review

The expanding family of innate lymphoid

cells: regulators and effectors of immunity
and tissue remodeling
Hergen Spits1 & James P Di Santo2,3
© 2011 Nature America, Inc. All rights reserved.

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.

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 cells
NK 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
1TytgatInstitute 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
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

nature immunology  VOLUME 12  NUMBER 1  JANUARY 2011 21

 review

Figure 1  The expanding family of ILCs. Distinct ILC subsets Innate lymphoid cells
Functions
develop from hematopoietic precursors in an Id2-dependent CD16++ CD16– or CD16+
way in a process orchestrated by transcription factors (Fig. 2). Intracellular pathogens, virus
ILCs can be grouped into three branches: NK, helper and RORγt. IFN-γ IFN-γ
Inflammation
IFN-γ
The IL-15-dependent NK branch includes conventional NK (cNK) cNK
ILC1
cells, which have spontaneous cytotoxicity, and the IFN-γ+ ILC1 (Thymic NK cells)
subset, which includes thymus and IL-7-dependent NK cells in IL-7
mice and a cytokine-polarized subset of CD56 hi cells in humans. IL-15 IL-15 Extracellular parasites
γc cytokine Allergy (asthma)
ILC1 cells function to protect against infection by viruses and IL-13
intracellular pathogens, and their activity is promoted through ILC2
(Nuocytes, NH cells)
IL-12 and IL-18. The helper branch contains the ILC2 subset LT-α–LT-β
IL-7
(including nuocytes and NH cells) that produce abundant IL-13 RORγt LN formation
ILCP Isolated lymphoid follicle
under the influence of IL-25 and IL-33. ILC2 cells are critical in
IL-7 LTi TNF formation
the control of extracellular parasites. The RORγt branch includes IL-7 T cells–independent B cell help
LTi cells, ILC17 cells and ILC22 cells. All of these ILC subsets
express and depend on RORγt and require IL-7 for their Extracellular bacteria
IL-22 RORγt
development. Signals that trigger secretion of cytokines from Extracellular bacteria Autoimmune disease
IL-17 (IBD)
Autoimmune disease RORγt
these cells vary; the RANK ligand triggers LTi cells to express IL-2 ILC17
cell surface heterotrimers of LT-α and LT-β, whereas IL-23 or ILC22
(NK22, NCR22, NKR+ LTi cells)
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.
© 2011 Nature America, Inc. All rights reserved.

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 cells
LTi 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 cells 33 (D. Withers and
P. Lane, personal communication). Collectively, these observations
would suggest that LTi cell functions are developmentally regulated.
Phenotype and function of ILC22 cells
An 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

22 VOLUME 12  NUMBER 1  JANUARY 2011  nature immunology


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
© 2011 Nature America, Inc. All rights reserved.
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 ILCs
An 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.
nature immunology  VOLUME 12  NUMBER 1  JANUARY 2011 23
review
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 cells
IL-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 T H2 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
 review
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
© 2011 Nature America, Inc. All rights reserved.
RORγt expression.
Plasticity of cytokine production by ILC subsets
Similar 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 subsets
Transcription 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).
24 VOLUME 12  NUMBER 1  JANUARY 2011  nature immunology
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
Tox
GATA-3
ILC2P
??
ILCP Id2+
Tox RORγt
LTiP
RORγt
RORγt
Ahr
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).

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-
© 2011 Nature America, Inc. All rights reserved.
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 subsets
All 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
nature immunology  VOLUME 12  NUMBER 1  JANUARY 2011 25
review
s­ eparate 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 states
Given 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
 review
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
© 2011 Nature America, Inc. All rights reserved.
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 considerations
There 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.
26 VOLUME 12  NUMBER 1  JANUARY 2011  nature immunology
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.
Acknowledgments
We 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 INTERESTS
The 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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