Innate Lymphoid Cells — How Did We Miss Them?

FOCUS ON The inbetweeners: innate-like lymphocytes REVIEWS

Innate lymphoid cells — how did we miss them?

Jennifer A. Walker, Jillian L. Barlow and Andrew N. J. McKenzie Abstract | Innate lymphoid cells (ILCs) are newly identified members of the lymphoid lineage that have emerging roles in mediating immune responses and in regulating tissue homeostasis and inflammation. Here, we review the developmental relationships between the various ILC lineages that have been identified to date and summarize their functions in protective immunity to infection and their pathological roles in allergic and autoimmune diseases.

Type 1 immune responses The orchestration of immune responses by T cells ILCs (comprising ILC3s and LTi cells) — based on their Type 1 immune responses are — through their release of a complex repertoire of ability to produce type 1, type 2 and TH17 cell-associated characterized by the cytokines — is crucial for adaptive immunity to infec‑ cytokines, respectively (FIG. 1). In this Review, we discuss production of cytokines such tion. Coffman, Mosmann and others defined different the developmental relationships of ILCs and summarize as interferon‑γ, interleukin‑2, T helper (T ) cell subsets based on their distinct cytokine their functional roles in protective immunity to infections tumour necrosis factor and H lymphotoxin‑α by various secretion profiles. TH1 cells, which produce interferon‑γ and their pathological roles in allergic and autoimmune immune cells, including (IFNγ), interleukin‑2 (IL‑2) and lymphotoxin-α (LTα), diseases. As the immune functions of NK cells and LTi T helper 1 cells, neutrophils, drive type 1 immune responses that protect against cells have been reviewed elsewhere, this article concen‑ macrophages and NK cells. intracellular pathogens, but they are also implicated in trates on the more recently defined LIN− ILCs, with a Such responses protect against intracellular pathogens and are various autoimmune diseases. By contrast, TH2 cells are particular focus on the ILC2 and ILC3 populations. also implicated in several typically defined by their secretion of IL‑4, IL‑5, IL‑9 autoimmune diseases. and IL‑13, and contribute to type 2 immune responses. ILC subsets Such responses are required for controlling extracellular Group 1 ILCs. The group 1 ILCs comprise ILCs such as Type 2 immune responses parasite infections, but they are also responsible for the NK cells that produce type 1 cytokines, notably IFNγ Type 2 immune responses are characterized by the secretion immunopathology that develops in patients with allergy and tumour necrosis factor (TNF). NK cells were first of cytokines such as and asthma. Furthermore, we now recognize that addi‑ identified in 1975 as innate effector lymphocytes that interleukin‑4 (IL‑4), IL‑5, IL‑9 T 17 cells T fol‑ 2,3 tional TH cell subsets — including H and exhibit cytotoxic activity towards tumour cells . Since and IL‑13 by various immune licular helper cells (T cells) — also have bespoke roles then, their roles in tumour surveillance, the elimination cells, including T helper 2 cells, FH eosinophils, basophils, mast in regulating different aspects of the immune response of virus-infected cells and the amplification of inflam‑ 4 cells and ILC2s. Such through their distinct cytokine secretion profiles. matory responses have been well documented . Crucial responses are required for Recently, it has become clear that, in addition to to their function is their capacity to induce granule- controlling extracellular these adaptive lymphoid cell sources of cytokines, mediated cytotoxicity through their expression of per‑ parasite infections, but they there are important innate lymphoid cell (ILC) sources. forin and granzymes, and their ability to secrete the are also responsible for the immunopathology that These previously unappreciated ILCs produce many pro-inflammatory cytokines IFNγ and TNF. NK cells develops in patients with TH cell-associated cytokines, but they do not express are dispersed in secondary lymphoid organs, blood and allergy and asthma. cell-surface markers that are associated with other peripheral organs, and they recognize and kill target cells immune cell lineages (TABLE 1). Furthermore, these through the expression of a series of activating and inhib‑ lineage marker-negative (LIN−) ILC subsets do not itory receptors on their surface, including the activating express a T cell receptor and thus do not respond in an receptors NKp46 (also known as NCR1) and NK1.1 (also

MRC Laboratory of Molecular antigen-specific manner. known as KLRB1C). Conventional NK cells arise in the Biology, Hills Road, A consortium of experts in the field has now proposed bone marrow, but NK cells can also develop in the thy‑ Cambridge, CB2 0QH, UK. that the term ‘innate lymphoid cell’ should be used to mus, and the two types of NK cell population have differ‑ Correspondence to A.N.J.M. encompass the LIN− ILCs, natural killer (NK) cells and ing growth factor requirements for their development5. e‑mail: lymphoid tissue-inducer (LTi) cells1. Furthermore, it has The diverse roles of NK cells have been reviewed exten‑ [email protected] doi:10.1038/nri3349 been suggested that ILCs be further divided into three sively elsewhere and so here we focus predominantly Published online subsets — group 1 ILCs (comprising ILC1s and NK on their developmental pathway and how NK cells are 7 January 2013 cells), group 2 ILCs (comprising ILC2s) and group 3 related to the more recently identified ILC lineages.

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Table 1 | Defining ILCs ILC group ILC Mouse Human Signature lineage cytokines Group 1 ILC1s LIN–RORγt–, RORγtfm+ (also LIN–CD56+NKp46+NKp30+ IFNγ ILCs THY1+SCA1+) NKp44+IL‑7Rα– NK cells NKp46+NK1.1+ (strain CD122+NKG2D+CD161+KIR+ IFNγ, TNF, cytotoxic dependent) CD122+NKG2D+ (REF. 99) effectors CD161+CD16+CD11b+ (REF. 5) Group 2 ILC2s LIN–ICOS+SCA1+IL‑7Rα+ST2var (also LIN–IL‑7Rα+CD45hiCD161+CRTH2+ IL‑5, IL‑9, IL‑13 and ILCs THY1+IL‑17RB+CD25+) small amounts of IL‑4 Group 3 ILC3s LIN–RORγt+NKp46+ (also LIN–CD56+NKp46+NKp30+ IL‑22 ILCs THY1+IL‑7RαintKITintCXCR5–CCR6–) NKp44+IL‑7Rα+ LTi cells LIN–RORγt+NKp46– (also LIN–IL‑7RαhiCD45intRORγt+ (also LTα, LTβ, IL‑17A, THY1+IL‑7RαhiKIThiCXCR5+CCR6+); CD7+CD161+CD4–CD94–) IL‑22 a proportion of LTi cells are CD4+ CCR6, CC-chemokine receptor 6; CXCR5, CXC-chemokine receptor 5; ICOS, inducible T cell co-stimulator; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; IL‑7Rα, IL‑7 receptor subunit-α; KIR, killer cell immunoglobulin-like receptor; LIN–, lineage marker-negative (defined in mice as negative for CD3, CD4, CD8, CD19, B220, CD11b, CD11c, FcεRI, GR1 and TER119 antigen; defined in humans as negative for CD1a, CD3, CD11c, CD34, CD123, TCRαβ, TCRγδ, BDCA2, FcεRI, CD19, CD14 and CD16); LT, lymphotoxin; LTi, lymphoid tissue-inducer; NK, natural killer; RORγt, retinoic acid receptor-related orphan receptor-γt; RORγtfm+, cells ‘fate-mapped’ for Rorc expression (that is, those expressing GFP from the ubiquitously active Rosa26 locus after a loxP-flanked STOP cassette has been excised by Cre recombinase that is expressed under the control of the Rorc locus on a BAC transgene); TCR, T cell receptor; TNF, tumour necrosis factor; var, variable.

