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Innate lymphoid cells (ILCs) represent a novel family of hematopoietic effectors that serve protective roles in innate immune responses to infectious microorganisms, in lymphoid tissue formation, in tissue remodeling after damage inflicted by injury or infection and in the homeostasis of tissue stromal cells. The prototypes of the ILC family are natural killer (NK) cells and lymphoid tissue–inducer (LTi) cells, which have different functions but have been shown to be develop-mentally related. Additional ILC populations with characteristics of both NK cells and LTi have been described (called NK22 cells, natu-ral cytotoxicity receptor 22 (NCR22) cells or NK receptor–positive (NKR+) LTi cells). Whereas NK cells generally produce interferon-γ (IFN-γ), LTi and NKR+ LTi-like cells produce interleukin 17 (IL-17) and/or IL-22, which suggests that these ILCs might represent innate versions of cells of the TH17 and TH22 subsets of helper T cells. Other novel ILC subsets in the mouse include ‘natural helper’ (NH) cells, or ‘nuocytes’, which have been shown to produce IL-5 and IL-13 associ-ated with T helper type 2 (TH2) responses. The remarkable functional diversity of the ILC family is reminiscent of that of T cells and seems to be under the control of an analogous transcription factor–directed regulation. Here we review the properties of these various ILC popula-tions in humans and mice, their developmental origins and the regula-tion of their effector functions.

Phenotype and function of NK cellsNK cells were the first ILC subset to be described with the characteristic property of prompt delivery of effector functions (including hardwired cytokine production) that help define the prototypic ILC. Humans and

mice with dysfunctional NK cells or deficiencies in NK cells are highly sensitive to viral infections, especially those caused by herpes viruses, which indicates that NK cells have a key role in limiting viremia before the initiation of the adaptive immune response1,2. NK cells can rec-ognize pathogen-induced ligands by using specific receptors and can eliminate infected or stressed target cells through the use of various effector pathways (such as perforin- and granzyme-containing cytotoxic granules, FasL, TRAIL, IFN-γ and tumor necrosis factor (TNF)3).

NK cells present in different tissues are phenotypically and func-tionally diverse4. In humans, two populations of NK cells can be dis-tinguished on the basis of CD56 expression5. It has been proposed that CD56lo cells that also express CD16 (the low-affinity receptor for immunoglobulin G, FcγRIII) have enhanced killing activity, whereas CD56hi cells are CD16− and are able to secrete large amounts of cytokines (IFN-γ, GM-CSF and TNF)6. Still, with the appropriate stimulus, CD56loCD16+ NK cells are abundant cytokine producers7. Similarly, mouse NK cell subsets that can be distinguished on the basis of their expression of CD27, CD11b, CD127 and KLRG-1 dem-onstrate distinct functional properties8. Some human CD56hiCD16− NK cells express CD127 and may be similar to thymic NK cells in the mouse that are Notch independent, characterized by expres-sion of the transcription factor GATA-3 and CD127 (IL-7 receptor α-chain), and show enhanced cytokine production9,10. On the basis of their notably T helper type 1 (TH1) cytokine-expression profile, we propose the designation ‘ILC1’ for these IFN-γ-biased (and mostly non-cytotoxic) NK cell subsets (Fig. 1). Although the developmental relationships among the various human and mouse NK cell sub-sets remain unclear, there is experimental evidence linking these subsets in a linear differentiation scheme11. Thus, NK cell subsets would represent different states of cellular activation or maturation that may be driven by tissue-specific environmental signals, notably trans-presented IL-15 and its receptor IL-15Rα on stromal cells, epi-thelial cells and dendritic cells (DCs) and by the proinflammatory

1Tytgat Institute for Liver and Intestinal Research, Academic Medical Centre, Amsterdam, The Netherlands. 2Innate Immunity Unit, Institut Pasteur, Paris, France. 3Institut National de la Santé et de la Recherche Médicale U668, Paris, France. Correspondence should be addressed to H.S. (hergen.spits@amc.uva.nl).

Published online 28 November 2010; doi:10.1038/ni.1962

The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodelingHergen Spits1 & James P Di Santo2,3

Research has identified what can be considered a family of innate lymphoid cells (ILCs) that includes not only natural killer (NK) cells and lymphoid tissue–inducer (LTi) cells but also cells that produce interleukin 5 (IL-5), IL-13, IL-17 and/or IL-22. These ILC subsets are developmentally related, requiring expression of the transcriptional repressor Id2 and cytokine signals through the common γ-chain of the IL-2 receptor. The functional differentiation of ILC subsets is orchestrated by distinct transcription factors. Analogous to helper T cell subsets, these evolutionarily conserved yet distinct ILCs seem to have important roles in protective immunity, and their dysregulation can promote immune pathology.

