selected commensal intes-tinal microbiota is not only part of a metabolic strategy toextract nutrients from food, but also acts as a defencemechanism. Interactions between epithelial cells, stromalcells, and innate and adaptive haematopoietic immunecells form the basis for a beneficial mutualistic relationwith commensal microbiota, and also for effective defencestrategies towards pathogens [1,2]. To promote appropri-ate immune responses in the gut, complex networks ofspecialised cells have evolved that act within the laminapropria and gut associated lymphoid tissue (GALT). With-in the GALT, cryptopatches (CP), isolated lymphoid folli-cles (ILFs), aggregated lymphoid follicles [such as Peyer’spatches (PPs)] and ILF-like tertiary lymphoid tissue (TLT)can be distinguished based on differences in cellular com-position, genetically predetermined developmental factors,as well as dependence on environmental triggers andresponse during tissue inflammation [3].

ILCs are an evolutionarily conserved and developmen-tally related family ofhaematopoietic cells that contribute tohost defence and immune homeostasis. Classical membersof the ILC family are natural killer (NK) cells and lymphoidtissue inducer (LTi) cells. However in the past three years,related but functionally distinct ILC populations have beendiscovered, engendering intense interest in their lineage

Corresponding author: Powrie, F. (fiona.powrie@ndm.ox.ac.uk).Keywords: gut-associated lymphoid tissue; innate lymphoid cell; lymphoid inducercell

1471-4906/$ – see front matter � 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.it.2

relationship and contribution to intestinal physiology andpathophysiology [4]. One of themost striking findings is thefunctional similarity between ILC subpopulations andadaptive CD4+ T helper cell subsets. ILC subsets are con-trolled by transcription factor modules and promptly pro-duce downstream effector cytokines such as IFN-g, IL-17,IL-22 and IL-13 that are homologous to defined Th subsets[4–7]. Based on these results, it is tempting to speculate thatcytokine-driven immune effector modules present in ILCsrepresent a primitive form of host defence subsequentlyacquired by the adaptive immune system.

To date, four distinct new ILC populations, termed LTi-like, NK22, CD4– NKp46– and nuocyte/natural helpercells, have been described and their properties are sum-marised in Box 1.

Alongside the established role of ILCs in GALT devel-opment, it is now evident that ILCs have additional func-tions throughout life beyond that of prenatal lymphoidtissue organisation. Here, we review the developmentallyprogrammed and environmentally driven functions of ILCpopulations; particularly their cytokine production andresponsiveness, which shape the gut microenvironmentand contribute to host protection and tissue inflammation.

LTi cells in the development of GALTLTi cells were originally identified in lymph nodes [8], andtheir function in the formation of lymphoid tissues, includ-ing GALT, is well described [9]. Classical LTi cells arefound prenatally and shortly after birth. A low frequency ofcells with a similar phenotype is also present in adults.However, it is not known whether these represent persis-tence of foetal populations or a developmentally distinctLTi-like population. Both foetal LTi and adult LTi-likecells express interleukin 7 receptor (IL-7R) a, and lympho-toxin (LT) as well as the transcription factor RAR-relatedorphan receptor (ROR)gt [10,11]. There are two RORg

isoforms, RORg and RORgt. The latter is shorter andexpression is restricted primarily to the thymus, Th17 cellsand certain ILC populations, including LTi cells. However,the two isoforms are very closely related, and it has notalways been possible to distinguish the relative contribu-tions of each isoform because cells expressing RORgt usu-ally also express RORg [12]. RORgt is absolutely requiredfor LTi development [10] and signalling via IL7–IL-7Ra

leads to RORgt as well as LT expression [13]. LT surfaceexpression on LTi cells signals to organiser stromal cells

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Box 1. ILC populations found within the gut

All gut ILC populations share a common requirement for the

transcriptional regulator inhibitor of DNA binding 2 (Id2). LTi, LTi-

like, NK22 and CD4–NKp46– cells also depend on expression of

RORgt. All ILCs are negative for lymphoid lineage markers B220,

CD11b, CD3 or T cell receptor (TCR)b, but do express Thy1 and the

IL-7 receptor.

LTi and LTi-like cells

Definition: LTi cells are present in the embryo and organise

lymphoid tissue during foetal development [8]. Recently, cells with a

similar phenotype have been described in adult individuals [55].

