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Innate lymphoid cells and innate-like

T cells in cancer — at the crossroads
of innate and adaptive immunity
Benjamin Ruf 1
, Tim F. Greten 1,2
& Firouzeh Korangy 1

Abstract Sections

Immunotherapies targeting conventional T cells have revolutionized Introduction

systemic treatment for many cancers, yet only a subset of patients Characteristics of ILTCs
benefit from these approaches. A better understanding of the complex and ILCs

immune microenvironment of tumours is needed to design the next The role of ILCs and ILTCs
in immunosurveillance
generation of immunotherapeutics. Innate lymphoid cells (ILCs) and
innate-like T cells (ILTCs) are abundant, tissue-resident lymphocytes Bridging adaptive and innate
immunity
that have recently been shown to have critical roles in many types of
ILC-based and ILTC-based
cancers. ILCs and ILTCs rapidly respond to changes in their surrounding cancer immunotherapy
environment and act as the first responders to bridge innate and
Conclusion
adaptive immunity. This places ILCs and ILTCs as pivotal orchestrators
of the final antitumour immune response. In this Review, we outline
hallmarks of ILCs and ILTCs and discuss their emerging role in
antitumour immunity, as well as the pathophysiological adaptations
leading to their pro-tumorigenic function. We explore the pleiotropic,
in parts redundant and sometimes opposing, mechanisms that underlie
the delicate interplay between the different subsets of ILCs and ILTCs.
Finally, we highlight their role in amplifying and complementing
conventional T cell functions and summarize immunotherapeutic
strategies for targeting ILCs and ILTCs in cancer.

Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Malignancies Branch, Centre for Cancer
1

Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 2NCI CCR Liver
Cancer Program, National Institutes of Health, Bethesda, MD, USA. e-mail: firouzeh.korangy@nih.gov

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Review article
Introduction
Peptide-reactive, conventional CD4+ and CD8+ T cells have been major
targets of cancer immunotherapy, improving therapeutic outcome
across various cancer types1. Nevertheless, recent clinical studies
have revealed the limitations of current adaptive immune cell-based
approaches1. This could, however, be overcome by targeting the innate
immune cell axis, namely, innate lymphoid cells (ILCs) and innate-like
T cells (ILTCs), in parallel2. ILCs3, including natural killer (NK) cells and
helper ILCs (termed ILC1s, ILC2s and ILC3s) and lymphoid tissue-inducing
(LTi) cells, are a heterogenous cell population of the innate immune
system that has emerged as pivotal regulators of inflammation, tissue
repair, barrier homeostasis and immune tolerance. Unconventional
T cells or ILTCs (used interchangeably) are composed of three key
subsets: natural killer T (NKT) cells4, mucosal-associated invariant T
(MAIT) cells5 and γδ T cells6. They have pleiotropic functions and react
rapidly to non-peptide antigens via their conserved T cell receptors
(TCRs). Although the importance of NK cells7 in the context of cancer
has been known for many years8, helper ILCs9 and unconventional T cells
have only recently gained attention as important regulators of cancer
immunology. ILCs as the most innate cells across the innate-adaptive
spectrum (with degree of innateness defined as transcriptional state of
each cell type with adaptive cells on the one end and ILCs on the other
end of the spectrum)10,11 share many characteristics with unconven-
tional T cells, although each cell type has distinct functional features
(Fig. 1). Although ILCs and ILTCs with varying degrees of innateness10,11
can have fundamental roles in cancer, their network of connection
and complex interplay within the tumour microenvironment (TME)
are not well understood.
Here, we provide an overview of our current understanding of ILCs
and unconventional T cells in the context of cancer with an emphasis
on antitumour immunity. We provide a summary of cell type defini-
tions, their role in tumour immunosurveillance, how these cells can
bridge innate and adaptive immunity and current concepts of how they
could be targeted for cancer immunotherapy. We aim to point out their
similarities, as well as relevant distinctions, and outline the TME factors
that govern ILC and ILTC antitumorigenic or pro-tumorigenic function.
With this, we discuss their potential synergistic and redundant roles,
as well as crosstalk with other cells of the innate and adaptive immune
system. We conclude with possible therapeutic strategies targeting
ILCs and ILTCs to enhance anticancer immunity.
Characteristics of ILTCs and ILCs
Innate-like T cells
Unconventional or ILTCs share phenotypical and functional similarities
of both conventional T cells and innate immune cells. Although conven-
tional, peptide-reactive T cells primarily recognize antigens presented
by polymorphic major histocompatibility complex (MHC) class I and II
molecules, ILTCs recognize a diverse range of self-molecules and non-
self-molecules. This includes lipid ligands for invariant natural killer
T (iNKT) cells, riboflavin derivatives for MAIT cells and phosphoanti-
gens for γδ T cell subsets. These are recognized through their semi-
invariant (MAIT, iNKT and some γδ T cell subsets, for example, Vγ9Vδ2+
T cells) or diverse (type II NKT and other γδ T cells) TCRs in an MHC-
unrestricted manner12 (Fig. 1). MHC class I-like molecules CD1d and MHC
class I-related gene protein (MR1) present antigens to NKT cells and
MAIT cells, respectively13–15. They are evolutionarily conserved, suggest-
ing that the ILTCs have a critical role in host physiology by mediating the
interaction with non-self, therefore maintaining tissue homeostasis11.
Together, MAIT, γδ T and iNKT cells have emerged as crucial players in
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
immunity against microbial pathogens and cancer4. ILTCs share typical
features of conventional T cells; they both develop in the thymus16 and
express TCRs that upon encounter with an antigen drive their activation
and oligoclonal expansion12. Beyond TCR-dependent activation, ILTCs
can rapidly sense changes in their environment through the expression
of various integrins, chemokine and cytokine receptors, enabling them
to promptly mount potent cytokine responses, a key feature shared with
innate immune cells12. Furthermore, similar to other innate immune
cells, ILTCs acquire an effector phenotype before thymic egress and
accumulate at barrier sites including the gut mucosa, lungs or liver.
The development of ILCs and ILTCs is further described in Box 1, and
their tissue distribution is described in Box 2. Although ILTCs share
the common features described thus far, NKT, MAIT and γδ T cells are
characterized by distinct attributes outlined subsequently.
Natural killer T cells. iNKT cells (also known as type I NKT cells) share
phenotypic and functional similarities with both T cells and innate
lymphocytes4. They were first described as a T cell subset expressing
a semi-invariant TCR, composed of an invariant α-chain and a limited
number of TCR β-chains13,17,18 (Fig. 1). This semi-invariant TCR is reactive
to self and foreign glycolipid ligands including α-galactosylceramide
(α-GalCer) presented by the MHC class I‐like molecule CD1d13.
A second broad type of CD1-restricted NKT cells, type II NKT, use
diverse αβ TCRs that do not conform to the semi-invariant motifs
described earlier and are reactive to self-lipid antigen sulfatide19.
iNKT cells can be further categorized according to the expression
of canonical transcription factors and cytokine profiles on the basis of
CD4+ helper T (TH) cell subsets TH1, TH2 and TH17 (Fig. 1) and are termed
iNKT1, iNKT2 and iNKT17 throughout the text16,20,21.
In general, the majority of iNKT cells are non-circulating, tissue-
resident lymphocytes, where their number varies between tissues
and species16. Recent work in mice suggests that there is considerable
heterogeneity also within the iNKT fraction consisting of a circulat-
ing subset with NK-like cytotoxic potential in vivo that is functionally
distinct from tissue-resident iNKT cells22. Type I NKT cells are abundant
in laboratory mice, representing between 1% and 3% of CD3+ T cells in
some tissues and up to 50% of hepatic T cells16. In humans, iNKT cells are
less frequent23,24, accounting for less than 1% of T cells in the blood, and
type II NKT cells outnumber iNKT cells in human peripheral blood
mononuclear cells (PBMCs).
Mucosal-associated invariant T cells. MAIT cells are ILTCs that sense
microbial metabolites derived from vitamin B2 (riboflavin) or vitamin B9
(folic acid) presented on MR1 (refs. 14,15,25,26). MAIT cells also have
a TCR and were initially identified through their highly restricted rep-
ertoire of TCR α-chains (consisting of TRAV1-2 and thus defined as
TRAV1-2+ T cells) and limited number of TCR β-chains27,28. Beyond this
definition of MAIT cells (MR1-riboflavin antigen-reactive, canonical
TCR+), another group of MR1-reactive T cells exists, which has recently
been shown to include cancer-specific (non-MAIT) T cells with the
potential as pan-cancer therapeutic29.
Much like CD4+ TH cells, several MAIT cell subsets have been
described (Fig. 1). MAIT cells sense a range of inflammatory triggers
through cytokine receptors such as IL-7 receptor (IL-7R), IL-12R, IL-15R,
IL-18R and IL-23R. This can enhance TCR-mediated signals or lead to
MAIT cell activation in a TCR-independent, innate-like manner30,31.
Following stimulation, MAIT cells rapidly produce various effector mole­
cules, cytokines and chemokines, including granzymes, granulysin,
perforin, GM-CSF, interferon-γ (IFNγ), tumour necrosis factor (TNF),
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Tumour Phosphoantigen
microenvironment

Key Key Key

APC
transcription cytokines cytokine
factors secreted receptors

CD1d MR1 MHC-II MHC-I

Type 1 Glycolipid Riboflavin BTN2A1– Peptide
metabolite BTN3A1 ligand T-bet • IFNγ • IL-12R
• TNF • IL-18R
• IL-15R
ILC1 CD4+ CD8+
NK iNKT1 MAIT1 γδ T CTL

Type 2
GATA3 • IL-4 • IL-33R
• IL-5 • IL-25R
• IL-9
ILC2 MAIT2 CD4+ • IL-13
iNKT2 (?) γδ T
• AREG

Type 17
RORγt • IL-17A • IL-23R
• IL-22 • IL-1RI
• TGFβR
ILC1
LTi ILC3 CD4+
iNKT17 MAIT17 γδ T

ILCs ILTCs Conventional T cells TCRVα24-Jα18 (human) TCRVγ9Vδ2

TCRVα14-Jα18 (mouse) (example)

Hours Reactivity Days TCRVα7.2-Jα33 Conventional

(human) αβ TCR
TCRVα19-Jα33 (highly variable)
Innateness Adaptiveness (mouse)

Fig. 1 | Subsets of innate lymphoid cells and innate-like T cells in the tumour
microenvironment. Innate lymphoid cells (ILCs), innate-like T cells (ILTCs) and
conventional CD4+ and CD8+ T cells are displayed according to their degree of
innateness as coined previously11. ILCs, namely, natural killer (NK) cells, ILC1s,
ILC2s, ILC3s and lymphoid tissue inducer (LTi) cells, have the highest degree
of innateness and thus do no express T cell receptor (TCR). Hereby, ILCs and
unconventional T cells react to environmental cues within hours, whereas adaptive
immunity requires days for potent TCR-specific activation. Although ILCs do
not express a TCR, invariant natural killer T (iNKT) cells and mucosal-associated
invariant T (MAIT) cells, subsets of ILTCs, express semi-invariant TCRs that are
restricted to recognizing CD1d presenting glycolipids or major histocompatibility
complex (MHC), class I-related gene protein (MR1) presenting riboflavin-derived
antigens, respectively. The figure displays subsets of type I NKT cells (or iNKT cells):
iNKT1, iNKT2 and iNKT17 cells, whereas type II NKT cells are not displayed to
improve clarity. The presence of type 2 MAIT cell subset is not clear to date. The
γδ TCR cells, another ILTC subset, are more diverse and because of this only one
mechanism for activating human Vγ9Vδ2+ T cells is displayed; this is through
phosphoantigens and butyrophilin (BTN) family members BTN3A1 and BTN2A1.
Conventional T cells, CD4+ and CD8+ T cells, express classical αβ TCRs that are
reactive to peptide ligands presented by MHC class II or MHC class I, respectively.
Here, antigen-presenting cells (APCs) are used as examples for MHC, CD1d and
MR1 expressing cells, but MR1 and CD1d are generally expressed in a broad range
of both haematopoietic and non‐haematopoietic cell types. Moreover, subsets of
ILCs and ILTCs can be categorized in accordance with major CD4+ helper T cell
subsets (type 1, type 2 and type 17 subsets are shown; type 9 and regulatory sub­
sets were omitted for brevity), sharing similar functional profiles as defined by
common transcription factor, effector molecules and cytokine receptors.
Type 1 ILCs and ILTCs express T-box expressed in T cells (T-bet) and secrete high
levels of interferon-γ (IFNγ) and tumour necrosis factor (TNF). Expression of
GATA3 represents a hallmark of type 2 subsets. These cells are characterized as
IL-4, IL-5, IL-9, IL-13 and amphiregulin (AREG) secreting cells. Type 17 phenotypes
are characterized by the expression of retinoic acid receptor-related orphan
receptor-γt (RORγt) and production of IL-17 and IL-22. CTL, cytotoxic T lymphocyte;
IL-12R, IL-12 receptor; TGFβR, transforming growth factor-β receptor.

