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Friend or Foe? The Ambiguous Role of Innate Lymphoid Cells in Cancer Development
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Review
Friend or Foe? The Ambiguous Role of Innate Lymphoid Cells in Cancer Development Jochen Mattner1 and Stefan Wirtz2,* The development of immunotherapies represents a major advance towards the effective eradication of malignant tumors. So far, therapeutic approaches have largely focused on T lymphocytes, but the innate arm of the immune system might be similarly important. Innate lymphoid cells (ILCs) are rapidly-responding cells that are functionally analogous to diverse T cell subsets. In recent years these cells have attracted enormous attention owing to their pleiotropic effects in early host defense to infection and organ pathologies. ILCs might also represent promising targets in the context of cancer therapy because they are an innate immune cell population endowed with potent immunomodulatory properties. In this review we discuss the impact of the three ILC subsets and the signature cytokines they release on cancer development and tumor growth. ILCs in the Tumor Microenvironment Most types of malignancies are characterized by substantial immune cell infiltrations, and complex mutual interactions between innate/adaptive immune cells and tumor cells are believed to profoundly affect tumor development and the success of therapeutic strategies. While the presence of some immune cells such as cytotoxic T cells is important for limiting tumor growth, the occurrence of others, including tumor-associated macrophages, correlates with invasiveness, metastasis, and poor prognosis [1]. Although many studies have focused on the interaction between immune cells and the tumor microenvironment, the role of ILCs remains largely unexplored. ILCs represent a heterogeneous group of developmentally related cells that in adults derive from shared lymphoid precursor populations including the common lymphoid progenitor (CLP) in the bone marrow, but typically lack recombination-activating gene (Rag)dependent rearranged antigen receptors [2]. Based on functional criteria, cytokine production characteristics, and transcription factor expression profiles, they were recently categorized into three major subgroups (ILC1, ILC2, and ILC3) that strikingly resemble the corresponding T helper cell subsets (Th1, Th2, and Th17) [3] (Table 1). The main biological function of ILCs is likely to center on their capacity to rapidly respond to environmental and inflammatory signals by the secretion of cytokines implicated in tissue repair and immune defense mechanisms. In addition, ILCs may substantially contribute to T cell polarization and effector functions by secretion of regulatory cytokines, antigen presentation, or direct cellular interactions [4]. Hypoxia and the acid milieu in the tumor microenvironment favor necrotic cell death and an abundance of immunostimulatory danger signals [5]. Given their activation characteristics and their importance in the context of tissue damage and repair, ILCs may be important players in tumor-associated host responses, and could represent interesting target cell populations for
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Trends ILCs are categorized into three major subpopulations (ILC1s, ILC2s, ILC3s) that have key and non-redundant functions during infections as well as in the pathogenesis of inflammatory and allergic diseases. Recent evidence suggests that ILC subsets may have important roles both in tumor immunosurveillance and cancer promotion. ILC1s include conventional NK and lin CD127+ cells that characteristically express IFN-g and the transcription factor T-bet. They most likely inhibit malignant transformation and tumor growth. ILC2s abundantly secrete the type 2 cytokines IL-5 and IL-13, and are strongly implicated with a microenvironment promoting tumor growth and blocking antitumor immunity. ILC3s display dependency on RORgt and secrete IL-17 and IL-22. While in the case of inflammation-driven cancers there is evidence for a tumor-promoting role of ILC3, other studies have indicated direct antitumor functions.
1 Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany 2 Department of Medicine 1, FriedrichAlexander University, Erlangen, Germany
*Correspondence: [email protected] (S. Wirtz).
http://dx.doi.org/10.1016/j.it.2016.10.004 © 2016 Elsevier Ltd. All rights reserved.