In addition to NK cells, other IFNγ-secreting ILCs have Following the discovery of ILC2s in mice, the search been described; the new nomenclature proposes that these for human equivalents began. The first report of human cells be referred to as ILC1s6. ILC1s are weakly cytotoxic ILC2s came from studies that screened human adult and and are closely related to, and appear to arise from, ILC3s, fetal gut tissue for the presence of LIN−IL‑7Rα+CD45int as described below. However, the provenance of such or LIN−IL‑7Rα+CD45hi cells23. A subsequent analysis additional group 1 ILC subsets remains to be established of the expression of RORγt (which is required for the rigorously to determine whether they simply represent development of other ILC lineages, as discussed below) an alternative differentiation state of ILC3s or NK cells. indicated that the LIN−IL‑7Rα+CD45int cells express high levels of RORγt, whereas the LIN−IL‑7Rα+CD45hi Group 2 ILCs. The existence of an innate immune cell cells expressed low levels of this transcription factor. type that produces type 2 cytokines was first postu‑ The LIN−IL‑7Rα+CD45hi population also expressed lated after the discovery that intranasal administra‑ transcripts encoding IL‑13, IL‑17 receptor B (IL‑17RB; tion of IL‑25 still induces the production of IL‑5 and a component of the IL‑25 receptor), ST2 (also known IL‑13 in Rag2−/− mice, which lack conventional B and as IL‑1RL1; a component of the IL‑33 receptor) and T cells7,8. Subsequently, a non‑B, non‑T cell population prostaglandin D2 receptor 2 (also known as CRTH2), that responded to IL‑25 and provided a crucial innate suggesting that they represent the human ILC2 equiva‑ source of type 2 cytokines at the onset of helminth lent. Indeed, such cells were also found in adult lungs infection was identified9. In 2010, three reports10–12 and blood and, notably, in elevated proportions in the further characterized these type 2 cytokine-producing nasal polyps of patients with chronic rhinosinusitis17,23. ILCs and showed that they are present in the mes‑ enteric fat-associated lymphoid clusters, mesenteric Group 3 ILCs: ILC3s. Almost simultaneously, three lymph nodes, spleen, liver and intestines. They were groups reported the existence of an intestinal lymphoid identified subsequently in the airways13–17 and Peyer’s cell population that expresses the NK cell activating

TH17 cells patches (J.A.W., unpublished observations). These cells receptor NKp46 but otherwise bears little functional A subset of CD4+ T helper cells are variably termed natural helper cells (NHCs), nuo‑ resemblance to conventional NK cells24–26. This NKp46+ that secrete predominantly the cytes and innate helper 2 (I 2) cells in the literature, population was dependent on the transcription factor pro-inflammatory cytokine H interleukin‑17A and have been but it was recently agreed that the term ILC2s should RORγt, lacked cytotoxic effectors (such as perforin, implicated in the pathogenesis be used to refer to all ILCs that produce predominantly granzymes and death receptors) and did not produce of many chronic inflammatory type 2 cytokines1. ILC2s require the transcription fac‑ IFNγ or TNF, but instead expressed the cytokine IL‑22 disorders. tors retinoic acid receptor-related orphan receptor-α (REF. 26). Fate-mapping approaches have demonstrated 18,19 20,21 T follicular helper cells (RORα) and GATA-binding protein 3 (GATA3) , that these cells are developmentally distinct from con‑ 27,28 + + + and they have key roles in antihelminthic responses and ventional NK cells . RORγt NKp46 cells are referred (TFH cells). A subset of CD4 helper T cells that interact with allergic lung inflammation. ILC2s express characteristic to in the literature as NCR22 cells, NKp46+ ILCs, ILC22s B cells within germinal centres surface markers (TABLE 1) and chemokine receptors, such and NKR-LTi cells, and additional names have been to provide co-stimulatory as CXC-chemokine receptor 6 (CXCR6), CXCR4 and used to describe equivalent populations in humans29–31. signals and regulate the development of antigen- CC‑chemokine receptor 9 (CCR9), which are involved Under the new unified nomenclature, it is suggested + specific B cell immune in the homeostatic distribution of lymphoid cells to that these cells be termed NCR ILC3s. This branch responses. specific organ sites22. of the ILC family resides predominantly in mucosal

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Notch, New New group Mediators Disease GATA3, nomenclature nomenclature produced Function association RORα ILC2 Group 2 ILCs IL-5, IL-9, IL-13, • Immunity to helminths Allergy IL-7, IL-33, amphiregulin • Wound healing and asthma IL-25, TSLP Nuocyte, NHC, IH2 cell

LTi cell Group 3 ILCs Lymphotoxin, • Lymphoid tissue development IL-17, IL-22 • Intestinal homeostasis RORγt LTi cell • Immunity to extracellular Notch bacteria AHR NCR+ ILC3 IL-22 • Homeostasis of epithelia IBD? IL-7, • Immunity to extracellular ID2+ ILC IL-23 NK22 cell, NCR22 cell, bacteria precursor NKR-LTi cell, ILC22

NCR– ILC3 IL-17, • Immunity to extracellular IBD IFNγ bacteria? ILC17 GATA3 NK cell Group 1 ILCs IFNγ (high levels) • Immunity to viruses and Inflammatory IL-7 intracellular pathogens conditions, Thymic NK cell • Tumour surveillance IBD E4BP4 NK cell IFNγ (low levels), • Immunity to viruses and Inflammatory IL-15 perforin, intracellular pathogens conditions, Conventional NK cell granzymes • Tumour surveillance IBD T-bet? RORγt ILC1 IFNγ • Inflammation? IBD?

Figure 1 | ILC subsets, functions and disease associations. Innate lymphoid cells (ILCs) differentiate from haematopoietic stem cells, via an ID2+ precursor cell, under the influence of cytokines such as interleukin‑7 (IL‑7), Nature Reviews | Immunology IL‑15, IL‑23, IL‑25 and IL‑33. These signals induce the expression of transcription factors that promote the differentiation of the various ILC subsets (group 1, 2 and 3 ILCs) and induce their expression of signature cytokines. The cytokines secreted by ILCs promote important physiological responses, such as wound healing, tumour surveillance and protection against infections. However, ILC-derived cytokines can also promote immunopathology in diseases such as asthma and inflammatory bowel diseases (IBDs). AHR, aryl hydrocarbon receptor; E4BP4, E4 promoter-binding protein 4

(also known as NFIL3); GATA3, GATA-binding protein 3; IFNγ, interferon‑γ; IH2, innate helper 2; LTi, lymphoid tissue-inducer; NHC, natural helper cell; NK, natural killer; ROR, retinoic acid receptor-related orphan receptor.