IL-17, IL-10, transforming growth factor-β (TGFβ), CC-chemokine ligand 3
(CCL3), CCL4 and CXC-chemokine ligand 16 (CXCL16)32–34.
MAIT cells are relatively scarce in human secondary lymphoid
organs, but account for up to 10% of the T cells in circulation35. They
are further enriched at mucosal sites and in the liver, where they are the
most common T cell subset36. By contrast, they are scarce in common
laboratory mouse strains C57BL/6 and BALB/c24. However, they are
more abundant in the CAST/EiJ strain, which allowed the generation
of an MAIThigh congenic (inbred) strain, a valuable tool for studying
MAIT cell biology37.
γδ T cells. Like the name might suggest, γδ T cells express TCRs with
γ-chains and δ-chains, rather than the canonical αβ TCR on conven-
tional T cells. Thus, they differ in their relative dependence on classical
MHC molecules. In human PBMCs, γδ T cells represent a fraction of up
to 10% of all T cells but can be markedly expanded at barrier sites and
peripheral, non-lymphoid organs38. In healthy adults, circulating γδ
T cells in the peripheral blood predominantly express TCR variable
(V) genes Vγ9 and Vδ2 (therefore Vγ9Vδ2+ T cells)39,40, whereas the Vδ1
subsets (paired with various γ-chains) are more abundant in peripheral
tissues41. Antigen recognition and specific ligands for γδ T cells are not

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Box 1
Innate lymphoid and innate-like T cells during development
Innate lymphoid cells (ILCs) and innate-like T cells (ILTCs) share
many phenotypic and functional properties, yet fundamentally
differ in their development. Both T cells and ILC lineages arise from
common lymphoid progenitor (CLP) cells with T cell precursors
migrating to the thymus for further development16. Studies in mice
have shown that γδ T cells develop at the fetal stage preceding
mucosal-associated invariant T (MAIT) and natural killer T (NKT)
cell development16. ILTC development is governed by the master
regulator promyelocytic leukaemia zinc-finger protein292–294,
and these cells acquire effector phenotypes that are governed
by lineage-defining transcription factors that orchestrate these
specialized differentiation programmes (listed in brackets after each
subset). This lineage commitment occurs before thymic egress,
and NKT1, MAIT1 (T-bet+), NKT2 (GATA3+), NKT17 and MAIT17 (retinoic
acid receptor-related orphan receptor-γt (RORγt+) cell subsets are
already found in the thymus. Importantly, CD1d and MR1 expression
on thymocytes is essential for NKT and MAIT cell development.
Beyond that, these ILTCs require early-life exposure to microbiota for
proper development 13,295,296 and shaping their contribution to barrier
immunity and tissue homeostasis 11,297.
ILC development, although closely shared by T cells, is different
as it is controlled by strict transcriptional factors, which guide the
ILC commitment pathway. All subsets of ILCs are derived from a
CLP cell in the adult bone marrow. ILC precursors in circulation
well understood and have been an intense area of research (reviewed
elsewhere42,43). The evolutionary divergence of γδ TCR genes across
species has hampered translation of functional studies, as the major
γδ T cell subsets in humans do not have orthologues in mice44. Addi-
tionally, the mechanisms of indirect phosphoantigen recognition by
human Vγ9Vδ2+ T cells through the butyrophilin family of molecules do
not apply to γδ T cells that are found in mice, as they lack the required
butyrophilin molecules45. Further studies are required to understand
how these cells are regulated in health and disease across species.
Furthermore, γδ T cells can rapidly respond to stimuli in an innate-
like way, indicating their potential to initiate an immune response as
first responders. Furthermore, γδ T cells can differentiate into TH1-like,
TH2-like, TH9-like, TH17-like and regulatory T (Treg)-like cells (Fig. 1).
Shaped by the local microenvironment as sensed through cytokine
receptors, they produce prototypical cytokines such as IFNγ, TNF, IL-4,
IL-10, IL-9, IL-17 and IL-22 (refs. 46–48).
Innate lymphoid cells
ILCs as the ‘most innate’ cells of the innate-adaptive continuum are a
heterogenous population of lymphocytes. They represent the innate
counterpart of T cells and are pivotal regulators of innate immunity,
inflammation and tissue repair. ILCs are largely tissue-resident cells,
sensing perturbations within their surrounding microenvironment and
rapidly responding by secreting cytokines, making them one of the first
responders in orchestrating an effective immune response49. All ILCs are
recombination-activating gene (RAG)-independent, lacking expression
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and tissues can differentiate at any stage in response to their
environmental signals to generate tissue-specific ILCs, that is, during
inflammation or perturbations in tissue homeostasis also referred
to as ‘ILC poiesis’298. CLP cells as influenced by transcription factors
such as nuclear factor interleukin-3-regulated (NFIL3), DNA-binding
protein inhibitor 2 (ID2), thymocyte selection-associated high-
mobility group box protein (TOX), T cell factor 7 (TCF7), GATA-
binding factor 3 (GATA3), zinc-finger and BTB domain-containing
protein 16 (ZBTB16) (in mice) and ID2, TOX, TCF7, GATA3, ROR-α and
ETS1 (in humans) will commit to the NK cell and ILC lineage through
a series of unipotent and multipotent ILC precursors such as
α-lymphoid precursor, early innate lymphoid precursor, common
helper innate lymphoid precursor and ILC precursor299. NK cells
and helper ILC1 subsets are both dependent on T-bet for their
development, but NK cells also require eomesodermin (EOMES)300
for their development. It is important to mention that among ILC1
subsets, liver ILC1 subsets are distinct and have been shown to
develop from fetal liver-derived haemopoietic stem cells via an
interferon-γ-dependent pathway301. Key transcription factors that
drive ILC2 differentiation are TCF1, BCL11B and GATA3, ensuring
effector function of ILC2 (ref. 302). ILC3 development is regulated
by RORγt mirroring the polarization of post-TH17 cells303. Several
outstanding reviews have discussed the constantly updating field
of ILC development in both mice and humans299,304,305.
of other immune cell markers and therefore known as ‘lineage-negative’
cells50. On the basis of the cytokine signatures and developmental
transcription factors that mimic TH cell subsets, five main groups of
ILCs have been classified: NK cells, ILC1s, ILC2s, ILC3s and LTi cells51.
NK cells. NK cells are circulatory, cytotoxic cells whose effector func-
tion is mediated by a combination of activating and inhibitory recep-
tors, leading to the secretion of pro-inflammatory cytokines such
as IFNγ and TNF7. NK cells are identified as CD3−CD56+ cells, which
are divided into two major subsets: (1) CD56dimCD16bright cells that are
mainly cytotoxic and circulatory and (2) CD56brightCD16− cells that pro-
duce cytokines and are tissue-resident52. The main activating receptors
of NK cells are natural cytotoxicity receptors (NCRs): NKp46, NKp44
and NKp30 (also known as NCR1, NCR2 and NCR3, respectively), as
well as NKG2D (also known as CD314) and CD16. Inhibitory receptors
include NKG2A, T cell surface glycoprotein YE1/48 (ly49) and killer
immunoglobulin-like receptors (KIRs) on mouse and human NK cells,
regulating their cytotoxicity, cytokine production and proliferation,
respectively53. Although NK cells play an important role in antitu-
mour immune responses, they can also gain a regulatory function in
inflammation, infection and cancer by suppressing T cell proliferation
through the production of IL-10 and adenosine54,55.
Helper ILCs. Helper ILC1 cells are tissue-resident, are T-bet-dependent
and, similar to NK cells, secrete type 1 cytokines such as IFNγ and TNF56,57.
ILC1s are responsive to IL-15, IL-12, IL-18, bacteria and viruses49,58–60 and
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parallel the TH1 arm of the adaptive immune system. Studies show that
in both mice and humans, ILC1s show extensive heterogeneity and differ
in number and function between lymphoid and mucosal sites, high-
lighting the impact of tissue localization on the phenotype and function
of these cells61,62. An important distinction between NK cells and ILC1
is that NK cells depend on eomesodermin (EOMES) and T-bet for their
development and differentiation, whereas ILC1s only require T-bet for
their development and differentiation63. Although ILC1s were originally
deemed to be weakly cytotoxic, several studies show that cytotoxic ILC1
subsets expressing granzyme B, granzyme C and perforin can mediate
cytotoxicity, contributing to immunosurveillance64–66.
The ILC2 subset as the innate TH2 counterpart produces type 2
cytokines IL-4, IL-5, IL-9, IL-13 and amphiregulin. They also depend on
GATA3 and retinoic acid receptor-related orphan receptor-α (RORα)
for their function and respond to IL-25, thymic stromal lymphopoi-
etin (TSLP) and IL-33 (ref. 67). Similar to ILC1, ILC2 has tissue-specific
phenotype and function68,69. In mice, ILC2s are the most homogeneous
of the other subsets and express GATA3, as well as the IL-33 receptor
(IL-1 receptor-like 1 (IL1R1), also known as ST2). Human ILC2s are defined
Box 2
Tissue distribution and layered immunity of innate lymphoid and
innate-like T cells
Most of our knowledge about the tissue distribution and
establishment of the innate lymphoid cell (ILC) and innate-like T cell
(ILTC) tissue lymphoid compartment during early development is
derived from studies in mice, in which the cells are found to populate
many tissues and barrier sites during fetal and perinatal periods11,306.
Beyond that, emerging studies show that the anatomical positioning
of ILCs in various tissues during development is explained by
waves of ILC development, in which multipotent and unipotent ILC
precursors give rise to circulating ILCs, known as layered immunity307.
The circulating ILCs can differentiate into any ILC subtype depending
on the tissue, and most tissue-resident ILCs are seeded during
the embryonic phase. Circulating ILCs are derived from the bone
marrow and can replace the different subsets of tissue-resident ILCs.
Therefore, the progenitor cells during the embryonic phase promote
generation of different ILC subsets in a site-specific manner described
as waves or layered immunity. The different waves of the various ILC
subsets lead to an effective and organized multilayered composition
of ILCs. As space in the tissues is limited168,308, ILTC occupation is
temporally restricted in tissue niches, as seen for mucosal-associated
invariant T cells in the skin295 and γδ T cells in the intestine309.
Immunological memory is typically considered a feature of
adaptive immunity and is characterized by a rapid response to
previously encountered antigens through the expansion of antigen-
specific individual immune cell clones310. Although the majority of
studies so far have focused on the innate function of ILCs, emerging
evidence shows the existence of memory features in these cells311.
For example, natural killer cells have been shown to have memory
responses to viruses and other small xenobiotic elements that
elicit immune response when combined with antibodies, termed
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by the expression of the chemoattractant receptor homologous mole­
cule (CRTH2) and CD161 and express IL1R1 in certain tissues upon
activation62.
Among ILCs, ILC3s are the most abundant subset in the intestinal
tract of both mice and humans and mirror TH17 cells70. The ILC3 subset
is defined by the expression of RORγt and KIT (also known as CD117),
as well as the ability to produce IL-17A and/or IL-22. They can be further
divided into NCR+ ILC3s, NCR− ILC3s and LTi cells71,72. Although both
NK cells and ILC1 subset have been shown to exert cytolytic function,
in vitro studies have demonstrated that human ILC3s can also have
cytotoxic activity73.
The role of ILCs and ILTCs in immunosurveillance
At the tumour initiation stage, cancer cells produce cytokines,
chemokines, alarmins and haematopoietic growth factors in response
to carcinogenesis, resulting in either pro-tumour or antitumour immu-
nity74. Successful immunosurveillance of malignant cells requires the
coordinated action of tissue-resident and circulating cells of the adap-
tive and innate immune system for induction of an effective antitumour
haptens312. Hapten sensitization has been shown to induce IL-7
receptor α+ ILC1 memory in the liver, such that they can migrate into
the draining lymph nodes to generate an allergic reaction313. Lung-
resident ILC2s have also shown recall responses against cytokines
such as IL-33 (ref. 314). The role of these memory-like ILCs in cancer
remains largely unknown.
Early-life and tissue imprinting of ILCs and ILTCs equips these
cells with unique features that are tailored to their tissue-specific
niche11. Such early-life signals include microbial-derived and
epithelial-derived signals that can shape the compartment of
unconventional T cells with lifelong consequences315. This has been
demonstrated for the development of mucosal-associated invariant
T cells, as they require exposure to commensal bacteria for proper
intrathymic development early in life295,296. The effect of this tissue-
specific functional imprint on the role of ILCs and ILTCs in the tumour
microenvironment remains unknown. Although ILTCs are considered
mainly tissue-resident, they have been shown to migrate from their
tissue of residence to their nearest lymph node, while retaining
the tissue of origin-specific transcriptional imprint. This emphasizes
their cooperative role as a functional network in shaping the adaptive
immune response316.
The notion that ILCs and ILTCs act as a cellular network through
coordinated interactions at barrier tissue or tumour sites rather than
individual subsets is derived from the observation that animal models
deficient for one ILTC subset compensate with increased numbers of
other ILTC subsets11,122,316. Nevertheless, the distinct localization and
stratification of ILCs and ILTCs in tissues indicate that each population
may contribute uniquely to tissue homeostasis and response to
tumours.
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a Tumour suppression Permissive microenvironment