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Table 1. Markers Commonly Used for Identification of Mouse and Human ILC Subsetsa Subset
Mouse
Human
cNK
Eomes, T-bet, NK1.1, NKp46, CD49b, IFN-g, TNF-/
Eomes, T-bet, NKp46, CD56, KIR, CD94, IFN-g, TNF-/
Helper ILC1
T-bet, NKp46, NK1.1, CD90, CD127, IFN-g, TNF-/
T-bet, NKp46, CD56+/ , NK1.1, CD90, CD117, CD127+/ , CD161, IFN-g, TNF-/
ILC2
Gata3, ROR/, IL-33R, IL-17RB, TSLPR, CD25, CD90, CD117+/ , CD127, SCA-1, ICOS, KLRG1, IL-4+/ , IL-5, IL-9, IL-13
Gata3, IL-33R, IL-17RB, TSLPR, NKp30+/ , CRTH2, CD25, CD117+/ , CD127, ICOS
NCR ILC3
RORgt, AhR, CCR6+/ , CD4+/ , CD25, CD90, CD117, CD127, IL-17, IL-22
RORgt, AhR, CCR6, CD117, CD127, CD161, IL-17+/ , IL-22+/
NCR+ ILC3
RORgt, T-bet, AhR, NKp46, CD90, CD117, CD127, IL-22
RORgt, Ahr, NKp30, NKp44, NKp46, CD117, CD127, CD161, IL-22, TNF-/
ILC1
ILC3
a
+ / indicates variability of expression in different subsets. Abbreviations: AhR, aryl hydrocarbon receptor; CRTH2, T helper 2 chemoattractant receptor; Gata3, GATA-binding protein 3; ICOS, inducible T cell costimulatory; KIR, killer cell immunoglobulin-like receptor; KLRG1, killer cell lectin-like receptor subfamily G member 1; NCR, natural cytotoxicity receptor; ROR/, RAR-related orphan receptor /; RORgt, RAR-related orphan receptor g; SCA-1, stem cell antigen-1; TSLPR, thymic stromal lymphopoietin receptor.
future therapeutic interventions. Indeed, the potent anticancer properties of natural killer (NK) cells, a well-known cytotoxic cell type that is nowadays assigned to the ILC1 family, are well established [6]. By contrast, the roles of the non-cytotoxic so-called helper ILC populations remain poorly understood. In the following we discuss how activation of these different ILC classes within tumors may provide signals modulating the tumor microenvironment and how the individual cytokine responses of the distinct ILC populations may overall impact on tumorigenesis (Figure 1).
Group 1 ILCs All members of the group 1 ILC family constitutively express the transcription factor T-bet, respond to interleukin-12 (IL-12) and release Th1 effector cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-/ [7,8]. Based on their developmental needs and their expression profiles for IL-7, IL-15, T-bet, and eomesodermin (Eomes), group 1 ILCs are currently subdivided into three subsets [2]: NK cells [9], CD127low CD103+ intraepithelial ILC1s [7], and CD127high ILC1s [8,9]. While the pivotal role of NK cells in cancer immunosurveillance and therapy is well established (reviewed in [6]), direct evidence for the involvement of helper ILC1s in tumor immunity is sparse. Nonetheless, a recent study reported cell transformation-induced expansion of an NK1.1+ CD49a+ CD103+ ILC1-like cell population which lysed tumor cells in a granzyme B- and TRAIL-dependent manner in an oncogene-induced cancer model [10]. Because the tumor also induced cytotoxic T cells, further studies will be necessary to dissect the impact of the different immune cell populations on cancer immunosurveillance. It is important to note here that the role of ILCs has generally been evaluated by comparing Rag-deficient mice, which lack adaptive cells but have ILCs, to Il2rg-deficient mice, which lack both populations. The indirect evidence for the antitumor functions of ILC1s includes their responsiveness to IL-12 and their identification as a cellular source for the effector cytokines IFN-g and TNF-/. IL-12, for example, which has emerged as one of the most potent antitumor cytokines [11],
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ILC1
ILC2
ILC3
IL-12
IL-33, IL-25, TSLP
IL-1β, IL-23
helper ILC1
NK IL-12R
NCR
IL-12R
NCR- ILC3 NCR
IL-1R
IL-23R
IL-25R
GATA3 RORα
T-bet IL-7Rα
IFN-γ, TNF-α, perforin, granzymes
IFN-γ, TNF-α
An-tumorigenic: • M1 macrophages, DC ↑ • CTL responses ↑ • Angen presentaon ↑ • Tumor cell proliferaon ↓ • Tumor cell apoptosis ↑ Pro-tumorigenic: • Angiogenesis ↑ (TNF) • EMT, Metastasis ↑ (TNF)
IL-1R
IL-23R CCR6
IL-33R T-bet
NCR+ ILC3
NCR
ICOS RORγt Ahr IL-7Rα
IL-13, IL-5, IL-9, IL-4, Areg
An-tumorigenic: • Eosinophilia ↑ (IL-5) Pro-tumorigenic: • M2 macrophages, MDSC ↑ (IL-13, IL-4) • Type 2 polarizaon of T cells (IL-4) • Treg ↑ (Areg) • Tumor cell proliferaon ↑ (Areg, IL-9) • Tissue remodeling ↑ (IL-13)
RORγt Ahr IL-7Rα
IL-17, IL-22, LT
IL-7Rα
IL-22, LT
An-tumorigenic: • TLS ↑ (LT) • Leukocyte invasion ↑ (NCRs) Pro-tumorigenic: • Tumor proliferaon ↑ (IL-22, IL-17) • Apoptosis ↓ (IL-22, IL-17)