tissues, particularly in the intestinal tract, where ILC3s Group 3 ILCs: LTi cells. LTi cells are an ILC subset have a crucial role in mediating the delicate balance that appears to be closely related to ILC3s. However, between the symbiotic microbiota and the intestinal as discussed below, the exact relationship between LTi immune system. cells and ILC3s is still controversial. LTi cells were first In humans, several cell types have been identified that identified as CD4+CD3− cells scattered among fetal share features of both LTi and NK cells. It was shown and neonatal lymph nodes32,33. These cells expressed that culturing LTi cells either with IL‑7 and FMS-related molecules required for the development of lymphoid tyrosine kinase 3 ligand (FLT3L) on stromal OP9 cells or tissues, including LTβ and the common cytokine + − with IL‑15 leads to the differentiation of a CD56 CD3 receptor γ-chain (γc; also known as IL‑2Rγ), and it NKp46+NKp30+NKp44+ population that does not express was therefore suggested that they might have crucial killer cell immunoglobulin-like receptors (KIRs) or per‑ roles in the generation of lymph nodes32 and Peyer’s forin and has only low levels of granzyme A expression30. patches34. Studies using a Rorc-EGFP (enhanced This population could be subdivided into ILC3‑like and green fluorescent protein) knock‑in reporter mouse ILC1‑like subsets based on IL‑7Rα expression. The LTi demonstrated that the transcription factor RORγt is OP9 cells cell-derived CD56+IL‑7Rα+ cells (the ILC3 subset) con‑ expressed by LTi cells and that, in its absence, LTi cells A bone marrow-derived tinued to express LTα and LTβ, retained the capacity are not generated and lymph nodes and Peyer’s patches stromal cell line used to 35 support haematopoietic stem to upregulate the expression of adhesion molecules on do not develop . LTi cells are therefore essential for cells and common lymphoid mesenchymal cells, and expressed IL‑22 and, to a lesser lymphoid organogenesis during embryogenesis, and progenitor cells during in vitro extent, IL‑17A. By contrast, the CD56+IL‑7Rα− cells failed they have subsequently been recognized as important culture. The OP9–DL1 to produce IL‑17A and IL‑22 and instead expressed IFNγ. regulators of lymphoid tissue architecture after birth. variation of this cell line This suggests that they fall into the category of group 1 Indeed, they have roles in the development of crypto ectopically expresses the 36 Notch ligand Delta-like 1, ILCs, although they could not be further differentiated patches , which are the precursors of isolated lym‑ 6 which promotes the into conventional NK cells and it is unclear whether such phoid follicles, and in the reconstruction of peripheral differentiation of T cells. cells arise under physiological conditions. lymph nodes following viral infection37. Furthermore,

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LTi cells have been linked to the maintenance of T cell be derived from a common ID2‑dependent progenitor memory38, and the production of IL‑17A and IL‑22 that is directed towards particular ILC phenotypes by by LTi cells has been shown to mediate immunity to the expression of lineage-specific transcription fac‑ enteric pathogens39,40. tors50. However, recent studies aimed at examining the Following the first description of LTi cells in mice, a developmental relationships between the ILC lineages recent report made a strong case for the existence of a have indicated that additional complexity and plasticity population of LIN−IL‑7RαhiCD45intRORγt+ LTi cells in exist within this arm of haematopoiesis (FIG. 2), the salient mesenteries isolated from 8–9 week human embryos and features of which are discussed below. in mesenteric lymph nodes from second trimester (13– 22 week) human embryos30. These cells also expressed The origin of group 3 ILCs. A close relationship between transcripts encoding LTα, LTβ and the transcription fac‑ the LTi cell and ILC3 lineages is suggested by their tor ID2, all of which are known to have roles in LTi cell shared requirement for several transcription factors, function and development. Although it is surprising that such as ID2, RORγt and the aryl hydrocarbon recep‑ these cells lacked CD4 expression, both CD4+ and CD4− tor (AHR)24–27,35,48,51–53. However, it is currently unclear LTi cells do occur in mice, possibly indicating that there whether LTi cells differentiate to acquire an ILC3 pheno‑ is diversity in LTi cell phenotypes. Importantly, human type or whether these two ILC subsets represent distinct LIN−IL‑7RαhiCD45intRORγt+ LTi cells were capable of lineages. Adoptive-transfer studies lend support to the Common lymphoid progenitor inducing the expression of the adhesion molecules vas‑ hypothesis that LTi cells are precursors to the ILC3 lin (CLP). CLPs are the earliest cular cell adhesion molecule 1 (VCAM1) and intercel‑ eage, as the transfer of lymph node or intestinal LTi cells progenitors of the lymphoid lular adhesion molecule 1 (ICAM1) on mesenchymal gives rise to NKp46+ cells in the intestinal lamina propria cell lineages, which include cells by producing lymphotoxin and TNF. The human of alymphoid recipients28. However, consistent with the B cells, T cells, NK cells and LTi cells also expressed transcripts encoding IL‑22 and alternative hypothesis, that ILC3s are a distinct develop‑ the newly described innate lymphoid cells. Bone marrow IL‑17A, suggesting that, similarly to mouse LTi cells, mental lineage, it was reported that ILC3s originate from − int low + CLPs are defined by their they may promote protective immunity during infection. CLP-like (LIN SCA1 KIT IL7Rα ) α4β7 integrin- expression of the IL‑7 receptor, negative fetal liver precursors, which are distinct from the FMS-related tyrosine kinase 3 ILC lineage relationships and plasticity ID2‑expressing α4β7+ cells that give rise to the LTi cell, (FLT3) and KIT, and the 42,45 absence of all conventional Studies of gene-targeted mice have provided a greater T cell, NK cell and dendritic cell lineages . Two addi‑ lineage markers. understanding of the transcription factors that are tional studies have recently corroborated these findings, required to instruct the development of the various ILC and these studies also examined the role of Notch sig‑ Notch signalling lineages (TABLE 2). However, we currently lack a com‑ nalling in specifying the fate of RORγt+ ILCs. Although The Notch signalling plete appreciation of the temporal regulation of these the Notch signalling requirements may differ between pathway regulates cellular differentiation in various transcriptional changes and of the extrinsic signals that fetal and adult CLPs, both studies concluded that Notch + tissues and at various stages are required for their induction. Furthermore, there is signals promote the generation of the α4β7 precursor, of development. During considerable controversy regarding the developmental but subsequently inhibit the upregulation of RORγt and lymphopoiesis, signals through relationships between the various ILC subsets and the the generation of LTi cells46,47. By contrast, the α4β7− pre‑ the Notch receptor modify gene expression patterns and degree of plasticity that exists within each lineage and cursor that gives rise to ILC3s does not require Notch have crucial roles in the permits the modification of its signature cytokine pro‑ or ID2 for its generation (although subsequent stages of development of T cells file. In this regard, it remains to be determined whether ILC3 development do require these factors52). Thus, the and the inhibition of B cell ILC subset plasticity exists, akin to that observed among ancestry of ILC3s remains a contentious issue, and there differentiation. In mammals, T cell subsets41. may be multiple routes by which these cells can be gener‑ there are four Notch receptors, which bind to ligands of the ated in vivo. For example, whereas Notch signals promote + Delta family (Delta-like 1, A common ILC precursor? The various branches of the the generation of α4β7 precursors, a Notch-independent Delta-like 3 and Delta-like 4) ILC family share a series of commonalities, which allude route might also exist in fetal haematopoiesis47. and the jagged family (jagged 1 to a common ancestry and interrelated developmental There are currently very few studies addressing the and jagged 2), which are typically expressed on pathways. ILCs are of lymphoid origin, and cell-transfer role of Notch signalling in the development of group 3 stromal cells. experiments have demonstrated that ILC2s and NK cells ILCs in vivo. Conditional deletion of the gene encod‑ arise from a common lymphoid progenitor (CLP; LIN− ing recombining binding protein suppressor of hairless Aryl hydrocarbon receptor IL7Rα+FLT3+SCA1lowKITlow), which in turn originates (RBPJ) — which is an essential mediator of Notch signal‑ (AHR). AHR is a cytosolic, from either the fetal liver or adult bone marrow19,42–45. ling — in haematopoietic cells resulted in fewer NKp46+ ligand-dependent transcription factor that translocates to the It remains to be determined whether the ILC popula‑ ILCs in the intestinal lamina propria, but led to a lesser 52 nucleus following the binding tions derived from fetal and adult haematopoiesis are reduction in the numbers of LTi cells . Interestingly, of specific ligands, which truly equivalent. With the exception of conventional NK AHR signals have been shown to induce the expression include dietary and microbial cells, all ILCs require IL‑7 signalling for survival under of Notch1 and Notch2 in ILC3s52, perhaps providing a metabolites. AHR participates Notch signalling in the differentiation of homeostatic conditions, and has also been means to modulate this signalling pathway in vivo. implicated in the development of the various ILC popu‑ regulatory T cells, TH17 cells and intraepithelial intestinal lations in vitro19,46,47. Notably, ILCs share a requirement Plasticity — ILC3 to ILC1 transition? ILC3s display no γδ T cells, and it is required for for the transcriptional repressor ID2 (REFS 10,27,46,48), cytotoxic activity and secrete IL‑22 rather than typical the secretion of IL‑22 by T 17 24–26 H which inhibits the activity of the E protein transcription NK cell cytokines such as IFNγ . However, it was cells. More recently, AHR has been shown to have crucial factors and is likely to antagonize B and T cell fates dur‑ recently reported that a proportion of ILC3s (which were 49 roles in the development and ing ILC development . These shared signalling require‑ fate-mapped for prior expression of RORγt) downregu‑ function of LTi cells and ILC3s. ments and reliance on ID2 suggested that ILCs might late RORγt and acquire the capacity to produce IFNγ in