IL-15 and IL-12 Dendritic cell Macrophage Monocyte

ILC2 NCR3+
ILC3 IL-12, IL-15 and IL-18 (IL-2 and IL-7)

ILC1-
like
TCR signalling Indirect tumour killing
↑ Plasticity
Dying
IL-12R IL-18R IL-15R
tumour cell ↑ Cytotoxicity
↑ Cytotoxicity
MAIT iNKT γδ T

Direct tumour killing

TCR
γδ T
TCR ligand

γδ T
ADCC NK
MAIT
CD16

FASL FAS ↑ Apoptosis

↑ MHC-I Tumour IFNγ
Perforin iNKT
cell
iNKT Granzymes ↑ Pyroptosis

↑ Fibronectin 1
NK

TRAIL
↑ Remodelling of the TME
ILC1
MAIT ILC1 ↓ Metastasis

b Tumour promotion

Type 2 immunity IL-17-dependent tumour promotion

TGFβ
MAIT IL-10
ILC2 MAIT2 NKT2 γδ T
↑ Immunosuppression ILC2
γδ T ↑ Plasticity

IL-4, IL-5 and IL-13

ILC1-
iNKT like IL-22
ILC3
↑ Plasticity ↑ Proliferation

IL-13Rα IL-4Rα ↑ Metastasis

↓ Cytotoxicity MAIT17
↓ T-bet
↓ NKG2D
NK
↓ IFNγ
↓ NKp30
IL-17R IL-17A iNKT17

Suppressive microenvironment

TGFβ γδ T

↑ Angiogenesis

Treg CAF ↑ Metastasis +/– TCR signalling

Platelets

TCRVα24-Jα18 (human) TCRVα7.2-Jα33 (human) TCRVγ9Vδ2

TCRVα14-Jα18 (mouse) TCRVα19-Jα33 (mouse) (example)