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cytokines IL-1β, IL-12 and IL-18. In some tissues (uterus and pancreas), these diverse NK cells seem to have roles unrelated to protection against microbial pathogens and instead promote vascular remodeling12 or tissue-specific pathologies13.

Phenotype and function of LTi cellsLTi cells induce the formation of lymph nodes during embryogenesis. In the mouse, these cells lack markers specific for T cells, B cells and myeloid cells but express CD4, lymphotoxin-α (LT-α) and LT-β, several chemokine receptors (CXCR5 and CCR7), cytokine receptors (CD127, CD117 or c-Kit), and the ligand for the receptor activator RANK. Developing lymph nodes collect CD4+CD3−LTα+LT-β+ cells that can differentiate into antigen-presenting cells or NK-like cells but not into T cells or B cells14,15. LTi cells stimulate lymph node formation through LTi cell–stromal cell clustering. Subsequent bind-ing of the receptor for LT-β on stroma-organizer cells to LT-α1LT-β2 on LTi cells upregulates expression of adhesion molecules such as VCAM-1, ICAM-1 and MADCAM-1 and induces the secretion of many chemokines, including CXCL13, CCL19 and CCL21. As a con-sequence, hematopoietic cells, including B cells, T cells and DCs, are recruited to form the lymph node15,16.

Mouse LTi cells are well characterized15–17. Notably, LTi cells are dependent on the transcriptional repressor Id2 (discussed below)18, the transcription factor RORγt (encoded by Rorc)19,20 and the cytokine IL-7 (ref. 21). The phenotype and characteristics of the human equivalent are known. Human LTi cells have been detected in human fetal mesenteric lymph nodes and, like mouse LTi cells, they express the transcriptional regulators Id2 and RORγt and several cell surface antigens (including CD127 and CD117, although only mouse (not human) LTi cells express CD4)22. Interestingly, cells very similar to fetal LTi cells persist after birth in both mice23 and humans22. Postnatal LTi cells are important for the formation of isolated lymphoid follicles in the gut in response to pathogen-associated patterns24,25. Evidence indicates that postnatal LTi cells are also involved in restoring damaged lymph nodes after acute viral infection in adult mice26.

Functional analysis of LTi cells has unexpectedly shown that these cells produce IL-17 and/or IL-22, which are involved in tis-sue remodeling and immunity27. LTi cells isolated from human fetal mesenteric lymph nodes express mostly IL-17 (ref. 22), and CD4+ LTi cells from post natal mouse spleen express both IL-17 and IL-22 (ref. 28), but whether or not the IL-17-producing mouse CD4+ LTi

cells also secrete IL-22 remains unclear. IL-17 is a proinflammatory cytokine that promotes neutrophil recruitment and the production of cytokines and antimicrobial peptides by epithelial cells and also has a role in angiogenesis. Furthermore, disruption of IL-17 affects the formation of germinal centers29,30. IL-22 is a member of the IL-10 cytokine family and acts on epithelial cells, such as gut epithelial cells and keratinocytes of the skin, and triggers the production of anti-microbial peptides such as β-defensin and the expression of genes involved in cellular differentiation and survival; therefore, IL-22 is thought to be involved in the homeostasis of epithelia and also in early host defense against microbial pathogens31. As production of these cytokines is not required for lymph node formation, the production of IL-17 and IL-22 by LTi cells suggests that these cells have roles in tissue immunity. Evidence from mouse studies suggests that LTi cells communicate not only with stromal cells but also with other cells of the immune response. In the gut, LTi cells support T cell–independent production of immunoglobulin A32. Moreover, research suggests that LTi cells are important for the maintenance of memory CD4+ T cell responses though interactions between the T cell–costimulatory molecule OX40, on memory T cells, and its ligand OX40L, which seems to be constitutively expressed on LTi cells33 (D. Withers and P. Lane, personal communication). Collectively, these observations would suggest that LTi cell functions are developmentally regulated.

Phenotype and function of ILC22 cellsAn additional ILC type (ILC22) has been identified that shares some characteristics with LTi cells and NK cells22,34–37. Thus far, these ILC22 cells have been found mainly at mucosal sites both in mouse and humans (for example, in the lamina propria of the intestine, Peyer’s patches, mesenteric lymph nodes and palatine tonsils). In humans, they express CD56 and NKp44 and have low NKp46 expression22,34, whereas in mice they express NKp46 but have low to no NK1.1 expression35–37. Similar to postnatal LTi cells in the mouse and human, these NKp46+ cells express IL-22 transcripts in situ. Because of the expression of NK cell markers and their ability to produce large amounts of IL-22, these cells were dubbed ‘NK22 cells’34 or ‘NCR22 cells’38. However, these cells are distinct from conventional NK cells, as they are noncytotoxic, lack killer inhibitory receptors (in humans) and Ly49 (in mouse), and produce little if any IFN-γ. Here we will refer to these IL-22-producing innate cells (including NK22, NCR22, NKR+ LTi and LTi-like NK cells) as ‘ILC22 cells’ (Fig. 1). Human IL-22-producing ILCs are defined by