Whether these LTi-like cells still mediate lymphoid inducer function

is unclear.

Location: All lymphoid structures including PPs, CPs and ILFs

throughout the intestinal tract.

Phenotype: LTi/LTi-like cells express IL-7 receptor a, LT a and b,

cKit, CCR6, CCR7 and chemokine CXC receptor (CXCR)5 [4]. In mice,

classical LTi cells coexpress CD4 and cKit. However a murine

population of CD4– LTis has been described and human LTis do not

express CD4 [55]. LTi-like cells primarily secrete IL-17 and IL-22

[5,55].

NK22 cells

Definition: NK22 cells express NK surface markers but lack

perforin and granzyme, and do not induce cell lysis. Human NK22

cells are IL-7-dependent and IL-15-independent [54].

Location: Small intestine and colon but have also been found in

the tonsils [6,54].

Phenotype: NK22 cells express NKp46 in mice, and NKp44 in

humans, and secrete IL-22 [6,18,53].

CD4– NKp46– cells

Definition: A phenotypically distinct ILC population that has a

pathogenic role in mouse models of colitis [39,50].

Location: Predominantly found in the colon and the caecum, and

less frequently in the small intestine [39,50].

Phenotype: This population lacks the expression of CD4, NKp46

and cKit, but expresses Stem cell antigen (Sca)-1 [39,50]. CD4–

NKp46– cells are IL-23-responsive and have been shown to secrete a

spectrum of cytokines such as IL-17A, IFNg and IL-22 [50].

Natural helper cells/nuocytes

Definition: Natural helper cells/nuocytes are IL-25- and IL-33-

responsive ILCs [61,62]. They are RORgt-independent but require

RORa and NOTCH signalling for their development. They do not

have in vitro LTi activity [62,63].

Location: Natural helper cells are found in FALCs in human and

mouse mesentery [62]. Nuocytes were described in mLNs and lungs

[61].

Phenotype: Natural helper cells and nuocytes express the IL-33R

and IL-25 receptor component IL-17RB. Murine cells are cKit+ and

Sca-1+ but RORgt–. Human IL-25- and IL-33-responsive ILCs express

the surface markers CD161 and CRTH2 and express no or low levels

of RORg [66]. After stimulation with IL-33 and IL-25, these cells

secrete IL-4, IL-5 and IL-13.

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that express LTbR leading to expression of adhesion mole-cules such as intercellular cell adhesionmolecule (ICAM)1,and subsequent secretion of chemokines involved in therecruitment of haematopoietic cells, particularly B and Tcells [13]. Thus, through a series of complex signallingevents, LTi cells play a pivotal role in orchestration oforganised lymphoid tissue development [14].

CPs

CPs (reviewed in [15]) are found after birth throughout thesmall intestine and colon. CPs contain dendritic cells(DCs), common lymphoid progenitor (Lin– cKit+ IL-7Ra+)cells, LTi-like cells [16] and a few NKp46+ NK22 cells[17,18] (Figure 1). In a mouse model lacking NK22 cellsand most LTi/LTi-like cells due to an absence of thetranscription factor aryl hydrocarbon receptor (Ahr), CPs

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were notably absent [19]. A more severe loss of ILCs occursin the absence of RORgt, resulting in the complete absenceof all forms of GALT [20]. CPs are present in recombinationactivating gene (Rag)-deficient hosts that lack T and Bcells, as well as in germ-free animals, demonstrating theevolutionarily ancient and genetically determined innatefunction of LTi/LTi-like cells.

Although CPs have been described in mice [21], theyhave not been found in humans. However, it was recentlyproposed that clusters of chemokine CC receptor (CCR)6-positive cells detected in the human intestine identify LTicells and CP-like aggregates [22]. The functional relevanceof CPs is a source of debate due to conflicting data onwhether they promote thymic-independent generation ofintraepithelialab and gdT cells [15,23,24], or whether theyare simply part of dynamic and interconvertible GALTstructures with other functions [25].