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Fig. 2 | Parallel and shared pathways prime pro-tumoural and antitumoural
functions of innate lymphoid and innate-like T cells. a, Regulation of
antitumour function. In a permissive type 1 cytokine (largely IL-12, IL-15 and IL-18)-
primed environment, innate lymphoid cells (ILCs) and innate-like T cells (ILTCs)
are antitumour-polarized. Direct tumour cell-killing mechanisms are mediated
through granzymes and perforin, as well as –FAS ligand (FASL), tumour necrosis
factor-related apoptosis-inducing ligand (TRAIL) or antibody-dependent cellular
cytotoxicity (ADCC). IL-15 and IL-12 have been shown to influence ILC plasticity
leading to transdifferentiation of NCR+ ILC3 and ILC2 to a more cytotoxic ILC1-like
phenotype, leading to tumour cell death. ILTCs can also be primed for enhanced
cytotoxicity through T cell receptor (TCR) signalling, leading to indirect tumour
killing through secretion of interferon-γ (IFNγ). They are primed through common
cytokine receptors, IL-12 receptor (IL-12R), IL-15R and IL-18R, leading to tumour
cell apoptosis, pyroptosis and tumour microenvironment (TME) remodelling
leading to decreased rates of metastasis. b, Pro-tumoural functions of ILCs and
ILTCs: type 2 primed ILCs and ILTCs can secrete IL-4, IL-5 and IL-13, which can
response75. ILCs and ILTCs as tissue-resident lymphocytes are abundant
at many tumour sites and can sense cytokines and alarmins through
cognate receptors, prompting the swift secretion of pro-inflammatory
or tissue-protective factors appointing them as the first responders
in the TME64,76. The functional properties of these cells in the TME are
context-dependent with key-shared adaptation programmes in
response to the microenvironment. Parallel pathways of activation
by tumour-derived signals and pro-inflammatory cytokines can trigger
antitumour transcriptional profiles in ILCs and ILTCs64. Alternatively, the
hostile TME can highjack their potential in tissue repair and homeostasis,
leading to their polarization towards tumour promotion3 (Fig. 2). The
complexity in dictating pro-tumour or antitumour function explains
why different clinical and preclinical studies have associated ILC and
ILTC subsets with seemingly paradoxical outcomes.
Subsequently, we outline how the balance between these con-
served signals within the TME influences ILCs and unconventional
T cells as a network of cellular mechanisms and highlight how each
subset serves a unique purpose in the cancer niche. We discuss subse-
quently how the presence of pro-inflammatory cytokines that affect
all subsets of ILCs and ILTCs can lead to effective antitumour responses
(Fig. 2). Tumour cells and other cells in the TME (for example, cancer-
associated fibroblasts or Treg cells)77 can secrete TGFβ, IL-17 and other
cytokines that can lead to phenotypic changes in ILCs and ITLCs, induc-
ing a tumour-promoting phenotype (Box 3). In Box 3, we outline the
concept of cellular plasticity, which enables these cells to act along
the full spectrum of the immunoregulatory axis.
Antitumour function
The response of ILCs and ILTCs to tumours is context-dependent, as
pro-inflammatory cytokines released in the TME induce a distinct
transcriptional profile and a characteristic effector function in these
cells. Numerous in vivo studies using genetically deficient mice or
depletion antibodies to NK, iNKT and γδ T cells have demonstrated the
tumour-protective effect of NK, NKT and γδ T cells78–80. Although
the function of these cells can generally span the continuum between
tumour promotion and destruction, respective cytotoxic ILC and ILTC
subsets are equipped with an arsenal of potent mechanisms that can
restrict tumour growth and spread of cancerous cells6,9,81,82. Subse-
quently, we outline microenvironmental determinants for successful
ILC-mediated and ILTC-mediated antitumour immunity, highlighting
shared mechanisms and pathways that lead to direct or indirect tumour
inhibition (Fig. 2a).
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contribute to general immunosuppression in the TME and act directly on tumour
cells through cognate receptors (IL-13Rα and IL-4Rα) to increase proliferation and
metastasis. Similarly, type 17-polarized ILC and ILTC subsets secrete IL-17A and
IL-22 to increase metastasis by acting on the stroma to promote angiogenesis or
directly on tumour cells to increase proliferation. Within the TME, transforming
growth factor-β (TGFβ) secreted by regulatory T (Treg) cells, platelets or cancer-
associated fibroblasts (CAFs) can act on ILCs and ILTCs, leading to further
immunosuppression (for example, enhanced secretion of IL-10) or loss of
cytotoxicity. In addition, TGFβ secreted by ILTCs themselves can further contribute
to an immunosuppressive microenvironment. One such immunosuppressive
effect of TGF-β is the conversion of cytotoxic natural killer (NK) cells into
non-cytotoxic ILC1s (also termed plasticity) as seen by the downregulation
of transcription factor T-bet, NKG2D, NKp30 and pro-inflammatory IFNγ.
DC, dendritic cell; iNKT, invariant natural killer T; MAIT, mucosal-associated
invariant T; MHC, major histocompatibility complex; NCR3, natural cytotoxicity
receptor (also known as NKp30); NKG2D, natural killer group 2, member D.
Direct tumour cell killing. ILCs and ILTCs utilize shared programmes
to mediate direct tumour cell killing. Unlike conventional cytotoxic
CD8+ T cells, ILCs and ILTCs do not require tumour-derived neoantigen
presentation on human leukocyte antigen (HLA) molecules to elicit
antitumour immunity82 and are therefore an attractive target for cancer
immunotherapy.
In general, ILCs and ILTCs induce tumour cell death after inte-
gration of activating and inhibitory receptor signals, shifting their
effector repertoire towards cytotoxicity83,84.One such pathway is
well studied for NK cells as they can eliminate cancer cells through
the so-called ‘missing-self’ mechanism. However, a unique feature
of ILTCs is that through the expression of their (semi-invariant) TCR,
ILTCs can exert TCR-dependent antitumour immunity, setting them
aside from ILCs84. Stimulation and activation of unconventional
T cells with activating ligands have been shown to induce potent
antitumour immune responses in iNKT cells85, MAIT cells86,87 and γδ
T cells88. For example, type I NKT cells can be activated by glycolipid
ligand α-GalCer to mediate in vitro and in vivo antitumour effects in
a CD1d-restricted manner85,89. We and others have shown that mouse
MAIT cells can be activated and expanded in vivo by the TCR-agonistic
bacterial vitamin B2-derivate 5-OP-RU (5-(2-oxopropylideneamino)-6-d-
ribitylaminouracil), leading to reduced tumour burden in models
of lung and liver metastasis86,87. γδ T cells and iNKT cells are capable of
recognizing tumour cells through their TCR or by expressing activating
receptors such as NKG2D, NKp30 or NKp44 (refs. 83,84,90).
NK, iNKT, MAIT and γδ T cells can directly lyse target malignant cells
in vitro through the release of preformed cytolytic granules such
as granzyme B and perforin85,91–93. Interestingly, studies of NK-cell-
mediated tumour cell killing in vitro show that granzyme-B-dependent
cytotoxicity occurs in the early events, whereas subsequent tumour
cell eliminations are preferentially induced through engagement
of death receptors expressed on NK cells such as FAS ligand (FASL;
also known as CD178) and TNF-related apoptosis-inducing ligand
(TRAIL)94.
TRAIL is a member of the TNF superfamily that is expressed on
NK cells, ILC1, ILC3 (ref. 95), iNKT, γδ and MAIT cells and can induce
apoptosis in tumour cells96,97. Intrahepatic and peripheral blood ILC3s
can directly kill hepatic tumour cells through increased IFNγ produc-
tion and TRAIL expression in vitro95. The TRAIL-dependent cytotoxic
capability of ILC1 and ILC3 cells suggests the potential of targeting
these cells as antitumour effector cells among the other cytotoxic ILC
and ILTC subsets.
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Box 3
Plasticity of innate lymphoid and innate-like T cells in the tumour
microenvironment
Specialized subsets of innate lymphoid cells (ILCs) and innate-like
T cells (ILTCs) are characterized by the expression of key transcription
factors determining their effector function. An adequate response
to tissue-derived stimuli requires flexibility and rapid adaptation of
each subset, which is achieved through transdifferentiation between
the cellular subsets and the acquisition of altered phenotypes and
function. This plasticity phenomenon has been studied for ILCs in
detail and reviewed comprehensively recently317,318. Although the
classical distinction of ILCs provides a useful framework, numerous
studies have confirmed that their heterogeneity and complexity
extend beyond the original classification (natural killer (NK) cell,
ILC1, ILC2 and ILC3 subsets). ILCs can change their function and
phenotype in response to signals in their microenvironment317,
similar to the differentiation of naive CD4+ T cells319, macrophages
and neutrophils320. Transcriptional analysis of ILCs has shown the
presence of transitional states between major subsets both under
homeostasis and pathological conditions321. Although most studies
investigating ILC plasticity have been carried out in the context of
inflammatory diseases, similar pathways and cytokines can dictate
ILC transdifferentiation in the tumour microenvironment (TME). For
example, in a mouse model of fibrosarcoma, transforming growth
factor-β induced the conversion of cytotoxic NK cells from the
surrounding non-tumour tissue into a non-cytotoxic ILC1 subset
in the tumour60,322, a phenotype that was later demonstrated in
ILCs and ILTCs can also directly kill tumour cells in vitro and in vivo
through FAS (also known as CD95–FASL) engagement94,98. Unlike
iNKT cells, NK cells mediate cytotoxicity through expression of perforin
and granzyme, highlighting the non-redundant roles of iNKT cells98,99.
Finally, the Fc regions of IgG antibodies on opsonized tumour cells
(antibody opsonization) bind to CD16 molecules expressed on NK
cells and unconventional T cells initiating antibody-dependent cellular
cytotoxicity (ADCC)100–103.
Indirect antitumour effects through interferon-γ. IFNγ is an impor-
tant pleiotropic cytokine with immunomodulatory effects on both
innate and adaptive immune responses104. In the local TME, respective
TH1-polarized subsets (NK cells, ILC1s, iNKT1 cells, MAIT1 cells and
γδ T cells) can produce large amounts of IFNγ as a rapid and first
response to tumour growth79,105–107. Such functional redundancy likely
allows for the robustness of defence against tumours and puts constant
pressure on cancer cells through maintained immunosurveillance. The
first-responder capacities of ILTCs and ILCs are consistent with what
has been reported in in vivo infection settings, in which ILC1s were the
main early source of IFNγ in response to IL-12 in virally infected tis-
sue limiting viral replication49. As a result, their IFNγ secretion in the
tumour niche likely establishes the antitumoural cytokine milieu at
early stages of tumour growth, creating a pro-inflammatory environ-
ment9. Similarly, acting directly on tumour cells, IFNγ signalling leads
to the upregulation of MHC-I and MHC-II, which can directly promote
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
patients with hepatocellular carcinoma154. In patients with squamous
cell carcinoma, IL-23 production by the tumour cells induced the
conversion of ILC1 into IL-17-producing ILC3 cells, promoting tumour
cell proliferation143. Pro-inflammatory IL-12 induced differentiation of
NKp46+ retinoic acid receptor-related orphan receptor-γt (RORγt)+
ILC3s into interferon-γ-producing ILC1 cells in a mouse model of
melanoma323. The transdifferentiation of ILC3s into ILC1 subsets was
also recently demonstrated in human colorectal cancer samples and
mouse models of colorectal cancer199.
As unconventional T cells egress the thymus with predetermined
memory or effector phenotypes and commitment to type 1, type 2
or type 17 lineage (Box 1), their potential for functional plasticity in
the TME is more restricted in comparison to ILCs16. However, several
studies point towards the likely ability of mucosal-associated invariant
T cells5, NK T cells324 and γδ T cells325 to alter their effector phenotype
upon microenvironmental changes. One might ask whether
unconventional T cells truly shift between TH17-like and TH1-like
phenotypes or whether the outgrowth of one subset versus the other
explains their altered functionality in the TME. Fate-mapping studies
in mice or the development of conditional knockout strains for these
ILTC subsets will provide further insight into this challenging question.
Nevertheless, in the context of many cancers, an understanding of the
developmental relationships and degree of plasticity of ILC and ILTC
subsets remain an important area for future investigations.
tumour cell apoptosis and pyroptosis108. The paramount role of IFNγ
in mediating both ILC and ILTC antitumour immunity has been dem-
onstrated in various settings and across different in vivo models and
tumour types86,109,110. Nevertheless, impaired IFNγ production of ILCs
and ILTCs is frequently observed in advanced cancers representing an
important immune escape mechanism111.
IL-15, IL-12 and IL-18 regulate ILC and ILTC antitumour function. ILC
and ILTC proliferation, maturation and antitumour function are tightly
governed by the presence of pro-inflammatory cytokines in the TME112.
The presence of IFNγ in the TME primes inflammatory myeloid cells
such as macrophages, monocytes and dendritic cells (DCs) to produce
IL-12, IL-15 and IL-18 cytokines driving TH1-type immunity112. This in turn
induces cytotoxicity in ILCs and ILTCs that express high levels of the
corresponding receptors9.
Although ILCs and ILTCs share the aforementioned common
response to a pro-inflammatory cytokine milieu, the unique features
of each subset allow for non-redundant responses in the TME. The
ability for simultaneous integration of TCR-mediated and cytokine-
induced signalling in ILTCs adds another layer of complexity to our
understanding of their function in the TME. Moreover, TCR ligands
(derived from bacteria or tumour cells) in synergy with cytokines can
promote distinct pro-inflammatory responses in vitro and in vivo87,113–115.
The response to pro-inflammatory cytokines is by no means uni-
form across ILC and ILTC subsets. IL-12 alone or in combination with
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IL-15 and IL-18 has been shown to activate NK cells9; however, IL-12 has
even more pronounced effects on helper ILC subsets, inducing IFNγ
production by ILC1 and ILC3 cells49,56,116.
Mechanistically, binding of IL-15 on its receptor expressed on
NK, iNKT, γδ T and MAIT cells induces an intracellular signalling
cascade, leading to the accumulation of T-bet in the nucleus30,117,118.
Consequently, IL-15-primed ILCs and ILTCs produce high levels of
granzyme B117 and IFNγ119,120, leading to enhanced cytotoxicity towards
cancer cells121.
In recent years, increasingly complex in vivo, ex vivo and in vitro
studies have shed light on how fine-tuned intercellular communica-
tion loops shape ILC and ILTC function and phenotype in inflamma-
tory, infectious and neoplastic processes122. The importance of the
IL-12–IFNγ circuit is shown by the plasticity of ILC subsets in which
in both in vivo and in vitro models, IL-12 and IL-18 mediate the trans-
differentiation of NCR+ ILC3s into ILC1-like cells (that is, ex-RORγt+
ILC3s) that secrete IFNγ and are cytotoxic58,123,124. It has been shown
that RORγt+ ILC3s, when cultured with IL-2, IL-15 and IL-23, secrete IFNγ,
along with upregulation of T-bet, gaining ILC1 characteristics125. In the
context of malignancy, IL-12 (ref. 126) secreted by antigen-presenting
cells (APCs) amplifies IFNγ production in NK and iNKT cells127, as seen
in 4T1 mouse models of breast cancer treated with recombinant IL-12,
which rescued the anti-metastatic function of NK cells128. In patients
with chromophobe renal cell carcinoma who showed prominent infil-
tration of tissue-resident CD56bright ILC1 cells, IL-15 secreted by the
chromophobe renal cell carcinoma tumour cells activated granzyme
cytotoxic ILC1 cells, emphasizing an IL-15-dependent, antitumour
function of ILC1 cells129. Given this, it is likely that ILCs and ILTCs act in
a synergistic manner as both initiators of an inflammatory response
at early neoplastic events and signal amplifiers of an already ongoing
protective, antitumour signalling cascade.
Tumour-promoting function
Successful immune evasion is characterized by the depletion of antitu-
moural immune cell subsets replaced by the accumulation of exhausted
and/or immunosuppressive leukocytes. Defects in antigen presentation
machinery, altered chemokine and cytokine milieu and metabolic alter-
ations negatively impact ILC and ILTC function in the TME. By secreting
distinct combinations of effector molecules (for example, IL-4, IL-10,
IL-17 or TGF-β), ILCs and ILTCs can dampen the innate and adaptive
immune responses against tumours. Subsequently, we highlight how
ILCs and ILTCs share a largely overlapping functional niche in tumour-
promoting immunity, emphasizing their unique and non-redundant
functions (Fig. 2b).
IL-17A-secreting ILCs and ILTCs promote tumour growth. High levels
of IL-17A in the blood of patients with cancer correlate with poor sur-
vival in various patient cohorts, highlighting its role in cancer develop-
ment and progression130. In mouse models of hepatocellular carcinoma
(HCC)131, as well as breast cancer132, lung cancer133 and colorectal cancer
(CRC)134, IL-17A deficiency has been shown to reduce tumour burden135.
Experimental findings derived from mouse models should be inter-
preted with caution, as there are important differences among species.
For example, in humans, in some contexts, tissue-resident Vδ1+T cells
have been found to preferentially produce IL-17A compared with their
circulating Vγ9Vδ2+ counterparts136, whereas in mice Vγ4+ or Vγ6+
T cells133 produce high levels of IL-17A. Furthermore, Vδ1+T cells can also
be found to predominantly secrete IFNγ, as demonstrated for human
studies of breast cancer and non-small-cell lung cancer (NSCLC)137,138.
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
Chronic IL-17 secretion by ILCs and ILTCs contributes to several
mechanisms in the TME, ranging from tumour initiation to tumour
progression. For instance, IL-17-secreting γδ T cells accelerated
pancreatic intraepithelial neoplasia in Mist1-CreERT2;KrasG12D mice
by direct signalling through the IL-17R on pancreatic acinar cells, which
accelerated pancreatic intraepithelial neoplasia initiation and progres-