Figure 1. Potential Functions of Innate Lymphoid Cell Subsets in Cancer. Except for NK cells, precise functional data demonstrating a direct involvement of the ILC subsets in cancer is limited. However, the studies available so far indicate that depending on the environmental context all subsets may have pro- and antitumorigenic roles. Helper ILC1s secrete IFN-g and TNF-/ that could orchestrate several antitumorigenic events. However, the role of TNF in particular is possibly ambiguous. ILC2 may promote the establishment of a pro-tumorigenic microenvironment via a variety of secreted factors. They may also have the capacity to support the development of infiltrating eosinophils attacking tumor cells. The release of IL-17 and IL-22 by the NCR and NCR+ (NKp46 in mice; NKp30, NKp44 and NKp46 in humans) ILC3 subsets might promote the growth of tumors. By contrast, via NCR-based interactions with malignant cells or their potential to drive the generation of TLS, they possibly also promote antitumor immunity. Abbreviations: Ahr, aryl hydrocarbon receptor; Areg, amphiregulin; CTL, cytotoxic T cell; DC, dendritic cell; EMT, epithelial–mesenchymal transition; IFN, interferon; ILC, innate lymphoid cell, LT, lymphotoxin; MDSC, myeloid-derived suppressor cell; NCR, natural cytotoxicity receptor; NK, natural killer; TNF, tumor necrosis factor; TLS, tertiary lymphoid structure; Treg, regulatory T cell; TSLP, thymic stromal lymphopoietin.
prevents melanoma growth only in ILC-containing hosts [12,13]. The IL-12-responsive and tumor-repressive cell population in this model was later identified as an ILC3 subset carrying the natural cytotoxicity receptor NKp46 [14]. Although presently used for the treatment of malignancies in clinical settings, IFN-g plays a more ambiguous role in tumor immunity [15]. Thus, IFN-g inhibits tumor cell proliferation [16] and angiogenesis [17], promotes the differentiation of Th1 cells [18,19], and boosts the response of macrophages, NK cells, and cytotoxic T lymphocytes (CTLs) against tumor tissues [20,21]. The upregulation of major histocompatibility complex (MHC) class I molecules on tumor cells by IFNg contributes thereby to the antitumor effects of this pleiotropic cytokine [22]. In addition, IFN-g can promote tumor cell apoptosis [23]. The pivotal role of IFN-g in the elimination of malignant cells was also confirmed in animals deficient for the receptor for this cytokine [24]. However, ILCs also promote inflammation in several mouse models and patient populations [2], and IFN-g released within these inflammatory conditions might even perpetuate tumor growth and/or malignant transformation. Furthermore, IFN-g is known to enhance cellular proliferation and might hamper the CTL- and/or NK cell-mediated lysis of tumor cells [15]. Subsets other than ILC1s might contribute to these adverse effects of IFN-g because, for example, RORgt natural
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cytotoxicity receptor (NCR)+ lymphoid tissue inducer cells (LTi) cells have been reported as an alternative cellular source for IFN-g which induced colitis [25]. TNF-/ is also involved in tumor immunity because it interferes with angiogenesis, cellular growth, and cell migration [26]. On the one hand, TNF-/ stimulates strong antitumor responses owing to the promotion of macrophage and dendritic cell (DC) recruitment and the generation of CTLs [26,27]. Because it selectively destroys tumor vasculature and induces apoptosis and necrosis of cancer cells, TNF-/ may even exert direct antitumor effects [28], and thus may serve as pivotal regulator of immune-mediated cancer cell destruction [29,30]. On the other hand, TNF-/ induces tumor formation, spread, and growth because of its versatile impact on the expression of angiogenic and growth factors, cytokines, adhesion receptors, and proteases [31,32]. Similarly to IFN-g, the proinflammatory functions of TNF-/ may favor tumorigenesis in some settings. Hence, blockade of TNF-/ in colitis-associated tumor and skin cancer models prevented tumor formation [33,34]. In summary, it appears that ILC1s primarily inhibit malignant transformation and tumor growth despite the ambiguous effects of their two signature cytokines, IFN-g and TNF-/, in tumor immunity. However, the protective function of ILC1s might be hampered in cancer patients. For example, patients with acute myeloid leukemia revealed shifts in the composition of ILC subsets and a reduced capacity of ILC1s to secrete IFN-g and TNF-/ compared to controls [35]. Bidirectional plasticity between ILC family members, which affects tumor immunosurveillance, has been also described in the gut in response to various environmental changes [36].
