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Development of Human ILCs and Impact of Unconventional Cytotoxic Subsets in the Pathophysiology of Inflammatory Diseases and Cancer


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Innate lymphoid cells (ILCs) were firstly described by different independent laboratories in 2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and ILC3, according to their cytokine and transcriptional profiles. Subsequently, also Natural Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to ILC3 subgroup. Starting from their discovery, significant advances have been made in our understanding of ILC impact in the maintenance of tissue homeostasis, in the protection against pathogens and in tumor immune-surveillance. However, there is still much to learn about ILC ontogenesis especially in humans. In this regard, NK cell developmental intermediates which have been well studied and characterized prior to the discovery of helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide an overview of the current knowledge about NK cells and helper ILC ontogenesis in humans. We will also focus on the newly disclosed circulating ILC subsets with killing properties, namely unconventional CD56dim NK cells and cytotoxic helper ILCs, by discussing their possible role in ILC ontogenesis and their contribution in both physiological and pathological conditions.
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Development of Human ILCs and
Impact of Unconventional Cytotoxic
Subsets in the Pathophysiology of
Inammatory Diseases and Cancer
Michela Calvi
, Clara Di Vito
, Alessandro Frigo
, Sara Trabanelli
, Camilla Jandus
and Domenico Mavilio
Department of Medical Biotechnologies and Translational Medicine (BioMeTra), University of Milan, Milan, Italy,
Unit of
Clinical and Experimental Immunology, IRCCS Humanitas Research Hospital, Milan, Italy,
Department of Pathology and
Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland,
Ludwig Institute for Cancer Research,
Lausanne, Switzerland
Innate lymphoid cells (ILCs) were rstly described by different independent laboratories in
2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T
helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and
ILC3, according to their cytokine and transcriptional proles. Subsequently, also Natural
Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were
attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to
ILC3 subgroup. Starting from their discovery, signicant advances have been made in our
understanding of ILC impact in the maintenance of tissue homeostasis, in the protection
against pathogens and in tumor immune-surveillance. However, there is still much to learn
about ILC ontogenesis especially in humans. In this regard, NK cell developmental
intermediates which have been well studied and characterized prior to the discovery of
helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide
an overview of the current knowledge about NK cells and helper ILC ontogenesis in
humans. We will also focus on the newly disclosed circulating ILC subsets with killing
properties, namely unconventional CD56
NK cells and cytotoxic helper ILCs, by
discussing their possible role in ILC ontogenesis and their contribution in both
physiological and pathological conditions.
Keywords: innate lymphoid cells (ILCs), natural killer (NK) cells, ILC-poiesis, cytotoxicity, unconventional subsets,
inammation, cancer
Starting from 2008, several independent laboratories around the world identied new players of
innate immunity in both humans and mice (14). These cells, named innate lymphoid cells (ILCs),
are a heterogeneous group of lymphocytes lacking recombination-activating gene (RAG)-
dependent rearranged antigen-specic receptors.
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142661
Edited by:
Rachel Golub,
´Paris Diderot,
Reviewed by:
Marina Cella,
Washington University in St. Louis,
United States
Kamel Benlagha,
INSERM U1160 Alloimmunite
Transplantation, France
Domenico Mavilio
Specialty section:
This article was submitted to
NK and Innate Lymphoid Cell Biology,
a section of the journal
Frontiers in Immunology
Received: 06 April 2022
Accepted: 28 April 2022
Published: 26 May 2022
Calvi M, Di Vito C, Frigo A, Trabanelli S,
Jandus C and Mavilio D (2022)
Development of Human ILCs and
Impact of Unconventional Cytotoxic
Subsets in the Pathophysiology of
Inammatory Diseases and Cancer.
Front. Immunol. 13:914266.
doi: 10.3389/fimmu.2022.914266
published: 26 May 2022
doi: 10.3389/fimmu.2022.914266
ILCs originate from the common lymphoid progenitor (CLP)
and require the common gchain of the interleukin (IL)-2
receptor and the transcriptional repressor ID2 for their
development (24). They are considered the innate counterpart
of adaptive T lymphocytes: ILCs share with T cells the
transcription factors governing their differentiation and the
same cytokines in response to inammatory insults (5,6),
which allows the classication of ILCs into different subsets.
Indeed, the ILC1, ILC2 and ILC3 subsets produce T helper (Th)
1-, Th2- and Th17/22-cytokines, respectively. Furthermore,
given their phenotypic, developmental and functional
similarities, Natural Killer (NK) cells, the innate counterpart of
cytotoxic T lymphocytes, are now grouped together with ILC1s,
whereas lymphoid tissue inducer (LTi) cells, belong now to
group 3 ILCs (5,7,8).
Despite these similarities, ILCs arise from distinct
developmental pathways and display unique epigenetic and
transcriptional programs with respect to T cells, thus
suggesting nonredundant roles of ILCs in immunity (9).
Differently from NK cells, that are mainly circulating
lymphocytes, helper ILCs are primarily tissue resident cells and
have been found in both lymphoid and non-lymphoid tissues.
Indeed, they are particularly enriched at the mucosal surfaces of
several organs, such as gut, lungs and skin, where they play a
pivotal role in tissue homeostasis and disease, by promoting
immune responses, inammation, tissue repair and tolerance to
commensal microbiota (1012). Despite being a rare population
in peripheral blood (PB), several lines of evidence indicate that
circulating helper ILCs are characterized by a unique pattern of
cytokine receptors, thus suggesting that they are not exclusively
tissue resident (9,13). Moreover, given their innate nature, ILCs
represent one of the primary sources of pro- and anti-
inammatory cytokines during the early stages of the immune
responses (1417).
Although progresses have been made in understanding the
role of ILCs in the maintenance of tissue homeostasis, in
immune-defense and in tumor immune-surveillance, there is
still much to learn concerning ILCs, especially in humans.
In this review we will summarize the principal features of
ILCs, focusing mainly on circulating subsets. Moreover, we will
provide an overview on ILC development in humans. In this
context, we will focus on the newly disclosed circulating ILC
subsets with cytotoxic properties, namely CD56
cells and cytotoxic helper ILCs, by discussing their possible role
in ILC ontogenesis and their contribution in both physiological
and pathological conditions.