sion to invasive disease139. γδ T cell-derived IL-17 has also been shown
to act directly on endothelial cells leading to enhanced angiogenesis,
which subsequently led to tumour development in mouse models of
CMS-G4 fibrosarcoma and HPV-induced K14-HPV16 (refs. 140,141).
Using mouse models of lung metastasis and subcutaneous tumour
growth, MAIT cells were found to exert tumour-promoting function
through IL-17A leading to a lower tumour burden in MAIT cell-deficient
Mr1−/− mice compared with WT mice 142. It seems possible that
IL-17-secreting iNKT type 17 cells show tumour-promoting effects in
a similar fashion4. Interestingly, studies in mouse models of HCC and
patients with squamous cell carcinoma show that in an IL-23-rich TME,
which induces IL-17 production, ILC1s can differentiate into RORγt-
expressing NCR− ILC3 cells143,144. Additionally, human KIT+CCR6+ ILC2
cells can differentiate into IL-17-secreting NCR− ILC3s and upregulate
RORγt when cultured with IL-1β, IL-23 and TGF-β145, pointing to the
effect of the TME on the plasticity of these cells. ILC3 and LTi cells are
major sources of IL-22, which has been shown to have a similar role to
IL-17 in the TME146,147. IL-22 protects tissue barriers against microorgan-
isms and plays an important role in intestinal homeostasis. However,
IL-22 can also induce inflammation and result in tumour growth, as
seen in a bacteria-induced colon cancer model146,148. Another study
showed that IL-22 can regulate cancer stem cells in CRC to promote
colon cancer149.
TGFβ-rich environments impair ILC and ILTC antitumour function.
TGFβ is an essential and pleiotropic cytokine produced within the
TME of many solid tumours by tumour cells, Treg cells, fibroblasts,
macrophages and platelets150. Although TGF-β is a regulator of homeo-
stasis, perturbations in its expression within the TME are an important
mechanism of tumour immune evasion and poor response to anti-
tumour therapy151. Within the local TME, ILCs and ILTCs can be both
effectors and producers of TGF-β. TGF-β signalling affects NK cells
in vivo at a transcriptional level by silencing T-bet expression and IFNγ
secretion152,153. Interestingly, in primary and metastatic mouse models
of fibrosarcoma and lung metastasis, as well as in patients with HCC,
TGF-β in the TME caused a shift of NK cells into less cytotoxic inter-
mediate ILC1 and ILC1-like cells, which were unable to control tumour
growth and metastasis. This demonstrated that a TGF-β-rich TME
can shape and modulate the cellular plasticity of the ILC subsets 60,154.
Similar mechanisms of immunosuppression by TGF-β were shown in γδ
T cells and iNKT cells, in which TGF-β also contributed to the induction
of IL-17-producing human Vγ9Vδ2+ T cells47 and to both the develop-
ment and peripheral responsiveness of type 17 iNKT cells155. In addition
to their response to TGF-β-signalling in the TME, ILTCs such as γδ T
and MAIT cells can secrete TGF-β in vivo contributing to an immuno­
suppressive feedforward loop156–158. Although the relative contribution
of ILC-derived and ILTC-derived TGF-β to immune evasion in cancer
remains to be determined, these findings provide further insights
on how the ILC and ILTC network could act as signal amplifiers in the
hostile TME. Dissecting the role of TGFβ in orchestrating the function
and plasticity of the ILCs and ILTCs in the TME will shed light on how
differential regulation of this pivotal cytokine leads to cooperation or
competition among the different innate subsets.
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Type 2 immunity leads to pro-tumorigenic ILCs and ILTCs. Type 2
immunity is characterized by the production of IL-4, IL-9 and IL-13 and
is responsible for maintaining homeostasis and tissue repair159. The
dysregulation of type 2 immunity has been shown to promote tumour
proliferation and metastasis160. The pro-tumoural role of ILC2s is attrib-
uted mainly to the production of IL-4, IL-5 and IL-13 upon stimulation by
epithelial cytokines such as IL-33, IL-25 and TSLP69,161. Mechanistically, in
the subcutaneous 15-12RM fibrosarcoma tumour model, iNKT-derived
IL-13 was found to activate the IL-4R–STAT6 axis and promote tumour
recurrence162. Chronic stimulation of MAIT cells, by CD3 and CD28 plus
IL-2 and IL-7, leads to IL-4, IL-5 and IL-13 production, which can induce
phosphorylation of the nuclear transcription factor STAT6 (pSTAT6)
signalling in CRC cells in vitro163.
The dichotomy of the pro-tumour versus antitumour effect of
TH2 signalling is organ site-dependent, stage-dependent and context-
dependent for ILC2s and type 2 ILTCs164,165. Whether ILC2s and type 2
ILTCs can promote or suppress tumour progression relies extensively
on their tissue environment. It is clear now that our current under-
standing of these cells underestimates the vast spectrum of effec-
tor functions and therefore does not yet sufficiently capture their
heterogeneity within this group.
TCR-dependent tumour promotion. The ability of unconventional
T cells to sense environmental and endogenous ligands through
their conserved TCR distinguishes them from ILCs and con­ventional
αβ T cells. TCR-derived signals are integrated with cytokine and
chemokine signals, prompting ILTCs to have pro-tumoural or
antitumoural effector functions87,113–115.
In mouse models of B16-F10 and LWT1 melanoma lung metastases,
MAIT cells show tumour-promoting functions when MR1 is expressed
on tumour cells, which can be reversed by inhibitory MAIT ligands
Ac-6-FP or MR1 blocking antibodies142. This indicates that endoge-
nous or microbial MAIT ligands exist in the TME, polarizing mouse
MAIT cells towards tumour promotion in a TCR-dependent manner.
Type II NKT cells were found to promote in vivo tumour growth in
several tumour mouse models166, and their activation by the proto-
typical agonist sulfatide suppressed tumour immunosurveillance167.
Functional studies on γδ T cells in mouse models of melanoma also
suggest that TCR engagement is essential for the tumour-promoting
effects of γδ T cell-produced IL-17 within the TME140.
These preliminary findings on ILTCs in the tumour setting high-
light how TCR signalling can shape their effector function through
poor or null agonists and antagonists. The relative proportion of these
agonistic and antagonistic signals, as well as co-stimulatory molecules,
may vary between different primary tumour sites, tumour stages and
microbial composition of the local TME.
Bridging adaptive and innate immunity
Even though ILCs and ILTCs can serve as direct effectors of pro-tumoural
or antitumoural immune responses, their predominant function lies
in their roles as orchestrators of tumour immunity. These cells bridge
innate and adaptive immunity by initiating, amplifying, inhibiting or
dampening the immune response in the TME. It is increasingly clear
that ILCs and ILTCs mount an innate immune response influenced by
the inflammatory signals in the TME. Thus, ILCs and ILTCs engage in
complex crosstalk with elements of the innate and adaptive immune
system, as well as the microenvironment122,168. In turn, innate and
adaptive immunity reciprocally regulates ILCs and unconventional
T cells, indicating that such cellular interaction networks are crucial
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
determinants of immune responses within tissues. In this section, we
aim to demonstrate the similarities and synergisms across the different
ILC and ILTC subsets during this crosstalk.
Antitumour response in a TH1 environment
ILCs and ILTCs, as a relay of potentially pro-inflammatory cells, can con-
tribute to successful immunosurveillance through initiation and ampli-
fication of an antitumour TH1-like immune response in a permissive
microenvironment64,86,87,107,169,170 (Fig. 3a).
ILCs and ILTCs can directly sense danger-associated molecular
patterns through cognate receptors. These appear in the TME dur-
ing immunogenic cell death, especially in early cancers171. Beyond
that, iNKT and MAIT cells show unique features as they can sense
pathogen-associated molecular patterns derived from intratumoural
microorganisms through their TCR, if presented by CD1d or MR1 mole­
cules, respectively172. Both in vitro and in vivo, the pro-inflammatory
cytokines polarize ILCs and ILTCs leading to an optimal antitumour
response characterized by high IFNγ expression and a TH1 transcrip-
tion factor profile6,173–175. IFNγ-signalling on macrophages and DCs in
the TME potentiates this antitumour effect through maturation of M1
macrophages and differentiation of DCs towards type 1 conventional
DCs (cDC1s)176. NK, γδ T and iNKT cells can recruit cDC1s177, which in turn
induces TH1 polarization of CD4+ helper T cells and thus leads to a CD8+
cytotoxic T cell response178. IFNγ as a central mediator in this circuit
then leads to improved antigen presentation capacities by the DCs
as seen in samples of patients and mouse models. This in turn leads
DCs to secrete more IL-12 and increases NK cell activity179. In a similar
fashion, iNKT, MAIT and γδ T cells have been found to enhance NK cell
cytotoxicity in an IFNγ or CD137-dependent way86,169,180,181.
Local conditioning of the TME cytokine milieu by ILCs and ILTCs
further shapes antigen-specific adaptive immunity demonstrated
in vivo and in vitro87,182. Further connections to adaptive immunity
with potential for an improved tumour control in human and mouse
exist, as ILCs and unconventional T cells can provide B cell help towards
class switching of immunoglobulins, formation of germinal centres
and shaping of peripheral B cell populations183–186. Although not yet
shown in the context of cancer, it seems likely that similar mechanisms
might apply to the TME.
ILCs and ILTCs recruit antitumour myeloid cells
Emerging studies show that ILC and ILTC subsets also share similar
pathways in recruiting innate immune cells such as macrophages or
DCs, which in turn are crucial for the induction of potent adaptive
antitumour immunity75. Tumour-infiltrating ILC2s secrete IL-5 and
GM-CSF recruiting eosinophils, which can subsequently secrete chemo-
attractants such as CCL5, CXCL9 and CXCL10. In both mouse models of
lung cancer187 and melanoma188, this was shown to facilitate the recruit-
ment of CD4+ and CD8+ T cells to the tumour site. In mouse models of
pancreatic ductal adenocarcinoma, tumour-infiltrating ILC2s were
shown to activate CD103+ DCs and CD8+ T cells to induce antitumour
immunity, which was amplified using anti-programmed cell death
protein 1 (PD-1) blockade189.
The strong interconnectivity between ILTCs and the myeloid cell
compartment is well established. For instance, MAIT cells are one of
the major and early producers of GM-CSF in infection settings, and
this MAIT cell-dependent GM-CSF release in vitro and in vivo induces
differentiation of CCR2+ inflammatory monocytes into DCs, leading to
activation of effector T cells. This can occur both in an MR1-dependent
and MR1-independent manner190. iNKT cells have also been shown to
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bridge the innate and adaptive immune system by modulating DC
and macrophage function in both infection and tumour settings.
Tumour cells engineered to secrete GM-CSF induced the expansion
of CD1d-restricted T cells and maturation of DCs. This was abolished in
CD1d− mice, emphasizing the role of NKT cells in interaction with DCs
and shaping the antitumour immunity191.
ILCs and ILTCs as APCs
ILCs and ILTCs as part of their fundamental role in connecting the innate
and adaptive arms of the immune system can also act as professional
APCs through expression of MHC-II to modulate conventional αβ CD4+
T cell responses. Changes in ILC3 expression of MHC and co-stimulatory
molecules induced by inflammatory cytokines can either reinforce
their regulatory role in tissue homeostasis or convert them into pro-
inflammatory ILCs inducing T cell activation192. In intestinal homeo-
stasis, MHC class II+ ILC3s limit inflammation through inhibition of
microbiota-specific effector CD4+ T cell proliferation192,193; however,
upon intestinal inflammation, ILC3s upregulate co-stimulatory
molecules to induce TH1 cell responses194. Recently, several groups
have shown that MHC II+ RORγt-expressing cells are responsible for
generating RORγt-expressing peripheral Treg cells in the gut, promot-
ing tolerance to microbiota. In human CRC tissue, intestinal ILC3s have
been shown to gain antigen-presenting function by upregulation of
HLA-DR and co-stimulatory molecules IL-1β and IL-18. This is inhibited
by TGFβ195–198.
ILC3-specific deletion of MHC-II in mice promoted CRC tumour
growth and resistance to anti-PD1 immunotherapy, suggesting that
MHC-II+ ILC3s provide a protective role in CRC199. γδ T cells can also
present microbial antigens and induce proliferation of naive CD4+
T cells, similar to DC activation of αβ T cells200. In patients with gastric
cancer, tumour-activated γδ T cells behaved as APCs, upregulating
MHC-II and expressing co-stimulatory molecules to induce CD4+ and
CD8+ αβ T cell proliferation and subsequent tumour cell death201. Taken
together, these studies underline the important role of MHC-II expres-
sion on ILCs and ILTCs in direct, cell-contact-dependent regulation of
the adaptive immune cells in tissue homeostasis and cancer.
Type 2-mediated immune evasion
Perturbations and inflammation in the context of cancer can induce the
release of alarmins such as IL-25, IL-33 and TSLP, which regulate type 1
and 2 immune responses202. The receptor for IL-33 and IL1R1 is present
on ILC2s, iNKT cells, MAIT cells and γδ T cells203–205 and can potentially
prime the cells to bridge inflammatory and danger signals in their sur-
roundings to the adaptive immune system (Fig. 3b). In several models
of lung metastasis, ILC2s activated by IL-33 recruit eosinophils in an
IL-5-dependent manner, resulting in tumour metastasis 206. IL-5
produced by iNKT cells207, γδ T cells208 and MAIT cells163 could poten-
tially follow a similar mechanism for the recruitment of eosinophils
and induction of type 2 immunity.
The pro-tumorigenic role of ILC2s and TH2 type ILTC subsets is
mainly through release of cytokines such as IL-5 and IL-13. ILC2s secrete
IL-13 to promote accumulation of suppressive myeloid derived suppres-
sor cell (MDSC), which has been shown to regulate antitumour immu-
nity in patients with acute promyelocytic leukaemia and mouse models
of breast cancer209,210. ILC2s also produce amphiregulin, which activates
Treg cells and therefore induces a suppressive microenvironment211.
NKT cells also trigger the IL-13–MDSC axis, in which secretion of
IL-13 by type II NKT cells induces TGF-β secretion by Gr1+CD11b+ MDSCs
through the IL-4R–STAT6 pathway. This results in suppression of CD8+
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
T cells and type I NKT cells19,212. Expression of IL-4 on both human and
mouse γδ T cells induced proliferation of IL-10-producing Vδ1 T cells, sup-
pressing the function of the Vδ2 T cells165,213. Tuft cells, epithelial chem-
osensory cells that reside in the epithelial lining of the intestine, express
IL-25 under homeostasis, infection and inflammation. IL-25 derived from
tuft cells has been shown to further activate ILC2s to secrete IL-13, which
can mediate type 2 immune responses in the intestine214,215. IL-25 has also
been shown to have a pro-inflammatory role in colitis, inducing secretion
of IL-13 by NKT cells214,215. This reinforces the notion that through differ-
ent pathways, TH2 cytokines, particularly IL-13, released by ILC2s and
ILTCs can have a role in mediating tumour progression. The differential
stimulation of ILC2s by IL-33 and IL-25 is tissue-dependent, suggesting
that the function of these cells differs between cancer types.
Type 17-dependent tumour promotion
Another multicellular programme in pro-tumour immunity, in which
the combined ILC and ILTC community bridges innate and adaptive
immunity, is the type 17 response, in which the canonical cytokine IL-17A
furthers tumour progression (Fig. 3c). The presence of TGF-β, IL-1β, IL-6,
IL-7 and IL-23 secreted by inflammatory macrophages, neutrophils or
DCs in response to endogenous or exogenous (microbial) signals in
the TME induces TH17 polarization of ILC3s, iNKT cells, γδ T cells and
MAIT cells133,216.
Blocking, depleting or genomically ablating these cytokines leads
to a reduction in the number of IL-17+ ILCs and ILTCs in mouse models
of breast cancer132,217, fibrosarcoma140,218, melanoma219 and gastric
cancer220,221. Notably, cellular crosstalk between IL-17A-producing
ILCs or ILTCs222 and myeloid cells is bidirectional, such that Vγ6+ T cells can
also mobilize peritoneal macrophages with pro-tumorigenic potential223.
Microbial dysbiosis, a common feature for many cancers224, is likely
another key factor for IL-17-mediated ILC and ILTC tumour promo-
tion. In a mouse model of lung adenocarcinoma, γδ T cells were the
predominant IL-17-producing cells driven by tumour-associated micro­
biota133. This increased the infiltration of tumour-associated neutro-
phils and led to enhanced tumour progression. Similarly, ILC2s have
been shown to recruit tumour-associated neutrophils to the TME in
either a CXCL1-dependent or CXCL2-dependent manner, albeit this
study does not link these findings to microbial dysbiosis225. Another
potential connection to the microbiome was recently demonstrated, as
IL-17A-producing MAIT cells promoted tumour growth in mouse models
of lung metastasis in an MR1-dependent manner142. Similarly, when
MAIT cells are co-cultured with Escherichia coli antigen-exposed human
breast cancer cell lines, they show enhanced MR1-dependent IL-17A
production142,226. Comparably, IL-17A from conventional CD4+ TH17 cells
and innate-like γδ17 T cells promoted tumorigenesis in bacteria-induced
CRC models227. ILC3s as the TH17 counterpart of the ILC family are par-
ticularly abundant at intestinal sites and have been shown to promote
colonic carcinogenesis partly through IL-17-mediated sustained inflam-
mation in response to IL-23R signalling220. Nevertheless, the question of
how local microbial communities within the TME shape phenotype and
function of ILCs and ILTCs remains an active field of research.
Finally, ILC-derived and ILTC-derived IL-17A has also been shown
to inhibit NK and cytotoxic CD8+ T cell function in vivo86,142,144, favour-
ing tumour progression. IL-17A production by ILC3 and γδ T cells also
promotes CXCL5 secretion by tumour cells, leading to the recruitment
of immunosuppressive CXCR2+ MDSCs131,144. Further indirect CD8+ T cell
inhibition could occur through the IL-17A-dependent recruitment of
tumour-associated neutrophils132, as demonstrated for γδ T cells in
mouse models of breast cancer132.
361
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a ↑ CD40 Immunogenic cell death,