Group 2 ILCs ILC2s share several developmental and transcriptional signatures with Th2 cells, suggesting a role in type 2 immune responses. Upon activation by cytokines such as IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), ILC2 cells produce large amounts of IL-5 and IL-13, and have been demonstrated to be a dominant innate source of these cytokines. By virtue of their strategic location and relative abundance in tissues harboring barrier functions such as gut, lung and skin, they have been shown to vitally contribute to early phases of host protection against helminths and viral infections [37]. In addition, ILC2s are enriched in visceral adipose tissue and fatassociated lymphoid clusters, and contribute prominently to steady-state metabolic homeostasis, for example by sustaining the pools of eosinophils and M2 macrophages [38–40]. Given that type 2-related immune responses and the presence of high levels of type 2 cytokines are strongly associated with a microenvironment promoting tumor growth and blocking antitumor immunity, ILC2 may most likely be considered as a somewhat pro-carcinogenic ILC subtype. Indeed, although virtually no robust mouse functional data about their precise role during carcinogenesis exist, and their accumulation in patients has only been reported in gastric cancer [41], this notion is clearly supported by evidence obtained in studies addressing their secreted downstream effector molecules or factors that trigger ILC2 proliferation and activation. Several studies have implicated IL-33, an important activator of ILC2, in the formation of tumors and metastasis [42–44]. Although several IL-33R+ cell types represent potential targets of the tumor-promoting properties of IL-33, the induction of IL-13 production seems to be particularly important in some settings. IL-13 via activation of signal transducer and activator of transcription (STAT) 6 signaling is an important mediator of macrophage polarization towards an immunosuppressive M2 phenotype. Similarly, IL-13 promotes functions of tumor-associated macrophages (TAM) that display similar characteristics to M2 macrophages and provide an immunosuppressive microenvironment for tumor growth [1]. Furthermore, IL-13 has been shown to be directly involved in the differentiation and activation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immune cells supporting tumor growth by multiple mechanisms including induction of T cell suppression via TGF-b and pro-angiogenesis
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via production of vascular endothelial growth factor (VEGF) [45]. Moreover, IL-13 production by ILC2 drives the development of tissue fibrosis in liver and lung [46,47]. Because such dysregulation of extracellular matrix homeostasis commonly culminates in cancer, the ILC2/IL-13 axis might contribute to the initiation of cancers linked to extensive tissue remodeling. By contrast, ILC2-derived IL-13 may also have direct pro-carcinogenic effects on tumor cells. Notably, IL-13 employs the IL-4 receptor / (IL-4Ra) subunit for signaling, and numerous studies implicate IL-4R activation in tumorigenesis. IL-4Ra seems to be particularly overexpressed by many epithelial cancers, and IL-4R signaling may support the proliferation and migration of epithelial cancer cells. Moreover, IL-4R activation has been shown to support tumor growth in vivo by mediating resistance to apoptosis [48]. Additional evidence for a potential role of ILC2-derived IL-13 in the context of cancer formation comes from in vivo studies of the pathogenesis of biliary atresia that demonstrated ILC2-dependent cholangiocyte hyperplasia [49]. In some settings, however, ILC2s have also been described as a key source of the Th2 master regulator IL-4, which shares with IL-13 the IL-4Ra chain for signaling [50,51]. Thus, IL-4 secretion by intratumoral ILC2 or other mechanisms of ILC2/T cell interactions may promote tumor growth and metastasis. Additional studies will be necessary to investigate these seemingly diverse and complementary functions of IL-4 and IL-13 cytokines during carcinogenesis. While the IL-4/IL-13 release by ILC2s rather supports the idea of a pro-carcinogenic role of these cells, some data indicate that ILC2-derived IL-5 might protect from cancer development. In the lung, tumor cells increased the local production of IL-5 by ILC2 in the B16F10 melanoma model, and this resulted in the recruitment and activation of eosinophils. Likewise, IL-5 signaling deficiency and neutralization of IL-5 led to decreased eosinophilia and increased lung tumor metastasis [52]. Eosinophilia is a common feature of many solid tumors and is associated with a better prognosis [53]. Given the pivotal role of IL-5 production by ILC2 for the homeostasis and migration