ILCs are subdivided into three main groups based on the
cytokine production, genetic signature and transcription
factors involved in their development (Figure 1A).
Total circulating ILCs, which comprises helper ILCs and NK
cells, are identied as lineage (CD3, CD14, CD15, CD19, CD20,
CD33, CD34, CD203c and FcϵRI) negative lymphocytes
(Figure 1B)(17). This lineage markers can be used also to
identify tissue resident ILCs, with the exception of human tonsil
ILC3s which express CD33 (18). Total helper ILCs are dened as
cells that constitutively express the IL-7 receptor-a
chain (CD127) and the different subsets are identied according
to the expression of CRTH2, cKit (CD117) and CD56: ILC1s are
, ILC2s are CRTH2
and ILC3s CRTH2
(Figure 1B)(5,17,19). However, it has been shown that
ILC2s, in some cases, can express CD56 (20). In addition,
among lineage negative cells, NK cell subsets are identied
according to the differential expression of CD56 and CD16
(Figure 1B)(21).
Despite the phenotype of the different ILC subsets is well
dened, their precise distribution across organs and species
needs to be rened. Indeed, ILC subset distribution and
cytokine production proles are impacted by environmental
cues, that enable a prompt response in case of inammatory
insults without the de novo recruitment of ILC subsets (22,23).
2.1 Group 1 ILCs
NK cells and ILC1s have been grouped together in group 1 ILCs.
Indeed, both subsets are characterized by the production of
interferon-g(IFN-g) and tumor necrosis factor a(TNF-a)in
response to IL-12 and IL-18 (5,7,24). Moreover, IL-15 is
required for the differentiation, homeostasis, and function of
both NK cells and ILC1s (25). Similarly to NK cells, ILC1s
express natural cytotoxicity receptors (NCRs) (26) and the T-box
transcription factor expressed in T cells (T-bet) (25,27). In
addition, both ILC1s and NK cells require the expression of
HOBIT TFs, encoded by Zfp683, for their differentiation and
functional programs (2830).
Despite their overlapping features, NK cells and ILC1s are two
phenotypically and functionally distinct immune cell subsets.
Indeed, while ILC1s require T-bet for their development, NK cells
need T-bet expression only for maturation and require the
expression of the transcription factor Eomesodermin (Eomes) for
differentiation (25,27). Moreover, ILC1s are fundamentally tissue-
resident lymphocytes, whereas NK cells can circulate across
lymphoid organs via the bloodstream and lymphatic system to
act as immune sentinels. Indeed, NK cells are unique in their ability
to recognize and kill virally infected and malignantly transformed
cells, through the balance of the signaling that NK cells receive from
their repertoire of activating and inhibitory NK cell receptors
Circulating NK cells comprise two main subsets, a cytotoxic
and a regulatory CD56
NK cell subset. CD56
NK cell subset accounts for up to the 90% of circulating NK cells.
They show high baseline perforin expression and are endowed
with cytotoxic abilities against target cells not expressing or
downregulating the major histocompatibility complex (MHC)
class I molecules on their surface. They preferentially produce
cytokines in response to direct target cell interactions rather than
via monocyte-derived cytokines stimulation (33). On the other
hand, CD56
NK cells represent the 10% of circulating NK
cells, while they are enriched in peripheral and lymphoid tissues
(34). Differently from CD56
NK cells, and similarly to ILC1s,
NK cells are poorly cytotoxic, can rapidly secrete
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142662
cytokines, including IFN-g, following stimulation by monocyte-
derived cytokines (35).
2.2 Group 2 ILCs
Group 2 ILCs are involved in several processes, including lipid
metabolism, protection against parasites and accumulate during
type 2 inammation in the airways (36). Among all ILCs, ILC2s
express the highest level of the transcription factor GATA3 and
are characterized by the production of Th2-associated cytokines,
such as IL-4, IL-5, IL-9, IL-13 and amphiregulin (AREG) in
response to IL-25, IL-33 and thymic stromal lymphopoietin
(TSLP). Hence, they are considered the innate counterpart of
Th2 cells (3739). Of note, several lines of evidence indicate that
ILC2s can produce higher levels of cytokine than T cells, thus
suggesting their primary role in the innate local immunity
against infections in different organs (40). Indeed, they have
been described in a variety of human tissues, including tonsils,
bone marrow (BM), spleen, skin, adenoids, adipose tissue, lung
lymph nodes (LNs) (41).
ILC2s are characterized by the expression of the transcription
factor BCL11B which controls their identity (42), the
prostaglandin D2 receptor 2 (CRTH2), the IL-33 receptor
(IL1RL1 also referred as ST2) and by variable levels of c-Kit
(43), all involved in ILC2 localization and function (4346).
More recently, it has been shown that ILC2s are further
characterized by the expression of the killer cell lectin-like
receptor subfamily G member 1 (KLRG1), a co-inhibitory
receptor previously reported in T and NK cells that binds to
FIGURE 1 | General features of ILC subsets. (A) Summary table showing the main transcription factors (TFs) governing the development, the cytokine production,
the functions and marker expression in the different ILC subsets. (B) Representative ow cytometry gating strategy to identify circulating ILC subsets among
lymphocytes. Yellow lines identify helper ILCs, while the blue lines identify NK cells.
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142663
the members of the cadherin family and is upregulated during
infection in response to IL-25 (20,47).
2.3 Group 3 ILCs
Group 3 ILCs, consisting of ILC3s as well as LTis, share features
with Th17 cells, including the expression of the transcription
factor retinoic acid orphan receptor isoform gt (RORgt), required
for their development and function, and the aryl hydrocarbon
receptor (AHR) (7,48). Group 3 ILCs are capable of producing
IL-17, IL-22 and granulocyte-macrophage colony-stimulating
factor (GM-CSF) in response to IL-23, IL-1b,ornatural
cytotoxicity receptor ligands (NCR-L), thus mirroring Th17
response (5,49). Regardless of the functional association with
ILC3s, LTis are considered a separate ILC lineage (49,50).