↑ Maturation early cancer and carcinoma in situ ↑ Antigen presentation
↑ CD80 ↑ cDC1 differentiation
↑ M1 > M2 polarization
↑ Antigen presentation ↑ GC formation
DAMPs, PAMPs, alarmins, intratumoural microbiome
↑ MHC class II Dendritic cell
Macrophage M1
IL-12 TH1-polarized IL-12 and IL-18
and
IFNγ
IL-18
ILC1 NK iNKT1 MAIT1 γδ T
↑ Maturation B cell
↑ Class switch
TCR
TH1 cytokines (IFNγ and TNF)
Tumour-specific
antibodies
↑ Leukocyte migration
↑ TH1 polarization
↑ Recruitment ↑ Effector function
↑ Cytotoxicity CD8 + Tumour Direct tumour
NK CTL cell
↑ Maturation killing ↑ T-bet
FAS FASL TCR
CD4+
Granzymes TH1
↑ TAA presentation
Perforin
↑ Apoptosis
Cancer death cell ↑ Pyroptosis

Tuft cell
b CAFs

↑ Recruitment IL-13
↑ M1 < M2 polarization IL-25
↓ Antigen presentation
Epithelial-derived alarmins (e.g., IL-33, IL-25 and TSLP)
↑ ARG1 chronic stimulation
PDL1 IL-13
↑ MMP9
↑ TGFβ

IL-13 and CD4+

Macrophage M2 ILC2 T cell
IL-4 iNKT2 MAIT2 γδ T

IL-4
B cell TH2 cytokines (IL-13, IL-4, IL-5, IL-9 and AREG)
IL-5 and
GM-CSF IL-13 IL-5, AREG
MDSC Treg
↑ Recruitment γδ1 T
IL-10
↑ Recruitment ↑ Recruitment
↓ Tumour cell killing
↓ Cytotoxicity
↓ IFNγ production CD8+
Eos NK CTL γδ2 T
Immune evasion

c
Dendritic cell Macrophage Neutrophil MDSC
CXCL5
CXCR2

↑ Recruitment
↑ Microbial dysbiosis ↑ T cell inhibition
IL-1β, IL-6, IL-7, IL-23 and TGFβ
5-OP-RU or
endogenous (?) CD8+
microbial (?) TCR CTL ↓ Tumour cell killing
ligands
Tumour IL-17 and IL-22 ↓ Cytotoxicity
ILC1 Tumour
MAIT17 NKT17 γδ T ↓ IFNγ production
cell cell
MR1 NK
Immune evasion
TH17 cytokines (IL-17A and IL-22) ↑ Tumour growth
↑ Metastasis
TAN
↑ Recruitment