of eosinophils, and potentially their cytotoxic responses [40], this axis may play an important physiological role in tumor biology, although this hypothesis remains to be formally tested. Among the factors secreted abundantly by ILC2, the cytokine IL-9 recently gained attention in the context of tumor biology. This cytokine has functional importance as a growth factor for ILC2 and is a survival factor for several other cell types [37]. So far, the cancer-related functions of IL-9 have been exclusively related to tumor-infiltrating Th9 cells [54]. However, studies with IL-9 fatereporter mice established that ILC2 are a major source of IL-9 in vivo, suggesting that the ILC2/ IL-9 axis may also account for at least some of the various in vivo functions of this cytokine [55]. In a melanoma model, IL9R deficiency or IL-9 neutralization led to increased tumor growth, while IL-9 treatment reduced metastasis [56,57]. Mechanistically, IL-9 may indirectly support the tumor-specific CTL response by enhancing the cross-presentation capacity of infiltrating DC [58]. Conversely, in some hematopoietic malignancies, high expression of IL-9 is associated with poor prognosis, indicating that its growth factor and anti-apoptotic properties on transformed cells promote tumorigenesis. Indeed, transgenic IL-9 overexpression in mice provoked the generation of lymphomas, and IL-9 production was associated with Hodgkin disease and T cell transformation in humans [59]. Epidermal growth factor receptor (EGFR) antagonists are used for treatment of some types of epithelial-derived metastatic cancer [60]. Interestingly, it was recently demonstrated that ILC2s are a significant source of the EGFR ligand amphiregulin (AREG) during polarized type 2 responses. While ILC2-derived AREG supported the proliferation and repair of the pulmonary epithelium after virus infection-induced injury [61], it has also been reported to endorse the growth of lung tumor cells and to provide resistance to apoptosis [62]. In addition, AREG production by ILC2s might limit antitumor immunity by stimulating regulatory T cells supporting the establishment of an immune-suppressive tumor microenvironment [63].
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Finally, a subset of human ILC2s were shown to express the NCR receptor NKp30 and respond to stimulation with the tumor-associated cognate ligand B7-H6. In vitro studies indicated, furthermore, that B7-H6-expressing tumor cell lines provoked cytokine secretion by ILC2, suggesting that ILC2 may directly interact via NKp30 with malignant or stressed host cells [64]. Collectively, ILC2 are associated with stress responses and tissue remodeling, and overall the evidence suggests that their signature cytokines are implicated in a tumor-promoting microenvironment in several malignancies. Given their relative abundance in barrier tissues, these potential pro-cancerogenic functions may be particularly relevant in the context of tumors of gut, skin, and the lung. Thus, functional studies addressing the precise role of ILC2s within the tumor microenvironment and novel options to modulate their effector functions in a way that can be used to treat cancers are urgently needed.
Group 3 ILCs ILC3s are a heterogeneous lymphocyte population depending on the retinoic acid receptor (RAR)-related orphan receptor (RORgt) and are predominantly locating in mucosal and mucosaassociated lymphoid tissues (MALT). In mice, expression of the chemokine receptor CCR6 and the NCR NKp46 discriminates between two prototypic ILC3 subsets which both produce IL-22. In humans, most NKp46+ ILC3 co-express NKp44, another NCR member, which is not conserved in rodents. The CCR6+ NKp46 ILC3 population consists of LTi cells that regulate the development of lymph nodes, Peyer's patches, and other organized lymphoid structures such as intestinal isolated lymphoid follicles (ILFs) and cryptopatches [65]. Conversely, NKp46+ ILC3 cells express and developmentally require the transcription factor T-bet and share with ILC1 the capacity to produce IFN-g [66]. In the steady-state, these NKp46+T-bet+ ILC3s are predominantly located in intestinal lamina propria and the skin. While ILC3s play pivotal roles during immunity against extracellular bacteria and fungi, as well as in epithelial tissue repair and homeostasis, dysregulated ILC responses have been implicated in chronic inflammation of skin, lung, and gut. These proinflammatory effects of ILC3 were primarily linked to the action of IL-23, a potent inducer of their IL-17 and IL-22 production [67]. In human colorectal cancer, elevated expression of IL-23, IL-23 receptor, and IL-17A has been correlated with progression to fatal metastatic disease [68]. In addition, IL-23 levels are increased in several solid