ILFs

ILFs have been described in both the small intestine andcolonofhumansandmice [26,27]. ILFsare composedmainlyof B cells surrounded by LTi/LTi-like cells, DCs and stromalcells [28,29]. B cells within ILFs preferentially differentiateinto IgA+ plasma cells facilitating local intestinal immunity[30]. ILFs may develop from CPs in a nucleotide oligomer-isation domain (NOD)-dependent manner through interac-tions with microbiota [21,31,32]. As NODs recognisepeptidoglycans from bacterial components, this provides afunctional link between commensal bacteria and the devel-opment of ILFs. Indeed, administration of antibiotics canreverse ILF formation [33], reinforcing the concept thatmicrobial elements can reversibly modulate the develop-ment of structural components of the GALT. As with CPs,ILF formation is completely abrogated inmice lackingNK22cells andwith reduced numbers of LTi-like cells [19]. Recentstudies suggest that CPs and ILFsmay be linked. Colonisa-tion of germ-free animals with commensal bacteria leads toadjustment of the solitary lymphoid clusters, shifting fromCP-like clusters towards mature isolated lymphoid follicles[31].Basedon thesedata, it shouldbenoted that theabsenceof ILFs in mice lacking NK22 cells may be secondary to alack of CPs rather than a direct requirement for ILCs inmaturation of ILFs.

PPs and PP-like aggregated lymphoid follicles

PPs are organised aggregated lymphoid follicles primarilylocated in the ileum [34]. However, there are well docu-mented equivalent lymphoid structures located at thecaecal tip in mice or the appendix vermiformis in humans,and colonic patches have been described [35]. Similar tolymph node development, PP anlagen are formed in thefoetus and do not require the presence of intestinal micro-biota. However, maturation and germinal centre formationdoes not occur until after birth in humans [36]. This isconsistent with the requirement of the intestinal micro-biota for PP maturation in mice [37].

The PP/PP-like aggregated lymphoid tissues function inintestinal luminal content surveillance, allowing rapidmucosal amplification of B and T cell responses. The folli-cle-associated epithelium contains specialised microfold orM cells as well as intra- and transepithelial DCs (Figure 1).

[(Figure_1)TD$FIG]

Cryptopatch

Peyer’s patch

Isolated lymphoidfollicle

Mesenteric LN

B cell

Key:

T cellILC

Dendritic cell

Monocyte/macrophage

M cell

Epithelial cell

Steady state Inflammation

Tertiary lymphoidtissue

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Figure 1. Gut lymphoid microenvironment in health and disease. Lymphoid tissue of the small intestine and colon. CPs are small structures consisting mainly of ILCs and

DCs. These can mature into ILFs that contain B cell zones and germinal centres. PPs are aggregated lymphoid follicles with organised T and B cell areas. During

inflammation, tertiary lymphoid structures can also form that resemble ILFs. GALT-associated epithelium lacks goblet cells and has reduced mucus layer thickness. The

epithelial layer contains specialised ‘microfold’ (M) cells as well as intraepithelial and interdigitating transepithelial DCs. Both M cells as well as DCs can sample luminal

content and present it to tissue-resident GALT lymphocytes, or, in the case of DCs, possibly migrate the mLNs to present antigen there.

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Both cell types are probably involved in sampling of anti-gens from the intestinal lumen, and pass antigen to follic-ular and conventional DCs, as well as to lymphocytes suchas B cells [38]. This sampling and antigen presentationwithin the PP provides a rapid means of local adaptation ofthe immune system under steady state conditions.

Although PP formation depends on RORg expression,prenatal PPs form almost normally in Ahr-deficient ani-mals that lack NK22 cells and have reduced numbers ofLTi-like cells [19]. This suggests that PP formation is lessdependent on NK22 and more dependent on LTi cells. Thisis consistent with a current model that different ILCpopulations may induce PP versus CP and ILF formation,directly reflecting the predominance of different ILC popu-lations before and after birth [39].

ILCs in generation of TLTIntestinal inflammation leads not only to increased num-bers of scattered inflammatory cells within the epitheliumand lamina propria but also to development of TLT. Al-though larger in size, TLT resembles ILFs in structure[40].

Given the similarity of TLT to ILFs, a functional rolefor LTi cells in TLT formation seems likely. However, thesituation is more complex, because unlike immatureILFs, de novo TLT induction is not only dependent onLTbR-expressing stromal cells but also on LT-positive Bcells [32]. Furthermore, TLT can form in a microbiota-dependent manner in RORgt-deficient animals that lackILCs [40]. This indicates alternative pathways of organ-ogenesis mediated by LT-expressing activated B cells[40]. These results demonstrate that, in contrast to

genetically predetermined lymphoid structures, ILCsdo not play a nonredundant role in induced TLT forma-tion. However, in the absence of RORgt+ ILCs, de novoTLT formation is associated with exacerbated inflamma-tion suggesting differences in the functional TLT-drivenresponse [38].