Figure 1 The expanding family of ILCs. Distinct ILC subsets develop from hematopoietic precursors in an Id2-dependent way in a process orchestrated by transcription factors (Fig. 2). ILCs can be grouped into three branches: NK, helper and RORγt. The IL-15-dependent NK branch includes conventional NK (cNK) cells, which have spontaneous cytotoxicity, and the IFN-γ+ ILC1 subset, which includes thymus and IL-7-dependent NK cells in mice and a cytokine-polarized subset of CD56hi cells in humans. ILC1 cells function to protect against infection by viruses and intracellular pathogens, and their activity is promoted through IL-12 and IL-18. The helper branch contains the ILC2 subset (including nuocytes and NH cells) that produce abundant IL-13 under the influence of IL-25 and IL-33. ILC2 cells are critical in the control of extracellular parasites. The RORγt branch includes LTi cells, ILC17 cells and ILC22 cells. All of these ILC subsets express and depend on RORγt and require IL-7 for their development. Signals that trigger secretion of cytokines from these cells vary; the RANK ligand triggers LTi cells to express cell surface heterotrimers of LT-α and LT-β, whereas IL-23 or IL-1β triggers the production of IL-17 and IL-22 from ILC17 and ILC22 cells, respectively. Dysregulation of the different ILC subsets may be associated with disease as follows: ILC1, inflammation; ILC2, allergy; ILC17, autoimmunity; and ILC22, autoimmunity. ILCP, Id2-expressing ILC precursor; IBD, inflammatory bowel disease.

FunctionsInnate lymphoid cells

CD16++

ILC1(Thymic NK cells)

cNKIFN-γIFN-γIFN-γ

Intracellular pathogens, virusInflammation

Extracellular parasitesAllergy (asthma)

IL-15 IL-15

IL-13ILC2

(Nuocytes, NH cells)

γc cytokine

LN formationIsolated lymphoid follicle formationT cells–independent B cell help

LTi

ILCP

LT-α–LT-β

Extracellular bacteriaAutoimmune disease(IBD)

IL-22

IL-7IL-7

IL-7

TNF

ILC17

RORγt

RORγt

RORγt

IL-17

ILC22(NK22, NCR22, NKR+ LTi cells)

Extracellular bacteriaAutoimmune disease IL-2

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their expression of CD127, CD117 and NKp44; many of these cells express CD56, although CD56− ILC22 cells exist as well39. ILC22 cells, like LTi cells, are able to induce expression of ICAM-1 and VCAM-1 on mesenchymal stem cells in vitro (which can be considered a surrogate assay for LTi activity)22,39. Still, formal proof that ILC22 cells have LTi activity in vivo is still lacking.

Evidence exists indicating that the presence of ILC22 cells in the mouse intestinal tract is modulated by the presence of gut commen-sals, as analysis of germ-free mice suggests that the homeostasis of ILC22 cells in such mice is compromised relative to that of ILC22 cells in conventional mice bearing microbial flora36,37. What particu-lar microbial species condition the development and homeostasis of intestinal ILC22 cells is not yet known. Microbiota, however, are not strictly required for the development of ILC22 cells, as these cells are found in the gut of germ-free mice40.

Several murine studies indicated that IL-22-producing cells in the gut mediate early protective innate immune responses to the colitis-inducing pathogen Citrobacter rodentium37,41. Although DCs were initially pro-posed as the source of IL-22 production in this context41, subsequent studies have convincingly demonstrated that ILC22 cells represent a potentially critical innate source of IL-22 in this model37,42.

Soluble factors (including IL-23) can regulate IL-22 production by ILC22 cells in mice35–37 and humans34. Cytokines of the com-mon γ-chain (γc) family (IL-2, IL-7 and IL-15) can also activate the proliferation and cytokine production of human ILC22 cells34,43. Combinations of IL-12 and IL-18 can also enhance IL-22 produc-tion by these cells in mice42. In contrast, crosslinking of cell surface receptors (NKp46, 2B4, NK1.1) on mouse ILC22 cells fails to elicit the secretion of IL-22, IL-17 or IFN-γ from these cells42.

Human ILC22 cells isolated from tonsils secrete many cytokines, including IL-2, IL-5, IL-8, IL-13 and TNF43. The large amount of IL-2 produced by human ILC22 cells is particularly striking and might suggest that these cells are involved in the recruitment of regulatory and/or effector T cells at mucosal sites43. Interestingly, human ILC22 cells also secrete large amounts of B cell–activation factor44 that may have a role in regulating T cell–independent antibody production.