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Review article
Fig. 3 | The role of innate lymphoid and innate-like T cells in bridging
adaptive and innate immunity in cancer. a, Orchestrating an antitumour
immune response in a T helper 1 (TH1) environment. Tissue perturbations such
as immunogenic cell death and tumour growth through released damage-
associated molecular patterns (DAMPs), pathogen-associated molecular
patterns (PAMPs), alarmins and the intratumoural microbiome are sensed
through macrophages and dendritic cells (DCs). This leads to the secretion
of IL-12 and IL-18, fostering type 1 polarization of innate lymphoid cells (ILCs)
and innate-like T cells (ILTCs). TH1-polarized ILCs and ILTCs then amplify this
signalling cascade by producing large amounts of type 1 cytokines (specifically
interferon-γ (IFNγ) and tumour necrosis factor (TNF)). These TH1 signalling
molecules can have pleiotropic beneficial effects on various immune cells
within the tumour immune microenvironment: IFNγ on macrophages leads
to maturation, M1 > M2 polarization, increased production of IL-12 and IL-18,
as well as enhanced antigen presentation capacities. Similarly, DCs show
preferred differentiation into type 1 conventional DCs (cDC1s) leading to higher
capacities of antigen presentation and the formation of germinal centres
(GCs), which is a crucial component of CD4+ helper T cell recruitment, effector
function and TH1 polarization. On B cells, these signalling cascades can lead to
maturation and class switch of tumour-specific antibodies. These mechanisms
lead to an increased infiltration of natural killer (NK) cells and cytotoxic CD8+ T
lymphocytes (CTL) and further enhanced tumour cell-killing capacities
of these effector cells. On tumour cells, TH1 effector molecules can induce
apoptosis and pyroptosis. Additionally, increase in major histocompatibility
complex (MHC)-I-related presentation of tumour-associated antigens (TAAs)
triggers enhanced direct tumour cell killing of CD8+ T cells. b, ILCs and ILTCs as
initiators and amplifiers of type 2-mediated immune evasion. Alarmins derived
from dying cells, pre-cancerous cells, cancer-associated fibroblasts, epithelial
tuft cells, chronic stimulation through epithelial cell damage or alternatively
M2-polarized macrophages can induce type 2 polarization of ILCs and ILTCs.
ILC-based and ILTC-based cancer immunotherapy
The capacity of conventional αβ T cells for antigen-directed cytotoxic-
ity has been the major focus in the development of cancer immuno-
therapies; however, clinical research has demonstrated that a large
proportion of patients do not benefit from these approaches1. It is
therefore necessary to explore alternative paths to engage the immune
system in the combat against cancer. The importance of NK cells for
cancer immunosurveillance has been recognized for many years8,228,
such that targeting them in cancer immunotherapy is ongoing in vari-
ous clinical studies. In comparison, helper ILCs and unconventional
T cells have only recently been recognized as important players in
cancer immunotherapy, and, thus, immunotherapeutic approaches
targeting these cells are less advanced. Subsequently, we highlight
the potential of ILCs and ILTCs as new targets for cancer immuno-
therapy along with future directions of harnessing their enormous
potential (Fig. 4).
Immune checkpoint inhibition
Therapeutic targeting of peptide-reactive conventional T cells through
inhibition of the cytotoxic T lymphocyte protein 4 (CTLA4) and PD1–
programmed death ligand 1 (PDL1) pathways has revolutionized treat-
ment outcomes for many types of cancers229. Similar to their adaptive
counterparts, ILCs and ILTCs in patients with cancer have also been
found to upregulate their cell surface expression of immune check-
point molecules such as PD1, CTLA4, T cell immunoglobulin and mucin
domain molecule 3 (TIM3), T cell immunoreceptor with immunoglobu-
lin and ITIM domains (TIGIT) and lymphocyte activation gene 3 protein
(LAG3)230–233 (Fig. 4a).
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
In response, type 2-polarized ILCs and ILTCs secrete TH2 cytokines (IL-13, IL-4,
IL-5, IL-9 and amphiregulin (AREG)) with negative impact on innate and adaptive
antitumour immunity. A positive feedforward loop has been described for
tuft cells in the gut and ILC2s through secretion of IL-25 and IL-13, respectively.
Secretion of type 2 cytokines can induce immunosuppression of antitumour
innate and adaptive immune responses, suppressing γδ2 T cells via activation
of γδ1 T cells, activation of regulatory T (Treg) cells, increasing myeloid-derived
suppressor cell (MDSC) and eosinophil (Eos) recruitment to suppress NK
function and polarizing M2 macrophages. Increased B cell recruitment through
IL-5 and GM-CSF, on the contrary, can have beneficial effects on tumour control.
c, ILCs and ILTCs as cornerstones of TH17-dependent tumour promotion. DCs,
macrophages, neutrophils and MDSCs can secrete key stimulators such as IL-1β,
IL-6, IL-7, IL-23 and TGF-β, induced through microbial dysbiosis in the tumour
immune microenvironment, which is sensed through Toll-like receptors on
these cells. In response, ILCs and ILTCs can acquire type 17 polarization and
secrete large amounts of IL-17A and IL-22 that can in turn have various negative
regulatory functions on neutrophils to promote tumour growth, enhanced
metastatic potential and inhibition of CD8+ T cell function. TH17 cytokines can
directly inhibit NK cells and CTLs, leading to tumour promotion. IL-17A signalling
on tumour cells can induce CXCL5 secretion and recruitment of MDSCs, which
are potent inhibitors of T cell recruitment and cytotoxicity. IL-17A in the tumour
immune microenvironment leads to an increased recruitment of tumour-
associated neutrophils (TANs) that can foster tumour growth and increase
metastasis and have the potential to negatively impact tumour-killing function
of CD8+ T cells. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil;
CAF, cancer-associated fibroblast; CXCL5, CXC-chemokine ligand 5; CXCR2,
CXC-chemokine receptor 2; FASL, FAS ligand; iNKT, invariant natural killer T;
MAIT, mucosal-associated invariant T; MMP, matrix metalloproteinase;
MR1, MHC class I-related gene protein; TCR, T cell receptor; TGFβ, transforming
growth factor-β; TSLP, thymic stromal lymphopoietin.
PD1 blockade in lymphoma models increased ADCC against
lymphoma cells232. γδ T cells have also recently been identified to
contribute to immune checkpoint blockade response in patients
with mismatch repair-deficient (dMMR), HLA class I-negative colon
cancer234. Similarly, in patients with melanoma, treatment with anti-
PD1 therapies induced higher numbers of circulating MAIT cells and
correlated with better overall survival235. Additionally, γδ T cells may
be important sources of immunosuppression, as PDL1 blockade on
γδ T cells enhanced CD4+ and CD8+ T cell infiltration in mouse mod-
els of pancreatic ductal adenocarcinoma236. In addition, alternative
checkpoint inhibitors such as targeting TIGIT may provide a prudent
strategy to target ILCs as TIGIT is expressed on NK cells and ILC1s233.
Nevertheless, whether ILCs and ILTCs have prognostic or predictive
value for cancer immunotherapy requires further evaluation across
various tumour types.
Monoclonal antibodies to immune receptors
ILCs and ILTCs overexpress NKG2A in human tumours, leading to inhibi-
tion of effector cells237 (Fig. 4b). A humanized anti-NKG2A monoclonal
antibody238, monalizumab, has been shown to block the inhibitory
function of NKG2A and is being evaluated in several phase II trials in
patients with solid tumours239,240.
Inhibitory KIRs negatively regulate NK cell function and are impor-
tant modulators of antitumour immunity241. Currently, IPH2102 and
lirilumab (IPH2102/BMS-986015) are monoclonal antibodies tar-
geting KIR2DL-1, KIR2DL-2 and KIR2DL-3 NK cell receptors that are
being evaluated either as monotherapy or combined with checkpoint
inhibitors in acute myeloid leukaemia, multiple myeloma, chronic
363
Review article

a Immune checkpoint inhibition b Targeting inhibitory signalling c BiKEs and TriKEs

Tumour antigen
Antibody
BiKE TriKE

NKG2A KIRs SLAMF7

CD16
NKp46
PD1 CTLA4 TIM3 TIGIT LAG3
or PDL1

ILTC ILC ILTC

ILC ILTC NK

↓ Inhibition ↑ Cytotoxicity ↑ Cytokine ↑ Cytotoxicity ↑ Cytokine ↑ Cytotoxicity

production production

d TCR ligands e Recombinant cytokines f Adoptive therapy

CAR ILC and CAR ILTC
IL-12 IL-18 IL-15 IL-2

CAR CAR
Phosphoantigens MAIT γδ T
5-OP-RU CD1d
α-GalCer IL-12R IL-18R IL-15R IL-2R
TCR
CAR

ILC ILTC CAR CAR

NK NKT
MAIT iNKT γδ T

↑ TH1 ↑ Cytotoxicity
↑ Proliferation ↑ Cytotoxicity polarization
Endogenous TCR

Fig. 4 | Strategies to harness innate lymphoid and innate-like T cells for
cancer immunotherapy. a, Innate lymphoid cells (ILCs) and innate-like T cells
(ILTCs) can be targeted by blocking inhibitory signalling pathways expressed
on the cell surface: programmed cell death protein 1 (PD1)– programmed
death ligand 1 (PDL1), cytotoxic T lymphocyte protein 4 (CTLA4), T cell
immunoglobulin and mucin domain-containing 3 (TIM3), T cell immunoreceptor
with immunoglobulin and ITIM domains (TIGIT) and lymphocyte activation
gene 3 (LAG3). b, Anti-NKG2A and anti-killer immunoglobulin-like receptor
(KIR) antibodies block inhibitory receptors on natural killer (NK) cells and
ILTCs. SLAMF7 can be activated with antibodies to prime antitumour immune
responses in NK cells and ILTCs. c, Bispecific or trispecific NK cell engagers (BiKEs
or TriKEs, respectively) consist of antibody-based constructs combining distinct
single-chain variable fragments that contain tumour-specific antigens on the
one side and NK cell or ILTC receptors (such as NKp46 or IL-15R, respectively)
on the other side, ultimately NK cells and ILTCs to attack tumour cells through
CD16-mediated antibody-dependent cellular cytotoxicity. d, Stimulation of ILTCs
with T cell receptor (TCR) ligands can increase proliferation and cytotoxicity
towards tumour cells. Proof-of-principle studies have demonstrated that
5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) can activate
MAIT cells, phosphoantigens such as aminobisphosphonates can activate
γδ T cells and α-galactosylceramide (α-GalCer) can activate invariant natural
killer T (iNKT) cells. To avoid anergy of NKT cells, antigen-presenting cells
such as dendritic cells can be loaded with α-GalCer and provide co-stimulatory
signalling. e, Recombinant cytokines in various formulations can be used for
TCR-independent stimulation of ILTCs and ILCs through cognate receptors,
increasing TH1 polarization and therefore tumour cytotoxicity. f, ILCs and ILTCs
can be used as platforms for adoptive cell transfer, by using chimeric antigen
receptor (CAR) constructs to target tumour-specific antigens.

lymphocytic leukaemia and breast and ovarian cancers with limited
success, particularly as a monotherapy242–244.
Signalling lymphocytic activation molecule family member 7
(SLAMF7) is a receptor present on ILCs and ILTCs245, which has been
shown to influence NK cell function and cytotoxicity246. Elotuzumab,
a humanized, immunostimulatory IgG1 monoclonal antibody, specifi-
cally activates NK cells by targeting the extracellular domain of human
SLAMF7 and binding to CD16 on NK cells via its Fc portion. This leads
to an increase in NK-cell-mediated ADCC through CD69 expression, as
well as IFNγ and granzyme B release245,247,248.
to maximize activation of ILTCs and NK cells249 (Fig. 4c). Given this, they
are also termed NK cell engagers. BiKEs and TriKEs that engage CD16 and
IL-15 along with tumour antigens such as CLEC12A, CD133 or epithelial
cell adhesion molecule have shown strong NK cell-mediated responses
against patient-derived acute myeloid leukaemia blasts250. Trifunctional
NK cell engagers were shown to target tumour antigen through two NK
cell receptors, NKp46 and CD16, with high efficacy both in vitro and in
a mouse model of lymphoma251,252. ILCs and ILTCs could be specifically
targeted through their semi-invariant receptor to direct them to tumour
sites and to enhance their cytokine secretion or cytotoxicity.