Together, the data paint a complex picture in whichspecific ILC subpopulations contribute to formation ofGALT anlagen during different phases of pre- and postna-tal development. Through their lymphoid inducer function,ILC populations contribute to the remarkable adaptabilityof the intestinal immune response. Further understandingof the host, microbial and dietary factors that control thedifferential activity and timing of lymphoid inducer func-tion by different ILC populations may provide a basis formore rational manipulation of the intestinal immune re-sponse.

ILCs as immune effector cellsBeyond their role in lymphoid organogenesis, recent stud-ies have highlighted the role of ILCs as immune effectorcells. ILC populations all develop from a communal Id2-dependent precursor, and similar to T cells, further differ-entiate into cells secreting different cytokines, dependingon genetic and environmental factors [4]. Like T cells, thedevelopment and persistence of ILCs is dependent on IL-7,a pro-survival homeostatic cytokine. IL-7 may also modu-late proinflammatory cytokine secretion by ILCs [41]. Thedescribed diversity of ILC populations mirrors that ofdifferentiated subsets of CD4 T helper (Th) cells withevidence for Type-1-, Type-2- and Type-17-like ILC popu-lations. These overlapping functions of innate and

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adaptive lymphoid cells are driven by commonality incytokine-driven transcription factor expression that allowsthe cells to respond to the cytokine milieu and adapt theireffector functions accordingly. It is now appreciated that Tcell differentiation is more flexible than previouslythought, and that differentiated subsets can acquire al-tered functions by acquiring or shutting down transcrip-tion factor expression in response to environmental cues[42]. Whether such flexibility of function occurs amongdifferentiated ILC populations is not known. In this sectionwe dissect the role of ILCs as cytokine-secreting effectorcells in the mucosal immune response.

RORgt-dependent IL-17- and IL-22-secreting ILCsThe signal transducer and activator of transcription(STAT)-3-driven RORgt-dependent Type 17 response playsa pivotal role in mucosal host defence [43]. Althoughoriginally described as a property of Th17 cells, a similarmodule of cytokine production including IL-17A, IL-17Fand IL-22 is produced by mucosal unconventional T cellpopulations including gd T lymphocytes and mucosallyassociated invariant T cells, as well as ILC populations.IL-17A, IL-17F and IL-22 are key players in the Type 17response [44]. IL-17A and IL-17F induce chemokines thatpromote neutrophil accumulation, whereas IL-22 is impor-tant in bolstering intestinal epithelial barrier function [45].Through binding to its receptor on intestinal epithelialcells, IL-22 promotes mucin production and upregulationof antimicrobial peptides such as RegIIIb and RegIIIg thatmediate direct antimicrobial activity (reviewed in [46]).

A characteristic feature of Type 17 cells is their RORgt-driven expression of the IL-23R [47]. Although not requiredfor the differentiation of Th17 cells, IL-23 plays an impor-tant role in enhancing cytokine secretion by Th17 and otherType 17 lymphoid cells. Recent studies indicate that IL-23playsa crucial role indriving IL-22-mediatedhostprotectiveimmunity to the mouse intestinal pathogen Citrobacterrodentium [48]. By contrast, overzealous IL-23-driven Type17 responses towards intestinal bacteria canpromote chron-ic intestinal inflammation [49,50]. Recent studies suggestthat distinct RORgt-dependent ILC populations (pheno-types summarised in Box 1) contribute to IL-23-mediatedmucosal host defence and immune pathology.

LTi-like cells

Under homeostatic conditions, a significant proportion ofadult LTi-like cells in the intestine can secrete IL-22 [51].In amousemodel, CD4+ adult LTi cells were found to be anessential source of host protective IL-22 required for clear-ance of C. rodentium infection [51]. IL-22-producing LTi-like cells may therefore provide important early innatedefence towards intestinal bacterial pathogens before theadaptive T and B cell immune response [51].

NK22 cells

Although LTi-like cells can produce IL-17 and IL-22, NK22cells primarily secrete IL-22. Quantitatively, these cells arean important source of intestinal IL-22 and have also beenimplicated in host defence towards C. rodentium infection[52,53]. Currently, it is not known whether the NK22 ILCpopulation arises from single or multiple progenitors.