Phenotype and function of IL-17-producing ILCsAn IL-17-producing ILC subset (ILC17) has been described in mice45. These IL-17-producing cells are present mainly in the intestinal tract (especially the colon) and express and require Rorc for development and function45. IL-17-producing ILCs are CD4−CD117−NKp46−, which dis-tinguishes them from CD4+CD117+ LTi cells and NKp46+ ILC22 cells. ILC17 cells also express CD90 (Thy-1), which is expressed by NK cells and ILC22 cells (S. Takayama and J.P.D., unpublished data). ILC17 cells are abundantly recruited to the intestine under inflammatory conditions, and IL-17 production by this ILC subset is regulated by IL-23 and is responsible for the induction of intestinal pathology45.

In humans, fetal CD127+CD117+ LTi cells express IL-17, whereas LTi cells express mostly IL-22 and very little IL-17 in the postnatal tonsil22. One interpretation of those data is that there is developmental regulation of IL-17 production versus IL-22 production, but it is also possible that there are distinct subsets of IL-17- and IL-22-producing ILCs. The latter idea is supported by the finding that ILCs expressing IL-17 and IL-22 in vivo differ phenotypically (T. Cupedo, personal communication). Moreover, clonal analysis of lineage-negative (Lin−) CD117+CD127+ cells from tonsil has identified the presence of cells that produce IL-17 and not IL-22, with the frequency of ILC17 cells being much lower than that of IL-22-producing ILCs, which explains the modest IL-17 expression in total tonsil Lin−CD127+ cells39. Interestingly, clones that produce both IL-17 and IL-22 can also be isolated39.

Collectively, the results so far suggest that ILC subsets with dedi-cated production of IL-17 or IL-22 exist (and may predominate), whereas IL-17+IL-22+ ILCs (which are reminiscent of TH17 cells) can also develop. The analysis of ILCs in human fetus seems to suggest that LTi cells and ILC17 cells are overlapping cell populations. Future research should be directed at elucidating the interrelationship and functions of these IL-17- and IL-22-producing RORγt+ ILC subsets.

Phenotype and function of ILC2 cellsIL-25 (an IL-17 family member; also called IL-17E) and IL-33 (an IL-1 family member) activate TH2 responses46,47. In addition to TH2 cells, non-T, non-B cells respond to IL-25 by proliferating and produc-ing TH2 cytokines, including IL-13. These IL-25-responsive innate cells lack lineage markers and are γc dependent47, but otherwise their phenotype is not firmly established. An IL-25- and IL-33-responsive ILC subset was found in a newly identified lymphoid structure associ-ated with adipose tissues in the mouse peritoneal cavity48. These ‘fat-associated lymphoid clusters’ are present in both human and mouse mesentery, express CD117, Sca-1 (Ly6a) and CD127 and are distinct from lymphoid progenitors. As innate cells associated with the fat-associated lymphoid cluster produce large amounts of TH2 cytokines (IL-5 and IL-13), these cells have been dubbed ‘natural helper’ (NH) cells. NH cells are distinct from LTi and ILC22 cells, as they are RORγt− and have no demonstrated in vitro LTi activity or IL-22 production48. In mice bearing a green fluorescent protein reporter inserted into the Il13 locus were an additional ILC called the ‘nuocyte’ was described49. Whereas NH cells were observed in fat-associated lymphoid clusters, nuocytes were detected in very small quantities in mesenteric lymph nodes. Although details of the cell surface pheno-type of nuocytes are not yet known, the observation that both NH cells and nuocytes express CD127 and are able to robustly produce IL-5 and IL-13 suggests that these cell types have analogous func-tions. That proposal is further supported by the observation that both NH cells and nuocytes express IL-33R (also known as ST2, IL1RL1, DER4, T1 and Fit-1) and IL-17RB, which are receptors for IL-33 and IL-25, respectively. These cytokines similarly stimulate NH cells and nuocytes to proliferate and to produce IL-5 and IL-13 (refs. 48,49). Another report has described cells in mesenteric lymph nodes that might represent precursors of TH2 cytokine–producing innate leukocytes, including basophils, mast cells and TH2 cytokine–producing ILCs50, which suggests a close relationship among NH cells, nuocytes and myeloid cells involved in type 2 immunity, an idea that is difficult to reconcile with the lymphoid nature of NH cells and nuocytes48,49.