Bi-specific and tri-specific killer cell engagers TCR ligands

Bispecific and trispecific killer engagers (BiKEs and TriKEs, respec- ILTCs can be potently stimulated and expanded in vivo and ex vivo
tively), are monoclonal antibody fragments that create a synapse through ligation of their semi-invariant TCR with prototypic ligands
between activating receptors on NK cells and tumour-specific antigens (Fig. 4d) such as glycolipids (to stimulate iNKT cells) and or riboflavin

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Review article

Glossary of clinical activity (1 pathological response and 14 with stable disease

in 35 enrolled patients)257.
Most clinical applications targeting TCR stimulation of ILTCs
αβ T cells with immunosuppressive features for improved cancer immunotherapy have focused on γδ T cells and
T cells expressing a lineage-specific that are often associated with poor polyclonal Vγ9Vδ2+ T cells specifically, with modest therapeutic
heterodimeric T cell receptor consisting outcomes in cancer. efficacy88,258–260. One mechanism that was recently introduced might
of an α-chain and a β-chain (as opposed explain the lack of efficacy of this approach, as phosphoantigen-
to γδ T cell receptors). ‘Missing-self’ mechanism stimulated γδ T cells can suppress NK cell-mediated antitumour
This describes a mechanism of response to missing-self261. Taken together, TCR-stimulated ILTCs
Antibody opsonization nature killer cell activation, in which may be potent effectors of anticancer immunity, yet require a greater
Antigen-specific antibodies can bind absence or altered expression of major understanding before broader application. Optimal polarization
to foreign pathogens or transformed histocompatibility complex I molecules through co-stimulatory signals might be needed for optimized
cells and mark them for elimination by (for example, on tumour cells) leads to cytotoxicity and antitumour function.
phagocyting cells. the recognition and elimination of these
target cells. Cytokine-based immunotherapies
Degrees of innateness As ILCs and ILTCs are stimulated by many overlapping cytokines and
This term was previously coined, for Pancreatic intraepithelial share numerous cytokine receptors, immunotherapeutic approaches
example, by Gutierrez-Arcelus et al.10 neoplasia involving cytokines could collectively target subsets of ILCs and ILTCs
and Constantinides and Belkaid11, to A precursor lesion to invasive ductal triggering overlapping or synergistic mechanisms (Fig. 4e). IL-2 is
describe where an immune response adenocarcinoma of the pancreas with not only known for its role in T cell survival and expansion but also
lies on the continuum that exists well-defined histological features. induces expansion and activation of ILCs262 and ILTCs30,263,264 in different
between innate and adaptive immunity. settings. IL-2 was one of the first cytokines used to enhance antitumour
Pyroptosis function of NK cells; however, it had limited efficacy owing to adverse
Graft-versus-host disease An inflammatory pathway leading to side effects and induction of Treg cells265. IL-15 was shown to be far more
(GvHD). A potentially fatal complication cell death as mediated by activation of efficient at ex vivo expansion and activation of NK and γδ T cells266. IL-15
after allogeneic cell transfer caused by caspase 1 and inflammatory cytokines alone or in combination with IL-12 and IL-18 led to increased cytotoxic-
a detrimental reaction of the transferred such as IL-1β and IL-18. ity by upregulating perforin, granzyme and IFNγ secretion. However,
allogeneic T cells (graft) against the continuous treatment with IL-15 induces characteristics of exhaustion
(host) healthy tissue of the recipient. T-bet in NK cells267.
The transcription factor T-bet Similarly, IL-12 and IL-15 together have been shown to enhance
Lymphoid tissue-inducing (encoded by TBX21) promotes the cytotoxicity by upregulating T-bet and increasing the secretion of
(LTi) cells differentiation programmes of innate perforin, IFNγ and granzymes A and B in iNKT cells268, γδ T cells and
These cells are members of the innate and adaptive lymphocytes towards MAIT cells30,269,270. In mouse models, IL-15 stimulation induces gran-
lymphoid cell family that promote interferon-γ-producing type 1 cells. zyme C expression in helper ILC1s65, inducing cytotoxicity, potentially
generation of secondary lymphoid contributing to antitumour immunity.
structures. Toll-like receptor
Pattern-recognition receptor expressed Adoptive transfer
M2 macrophages on innate immune cells that recognizes The ability of ILCs and ILTCs to recognize tumours in an MHC-
Although defining macrophage structurally conserved molecules (also independent manner gives them a unique advantage over conventional
polarization is oversimplistic, this called pathogen-associated molecular T cells. This allows them to be used as effector cells without the risks of
is commonly used to describe patterns) derived from microorganisms. alloreactivity271,272. Therefore, designing immunotherapeutic strategies
alternatively activated macrophages targeting ILCs and ILTCs as universal ‘off-the-shelf’ or universal agents
for different types of tumours is an appealing pan-anticancer strategy.
Adoptive cell therapy is an immunotherapeutic approach, in which
derivates (to stimulate MAIT cells). γδ T cell TCRs can be indirectly lymphocytes are typically separated from the blood of patients through
activated by aminobisphosphonates or synthetic phosphoantigens. a process termed apheresis, then ex vivo expanded and reinfused back
To avoid the potential tumour-promoting functions of TH2 or TH17 into patients with cancer, also termed autologous cell transfer. Adop-
subsets, it must be established how to polarize these cells towards tive transfer of NK cells in immunotherapy takes advantage of their allo-
a TH1 phenotype. Priming potential APCs through the addition of reactivity, which is derived from a mismatch between donor NK cells
adjuvants such as Toll-like receptor ligands might be a practical strat- expressing KIR and HLA ligands on tumour cells. The majority of the
egy to maximize TH1 polarization and optimize the cytotoxicity of NK cells used for adoptive transfer use PBMC collected by apheresis;
ILTCs87,253. Eventhough glycolipid ligand α-GalCer has demonstrated however, other sources such as cord blood and bone marrow have also
promising antitumour efficacy in mouse tumour models89, repetitive been used273. These allogeneic NK cells have demonstrated safety, effi-
administration of α-GalCer induces a strong and long-lasting anergy cacy and antitumour activity in various tumours such as acute myeloid
of iNKT cells254. To further circumvent stimulation-induced anergy, the leukaemia274, colon carcinoma275 and non-Hodgkin’s lymphoma276
concept of combined adoptive transfer of iNKT cells and α-GalCer- with little toxicity such as graft-versus-host disease (GvHD). Adoptive
pulsed DCs has been applied and tested clinically in small phase I stud- iNKT cell-based therapies have also been explored in phase I and II trials,
ies255,256. In a phase II trial in patients with advanced or recurrent NSCLC, which involve pre-expansion and activation of iNKT cells before admin-
α-GalCer-pulsed APCs showed an acceptable safety profile with signs istration in patients. These approaches were tested in patients with

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Review article
NSCLC, melanoma and HCC277–279. These studies showed that ex vivo
expansion of iNKT cells is feasible and well tolerated, and induction of
TH1 responses could have antitumour potential. Although not tested
clinically, expansion of MAIT cells in vitro was shown to be feasible, as
they retain their function and cytotoxicity280, making them a suitable
candidate for adoptive cell transfer protocols.
Chimeric antigen receptors (CARs) are receptors that are geneti-
cally engineered to target specific antigens and mediate cytotoxicity
against tumours281. Currently approved CAR T cell therapies are based
on αβ T cells recognizing antigen–MHC complexes and are therefore
HLA-matched to avoid GvHD. This highlights once again that ILCs and
ILTCs as MHC-independent cell types are strong candidates for CAR
therapies. This notion was further substantiated by a recent longitu-
dinal study from two patients with chronic lymphocytic leukaemia
who received CD19-targeted CAR T cells (CTL019) therapy and
showed an initial response phase dominated by CD8+ CAR T cells and γδ
CAR T cells. This indicates that these ILTCs are likely important effector
cells in this therapy282.
CAR NK, γδ CAR T and CAR NKT cells have been used in various
settings, showing both antitumour function and enhanced cytotox-
icity272. CAR NK cells having the advantage of not causing GvHD in an
allogeneic setting have been used successfully in different haemato-
logical malignancies and solid tumours283,284. γδ CAR T cells have shown
superior cytotoxicity against leukaemia xenografts when compared
with unselected CAR T cells285. In neuroblastoma cell lines express-
ing GD2 ganglioside, transducing γδ CAR T cells with GD2-specific
CAR constructs enhanced GD2-specific killing while retaining the
innate cytotoxic capacities of γδ T cells. These cells also retained their
capacity to cross-present tumour antigens to αβ T cells, which could
have additional antitumour effects286. Similarly, iNKT cells expanded
ex vivo and engineered to express GD2-targeted CAR more efficiently
infiltrated tumours in xenograft models of neuroblastoma than unse-
lected CAR T cells287. MAIT cells are also promising candidates for
CAR therapeutics, as they are MR1-restricted and therefore unlikely
to induce GVHD and are being explored in different settings271,288.
Interestingly, co-expansion of iNKT cells and Vγ9Vδ2+ T cells, using
IFNγ, IL-2, anti-CD3 antibody and feeder cells, showed higher cytotoxic
activity in vitro compared with αβ CAR T cells and enhanced survival
in xenograft models289.
Although the aforementioned approaches are promising, they
are limited by proliferation efficiency, difficulties in activation ex vivo
before administration and short persistence of response in vivo. How-
ever, as discussed earlier, ILCs and ILTCs offer many advantages such as
lack of alloreactivity, antigen-presenting as well as immunomodulatory
abilities and rapid secretion of cytokines, which place these cells as the
new milestones providing new and exciting opportunities for the next
generation of adoptive cell therapy in cancer.
Conclusion
The high frequency of ILCs and ILTCs in tissues and their pleiotropic
effector functions appoint them as master regulators and pivotal
orchestrators of tumour immunity. As the complex and dynamic micro-
environment of the tumour is shaped during tumorigenesis, ILCs and
ILTCs dance to a delicately choreographed programme as the first
responders in the TME during tumour initiation, immunosurveillance
and tumour progression64.
It is becoming increasingly clear that unlike conventional T cells,
owing to the ability of ILCs and ILTCs to rapidly integrate and respond
to signals from their environment, they are attractive candidates for
Nature Reviews Cancer | Volume 23 | June 2023 | 351–371
immunotherapeutic strategies. ILCs specifically will be of particular
importance, because of their high degree of innateness and therefore
most rapid responses10,49,290.
Given the complex dynamics between their cellular frequency in
tissues and their associated cytokine milieu and receptors, ILCs and
ILTCs can be activated by the TME to jointly either through synergy
or compensation elicit a robust antitumour immune response3. Their
relationship with surrounding cells of the TME during carcinogenesis
and any conserved functional programmes (for example, expression
of cytokine receptors, transcription factors and effector molecules)
between ILCs and ILTCs remains poorly understood. In the light of this,
the specific mediators that shape ILC and ILTC function throughout
carcinogenesis, and whether these cells reach a ‘tipping point’ switch-
ing from antitumorigenic to pro-tumorigenic signalling remains largely
unknown. What influence does the stage of disease, potential underly-
ing chronic inflammation or tumour intrinsic factors have on ILC and
ILTC plasticity, phenotype and function? Investigating pre-cancerous
lesions from patients and monitoring ILC and ILTC plasticity during
tumour growth in mouse models of cancer will help to provide the
necessary insight into the dynamics and hierarchies of ILCs and ILTCs
for targeted immunotherapeutic intervention291. The capability of ILCs
and ILTCs to modify and shape the adaptive immune system under
homeostasis, as well as in tumour settings, puts them in the spotlight
for sharing or synergizing potential effector mechanisms and as new
targets for cancer immunotherapy.
More studies are needed to further understand and delineate the
regulatory mechanism that affects ILCs and ILTCs, as well as the clinical
consequences of ILC-directed and ILTC-directed therapies to approve
them as off-the-shelf or universal agents. Taking into account that
ILC subsets are both tissue-dependent and tumour-type-dependent,
identification of additional markers to better distinguish ILC subsets
in both mice and humans will also aid in developing better targeted
therapies. Additionally, the use of genetically engineered mice and
development of tools to manipulate these cells in mouse models will
be important for immunotherapeutic interventions.
Together, our understanding of ILCs and ILTC biology, as well as a
deeper knowledge of the mechanisms that support the innate–adaptive
relationship, will further the development of the next generation of
immunotherapeutics. The future of immunotherapy targeting ILCs and
ILTCs will certainly generate a resounding applause from its audience.
Published online: 20 April 2023
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Acknowledgements
The authors thank J. Zhu (NIH) for critical reading of the manuscript and helpful suggestions
and comments. The authors apologize to all the scientists whose work could not be cited
owing to space limitations. B.R. was supported by the International Liver Cancer Association
Fellowship Award 2021. T.F.G. was supported by the Intramural Research Program of the NIH,
NCI (ZIA BC 011345).
Author contributions
F.K. and B.R. researched data for the article. All authors contributed substantially to discussion
of the content, as well as writing, reviewing and editing the manuscript before submission.
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Reviews Cancer thanks Hergen Spits, Pamela Ohashi and
Nicolas Jacquelot and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work.
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