Indeed, LTis are involved in the secondary lymphoid organ
formation during embryogenesis and adulthood and in their
restoration following infection (51), whereas ILC3s mainly
contribute to the immune responses against specic
extracellular pathogens and in the maintenance of tissue
homeostasis at mucosal sites, where they are mainly
localized (48).
ILC3s can be further subdivided according to the expression
of the NCR NKp44: human NKp44
ILC3s, largely co-
expressing NKp46, are the majority in adult tonsil and
intestine and represent an exclusive source of IL-22, while
ILC3s are the major population in fetal LNs and
preferentially express IL-17 transcripts (48).
Differently from tissues, ILC3s are under-represented in the
circulating lymphocyte pool. However, recently, a subpopulation
of cells phenotypically resembling to NKp44
ILC3s has been
described in cord and adult PB. However, most peripheral-blood
ILCs express low levels of RORgt, do not
produce any of the ILC cytokine signatures following stimulation
with IL-1band IL-23 and their transcriptome is different from
that of mature ILC3s present in secondary lymphoid organs (52,
53). Circulating CD127
ILCs are instead multi-
potent ILC precursors (ILCPs) that retain the ability to give
rise to functionally mature helper ILC subsets, as well as to
NK cells, after in vitro culture with appropriate
cytokine mix or after transfer in vivo into immunodecient
mice (49,53).
ILC-poiesis has been a topic of ardent research in the last several
years. Although many issues remain to be disclosed, including
the transcriptional regulators that dictate the choice of mature
ILC subset fate, the ILC differentiation in mouse has been deeply
investigated, thanks to genetically modied rodent models. On
the contrary, the study of the human ILC differentiation is still in
its infancy because of different reasons. Firstly, the lack of
comparable genetic and tracing tools that are available in
animal models of lymphoid development. Secondly, the
heterogeneity of the studies conducted in terms of denition of
progenitor, identication of lineages based solely on cytokine
secretion or on few surrogate markers.
Despite these limitations, over the past years a map of human
ILC development has been under construction (Figure 2)by
taking advantage of both murine ILC-developmental model and
the pre-existing model of human NK cell development, based on
the identication and characterization of human NK cell
developmental intermediates (NKDIs) prior to the discovery of
helper ILCs (54).
3.1 Human NK Cell Development
Since their discovery in 1970s, NKDIs have been well
characterized in both mice and humans as well as the site for
NK cell development.
Human NK cells were originally thought to develop strictly
within the BM (33,55). Indeed, CD34
with ex vivo potential for NK cell differentiation were rstly
identied in the BM, and then were also found in PB and in
extramedullary tissues, including thymus, secondary lymphoid
tissues (SLTs), liver, and uterus, at steady state conditions (56
58). Noteworthy, IL-15-responsive CD34
precursors comprise a relatively higher proportion of total
progenitor cells (5-10%) in the blood compared to
bone marrow (<1%), and in SLT they comprise the major subset
of CD34
progenitor cells (>90%) (59,60). In this regard, several
lines of evidence demonstrated that CD34
precursors originate in the BM and trafc to extramedullary
tissues where later stages of NK cell differentiations can take
place, giving rise to tissue-specic and functionally distinct
mature NK cell subsets. In particular, tonsils, spleen, and lymph
nodes are considered those SLTs hosting the main extra-medullary
sites of NK cell development and maturation (57,5962).
Originally, ve main sequential stages of NK cell maturation
were identied: NK cell progenitors (NKPs, stage 1), pre-NK
cells (stage 2), immature NK cells (iNK, stage 3) (6365) and the
mature CD56
(stage 4) and CD56
(stage 5) NK cell
subsets [reviewed in (62)].
Briey, NKPs and pre-NK cells still express CD34 and retain
the ability to differentiate into T cells, dendritic cells (DCs) and
other ILCs. Subsequently, the expression of CD122 (IL-2Rb),
together with the downregulation of CD34, marks the
irreversible fate decision into NK cell lineage. The commitment
of NKPs towards pre-NK cells also required the acquisition of
CD117 expression (64,66). Recently, the stage 2 has been further
subdivided into two additional stages: the IL-1R1
stage 2a,
mainly enriched in SLTs and PB, that still retains the ability to
give rise to T cells and DCs, and the IL-1R1
stage 2b, with a
commitment restricted to the generation of ILCs, including NK
cells (62,67). IL-1R1
stage 2b give rise to iNKs whose features,
including AHR, CD127, RORgt, IL-1R1, and IL-22 expression,
mirror those of Group 3 ILCs (5,68,69). Indeed, it is not yet clear
if iNKs and ILC3s are entirely overlapping at least in their
phenotypic characteristics and further investigation is needed.
The nal transition of iNK into mature NK cell subsets is
marked by the appearance of CD56 expression and the main
functional properties, including cytokine secretion (i.e. TNF, IL-8,
GM-CSF, CXCL12 and IL-13, together with IL-22), IFN-grelease
and then cytolytic activity (58,7072). Two distinct stages 4
NK cells have been described: the NKp80
stage 4a
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142664
cells, that, despite the bright expression of CD56, are more similar
to iNKs cells due to their higher expression of transcription
factors RORgt and AHR, their higher production of IL-22 and
their preferential localization in SLTs, the NKp80
stage 4b cells,
expressing higher levels of T-bet and Eomes and producing IFN-g
(73). Subsequently, through the acquisition of CD16, Killer
Immunoglobulin-like receptors (KIRs) and cytotoxic granules,
the fully mature CD56
NK cells, endowed with cytolytic
potential and able to perform antibody-dependent cell-mediated
cytotoxicity (ADCC) are generated.
Despite the process of NK cell-poiesis is well dened, with the
identication and characterization of other subtypes of ILCs, the
old model of NK cell ontogenesis needs to be reassessed and
rened in the context of new data about helper ILCs (74).
3.2 Murine ILC Development
Murine ILC differentiation is regulated by a wide range of
transcription factors, including Id2,Nl3,Zbtb16,Tcf7,Gata3,
Ets1, and Tox [reviewed in (75)].