The administration of IL-25 promotes TH2-type immune responses46, and IL-25-deficient mice have an impaired TH2 response to infection by the parasitic helminthes Nippostrongylus brasiliensis and Trichuris muris, which results in greater susceptibility to infection and chronic inflammation51,52. IL-25 also mediates pulmonary antigen–induced TH2 responses in the lung. Both NH cells and nuocytes mediate expul-sion of N. brasiliensis by inducing goblet cell hyperplasia, a critical first step in worm expulsion48,49. This effect is IL-13 dependent, as IL-13-deficient nuocytes are unable to induce worm expulsion49. Interestingly, nuocytes engage in crosstalk with T cells, as they promote the gen-eration and population expansion of IL-13-producing T cells, whereas T cells are needed to maintain the number of nuocytes via an as-yet-unknown mechanism49. NH cells promote the self-renewal of B-1 cells and promote the production of immunoglobulin A, presumably in an IL-5-dependent way48.

In IL-4 and IL-13 reporter mice, IL-25- and IL-33-responsive cells are more widely distributed in tissues in the mouse than suggested by earlier

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studies53. These cells express CD117, CD90.1, CD44 and CD122 (IL-2 receptor β-chain) and are particularly prevalent in mesenteric lymph nodes, spleen and liver. Whether these different IL-25-responsive (and IL-33-responsive) cells in different tissues are identical remains to be firmly established, but given the similarity of these cell types and the fact that they seem to be dedicated to producing TH2 cytokines, we propose that this subset be called ‘ILC2’.

Human ILC2 cells are not yet precisely defined. A possible can-didate is a Lin−CD161+CD56− cell population that develops in vitro from cord blood CD34+ progenitor cells in the presence of IL-2; this population produces IL-13 but not IFN-γ and includes an IL-5+ subset54. Lin−CD127+CD117+ cells are able to produce IL-5 and IL-13 after stimulation with Toll-like receptor 2 ligand and IL-2 (ref. 43). In contrast to mouse ILC2 cells, these IL-13-producing human ILCs express RORγt and IL-22. Clonal analysis has shown that most of those clones coexpress IL-22 and IL-13, although some clones have been identified that produce IL-13 and IL-5 but not IL-22. Interestingly, the IL-5+IL-13+IL-22− clones still express transcripts of the gene encoding RORγt (RORC)43; this suggests that these cells may not be the human equivalent of mouse ILC2 cells, which lack RORγt expression.

Plasticity of cytokine production by ILC subsetsSimilar to CD4+ helper T cell subsets, several ILC subpopulations seem to be polarized toward a restricted cytokine-production pro-file. However, just as there is a growing realization that CD4+ helper T cells are more ‘plastic’ in their cytokine production than previ-ously thought55–57, evidence is accumulating indicating that ILCs have substantial plasticity in cytokine production as well. Exogenous triggers can change the ILC cytokine–production profile. Whereas ILC22 cells freshly isolated from human tonsil produce IL-22 (but not IL-17 or IFN-γ), these cells produce IL-17 after culture with IL-1β and IL-7 (ref. 44). Human ILC22 cells also make IL-5 and IL-13 in addition to IL-22, although the signaling requirements for the synthesis of these cytokines seem to be different, as stimula-tion with IL-23 plus IL-2 results in the induction of IL-22 but not of IL-13, whereas stimulation with IL-2 and the Toll-like receptor 2 agonist Pam3Cys results in the production of both IL-22 and IL-13. Studies using specific inhibitors have shown that IL-13 production is dependent on the transcription factor NF-κB, which indicates that plasticity may result from the use of different signaling pathways43. Although freshly isolated ILC22 cells do not express IFN-γ, culture of these cells in IL-2 (ref. 44) or together with irradiated peripheral blood mononuclear cells43 induces IFN-γ production. Similarly, mouse ILC22 cells stimulated with IL-12 and IL-18 express IFN-γ (but not IL-17), with some cells co-expressing IL-22 and IFN-γ42. It is likely that environmental cues orchestrate epigenetic alterations that account for changes in cytokine outputs. The future challenge will be to understand how transcriptional programs, epigenetic mechanisms and microRNA-mediated control of cytokine expression determine the functionality of the different ILC subsets.

Development of various ILC subsetsTranscription factors and cytokines represent two classes of signals that have dominant roles in the specification of hematopoietic lineages from multipotent progenitors. Responsiveness to transcription factors and responsiveness to cytokines are frequently linked, as transcription factor targets include the cytokine and growth factor receptors that allow the survival, proliferation and differentiation of lineage-specific precursor cells. Several transcription factors have been identified that induce or modulate the development of ILCs (Fig. 2).