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Table 2 | Signals and transcription factors that specify ILC lineages Transcription Associated Consequence of deletion Proposed function Role in other lineages Refs factor ILC lineage ID2 NK cells, All ILC lineages absent Antagonism of E protein Required for CD8α+ and CD103+ 10,27, LTi cells, activity; suppression of B and dendritic cell lineages 48,49, ILC2s, ILC3s T cell differentiation 100 E4BP4 NK cells Failure to develop conventional NK Induction of ID2? (In the Currently untested; lymph 101,102 cells absence of E4BP4, a block in node development is normal in development occurs prior to animals lacking E4BP4 ID2 expression; this defect is partially restored by ectopic expression of ID2) RORα ILC2s Failure of ILC2 populations to Regulator of development Required, in conjunction 18,19, develop and/or expand in response and proliferation or survival; with RORγt, for TH17 cell 103 to IL‑25 or IL‑33 specific gene targets remain development and IL‑17 to be identified production; mice lacking RORα have normal NK cell and ILC3 frequencies, and normal lymph node development GATA3 NK cells, Lack of thymic NK cell development Required for NK cell Crucial for T cell development 20,21,

ILC2s and IFNγ production by maturation and polarization to a TH2 58,104 conventional NK cells; conditional phenotype; its role in deletion in ILC2s results in defective RORγt-dependent ILCs is development and IL‑13 production untested RORγt LTi cells, Absence of LTi cells and ILC3s; failure Regulator of development Crucial for the development of 24–26, ILC3s to develop lymph nodes, Peyer’s and proliferation or survival; TH17 cells, γδ T cells and some 35 patches, cryptopatches and ILFs specific gene targets remain invariant NKT cells to be identified

AHR LTi cells, Embryonically imprinted lymphoid Required for the proliferation Promotes the expansion of TH17 51–53, ILC3s structures (for example, lymph nodes and maintenance of RORγt+ and γδ T cell populations and 105 and Peyer’s patches) are intact, ILCs their production of IL‑22 but postnatal lymphoid tissues (for example, cryptopatches and ILFs) are severely diminished; ILC3s are absent TOX NK cells, Impaired NK cell development; ID2 expression is reduced Required at several stages 106 LTi cells reduced frequency of fetal and adult in Tox −/− NK cells, but the of T cell development; LTi cells developmental defect is NKp46+RORγt+CD3− cells are not restored by ectopic ID2 present in the intestine of Tox −/− expression; specific roles in mice, suggesting that ILC3s may LTi cells are unknown develop independently of TOX; its role in ILC2s is untested RUNX proteins NK cells, Impairment of NK cell maturation Required for the expression Required for the differentiation 107,108 LTi cells (especially in the absence of RUNX3) of CD122 (the common of multiple lineages, including and LTi cell development β-subunit of the IL‑2 and T cells; required for α4β7+ CLP IL‑15 receptors); required for development, suggesting a role the generation of α4β7+ CLPs in other ILC populations AHR, aryl hydrocarbon receptor; CLP, common lymphoid progenitor; E4BP4, E4 promoter-binding protein 4; GATA3, GATA-binding protein 3; ID2, inhibitor of DNA binding 2; IL, interleukin; ILC, innate lymphoid cell; ILF, isolated lymphoid follicle; LTi, lymphoid tissue-inducer; NK, natural killer; ROR, retinoic acid receptor-related

orphan receptor; RUNX, RUNT-related transcription factor; TH, T helper; TOX, thymocyte selection-associated high-mobility group box protein.

response to IL‑12 and IL‑23 (REF. 28), although this find‑ RORγt+ ILCs that were transferred to IL‑7‑transgenic ing awaits confirmation. Similar plasticity exists within recipients maintained RORγt expression, whereas the the human ILC3 population, which when cultured with expression of RORγt was lost when these cells were IL‑7 plus IL‑2 expressed increased levels of IFNγ in transferred to recipients receiving IL‑7‑specific blocking response to IL‑23 stimulation, despite retaining RORγt antibodies28. Interestingly, RORγt expression is reported expression54,55. The resulting cells, which were analogous to be more stable in ILCs isolated from the small intestine in phenotype to ILC1s, were potent inducers of disease than in ILCs isolated from the colon, spleen or lymph in a colitis model in which Rag2−/− mice were dosed sys‑ nodes28,45,57, suggesting that the stability of RORγt is organ temically with CD40‑specific antibodies28, and they prob‑ specific and influenced by environmental factors, such as ably correspond to the RORγt-dependent colitogenic diet and the commensal microbiota. Consistent with this, ILCs that were identified by another group56. The factors commensal flora were shown to promote the production determining the stability of RORγt expression are poorly of IL‑7 by intestinal epithelial cells, and this correlated understood, but IL‑7 has been identified as a key determi‑ with increased stability of the RORγt+ ILC population. nant in the maintenance of the ILC3 phenotype28. Indeed, Furthermore, the transcription factor AHR, which