FIGURE 2 | Human ILC developmental stages. (A) Summary table of the main transcription factors (TFs), marker expression and tissue localization of common
precursors/progenitors. (B) Schematic representation of different stages of human ILC-poiesis starting from the early tonsillar progenitors (ETPs), that still retain the
ability to give rise to T cells and dendritic cells (DCs), to ILC precursor (ILCPs) that branches into progenitors with restricted differentiation potential and give rise to
different mature ILC subsets. Dashed lines indicate the hypothetic developmental pathways of unconventional cytotoxic ILC subsets.
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142665
Two distinct progenitors downstream of the murine CLP
have been identied, each with restricted ILC potential. These
include the CXCR6
a-lymphoid precursor
(aLP) and the Lin
early innate
lymphoid progenitor (EILCP) that along with the other
downstream progenitors are most prevalent in murine BM (54,
76). The transcription factor Nl3 is required for the generation
of aLP. Indeed, Nl3 knock out mouse models lack mature ILCs,
including NK cells (7678).
On the other hand, EILCPs, requires the transcription factor
Tcf7 (79). Given the drastic reduction of aLPs together with
EILCPs in Tcf7-decient mice, it has been also proposed that
aLP could constitute an intermediate stage of maturation
between CLP and EILCP (76).
Two additional EILCP subsets with different commitment
potential have been identied: the early-stage EILCPs (EILP1s),
that can give rise to DCs as well as cytotoxic and helper ILCs, and
the committed EILPs (cEILCP) expressing TCF-1 and losing the
ability to differentiate in DCs (80).
The EILCP can further differentiate in Id2-dependent
common helper-like ILC
progenitors (CHILPs), which can give rise to helper-like ILCs
(ILC1s, ILC2s, and ILC3s) and LTis but not to NK cells (79,81)
or in Id2-independent NK1.1
NKPs (82,83).
CHILPs can be separated into two different subsets based on
the expression of Zbtb16. The Zbtb16
common ILC precursor
(CILCP) which can no longer produce LTi cells (83,84) and the
ILC precursor (ILCP) capable of potentially giving rise
to conventional NK cells, ILC1s and ILC2s, but with a reduced
ability to differentiate into ILC3s (8386).
3.3 Human ILC Development
According to NK cell developmental model, the most immature
ILC progenitors identied in humans are mainly localized in
SLTs and were originally described as stage 1 and stage 2 NKIDs
and are now dened as early tonsillar progenitors (ETPs) in the
context of ILC-poiesis (Figure 2A). In particular, ETPs are
subdivided into Lin
ETP1 and
ETP2 (54,63). Both ETP1s
and ETP2s are multipotent and could also give rise to T cells
and DCs in vitro (54,63). ETP2s are heterogeneous in terms of
IL-1R1 expression. IL-1R1
ETP2s have a residual T-cell and
DC potential, whereas IL-1R1
ETP2s are ILC restricted. These
population are also characterized by a unique transcription
factor signature: ETP1 are RAG1
and express low levels of
ID2 and RORgt, IL-1R1
ETP2s are ID2
and retain
a low expression of RAG1, whereas IL-1R1
ETP2s are
(Figures 2A, B)(67). Hence, IL-
ETP2s are the earliest committed human CILCP
identied to date.
In addition, a Lin
ILC progenitor (ILCP), with phenotypic features that
overlap those of stage 3 NKIDs endowed with a restricted potential
for ILC generation, has been recently identied in human cord and
adultPBaswellasfetalliverandseveraladulttissues.Uponin vitro
culture with an appropriate cytokine environment or after transfer
in vivo to immunodecient mice, these human ILCPs demonstrate
their potential for generating all mature helper- and cytotoxic-
ILCs (53).
Consistent with their differentiation potential, ILCPs express
high levels of transcription factors that have been shown to be
essential for mouse ILC development, such as ID2, GATA3,
TCF-7 and ZBTB16 (Figure 2A). In contrast, moderate to low
levels of the lineage-determining transcription factors RORgt, T-
bet, Eomes, cytokine receptors (including IL-1R1), and signature
cytokines have been found (53). ILCPs show a migratory prole
including the expression of L-selectin (CD62L) and b2-integrin
(CD18), which would allow these cells to populate the mucosal
tissues where they terminally differentiate into mature ILCs in
response to local and environmental triggers, during both
homeostatic and inammatory conditions (54,87,88).
Consistent with this idea, ILCPs were found at mucosal sites
where they mature (53).
In light of this evidence, ILCPs might be the equivalents of
naïve CD4
T cells. Indeed, they both express CD45RA and
CD62L and the development of ILC subsets from ILCP parallels
that of CD4
T cell subsets from naive CD4
T cells, with
similar polarizing cytokines and transcription factors being
required for their differentiation, although the differentiation of
naïve CD4
T cell subsets also depends on TCR signaling and
CD28 co-stimulation (22).
Different ILCP populations with a restricted differentiation
potential have been recently described and can be identied
based on the expression of CD56, NKp46 and KLRG1. CD56
ILCPs show a restricted potential for NK cells, ILC1s and ILC3s,
ILCP predominantly differentiate into ILC3s, whereas
ILC precursors mostly develop into ILC2s (Figure 2B)
Adding complexity to this scenario there is the so called ILC
plasticity phenomenon. Indeed, it has been demonstrated in both
humans and mice, that ILC subsets can switch into another
subset depending on the presence of cytokines and NOTCH
ligands in their environment. This process is regulated by a
complex network of transcription factors. Briey, ILC2s and
ILC3s transdifferentiate into ILC1s in response to IL-1band IL-
12, whereas IL-1band IL-23 can drive the plasticity of ILC1s and
ILC2s towards ILC3s. Despite ILC2s lack the expression of IL-23
receptor, IL-1 bis known to induce ILC2s responsiveness to IL-
23 by STAT3 phosphorylation (90). The transdifferentiation of
ILC2s into ILC1s or ILC3s can be reversed by IL-4. ILC2s
requires TGF-bin addition to IL-1band IL-23 to differentiate
into ILC3s (22). Moreover, NK cells, in a TGF-b-rich tumor
environment, transdifferentiate into ILC1-like cells devoid of
cytotoxic activity (91,92). Likely, the plasticity of ILCs is the
mechanism of tissue-resident ILCs to dynamically adapt to a
given stimulus, such as an inamed state (23).