The development of T cells, B cells and DCs critically requires members of the E2A transcription factor family (which includes E12, E47, E2-2 and HEB)58. In contrast, the transcriptional activity of E2A proteins acts to inhibit the development of several ILC subsets, an effect that is overcome by the activity of Id (‘inhibitor of DNA bind-ing’) proteins that form heterodimers with E2A proteins, rendering them functionally inactive. Of the four Id proteins (Id1–Id4), Id2 is critical for the normal development of mouse NK cells and LTi cells, as Id2-deficient mice have considerably fewer NK cells and lack LTi cells18. Ablation of E47 in Id2-deficient mice restores the develop-ment of NK cells, lymph nodes and Peyer’s patches59, which demon-strates that Id2 acts to ‘titrate’ E47 activity. In human hematopoietic precursor cells, Id proteins similarly promote the development of NK-like cells while inhibiting the development of T cells, B cells and plasmacytoid DCs60,61. Other ILC populations, including NH cells or ILC2 and ILC22 cells, depend on Id2 (refs. 38,48). Thus, the data so far indicate that all ILCs require Id2 for their development, which provides a rationale for classifying these cells in one family.

NFIL3 (E4bp4) is a bZIP family transcription factor critical for the development of NK cells62,63. Mechanistically, NFIL3 is thought to regulate Id2 expression in NK precursor cells and immature NK cells. Although no other ILC subset deficiencies have been reported in NFIL3-deficient mice (their lymph node formation seems to be normal), the observation that these mice develop an intestinal inflammatory syn-drome (H. Brady, personal communication) warrants detailed analysis of other ILC subsets.

The transcription factor RORγt was initially identified for its role in thymocyte survival, for which it acts through its transcriptional target Bcl-xL (ref. 19). Subsequently, RORγt was shown to be critical for LTi activity, although the transcriptional targets of RORγt in LTi cells are not known and do not involve Bcl-xL, as Bcl-xL overexpression does not restore LTi function in Rorc-deficient embryos20. ILC22 cells in the mouse require Rorc for their development35–37. RORγt conditions the expression of IL-17 and IL-22 in T cells64 and may have a similar role in differentiated human and mouse ILC17 and ILC22 cells. In contrast, the development of conventional NK cells and ILC2 cells seems to be Rorc independent36,37,48.

bmNKP

thyNKP

??

NFIL3

GATA-3Tox

Id2+

ILC2P

Tox RORγt

RORγtRORγt

Ahr

ILCP

LTiP

ILC17P

ILC22P

Figure 2 Transcription factors regulate the differentiation of distinct ILC subsets. Different transcription factors are essential for the development of different ILC subsets, including bone marrow NK precursors (bmNKP), thymic NK precursors (thyNKP), ILC2 precursors (ILC2P), LTi precursors (LTiP), ILC17 precursors (ILC17P) and ILC22 precursors (ILC22P), from Id2-expressing ILC precursors (ILCP).

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The ligand-dependent transcription factor aryl hydrocarbon recep-tor (AhR) has a critical role in regulating IL-22 production by mouse T cells65 and human T cells66. AhR binds to halogenated and non-halogenated polycyclic aromatic hydrocarbons (such as the synthetic compound β-naphthoflavone and the prominent environmental toxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)). Endogenous ligands of AhR include the tryptophane metabolite FICZ (6-formylindolo(3,2-b)carbazole)67. AhR is expressed by human and mouse ILC22 cells34, and preliminary results suggest that AhR-deficient mice have fewer ILC22 cells that produce less IL-22 after stimulation with IL-23 (M. Colonna, personal communication, and Satoh-Takayama et al., personal communication).

Tox was initially identified for its role in the positive selection of thymocytes68. Remarkably, Tox-deficient mice manifest a complete absence of lymph nodes, a much smaller size and number of Peyer’s patches and an abrogation of NK cell development69. Although Tox-deficient hematopoietic precursors have lower Id2 expression, over-expression of Id2 in these cells does not restore the developmental potential of NK cells. The effect of Tox deficiency on other ILC subsets is not yet known, although NKp46+ cells in the gut have been identi-fied in Tox-deficient mice, which suggests a Tox-independent pathway of ILC22 development.

As ILC2 cells are characterized by TH2 cytokine–production pro-files, it might be expected that these cells developmentally depend on transcription factors known to influence TH2 differentiation (GATA-3, c-Maf and NFAT2). Curiously, transcriptional profiling of NH cells has not identified a dominant TH2 cell–related transcription factor, except Aiolos, which is shared by TH2 cells and ILC2 cells53. Clearly more work is needed to decipher the developmental pathways that lead to the formation of the ILC2 subset.