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Lineage tracing experiments with cells isolated from thehuman tonsil (Lin– CD45int CD127hi) and mouse intestine(Lin– NKp46– RORgt+) have shown that adult LTi cells cangive rise to NK22 cells (CD56+ CD3– in humans, Lin–

NKp46+ RORgt+ in mice) [41,54,55]. However, it cannotbe excluded that the starting population in those experi-ments also contains ILC precursors, allowing both popula-tions to develop independently and in parallel. Indeed,another studywasunable to replicate the transition of adultLTi cells to NK22 cells using RORg/gt fate-mapping in vivoor following in vitro culture [39], favouring the idea thatthese are two separate populations.

Once differentiated, cytokines including IL-1b, stem cellfactor (SCF), IL-2 and IL-7, appear to play an important rolein controlling ILC function. For example, a combination ofIL-2 and IL-7 stimulates proliferation of human NK22 cellsin vitro. However culture with IL-7 alone induces IL-22secretion, whereas addition of IL-2, or IL-2 alone, altersthe cytokine profile in favour of interferon (IFN)g [56].

Recently it has been shown that IL-1 receptor and thedownstream adaptor myeloid differentiation primaryresponse gene (88) (MyD88) are required for basaland IL-23-induced IL-22 production by NKp46+ andNKp46– ILCs. ILC populations express high levels ofIL-1R and addition of IL-1b promotes IL-22 production[16]. These data highlight IL-1b, alongside IL-23, as a keyfactor in the intestinal innate IL-22 response.

CD4– NKp46– ILCs

Recently, a phenotypically distinct IL-7Ra+ Thy1+ CD4–

NKp46– ILC population that can induce tissue inflamma-tion in the gut has been described [50]. This IL-23-respon-sive and IL-17A, IFNg and IL-22-secreting inflammatorypopulation is present in the colon and preferentially accu-mulates during Helicobacter hepaticus-induced colitis inRag-deficientmice.Aphenotypically similarpopulationalsoaccumulates in an anti-CD40 model of innate colitis [50].Depletion of Thy1+ cells blocks the onset of colitis in bothmodels [50], indicating a functional role for these cells ininnate intestinal pathology. RORg/gt fate-mapping showedevolution of the ILC response in anti-CD40-induced colitis,with reductions in RORgt expression and IL-22 secretionand reciprocal increases in T-bet, a Th1-specific transcrip-tion factor, and IFNg production [41]. A similar functionalshift has been observed in the T cell transfermodel of colitisandmay reflect the adaptation of a pathogenic rather thanahost protective Type 17 response [49].

Studies in humans have validated those in the mousewith the identification of an IL-7Ra+ CD56– IL-23-respon-sive ILC population that resembles IL-7R a+ Thy1+ CD4–

NKp46– ILCs in mice. IL-7R a+ CD56– ILCs are selectivelyincreased in the ileumand/or colon of patients with Crohn’sdisease compared with control or ulcerative colitis patients[57]. Differential increases in IL-7Ra+ CD56– ILCs in CDare correlated with increased expression of IL17A andIL17F but not IL22 transcripts, suggesting these cellsare distinct from human NK22 cells but may constitutean additional source of proinflammatory IL-17. Althoughprovocative, these results are correlative and further stud-ies are required to prove a functional role of ILCs in humaninflammatory bowel disease (IBD) pathogenesis.

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RORgt and Ahr in ILC populations – role of the

microbiota and diet

Although initial studies showed that NK22 ILCs areabsent in germ-free mice [53], more recent studies havefound no role for the microbiota in ILC development,including NK22 cells [19,58]. Treatment of adult mice withbroad-spectrum antibiotics, however, does decrease thenumber of ILCs expressing RORgt in the small intestineand colon, suggesting either that the maintenance of ILCsis dependent on signalling from commensal microbiota, orthat the expression of RORgt itself is stabilised by micro-bial signals [41]. Relevant to this, it has been suggestedthat IL-7 acts to stabilise RORgt expression, promotingIL-22-expressing ILCs [41]. Whether the effects of themicrobiota are mediated through alterations in IL-7production remains to be determined.