Until recently, NK cells were considered the only cytotoxic
innate lymphocytes, being functionally associated with CD8 T
cells (7). However, owing to the highly plastic nature of ILCs,
Calvi et al. ILC Ontogenesis and Unconventional Subsets
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increasing evidence shows that beside their ability to
transdifferentiate between helper subpopulations, these cells
can also acquire cytotoxic capacities upon dened
environmental conditions (93). Indeed, it has been recently
reported that, upon exposure to cytokine cocktails, ILC3s or
ILC1s, isolated from human tonsils, secondary lymphoid organs
(e.g., spleen) and intestinal tissues of humanized mice, give rise
to cytotoxic lymphocytes resembling stage 4a NK cells (94,95).
In particular, hallmark NK cell genes, such as NCAM-1,KLRD1,
KLRC1, CD2, CD226 have been reported to be signicantly
upregulated in ILC3s or ILC1s exposed to IL-12/IL-15, and to
be paralleled by the acquisition of cell surface expression of at
least some of these markers (e.g. NKG2A, NKG2C, CD2).
Moreover, these cells show a weak IFN-gsecretion in response
to K562, but an efcient perforin- and granzyme-dependent
cytotoxicity, primarily mediated by the Eomes
fraction of stimulated ILC3s. Nevertheless, these cytotoxic
responses remain weaker and of slower kinetics as compared
to that of conventional NK cells.
Phenotypically, the distinction between human cytotoxic
helper ILCs and NK cells is complicated by the fact that many
markers are shared, including CD56, CD161, NKp44 and
NKp46. The same stands for mouse ILCs and NK cells that
share the expression of NKp46 and NK1.1. Possible
discriminators to distinguish cytotoxic helper ILCs, or helper
ILCs in general, from NK cells include CD127, which is
constitutively expressed by both human and murine helper
ILC subsets but not by terminally-differentiated CD56
cells (67) and only at intermediate levels on human peripheral
blood CD56
NK cells (96). Noteworthy, in mice, ILCs with
cytotoxic potential were also described within NK1.1
cells, lacking CD127 expression (26).
Another marker enabling the discrimination between helper
ILCs and NK cells might be the inhibitory receptor CD200R1. The
expression of CD200R1 has been described to be specicforILCsin
human blood and tonsils (20), and in mice (84,97). However,
intestinal NK cells have been recently described to express
CD200R1, although at lower levels as compared to conventional
ILCs (95). Inversely, CD200R1 is poorly expressed by intraepithelial
ILC1s in the small intestine, lamina propria and visceral adipose
tissue (98). Overall, these results highlight the difculty to identify a
universal marker enabling ultimate discrimination between helper
ILCs, including cytotoxic ones, and NK cells. Nevertheless, despite
potential overlapping phenotypes and functions, the distinct
anatomic distribution of cytotoxic helper ILCs and NK cells
argues for complementary roles in the protection against
infections or emerging malignancies.
Evidence of in vivo ability of helper ILCs to acquire cytotoxic
features has been recently shown in different districts, as detailed
in the next paragraphs.
4.1 Unconventional CD56
NK Cells
In the context of NK cell development, an additional NK cell
subset has been recently identied. This subset, named
unconventional NK cells (unCD56
), displays a
phenotype. Barely
detectable in the PB, this NK cell subset is mainly enriched in
the BM (99,100). On the other hand, their presence in
extramedullary tissues has not been investigated so far.
NK cell subset expresses surface markers of
mature NK cells, such as NKG2D and NKp30 (99). Moreover,
this NK cell subset is equipped with lytic molecules, thus
suggesting its putative role in mediating cytotoxic responses.
However, their phenotype and transcriptional prole suggest
that unCD56
NK cells are a bona de NK cell subset distinct
from activated CD56
that underwent CD16 shedding
mediated by the metalloproteinase-17 (101). Indeed, compared
to CD56
NK cells, unCD56
NK cells show higher levels of
CD27, whose expression was described to decline during NK cell
development in mice, and lower levels of markers of terminally
differentiated and licensed NK cells, namely CD57 and KIRs,
which are acquired at late stages of NK cell differentiation (100).
Moreover, unCD56
NK cells display higher levels of CD25,
CD122 and CD127, the receptor chains for IL-2, IL-15 and IL-7
cytokines, which play a major role in controlling NK cell
development, homeostasis, survival and activation (100). The
chemokine receptor expression pattern of unCD56
consistent with a less differentiated phenotype compared to
NK cells. Indeed, they are characterized by
undetectable CX3CR1 expression, which is usually acquired
during NK cell development, and by a higher expression of
CXCR4 compared to the other conventional NK cell subsets, in
line with their preferential BM localization (100).
In the context of the lymphopenic environment of patients
affected by hematologic malignancies and which underwent
haploidentical hematopoietic stem cell transplantation (haplo-
HSCT), others and we reported that unCD56
NK cells are by
far the largest subset of NK cells immune-reconstituting in the
rst 2-4 weeks after the transplant, compensating the low
frequency of the conventional cytotoxic CD56
NK cells
(99,102). These data, together with the transcriptional
and phenotypic characteristics of unCD56
NK cells,
intermediate between that of CD56
and CD56
NK cells,
suggest that this subset could represent an additional or
alternative stage of NK cell differentiation that drives the NK
cell maturation process (Figure 2B)(99,100).
In vitro experimental evidence also suggests that unCD56
NK cells possess multifunctional ability and superior effector-
functions. Indeed, despite poorly present under homeostatic
conditions in the PB, they are endowed with potent
cytotoxicity, signicantly higher than that of CD56
, and an
IFN-gproducing capability comparable to that of CD56
response to cytokine stimulation (99,100,103). On the contrary,
immune-reconstituting unCD56
NK cells, highly expanded
early after haplo-HSCT, are anergic due to a high expression of
CD94/NKG2A, an inhibitory receptor involved in NK cell
differentiation and education, thus further supporting the
assumption of unCD56
NK cells as a distinct NK cell subset
and highlighting their key role in NK cell development.