Another unifying characteristic of ILCs is their dependence on cytokines that use γc of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Distinct ILC subsets require different members of this fam-ily of hematopoietic cytokines (Fig. 1). In the mouse, NK cells require trans presentation of IL-15 by IL-15Rα-expressing cells for optimal development70–72. Human NK cells are considerably fewer in number in patients carrying mutations of the gene encoding γc, its associated tyrosine kinase Jak3, or CD122 (the IL-2 receptor β-chain shared by the IL-2 and IL-15 receptors)11, whereas they are present in patients deficient in CD127 (the IL-7 receptor α-chain)73, which suggests that IL-15 is also required for the development of human NK cells. That idea has been confirmed by studies of mice carrying a human immune system, which have shown that the human IL-15–IL-15Rα complex promotes the development of human NK cells74. Although all NK1.1+ cells require IL-15 for their development, thymus-depend-ent CD127+ IFN-γ-producing NK cells have an additional require-ment for IL-7. This characteristic brings them closer to the other ILC subsets (ILC2, ILC22 and LTi) that critically require IL-7 for normal homeostasis18,38,48. The signals that regulate the trans presentation of IL-7 and IL-15–IL-15Rα required for the homeostasis of ILC subsets are poorly understood, but it is likely that under inflammatory situ-ations or during infection, the concentrations of these γc-dependent cytokines may be increased to maintain or expand ILC function.

Developmental relationships of various ILC subsetsAll of the ILC subsets described so far are derived from Id2-expressing hematopoietic progenitors, but the developmental relationships between distinct ILC subsets are not completely defined. There is some evidence that the ILC subsets described above represent distinct hematopoietic lineages and do not simply represent activation states of cells from a single lineage. NK cells (including ILC1 cells) form a

separate branch of the Id2-dependent ILC tree, given their depend-ence on NFIL3 and IL-15. Several independent lines of evidence support the idea that RORγt+ ILC cells are not related to conven-tional NK cells. First, mouse LTi cells and IL-22-producing NKp46+ cells are noncytotoxic, lack other NK cell surface markers (includ-ing Ly49 family members, NKG2D, CD122 and CD49b) and develop independently of IL-15 (ref. 38). Moreover, whereas LTi cells and IL-22-producing NKp46+ cells require Rorc for their development, conventional NK cells do not. Finally, elegant fate-mapping studies in mice have further shown that conventional NK cells are not derived from Rorc-expressing precursor cells38.

Although human fetal LTi cells acquire CD56 and other NK cell markers such as NKp44 when cultured in IL-2 (ref. 22), these LTi cell–derived CD56+ cells continue to express RORγt and lack the cytotoxic effector molecules perforin and granzyme, which suggests that these CD56+NKp44+ cells are distinct from NK cells. A model of human NK cell development in lymph nodes has been proposed that includes stage-3 immature NK cells, with the CD56−CD34−CD117+ pheno-type75,76. Fetal mesenteric lymph nodes, as well as postnatal pala-tine tonsil, contain such stage-3 cells that express IL-22 (refs. 22,77). This stage-3 immature NK cell population is heterogeneous, given its expression of CD127 (ref. 39) and IL-1 receptor (IL-1R)78. CD127+ cells constitute the majority of immature NK cells (<90%), and all express RORC, whereas the minority (CD127−CD117+ cells) lack RORC39. Clonal analysis of CD127+CD117+ cells has confirmed that these cell populations lack precursors of conventional NK cells39. In contrast, CD127−CD117+ cells in the immature NK cell population differenti-ate into cytotoxic, IFN-γ-producing CD56+CD117− conventional NK cells39. There is high expression of IL-1 receptor type 1 (IL-1R1) on 80% of the immature NK cells78, and IL-22 and AhR are expressed only in IL-1R1hi cells78. IL-1β supports the maintenance of expression of IL-22 and AhR in vitro while impeding the differentiation of immature NK cells into conventional NK cells, which suggests an important role for IL-1β in regulating development of IL-22-producing cells78. IL-1R1lo cells express RORC transcripts, as determined by RT-PCR; this raises the possibility that some precursors of human conventional NK cells may express RORC78, although this idea is not compatible with mouse studies38. Further analysis of the IL-1R1lo and IL-1R1hi cells, and in parti-cular of CD127+ and CD127− cells in those populations, will be needed to fully decipher the developmental pathways and common precursor for human conventional NK cells and CD56+RORγt+ cells.

Roles of ILC in pathological statesGiven the biological roles of the cytokines produced by the various ILC populations, it is likely that these cells condition pathological processes, either by preempting their development or by exacerbating their clinical course. Defective regulatory T cell activity and highly polarized TH17 and TH1 responses underlie the inflammatory bowel disease of many mouse models of colitis79. Studies suggest that diverse ILC subsets may also be involved in the pathogenesis of inflammatory bowel disease in the mouse. Treatment of recombination-activating gene 1–deficient mice with antibody to the costimulatory molecule CD40 results in an IL-23-induced colonic inflammation accompanied by local increases in the production of IL-17, IFN-γ and TNF, which elicits an intestinal wasting syndrome80. Analysis of such mice indi-cates that the disease-causing cell is a Thy-1+CD127+ ILC that acts via secretion of IFN-γ (but not of IL-17)45. In contrast, in the colitis model elicited by the pathogenic bacterium Helicobacter hepaticus, an IL-17-producing ILC (bearing the same Thy-1+CD127+ pheno-type) mediates this disease via the production of IL-17 and IFN-γ45. These observations indicate that distinct ILC subsets can mediate

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intestinal inflammation in different manners involving different cytokines. Whether different ILC subsets are operational in these disease models or whether microenvironmental signals modulate the cytokine production of a single but plastic subset of ILC remains to be determined.