The proportions of lamina propria RORgt+ ILCs varythroughout life. Before birth, the LTi population is mostabundant, but quickly after birth NK22 or CD4– NKp46–

cells dominate [39]. Analysis of precursor–product rela-tions has shown that culture of CD4+ LTi-like cells does notlead to development of different lineages of ILCs in vitro,suggesting that LTi cells are a fixed population [39]. Theseresults are consistent with the view that NK22 cells do notderive from LTi-like cells but rather from an alternativeprecursor.

Several recent papers have revealed a role for dietaryfactors, through Ahr activity, in modulating IL-22-produc-ing ILCs and induced lymphoid structures. Ahr is a basichelix–loop–helix transcription factor with natural andsynthetic ligands that is involved in responses to environ-mental triggers [59]. Ahr�/� mice lack CPs, ILFs andNKp46+ IL-22-producing cells in the small intestine andcolon. The dependence of LTi and LTi-like cells on Ahrremains unclear from the data [19,60]. Ahr maintainsIL22-producing cells and GALT development via Notch-dependent and-independent mechanisms [19]. Dietaryantigens from cruciferous vegetables, such as broccoli,contain Ahr ligands such as indole-3-carbinol (I3C) andare able to reconstitute CP and ILF formation in mice fed adiet from non-plant sources [60], although this was notconfirmed by another study [19]. These studies provide amolecular explanation for how dietary factors can directlymodulate intestinal ILC function and lymphoid structureformation.

RORgt-independent ILCs – nuocytes and natural helpercellsType 2 cytokine-secreting RORgt-independent ILCs, so-called nuocytes [61] and natural helper cells [62], havebeen identified within gut tissues and elsewhere [61,62],and appear to represent Th2-equivalent cells. Both of thesepopulations respond to IL-25 and IL-33, and induce expul-sion of the helminth Nippostrongylis brasiliensis in amouse model. Nuocytes produce IL-13 and are located inthe mesenteric lymph nodes (mLNs) [61]. Natural helpercells also respond to IL-25 by secretion of IL-13, IL-4 andIL-5, and are located in fat-associated lymphoid clusters(FALCs) within the peritoneum [62]. Unlike other ILCs,these populations are not dependent on RORgt, but doexpress IL-7Ra, confirming their lymphoid origin. A recent

study suggests that nuocytes also express (and are func-tionally dependent on) RORa for worm expulsion [63]. Aslightly different IL-25-responsive ILC population facili-tates worm expulsion in experimental Trichuris murisinfection. This latter population has multipotent progeni-tor capacity giving rise to monocytes/macrophages or baso-phils [64].

In the lung, depletion of natural helper cells using adepleting Thy1 antibody causes exaggerated pathology inresponse to influenza. These cells show selective expres-sion of wound repair genes, particularly amphiregulin,compared with splenic LTi-like cells [65]. Indeed, amphir-egulin is sufficient to restore tissue repair, and it will beinteresting to assess the role of such tissue repair path-ways in the gut in the context of IBD. Recently, human IL-25- and IL-33-responsive type 2 innate lymphoid cellsdefined by expression of CRTH2 and CD161 have beendetected in the human foetal gut (0.5–2.3% Lin�IL-7Ra+CRTH2+ cells among CD45+ cells, with low or nega-tive RORg expression) but are less frequent in adult ileum[66].

Interestingly after birth, as the gut is colonised withbacteria, the constitutive IL-22 production by ILCs (bothLTi-like and NK22) becomes reduced as microbiota-stim-ulated epithelial cells secrete IL-25 [58]. This raises theintriguing possibility that microbial-dependent IL-25production may induce IL-25-responsive Type 2 ILCs(Figure 2). Interactions between RORgt-dependent andindependent ILCs have not been well studied, and it ispossible that under conditions of high IL-25 concentra-tion there is functional plasticity allowing an IL-22-secreting RORgt+ ILC to become a RORgtlo/– ILC thatsecretes IL-5 and IL-13. It will be of interest to establishthe role of Type 2 ILCs in human inflammatorydiseases.