Moreover, this observation allowed us to develop a phase II
clinical trial (ONC-2020-001) by using an anti-NKG2A
humanized monoclonal antibody (humZ270 mAb, IPH2201,
monalizumab, AstraZeneca) to block this inhibitory
checkpoint, unleashing alloreactive unCD56
NK cells, thus
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142667
potentially improving the clinical outcome of haplo-HSCT early
after transplant (99,104,105).
4.2 Cytotoxic Helper ILCs
4.2.1 Tonsillar Cytotoxic ILCs
The ex vivo analysis of human tonsil ILCs has shown the
existence of a population of CD94
cells, that co-expressed
CD200R1, while negative for CD16, NKp80 and KIRs. In terms
of transcriptomic proles, these cells cluster close to NKp44
ILC3s, sharing with them the expression of RORgt, but being
distinct in terms of cytotoxic gene expression (e.g., Eomes,
Granzymes,Granulysin)(106). This gene expression pattern
has been also correlated with direct cytotoxic activity against
the target cell line K562, which is predominant for the
ILC3 subpopulation. Additional evidence of
the in vivo generation of cytotoxic helper ILCs in human
inamed tonsils has been recently reported by combining bulk
and single cell RNA sequencing (scRNAseq), ow- and mass-
cytometry studies (107). In particular, it has been demonstrated
that ILC3s and ILC1s reside at the terminal ends of a
differentiation spectrum. Between these two extremes, four
ILC3-ILC1 intermediates, with a NKp44
emerge by RNA velocity analyses. These intermediates are
characterized by the gradual acquisition of genes expressed in
conventional NK cells, such as KLRD1,KLRC2,GZMB and
GZMK, loss of RORG,CD200R1,KIT,IL7R and upregulation
of Tbx21 and IKZF3.
4.2.2 Circulating Cytotoxic ILCs
We recently identied a CD56-expressing subset of circulating
ILCs with high cytotoxic potential that belong to conventional
ILC1s, being characterized by the lack of lineage markers, the
expression of CD127 and the absence of CD117 and CRTH2
(108). RNA-sequencing analysis revealed a transcriptional prole
closer to ILCPs and NK cells rather than ILC1s. These
ILC1-like cells showed distinct nutrient uptake and
mitochondrial activity in comparison to helper ILCs and NK
cells, low expression of NKp46 and the capacity to produce IL-8,
beside IFN-g, when stimulated with IL-12, IL-15 and IL-18.
Through comparison with NK developmental intermediates in
terms of phenotype (73), by verifying their presence in patients
with severe combined immunodeciency, in human fetal tissues
and during immune reconstitution in humanized mice, and by
assessing their capacity to differentiate into conventional ILCs/
NKs when cultured on OP9 mouse stromal cells, we concluded
that they are related to the developmental stage 4a of NK cells. The
cytotoxic machinery of CD56
ILC1-like ILCs comprised
DNAM-1, NKp30, NKp80 and TRAIL, as well as the ability to
produce perforin, granzyme A, B, K, M and granulysin.
Functionally, CD56
ILC1-like ILCs can kill both the MHC-
K562 cell line and MHC-I
cell lines, such as BJAB and
U937, in accordance with the absence of KIRs. Their cytotoxic
capacity is dependent on the expression of NKp30, NKp80 and
TRAIL, since the addition of specicblockingantibodiescan
inhibit their killing ability. We further investigated their presence
and function in acute myeloid leukemia (AML), a hematologic
malignancy characterized by a dysfunctional helper ILC
compartment (109,110). The data obtained demonstrated that
in AML patients at diagnosis, the cytotoxic machinery of
ILC1-like ILCs is completely impaired, resulting in their
the other hand, AML patients that achieved remission showed a
completely restored function of CD56
ILC1-like cells.
Interestingly, CD56
ILC1-like ILCs but not conventional NK
cells from AML patients at diagnosis had a reduced expression of
TRAIL, NKp80 and granulysin, thus suggesting that despite their
relatedness they are distinct populations able to differentially react
to the microenvironment. Further studies are needed to determine
whether this population is an intermediate between helper ILCs
and NK cells, or a specic cytotoxic circulating helper ILC subset.
4.2.3 Intestinal Cytotoxic ILCs
In a recent study aimed at creating a high-dimensional tissue map of
human NK cells across multiple tissues, cytotoxic helper ILCs have
been also identied (111). Indeed, CD56
cells expressing
CD127, CD56 and CD161 have been found to be enriched in the
intestine, as well as in lung-draining lymph nodes and mesenteric
lymph nodes. These cells secreted IFN-gupon stimulation and
upregulated CD107a when co-cultured with MHC-I
suggesting that they might represent either immature NK cells or
tissue-resident cytotoxic helper ILC3s, present at selected
anatomical sites. These cells might be key in maintaining
intestinal homeostasis by cytokine secretion. In that regard,
intraepithelial cytotoxic ILCs, very much resembling NK cells,
have also been identied by others in the intestinal mucosa (26).
These cells are characterized by the expression of CD56, Eomes, T-
bet, variable levels of NKp44 and CD103, low levels of CD94 and
CD127. This phenotype has not been observed in other anatomic
locations, suggesting a selective localization of this subset in the
mucosal epithelium of the gut. In vitro characterization of these cells
revealed that they are able to respond to IL-12 and IL-15 stimulation
by secreting IFN-g, to produce Granzyme B and to express CD107a
when exposed to cell targets. Studies in mice showed that these
intraepithelial cytotoxic ILCs rely on Nl3-andTbx21-transcription
factors, while IL-15 is dispensable for their development, suggesting
that, at least in mice, they are distinct from conventional NK cells.