Polymorphisms in the IL-23 receptor have been associated with a higher or lower risk of inflammatory bowel disease (Crohn’s disease and ulcerative colitis) in humans81, which suggests involvement of the IL-23–IL-17–IL-22 axis in these diseases. It is therefore noteworthy that IL-23-responsive IL-17-producing CD127+ ILCs have been found in inflamed tissues of human inflammatory bowel disease patients45 (J.M. Mjosberg, C.P. Peters and H.S., unpublished data). Whether these cells contribute to the disease remains to be established. A disease-causing role for ILC2 cells is yet to be demonstrated, but we speculate that aberrant stimulation of these cells might contribute to chronic allergic diseases with a distinct TH2 cytokine etiology, such as asthma and atopy.

A role for ILCs in antitumor immunity has been reported. Studies of a transplantable melanoma tumor model have shown that small numbers of adoptively transferred splenic NKp46+ cells protect against tumor formation in an IL-12-dependent way82. The ILC subset is distinguished from NK cells by its presence in IL-15-deficient mice and by its expression of RORγt. After in vitro culture with IL-7 and stimulation with IL-12, these antitumor ILCs express transcripts for both IL-22 and IFN-γ but secrete only IFN-γ protein and little to no IL-22. Whether these cells have the same cytokine-secretion pattern in vivo was not determined82. The precise relationship of antitumor ILCs with other ILC populations remains unclear, although it is pos-sible that they may be similar to intestinal ILC22 cells that express IFN-γ after stimulation with IL-12 and IL-18 (ref. 42). Overall, these results indicate that these newly identified ILC subsets may be useful targets for clinical intervention in diseased states.

Evolutionary considerationsThere is considerable similarity between the cytokine outputs gener-ated through helper T cell differentiation and the effector functions of ILC subsets. Moreover, some of the transcriptional regulators known to condition cytokine production profiles in helper T subsets seem to also control cytokine production by ILCs, although knowledge of this is still incomplete. It is possible that evolutionary pressures, perhaps mediated by pathogens, have selected a diverse set of innate lymphocytes that have roles in immune protection, as they have also shaped the ability of adaptive lymphocytes to tailor their effec-tor functions. With functional diversification of the innate immune systems, a coherent polarized cytokine production can be achieved at an early innate stage of host defense that can then be maintained and focused after clonal selection of polarized T cells. Such organiza-tion would be advantageous in reinforcing the initial orientation of immune responses and would suggest that initial pathogen encounter by immune system sentinels (DCs and macrophages) has a determi-nant and continuing role in polarizing immune reactions.

There are several important differences between the innate and adaptive systems of effector diversification. First, in the mouse, most ILCs seem to be short-lived (about 2 weeks)40 and therefore must be constantly renewed. Moreover, because cytokines maintain ILC homeostasis, cell-autonomous renewal might be limited, although there is possibly an innate regulatory cell that controls the prolifera-tion and effector functions of ILCs. In contrast, adaptive lymphocytes have intrinsic mechanisms to expand their populations, either clonally in response to antigen, and therefore additional mechanisms may have evolved to control T cell proliferation.

It can be speculated that the ILC system is an ancient one that predates the appearance of the recombination-activating genes that allowed the emergence of adaptive immunity. Avian species lack the receptor for lymphotoxin-β required for lymph node formation83. The appearance of the receptor for lymphotoxin-β in mammals may have allowed ILCs, perhaps ILC17 cells, to communicate with stroma-organizer cells to form lymph nodes. As IL-17 was already present in primitive fish such as lamprey, an ancient jawless fish84, it might be possible that ILC17 cells were the very first ILCs to appear in evolution.

AcknowleDgmentSWe thank T. Cupedo, N. Crellin, S. Trifari and C. Kaplan for contributions and collaborations; and G. Eberl, N. Satoh-Takayama and C. Vosshenrich for collaborations. Supported by the Institut Pasteur (J.P.D.), Institut National de la Santé et de la Recherche Médicale (J.P.D.) and the Agence National de Recherches (J.P.D.).

comPetIng FInAncIAl InteReStSThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureimmunology/. reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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