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athymic mice, it is probable that they can complete their 19 B cell T cell development in the bone marrow in vivo . Consistent with this, CLPs can be differentiated in vitro into ILC2s HSC by culturing them on OP9–DL1 stromal cells19. This sys‑ Notch tem, in conjunction with adoptive cell transfers, has also E4BP4 GATA3 NK cell been used to identify a committed LIN– ILC2 precursor, which is characterized by the expression of GATA3, ID2 and stem cell antigen 1 (SCA1)20. ILC2s require the trans Notch Notch RORα cription factor RORα, the expression of which appears to 18,19 ID2 GATA3 be instrumental in driving the phenotype of these cells . CLP α4β7+ precursor Similarly, GATA3 has been shown recently to facilitate the secretion of IL‑13 by ILC2s58 and, analogously to its role in

the development of TH2 cells, this transcription factor has a key role in promoting ILC2 development20,21. A minor ILC2 RORγt Notch role has also been identified for signal transducer and Notch activator of transcription 6 (STAT6) in promoting the α4β7– CLP independent? expansion of ILC2 populations following infection by parasitic worms58. To date, there is no report of ILC2s Notch ID2 converting to any of the other ILC subsets. RORγt ILCs: an evolutionary perspective The evolutionary origin of ILCs is as yet unexplored, T-bet? although their relationship to T cells is an interesting one. Current evolutionary models of the adaptive immune ILC3 LTi cell system show that T cells capable of V(D)J rearrange‑ Loss of ment first arose in jawed vertebrates that evolved 450 IL-7 59 RORγt million years ago . In addition, T-like cells that use gene conversion to generate a diverse receptor repertoire60,61 have been identified in jawless fish (which evolved ~500 million years ago). Jawless fish also possess a thymus- like structure that expresses a forkhead box N1 (FOXN1) ILC1 orthologue and a network of Notch and Delta-like Figure 2 | A model for ILC development. Following the receipt of Notch signals, proteins62. The existence of these two gene rearrange‑ Nature Reviews | Immunology common lymphoid progenitors (CLPs) in the bone marrow or fetal liver undergo ment systems in lymphocytes suggests that these dis‑ transcriptional changes that restrict their developmental potential to the T cell, natural tinct populations may have developed from a common killer (NK) cell, group 2 innate lymphoid cell (ILC) and lymphoid tissue-inducer (LTi) cell ancestral ILC. Thus, the recent discovery of an extended lineages. These changes coincide with the expression of ID2 and α4β7 integrin. Further family of specialized ILCs raises the question of whether ILC differentiation requires the expression of lineage-specific transcription factors, such an ILC was the primordial T cell precursor. as retinoic acid receptor-related orphan receptor- (ROR ), ROR t or E4 promoter- α α γ LTi cells and ILC2s develop independently of the binding protein 4 (E4BP4), and is regulated by the availability of Notch signals. ILC3s are 19,63 − thymus in Foxn1‑null mice and may therefore reported to be derived from α4β7 CLPs and require Notch signals and ID2 expression for full maturation, but they also arise from LTi cells in the periphery. In turn, ILC3s may lose pre-date the existence of this structure. Similarly to RORγt expression in the absence of stabilizing signals — such as interleukin‑7 (IL‑7) — modern-day ILCs, primordial ILCs might have had the and give rise to interferon‑γ (IFNγ)-producing ILC1s. GATA3, GATA-binding protein 3; capability to orchestrate immune responses via cytokine HSC, haematopoietic stem cell. secretion, but lacked the ability to respond to specific antigens. The IL‑17 family of cytokines is important for the functions of ILC2s and ILC3s, and homologues binds to dietary and microbial metabolites, is reported of these cytokines have been described in sea urchins, to influence the homeostasis of RORγt-dependent ILC sea squirts and oysters. Two new homologues that populations51–53. Thus, ILC3s retain the plasticity to divert are phylogenetically related to Il17c and Il17d were to an ILC1 phenotype, and their stability appears to be described recently in Oncorhynchus mykiss (rainbow regulated by specific environmental cues. trout)64. Furthermore, the lamprey homologue of IL‑17 is expressed by basal-layer epithelial cells in the lamprey Development of ILC2s. Similarly to the other innate skin and is upregulated in response to lipopolysaccha‑ lymphoid lineages, ILC2s are dependent on the trans ride stimulation65. However, the cytokine gene families Forkhead box N1 cription factor ID2 (REF. 10) and, at least in vitro, require that characterize mammalian ILCs were largely absent (FOXN1). A winged-helix Notch signals for their generation19. In the presence before the evolution of jawed fish66. Nonetheless, an transcription factor that is of IL‑7 and IL‑33, ILC2s can be derived from double- Il4 or Il13 orthologue has been identified in Tetraodon thought to regulate keratin negative 1 (DN1) and DN2 thymic precursors in vitro19, nigroviridis (green spotted pufferfish) based on linkage gene expression. Mutations in 66 the Foxn1 gene result in a and it seems likely that, similarly to T cells and NK cells, to the Rad50 gene . Similarly, Il22 has been found in the + + hairless (‘nude’) phenotype and the ILC2 lineage might diverge from an ID2 α4β7 pre Danio rerio (zebrafish) genome clustered with Il26 and athymia. cursor. However, given that ILC2s continue to develop in Ifng, as it is in mammals67.

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The major transcription factors responsible for ILC parasite elimination11. Moreover, the role of ILC2s as a development — RORγ6,24–26 and RORα19 — have not crucial source of IL‑13 was demonstrated by the adop‑ been reported in organisms lower than vertebrates. tive transfer of Il13−/− ILC2s, which failed to elicit worm However, related retinoic acid receptors are evolution‑ expulsion. Finally, transferring wild-type ILC2s into arily ancient and are found in Drosophila melanogaster, IL‑13‑deficient mice confirmed that IL‑13 secretion Caenorhabditis elegans and Danio rerio68. The evolu‑ from ILC2s alone is sufficient for worm expulsion11. tionary origins of ILCs are therefore unclear, but with Worm expulsion coincided with goblet cell hyperplasia the identification of additional factors involved in ILC and mucus production, which were also consequences development the field will be better placed to exam‑ of the adoptive transfer of ILC2s into Il2rg−/−Rag2−/− mice ine the contribution of ILCs to lymphocyte evolution. infected with N. brasiliensis10,12. It has been suggested that Examination of lower vertebrates for the existence the IL‑25 required for worm expulsion is derived from the of ILCs will help to refine our understanding of the epithelium and may amplify its own expression through evolution of these newly defined cells. paracrine or autocrine feedback74. A recent study also used helminth infection to inves‑ ILC functions in infection tigate the potential roles of ILC2‑expressed transcrip‑ The existence of different ILC populations that can rapidly tion factors in protective immunity. GATA3 is expressed secrete immunoregulatory cytokines suggests that these by all ILC2s, and intracellular staining demonstrated cells may have evolved to provide immunity to infections. that GATA3hi ILC2s express IL‑13 (REF. 58). By cross‑ Indeed, it is notable that ILC subsets seem to be particu‑ ing Il13‑Cre mice with Gata3flox/flox mice, this study also larly prevalent at mucosal surfaces, which are constantly revealed that GATA3‑regulated IL‑13 expression is cru‑ exposed to infectious agents in the external environment. cial for worm expulsion, but the relative contributions of The following sections discuss the growing evidence that T cells and ILCs could not be determined. ILCs contribute to immune responses to infection and, To date, there are limited reports on the roles of ILC2s although the present focus is on their described roles in other parasitic worm infections, and it will be of inter‑ in the intestine, it is likely that future investigations will est to determine whether specific parasites have evolved unearth more diverse roles in other tissues. mechanisms to evade ILC2 activities.