The investigation of their putative role in gut inammation revealed
that their frequency is increased in patients with Crohnsdiseaseas
well as in in vivo models of colitis, arguing for a direct involvement
in pathology. In a more recent study, amplied CD94
ILCs has been reported in the intestinal lamina propria in adults, but
not in the epithelium, as opposed to the previous study (95).
scRNAseq analysis of healthy and Chrons disease patientstissue
specimens revealed the existence of two clusters of lamina propria
cells, with cytotoxic attributes. These cells express
Eomes and CD200R1, but lacked CD16 expression. Heterogeneity
was observed in terms of CD161, CD117, CD18, cytotoxic
molecules (e.g., granzymes and perforin) and granulysin
expression. Specically, the granulysin
subpopulation is highly amplied in Chrons disease patients,
arguing for its induction during the inammatory process. This
hypothesis is supported by the observation that CD94
ILCs are absent in fetal intestine, where instead NK cells are
abundant. The activation of these CD94
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142668
ILCs in patients with bowel inammation might have opposite
effects: sustain inammation on the one hand and fulll bactericidal
activities on the other hand. However, additional experimental
investigations are needed to conrm this hypothesis. Given the
localization of ILC3s at mucosal barriers, these in vitro observations
argue for a potential role of cytokine-induced NK-cell like ILC3s in
providing cytotoxic protection at mucosal sites, where NK cells are
low abundant. If and how this NK cell-like activity of ILC3s could be
exploited in vaccination settings against viruses or cancer remains to
be studied (94).
4.2.4 Liver Cytotoxic ILCs
A very recent work, investigating the immune phenotype of ILCs
in hepatocellular carcinoma by scRNAseq, has demonstrated the
existence of a cluster of liver-resident ILC1s endowed with
cytotoxic potential (112). This cluster is characterized by the
expression of cytolytic effector genes, including FGFBP2,
FCGR3A,CX3CR1,GZMB,GZMH, and PRF1. Moreover, these
cytotoxic ILC1s are mainly enriched in non-tumor tissues, while
in tumor samples ILC1s are characterized by higher levels of
exhaustion markers, such as LAG3, thus suggesting that ILC1s
could undergo functional conversion during liver cancer
progression. Further, a high accessibility to the granzyme C
gene locus and high GrzmC transcripts were recently observed
in ILC1s puried from murine liver and salivary glands.
Granzyme C
ILC1 could be differentiated from ILC
precursors, are ontogenetically distinct from NK cells and do
not convert into ILC2 or ILC3. Granzyme C expression is
dependent on T-bet, while sustained TGF-bsignaling is
required for the maintenance of granzyme C
ILC1 in the
salivary gland, but not in the liver. Using the PyMT breast
carcinoma model, the authors show that these cells expand and
contribute to tumor anti-tumor functions in a TGF-b-dependent
manner. If a similar ILC1 subset exists in humans remains to be
tested (113).
Overall, cytotoxic helper ILCs have been so far identied
within the ILC1 and/or the ILC3/ILCP subsets, but not within
ILC2 compartment (Figure 2B). However, CD56
(20) and NKG2D
(114) ILC2s have been reported
in different settings, either in vitro upon cytokine exposure or in
vivo in human peripheral blood. It remains to be veried if these
cells have direct cytotoxic functions, like their cytotoxic ILC1 and
ILC3 counterparts.
Since their discovery, many efforts have been done to
characterize the origin, the function and identity of different
ILC subsets. The knowledge regarding ILC biology is continuing
to expand and includes the identication and characterization of
progenitors, the renement of mature ILC identities as well as
the denition of additional ILC subsets. However, it is of utmost
importance to understand if these novel ILC subsets coincide to
additional developmental intermediate stages or they are the
result of ILC plasticity to adapt to environmental stimuli.
Furthermore, emerging evidence highlights the existence of
circulating and tissue-resident helper ILCs endowed with
cytotoxic potential. These cells, with a phenotype resembling to
consequence of environmental and/or inammatory triggers
and could provide early innate defenses against different
pathogens, particularly in mucosal tissues, where NK cells
are underrepresented.
In the present review, we gave an overview of the current
knowledge of ILC biology, mainly focusing on their
developmental process. We further summarized the possible
developmental pathways of the unconventional cytotoxic ILC
subsets recently identied (Figure 2B).
Nevertheless, further studies are also needed to deeper
characterize the pathways of human ILC development and to
understand the differences and similarities with murine ILC-
poiesis. In this context, studying the immune-reconstitution of
ILC subsets after HSCT certainly represents an important
strategy to shedding light on the in vivo ILC developmental
trajectories at least in periphery. Moreover, the understanding of
ILC-poiesis and homeostatic mechanisms driving donor-derived
ILC immune-reconstitution as well as determining the
acquisition of cytotoxic features, could be of clinical utility.
Indeed, it will allow the development of protocols to
ameliorate the HSCT outcome based either on adoptive ILC
transfer therapies of ex vivo generated alloreactive ILCs or on
systemic cytokine infusion/blocking antibodies to boost in vivo
ILC effector-functions. Moreover, given the important role
of helper ILCs in tissue immune-surveillance, these
novel therapeutic options will nd application in the
management of solid as well as hematologic cancers and of
inammatory disorders.
MC, CDV, AF, ST, CJ, and DM wrote and critically reviewed the
manuscript. AF and MC draw the gures. All authors gave the
nal approval to the manuscript.
This work was supported by Associazione Italiana per la Ricerca
sul Cancro (IG 2018-21567 to DM), Intramural Research Funding
of Istituto Clinico Humanitas (to DM), the Italian Ministry of
Health (Bando Ricerca Finalizzata PE-2016-02363915 to DM),
SNSF PRIMA fellowship (PR900P3_17972729 to CJ), the Swiss
Cancer Research Foundation (KFS 5250-02-2021 to CJ) and the
Geneva Cancer League (GCL, 2007 to CJ). MC is a recipient of the
Leonelli AIRC fellowship (26580). MC and AF are recipients of
competitive fellowships awarded from the PhD program of
Experimental Medicine from University of Milan. ST is recipient
of a Dr Henri Dubois-Ferrière Dinu Lipatti Foundation research
fellowship. We also thank the nancial support from Fondazione
Romeo ed Enrica Invernizzi.
Calvi et al. ILC Ontogenesis and Unconventional Subsets
Frontiers in Immunology | May 2022 | Volume 13 | Article 9142669
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Full-text available
Tissue-resident immune cells reside in distinct niches across organs, where they contribute to tissue homeostasis and rapidly respond to perturbations in the local microenvironment. Innate lymphoid cells (ILCs) are a family of innate immune cells that regulate immune and tissue homeostasis. Across anatomical locations throughout the body, ILCs adopt tissue-specific fates, differing from circulating ILC populations. Adaptations of ILCs to microenvironmental changes have been documented in several inflammatory contexts, including obesity, asthma, and inflammatory bowel disease. While our understanding of ILC functions within tissues have predominantly been based on mouse studies, development of advanced single cell platforms to study tissue-resident ILCs in humans and emerging patient-based data is providing new insights into this lymphocyte family. Within this review, we discuss current concepts of ILC fate and function, exploring tissue-specific functions of ILCs and their contribution to health and disease across organ systems.