Direct cell–cell interactions between ILCs, B and T cellsIn addition to cytokine secretion, there is also evidence thatILCs can directly modify adaptive B and T cell responsesvia cell–cell interactions. Indeed RORgt+ ILCs coordinateT cell independent IgA class switching via follicular DCsand plasma cell differentiation via DCs in ILFs [29]. It hasbeen proposed that LTi cells were originally innate im-mune cells with the capacity to secrete proinflammatorycytokines and IL-22, and that these cells clustered togetherin CPs to promote epithelial integrity via secretion of IL-22[67]. Over time, this cluster of LTi cells may have acquiredadditional signalling pathways including LT signalling,which provides the ability to recruit B cells that can thenundergo T cell independent class switching. LTi-like cellsalso interact with CD4 memory cells through OX40L andCD30L expression [68,69]. A functional role for this directinteraction has been proposed based on the findings thatCD4 memory cells fail to persist when transferred intoRORg-deficient hosts that have no LTi cells. Whether thisis attributable to a lack of an LTi-mediated OX40 or CD30signal, or if it occurs indirectly due to the absence oforganised lymphoid structures in RORg�/� mice remainsto be established. Further examination of the interactionsof LTi-like cells or other ILC populations with adaptive Tand B cell populations will establish the functional

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[(Figure_2)TD$FIG]

Key:

Epithelial cell

Fibroblast

Dendritic cell

B cell

Th1

Th2

Th17

Eosinophil

Mast cell

Neutrophil

IL-23

IL-17A / IL17FIL-22

RORγ

IL-7

IL-7R

LTβR

Lymphoid tissue inducer

IL-25

IL-23R

IL-13 / IL-5

IL-33

IL-15R

IL-2

IL-2R IL-25RIL-1RL1/IL-1RAP

NFkB/MAPK ?

kynurenine

AhR

LT

Environmental trigger(food, bacteria, worms)

Inflammatory trigger

ILC signal integrationand response pattern

Influence onmicroenvironment

ICAM

LTi / LTi-like

NK22

CD4-NKp46-

Natural helper

Helicobacter hepaticus Nippostrongylis brasiliensisTrichuris muris

Epithelialprotection

Type 17 dominantinflammation

Type 2 dominant inflammation

RegIIIβRegIIIγ

NOTCH RORα

ILC cell types

t-bet NOTCH

IL-15

Citrobacter rodentiumCruciferousvegetables dioxin

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Figure 2. Functional response modules of ILCs. ILC (e.g. LTi/ILC-like cells, NK22, CD4–NKp46–, natural helper/nuocytes) populations apply a set of partially shared response

modules towards different stimuli including pathogens, food or inflammatory triggers (cytokines). Each ILC population can respond with several response modules in

parallel. Basic response modules are lymphoid inducer function (green), IL22-driven epithelial protection (blue), Type-17-mediated inflammation (pink) or Type 2

inflammation (purple). Exogenous Ahr ligands within food or the environment (such as cruciferous vegetables or dioxin) as well as endogenous ligands (including

kynurenines) enable LTi and LTi-like cells to interact with LTbR+ stromal cells to form lymphoid structures, but can also stimulate IL-22 secretion. RORg-dependent ILCs are

stimulated by intestinal microbiota-driven IL-7, IL-15 or IL-23 signals. This can trigger an IL-22 response promoting epithelial integrity and antimicrobial peptide secretion.

RORg-dependent cells can mount antibacterial or antifungal IL-17 responses leading to neutrophil recruitment. Worm or parasite infection can activate an IL-25 or IL-33-

dependent Type 2 response, possibly through the nuclear factor kB pathway. This Type 2 response may influence T cells, eosinophils or mast cell responses. Arrows show

stimulation and lines show repression pathways.

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relevance of ILCs beyond lymphoid tissue formation inorganisms with an intact adaptive immune system.

Concluding remarksIn this review, we have highlighted the multiple activitiesof ILCs that influence the intestinal microenvironment,providing an explanation for how a relatively small num-ber of ILCs may play a substantial role in shaping theadaptive immune system. First, LTi cells function pre andpostnatally to generate organised lymphoid structures inthe gut. Second, ILCs establish rapid cytokine production

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and can contribute to the polarisation of the subsequentadaptive immune response. Third, ILCs contribute to T cellindependent IgA production as well as the maintenance ofmemory T cells.

Despite rapid advances, several questions remain. ILClineage relations and plasticity are not fully understood.The functional role of ILCs in human intestinal lymphoidtissue generation, tissue remodelling, epithelial barrierfunction, and their pathogenic contribution to human in-testinal inflammatory diseases and the onset of cancer alsoneeds further clarification.

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AcknowledgementsF.P. and C.P. are funded by the Wellcome Trust.

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