ILCs in protective immunity to helminths. Type 2 immu‑ ILCs in responses to enteric pathogens and commensals. nity probably evolved to combat intestinal worm infec‑ The intestinal immune system comprises a complex tions. Indeed, IL‑13 is indispensible for the efficient array of lymphocytes, which are distributed throughout expulsion of the helminth parasite Nippostrongylus the lamina propria and intraepithelial layer and are also brasiliensis, as this cytokine upregulates crucial physio arranged in specific structures such as Peyer’s patches logical responses, such as goblet cell mucus secretion (FIG. 3) and cryptopatches. Peyer’s patches and crypto‑ and the contraction of intestinal smooth muscle69,70. patches recruit other cells of the immune system, par‑ Although T cells are also essential for the expulsion of ticularly B cells, to counteract microbial invasion. In this helminth worms71,72, it was shown that T cells are not the environment, there is a precarious equilibrium between crucial source of IL‑13, as N. brasiliensis was expelled the intestinal immune system and the estimated 1 × 1014 from Rag2−/− mice reconstituted with CD4+ T cells from microbial symbionts that make up the mammalian Il4−/−Il13−/− mice73. Furthermore, wild-type CD4+ T cells microbiota. Cytokine expression and regulation con‑ were incapable of inducing worm expulsion when trans‑ tribute to this balance, with IL‑17A and IL‑22 having ferred into Rag2−/− mice that were deficient in IL‑4 and key roles. IL‑13. Thus, the essential source of IL‑13 required for Both IL‑17A and IL‑22 are crucial for the immune the expulsion of N. brasiliensis must originate from the response against enteric bacterial pathogens such as innate immune system. Citrobacter rodentium, which causes acute colitis in The first reported role for ILC2s in protective mice (FIG. 3). IL‑22 is upregulated early in the response immunity was in the response of IL‑25‑deficient mice to C. rodentium, and neutralization of IL‑22 during this to N. brasiliensis infection9 (FIG. 3). The relevant ILC2 early phase, but not later in infection, results in increased population was characterized as LIN−KIT+THY1.2+ mortality75. This is due in part to IL‑22 regulating the IL‑13+IL‑5+, and subsequent investigation using secretion of antimicrobial proteins, such as those of Il13‑EGFP reporter mice infected with N. brasiliensis the REG family of C‑type lectins, from epithelial cells75. showed that these LIN− cells were the major source of Importantly, IL‑22 also has multiple roles in the main‑ early IL‑13 production, before the initiation of T cell tenance of epithelial cell integrity and thus prevents the responses. These ILC2s were readily detected in the dissemination of pathogenic bacteria76. REG family of C‑type lectins −/− Members of the REG3 spleen, mesenteric lymph nodes, peritoneal cavity, Studies of Rag2 mice demonstrated that, in the early subgroup of the C‑type lectin lungs and blood following exogenous administration of phase of C. rodentium infection, IL‑22 is produced from family are antimicrobial IL‑25, and their population expansion correlated with an innate cell source. Furthermore, Rag2−/− mice were peptides that interact with the increased worm clearance. shown to eventually die as a result of chronic inflamma‑ peptidoglycans present on the The adoptive transfer of ILC2s into N. brasiliensis- tion, rather than as a result of a failure to maintain epi‑ surface of Gram-positive −/− −/− 77 bacteria. They can be released infected Il17rb Il1rl1 mice — which are deficient in thelial integrity and contain the infection . The innate into the intestinal lumen from IL‑25 and IL‑33 signalling and therefore severely impaired IL‑22‑producing cell population was originally proposed multiple epithelial cell lineages. in their ability to expel worms — resulted in efficient to comprise dendritic cells75, but recently it has been

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demonstrated that ILC3s are the predominant source of Analogously to their role in the response to bac‑ IL‑22 during C. rodentium infection52. In fact, the pheno‑ terial pathogens, ILC3s also provide an early source type of the originally reported protective ‘dendritic cell’ of IL‑22 during Candida albicans fungal infection78, (CD11c+LY49B−) is consistent with that of an ILC3. and a recent study indicated that they promote the Mice lacking the NKp46+ and NKp46− ILC3 sub‑ anatomical containment of lymphoid tissue-resident sets26,40 (owing either to AHR deficiency52 or to combined commensal bacteria79. ILC3s therefore provide a rapid 26 RAG2 and γc deficiency ) fail to contain C. rodentium source of IL‑22 to strengthen the epithelial barrier in infection in the gut. Similarly to the phenotype of Il22−/− response to epithelial stress or breach. However, cells

mice, the intestines of infected AHR-deficient mice of the adaptive immune system (probably TH1 and

exhibit an increased infiltration of inflammatory cells, TH17 cells) are still required to completely eliminate mucosal hyperplasia and erosion of the epithelium52,75. infection. This rapid innate response is indicative of

a LTi cells b Defensins and Intestinal lumen Lymphotoxin and Microorganism TNF for development antibacterial of Peyer’s patch peptides Microbiota IL-17 IL-22 Helicobacter Citrobacter Peyer’s patch Intestinal Mucus layer epithelial cell

B cells

IL-6 IL-13 IL-5 IL-22 IL-17 IL-25 Parasitic helminth ILC2s IL-23

Goblet cell Homeostasis hyperplasia Response to ? NCR– bacterial infection NCR+ Mucus DC hypersecretion ILC3s ILC3s c Allergen Goblet cell Parasitic Mucus layer helminth Fibrosis Figure 3 | Schematic of the roles for ILCs in intestinal immune function. a | Innate lymphoid cells (ILCs) have various immune functions in the intestine. Lymphoid tissue-inducer (LTi) cells express lymphotoxin and tumour necrosis factor (TNF), thereby upregulating Amphiregulin the expression of adhesion molecules on the epithelium Smooth promotes Smooth muscle muscle wound repair IL-25, IL-13 IL-25, contraction and inducing the development of lymphoid tissues, such cell IL-33 IL-33 as Peyer’s patches. Peyer’s patches harbour ILC2s that ILC3 IL-13 can provide interleukin‑5 (IL‑5), IL‑6 and IL‑13 to B cells. IL-22 b | ILC3-mediated production of IL‑22 maintains ? homeostasis with the intestinal microbiota and is IL-13 modulated by dendritic cells (DCs), which are in turn Undeﬁned ILC2s Alternatively regulated by IL‑25 released by the epithelium. During activated interaction and bacterial infection, IL‑22 expression is elevated maintenance factors ? macrophage following termination of the IL‑25 signal, resulting IL-4 IL-5 in the increased release of antimicrobial peptides and defensins. c | Parasitic worm infection results in the IL-5, release of IL‑25 and IL‑33 from epithelial cells. These IL-6 MHC class II factors induce the proliferation of ILC2s and their antigen expression of IL‑5, IL‑6, IL‑13 and possibly IL-4. These presentation Eosinophil cytokines in turn drive type 2 effector responses, including TH2 cells mucus hypersecretion, the alternative activation of Antibodies IL-4, IL-5, macrophages, eosinophilia and B cell proliferation. IL-9, IL-13 A question mark denotes suspected pathways that B1 cells have not been proven formally. TH2, T helper 2.