Full-text available
Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.
Full-text available
Innate lymphoid cells (ILCs) participate in tissue homeostasis, inflammation, and early immunity against infection. It is unclear how ILCs acquire effector function and whether these mechanisms differ between organs. Through multiplexed single-cell mRNA sequencing, we identified cKit⁺CD127hiTCF-1hi early differentiation stages of T-bet⁺ ILC1s. These cells were present across different organs and had the potential to mature toward CD127intTCF-1int and CD127⁻TCF-1⁻ ILC1s. Paralleling a gradual loss of TCF-1, differentiating ILC1s forfeited their expansion potential while increasing expression of effector molecules, reminiscent of T cell differentiation in secondary lymphoid organs. The transcription factor Hobit was induced in TCF-1hi ILC1s and was required for their effector differentiation. These findings reveal sequential mechanisms of ILC1 lineage commitment and effector differentiation that are conserved across tissues. Our analyses suggest that ILC1s emerge as TCF-1hi cells in the periphery and acquire a spectrum of organ-specific effector phenotypes through a uniform Hobit-dependent differentiation pathway driven by local cues.
Full-text available
Innate lymphoid type-2 cells (ILC2) are a population of innate cells of lymphoid origin that are known to drive strong Type 2 immunity. ILC2 play a key role in lung homeostasis, repair/remodeling of lung structures following injury, and initiation of inflammation as well as more complex roles during the immune response, including the transition from innate to adaptive immunity. Remarkably, dysregulation of this single population has been linked with chronic lung pathologies, including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrotic diseases (IPF). Furthermore, ILC2 have been shown to increase following early-life respiratory viral infections, such as respiratory syncytial virus (RSV) and rhinovirus (RV), that may lead to long-term alterations of the lung environment. The detrimental roles of increased ILC2 following these infections may include pathogenic chronic inflammation and/or alterations of the structural, repair, and even developmental processes of the lung. Respiratory viral infections in older adults and patients with established chronic pulmonary diseases often lead to exacerbated responses, likely due to previous exposures that leave the lung in a dysregulated functional and structural state. This review will focus on the role of ILC2 during respiratory viral exposures and their effects on the induction and regulation of lung pathogenesis. We aim to provide insight into ILC2-driven mechanisms that may enhance lung-associated diseases throughout life. Understanding these mechanisms will help identify better treatment options to limit not only viral infection severity but also protect against the development and/or exacerbation of other lung pathologies linked to severe respiratory viral infections.
Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C–expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C–expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor–β (TGF-β) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C–expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond “helper-like” lymphocytes.
Background & aims: Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that play critical roles in cytokine-mediated regulation of homeostasis and inflammation.However, relationships between their immune phenotypic characteristics and hepatocellular carcinoma (HCC) remain largely unexplored. Approach & results: We performed single-cell RNA sequencing (scRNA-seq) analysis on sorted hepatic ILC cells from human HCC patientsand validated using flow cytometry, multiplex immunofluorescence staining, and functional experiments. Moreover, we applied selection strategies to enrich ILC populations in HCC samples to investigate the effects of B cells on the immune reaction of ICOS+ ILC2 cells. Dysregulation of ILCs was manifested by the changes in cell numbers or subset proportions in HCC. Seven subsets of 3,433 ILCs were identified with unique properties, of which ICOS+ ILC2a were preferentially enriched in HCC and correlated with poor prognosis. Mechanistically, we report that B cells, particularly resting naïve B cells, have a previously unrecognized function that is involved in inflammatory differentiation of ILC2 cells. B cell-derived ICOSL signaling was responsible for exacerbating inflammation through the increased production of IL-13 in ICOS+ ILC2a cells. HSP70 genes HSPA1A and HSPA1B were highly expressed in ILC2s in late-stage HCC, and targeting to ICOS and its downstream effector HSP70 in ILC2s suppressed tumor growth and remodeled the immunosuppressive tumor microenvironment. Conclusions: This in-depth understanding sheds new light on B cell-driven innate type 2 inflammation and provides a potential strategy for HCC immunotherapy.
Significance Innate responses against viral infection and other intracellular pathogens rely on immune cells that are capable of lysing infected cells and producing interferon-gamma (IFNγ). These cells encompass two major cell lineages: natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s). While NK cells have been extensively characterized, identification of ILC1s and their distinction from NK cells are less clear. The transcription factor Hobit encoded by Zfp683 has been put forth as a prototypic feature of ILC1s. By analyzing Zfp683 reporter, fate-map, and -deficient mice, we demonstrate that the impact of Hobit on ILC1 identity and transcriptional and functional programs is tissue- and context-dependent. Thus, ILC1s adapt to local stimuli and tailor their responses to the tissue niche.
Eric Vivier describes the unexpected discovery of new populations of innate-like lymphocytes and the development of the innate lymphoid cell nomenclature.
Innate lymphoid cells (ILCs) are critical effectors of innate immunity and inflammation, whose development and activation pathways make for attractive therapeutic targets. However, human ILC generation has not been systematically explored, and previous in vitro investigations relied on the analysis of few markers or cytokines, which are suboptimal to assign lineage identity. Here, we developed a platform that reliably generated human ILC lineages from CD34⁺ hematopoietic progenitors derived from cord blood and bone marrow. We showed that one culture condition is insufficient to generate all ILC subsets, and instead, distinct combination of cytokines and Notch signaling are essential. The identity of natural killer (NK)/ILC1s, ILC2s, and ILC3s generated in vitro was validated by protein expression, functional assays, and both global and single-cell transcriptome analysis, recapitulating the signatures and functions of their ex vivo ILC counterparts. These data represent a resource to aid in clarifying ILC biology and differentiation.
Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin⁻NK1.1⁺NKp46⁺ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1⁺NKp46⁺ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1⁺NKp46⁺ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.