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crosstalk between ILCs and epithelial cells, which is and exacerbate allergic asthma83. However, the mecha‑ echoed in the response to helminths. The nature of these nisms by which this occurs are unclear. One group signals is not well understood but clearly requires epithe‑ demonstrated that infection of mice with influenza A lial cells to sense microbial products (possibly indirectly) virus (strain H3N1) caused airway hyperreactivity in and to release factors that promote the appropriate ILC an IL‑33R- and IL‑13‑dependent manner that was inde‑ response. pendent of B and T cells15. The development of H3N1 influenza A virus‑induced airway hyperreactivity was ILCs in asthma and allergy preceded by the production of IL‑33 by alveolar macro In addition to the crucial roles identified for ILCs in phages and increased numbers of ILC2s in the lungs. response to pathogens, recent studies have highlighted Treatment of mice with THY1‑specific depleting anti‑ the involvement of ILC subsets in regulating tissue bodies to remove ILC2 populations (THY1 is expressed homeostasis and pathology. Allergic responses are by all ILCs, but also by basophils and numerous other characterized by the production of type 2 cytokines, cell types) resulted in resistance to virus-induced airway leading to eosinophil and mast cell degranulation, hyperreactivity. By contrast, the transfer of ILC2s into goblet cell hyperplasia, mucus production, increased Il13−/− mice — which are normally resistant to virus- serum IgE levels and smooth muscle contraction. Such induced airway hyperreactivity — resulted in the devel‑ responses can develop following repeated low-dose opment of airway hyperreactivity in the mice following exposure to normally innocuous allergens, such as virus infection15. Similar results were reported by another bacterial products, food items, plant pollens or fungal group, who went on to show that amphiregulin is expressed spores at mucosal barriers. by ILC2s during viral lung infection, and it was suggested The preconception that allergic lung responses are that this is important for tissue repair17.

solely TH2 cell-mediated is now being questioned fol‑ Given the recent identification of human ILC2s in 23 lowing the discovery of ILC2s. Although TH2 cells are a samples from patients with chronic rhinosinusitis , it is major source of type 2 cytokines during allergic asthma, important that future studies investigate the role of these ILC2s also contribute to disease pathology. Using a com‑ cells in other allergic disorders, such as atopic dermati‑ pound mouse strain expressing EGFP as a reporter for tis and allergic asthma. Furthermore, studies of influ‑ IL‑4 and the fluorescent protein tdTomato as a reporter enza virus infection in mice highlight potential roles for 15,17 for IL‑13, TH2 cells were identified as the major cellu‑ ILC2s in virus-induced asthma exacerbation . lar source of these cytokines in an ovalbumin-induced mouse model of allergic lung inflammation13,80. Strikingly, ILCs in inflammatory bowel diseases however, ILC2s also expressed Il13‑tdTomato but not Inflammatory bowel diseases (IBDs), such as Crohn’s

Il4‑EGFP. Taken together, TH2 cells and ILC2s accounted disease and ulcerative colitis, are chronic inflammatory for the majority of IL‑13 expression in the lungs during disorders of the gastrointestinal tract. Crohn’s disease ovalbumin-induced lung allergy. ILC2s also accounted for is characterized by transmural, discontinuous inflam‑ the majority of IL‑13 expression in the lungs in response mation along the intestinal tract, whereas ulcerative to both IL‑25 and IL‑33 (REFS 13,16,80), and they have colitis involves inflammation of the superficial mucosal subsequently been shown to express IL‑5 (in Il5‑Venus and submucosal tissue layers of the colon. These dis‑ reporter mice) and to respond to thymic stromal eases have a highly complex aetiology that may include lymphopoietin (TSLP), in synergy with IL‑33, in vitro81,82. mutations in single crucial genes or in a combination In studies using Il13−/− mice, which are refractory of multiple disease susceptibility alleles. These genetic to the induction of allergic lung inflammation, it was aberrations lead to the disruption of intestinal homeo‑ shown that the transfer of IL‑13‑expressing ILC2s was stasis, including effects on epithelial barrier function, sufficient to restore airway hyperreactivity, eosino‑ local immune cell responses and the diversity of the gut philia and cytokine production in an IL‑25‑induced microbiota84. lung inflammation model (REF. 13 and J.L.B., unpub‑ IL‑17A, IL‑17F, IL‑22 and IFNγ have essential roles lished observations). ILC2s were also sufficient to drive in IBDs84, whereas IL‑13 has been reported to play a sig‑ allergic responses in glycolipid-induced lung allergy16. nificant part in ulcerative colitis85,86. Although the secre‑ Therefore, IL‑13 production by ILC2s alone can restore tion of IL‑17A, IL‑22 and IFNγ is principally ascribed + allergic responses, even when IL‑13 from CD4 T cells to TH cells, additional sources of these cytokines include is absent. Similarly, intranasal treatment of mice with γδ T cells, natural killer T (NKT) cells and NK cells84. a fungal allergen from Alternaria alternata also led to Notably, recent studies have also implicated ILCs in the increased production of the ILC2‑inducing cytokine development of IBDs. One group reported the exist‑ IL‑33. Maximal IL‑33 production occurred at ~12 hrs ence of a CD11b−B220−GR1−THY1+ ILC population after treatment and resulted in IL‑33R‑dependent ILC2 that arose in Rag2−/− mice in response to Helicobacter Amphiregulin population expansion in the lungs, eosinophilia and hepaticus infection and that secreted IL‑17A, IL‑22 and A member of the epidermal IL‑5 and IL‑13 production14. IFNγ in response to IL‑23 (REF. 56). A functional role for growth factor family that drives The important role of ILC2s in virus-induced experi‑ these cells was determined by showing that antibody- the proliferation of epithelial mental models of airway hyperreactivity has been rec‑ mediated depletion of THY1+ cells in H. hepaticus- cells and fibroblasts to −/− promote tissue repair and ognized recently. Several viral respiratory infections infected Rag2 mice ameliorated disease development, remodelling in response to (namely rhinovirus, respiratory syncytial virus and although once again the caveat remains that THY1 is not epithelial injury. influenza virus infections) promote type 2 responses specifically expressed by ILCs.

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Similarly, abrogation of colitic pathology was ILCs may also maintain intestinal homeostasis dur‑ observed when THY1+ ILCs were depleted in a mouse ing colonization with the intestinal microbiota by pro‑ model of colitis that is induced by the administration of moting the formation of isolated lymphoid follicles. In CD40‑specific antibodies, and the decreased pathology the absence of RORγt (which results in the loss of LTi

following ILC depletion coincided with decreased levels cells, isolated lymphoid follicles and TH17 cells), large of IFNγ, IL‑22 and TNF56. The colitogenic ILCs did not numbers of B cell-rich tertiary lymphoid tissues form produce IL‑17A, possibly indicating an element of plas‑ in response to the natural endogenous load of intes‑ ticity that is dependent on the specific stimuli. Although tinal bacteria88. Following dextran-sulphate sodium these cells appear to be phenotypically distinct from the (DSS)-induced epithelial barrier damage, Rorc−/− IFNγ-producing NK receptor-expressing (NKR+) LTi mice develop severe intestinal inflammation charac‑ cell population that was previously shown to promote terized by the infiltration of neutrophils and B cells. colitis28, it is likely that such RORγt−IFNγ+NKR+ LTi These pathological consequences were reversed by cells reside within this heterogenous THY1+SCA1+ antibiotic treatment or the administration of intra population56. Thus, ILCs can contribute to the devel‑ venous IgG. Thus, the depletion of RORγt-dependent

opment of intestinal inflammation by secreting IL‑17A cells — which include LTi cells, ILC3s and TH17 cells and IFNγ in response to IL‑23. A role for ILCs has also — results in compromised homeostasis with the been demonstrated recently in the Tbx21−/−Rag2−/− intestinal microbiota. ulcerative colitis (TRUC) disease model87. These mice ILCs have also been detected in the human intes‑ have a predominance of ILC3s secreting IL‑17A, but tine and accumulate under the inflammatory condi‑ very few IFNγ‑expressing cells, and treatment with a tions that occur in Crohn’s disease89. In this study, THY1‑specific antibody ameliorated disease. LIN−CD45+ cells from the ileum and colon of patients with Crohn’s disease were sorted on the basis of their expression of CD56. The LIN−CD45+CD56+ cells Adaptive lymphoid cells Innate lymphoid cells could be induced to express mRNA encoding IL‑22 and IL‑26 in response to IL‑23 stimulation, whereas the LIN−CD45+CD56− cells expressed IL‑17A and T 2 H ILC2 Group 2 ILCs IL‑17F, and this was not altered by IL‑23. IFNγ was cell GATA3 expressed by both subsets. Another study also detailed RORα distinct NKp44+ and NKp46+ ILC populations in the GATA3 Notch intestines of patients with Crohn’s disease90. The NKp44+NKp46−CD56+CD122+IL‑7Rα+ cells expressed IL‑22 (suggesting that they are a type of ILC3), and the LTi cell authors reported that the numbers of these cells were RORγt decreased in patient samples compared with controls. − + + + − TH17 By contrast, the NKp44 NKp46 CD56 CD122 IL‑7Rα cell RORγt cells responded to IL‑23 by secreting IFNγ, which is Lymphoid RORγt – consistent with an ILC1 phenotype, and the numbers NCR Group 3 ILCs precursor ILC3 of these cells were increased in patients with Crohn’s AHR disease. It is clear from these reports that extensive RORγt phenotyping is necessary to more accurately categorize the ILC populations that are present in the inflamed + TH22 NCR human intestine. ? cell ILC3 Unlike Crohn’s disease, ulcerative colitis is dis‑ tinguished by a type 2 immune phenotype, and the levels of IL‑4, IL‑5 and IL‑13 associate with the sever‑ T-bet ity of intestinal pathology in patients85,91. Recently, ILC1 IL‑13‑producing ILC2s have been described in an oxazolone-induced mouse model of colitis, which T 1 H E4BP4 Group 1 ILCs is characterized by a type 2 inflammatory response. cell This correlated with the expansion of IL‑13‑producing NKT cell populations, in keeping with previous NK cell reports86. Notably, at the time points analysed, the IL‑13‑secreting NKT cells were localized almost entirely in the mesenteric lymph nodes, whereas the Figure 4 | Comparison of T helper cell and ILC subsets. The innate lymphoid cell ILC2s were distributed in the lamina propria of the (ILC) subsets (shown on the right) broadly parallel the known T helper (T ) cell subsets Nature ReviewsH | Immunology (shown on the left) in terms of their signature cytokine secretion profiles. An intestinal mucosa. This study also demonstrated that overlapping series of transcription factors is also used to drive the differentiation of neutralizing IL‑25‑specific and IL‑17RB‑specific antibodies effectively blocked pathology in this the various ILC and TH cell subsets. AHR, aryl hydrocarbon receptor; E4BP4, E4 mouse model. Further investigations will be neces‑ promoter-binding protein 4; GATA3, GATA-binding protein 3; IH2, innate helper 2; LTi, lymphoid tissue-inducer; NHC, natural helper cell; NK, natural killer; ROR, retinoic acid sary to elucidate the functional importance of ILC2s receptor-related orphan receptor. in human IBDs.

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Box 1 | Challenges facing the ILC field • The elucidation of the physiological roles of innate lymphoid cells (ILCs) in health and disease requires the development of more sophisticated mouse models in which the various ILC lineages can be deleted specifically, rather than relying on antibody-mediated depletion strategies that invariably result in ‘collateral damage’ to other cell populations. Such mice will also enable the distinct roles of ILCs and T cells during immune responses and wound healing to be dissected, which will in turn clarify the immunological significance of having these two, distinctly regulated sources of cytokines. • The current knowledge of the tissue localization of ILCs, their chemokine-regulated migration and their interactions with other immune and stromal cells is in its infancy. Progress will be aided by the development of new cell-lineage-reporter mouse strains to enable the marking of ILCs in the context of other adaptive and innate cell populations during immune responses. • Future investigations need to better define the interactions of ILCs with the adaptive immune system in order to understand the initiation, maintenance and termination of immune responses. A role for regulatory ILC subsets in the resolution phase of immune responses remains an unexplored possibility. • Despite significant progress in understanding the development of ILCs, investigation of the temporal regulation of crucial transcription factors and the signals required to direct differentiation is lacking. Such studies may begin to define the degrees of overlap and the subtle differences between ILC and T cell development.

ILC crosstalk with other cells An important question with regard to ILCs is how in Rag2−/− mice, it was noted that ILC2 numbers were these cells interact with other cells in their surround‑ not maintained, suggesting that T cells provide survival ing environment. Several studies suggest that epithelial signals to ILCs, for example through the production of cell-derived factors, and signals from adaptive immune IL‑2 (REFS 11,98). Although it remains to be determined cells, may be important for regulating ILCs. In the intes‑ whether ILC2s can process and present antigens, the tine, it was noted that IL‑25 levels, which were increased expression of MHC class II molecules by ILC2s indicates by age and microbial colonisation, correlated inversely that MHC–T cell receptor interactions might facilitate with those of IL‑22, and that exogenous administration dialogue between these cell populations11. Physiologically, of IL‑25 suppressed the expression of IL‑22 by ILC3s92. this could provide a means for ILCs to contribute to the This effect was not direct and required an intermediary initiation of a T cell response through antigen presenta‑ IL‑25‑responsive dendritic cell population. Supporting tion, and it suggests a mechanism for the termination of these findings, Il25−/− mice had more ILC3s. In the the ILC response as the antigen is cleared and the T cell lungs, IL‑22 is required for the onset of airway inflam‑ population contracts. mation, but also has a protective effect during established inflammation93,94. The predominant source of IL‑22 in an The future — the known unknowns allergic asthma model was LIN−THY1.2+SCA1+RORγt+ Research over the past few years has revealed a previously ILC3s95, a proportion of which also produced IL‑17A. unappreciated family of ILCs with diverse physiological Interestingly, the abundance of IL‑25 in bronchoalveolar roles, ranging from immune protection to wound repair lavage fluid correlated inversely with the levels of IL‑22, and homeostasis. Remarkable progress has been made and treatment with IL‑25‑specific blocking antibodies in this field, with the discovery not only of distinct sub‑ was sufficient to reverse the pro-inflammatory effect sets of ILCs but also of the transcription factors that are of IL‑22‑specific antibodies95,96. Thus, IL‑22 and IL‑25 responsible for their development. Indeed, a comparison have antagonistic roles in controlling immune responses of T cell and ILC subsets now reveals striking similari‑ in mucosal tissues. There is also evidence that IL‑17A ties (FIG. 4). However, many challenges remain (BOX 1), not and IL‑25 may interact to influence lung inflammation in least the identification and more detailed characterization mice97. The ability of IL‑25‑specific antibodies to prevent of the roles of ILCs in human health and disease. airway hyperreactivity is dependent on increased produc‑ So, ILCs — how did we miss them? Well, we had just tion of IL‑17A in the lungs, and simultaneous blockade of gated them out, assuming that we already knew all the IL‑17A inhibits the therapeutic efficacy of IL‑25‑specific players. Who knows what other unknowns wait to be treatment. In this model, IL‑17A was shown to be derived discovered within the LIN– population? from an undefined non‑B cell and non‑T cell source97. In addition to the crosstalk that exists between ILCs Note added in proof and the epithelium, ILCs might also be influenced by the A recent report has indicated a central role for T-bet in adaptive immune system. During helminth infection ILC3 development109.

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