human cancers, suggesting that IL-23 links tumorpromoting chronic inflammation to a microenvironment providing insufficient immunosurveillance. Given the ample evidence of a pro-tumorigenic role for IL-23-related signaling pathways in colorectal cancer, ILC3 may also play a direct role in carcinogenesis. Accordingly, minicircle-based overexpression of IL-23 was sufficient to rapidly induce duodenal adenoma formation in wild-type and Rag1 / mice, whereas Rag1 / mice lacking the common g-chain and thus ILCs proved to be resistant. IL-17 production by ILCs was important in this model because Rag1 / Il17 / double-deficient mice were also resistant [69]. Further studies will need to address whether ILC3 activation by endogenous IL-23 is sufficient to contribute to intestinal tumor formation. In a model of bacterial inflammationinduced colorectal cancer in Rag / mice, ILC3 were essential for the formation of invasive colonic tumors. However, in this case IL-22 activation of epithelial cells via STAT3 signaling, rather than the function of IL-17, was reported to be crucial for the phenotype [70]. This study also demonstrated the presence of IL-22-expressing cells within human colorectal tumors, and these comprise CD3+ T cells and to a lesser degree lineage-negative cells, most likely ILC3s. Thus, while ILC3-derived IL-22 is important for epithelial regeneration and tissue repair after injury, its proliferative and anti-apoptotic capacities may support malignant transformation in chronic inflammatory diseases. However, whether IL-22 release by ILC3s supports colorectal tumor growth in non-lymphopenic mice was not addressed in this study. Interestingly, the pro-carcinogenic role IL-22 was also established in a colitis-associated
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colon cancer model, where deficiency in IL-22 bp, a neutralizing soluble IL-22 receptor released by colonic dendritic cells, was associated with increased tumorigenesis [71]. Although there is a wealth of evidence supporting a tumor-promoting role for ILC3s in the case of cancers related to chronic inflammation, a recent study also indicated direct antitumor functions of human NCR+ ILC3s. In this report, increased frequencies of this ILC3 subset, but not of NCR ILC3, ILC2, or ILC1 cells, were present in lungs of patients with non-small cell lung cancer. These cells were located near intratumoral tertiary lymphoid structures and had the capability to secrete patterns of proinflammatory cytokines and chemotactic factors that suggest an LTi-like involvement in the formation of protective tertiary lymphoid structures. This active role in immunosurveillance may be linked to their potential ability to recognize transformed cells via the NKp44 receptor [72]. Further evidence for an antitumor activity of ILC3 was provided in mice injected with IL-12-overexpressing B16F10 melanoma cells [14]. Here, NKp46+, RORgt fate-mapped ILC3, but not T cells or NK cells, were crucial for IL-12-dependent suppression of tumor growth. Notably, tumor suppression seemed to be independent of the ILC3 effector cytokines IL-17 and IL-22 and, moreover, a non-redundant role of IFN-g production was not observed. Conversely, this study indicated that ILC3 may favor immune cell recruitment by inducing the upregulation of adhesion molecules in the tumor microvasculature. More recently, CD90+ NK1.1 RORgt+ ILCs were reported to be responsible for the effects of a combinational therapy in a melanoma model in lymphopenic Rag1 / mice [73]. In summary, although some data point to an important role of ILC3s during tumor development, their ambiguous functions in humans are incompletely understood. However, therapeutic targeting of ILC3 may be beneficial in the setting of inflammation-driven cancers.
The Plasticity of ILCs May Impact on their Role in Tumor Immunity Diverse signals within tumors (e.g., cytokines, danger signals, metabolic signals) can shape the functional polarization of immune cells. In the context of cancer, this plasticity towards cells with either anti-inflammatory or tumor-promoting abilities might be of particular importance for antitumor immunity. Similarly to some of their T cell counterparts (e.g., Th17 and Treg cells), ILC subsets display the noteworthy capacity to convert into one another within distinct cytokine and environmental milieus and tissue locations. In particular, plasticity has been reported between group 1 and group 3 ILCs upon engagement of distinctive signals in mice and humans. The numbers of CD127low ILC1s, for example, increased in inflamed intestinal tissues of patients with Crohn's disease [36]. These ILC1s originated from ILC3s upon downregulation of RORgt and concomitant upregulation of T-bet expression and production of IFN-g [8,9,25,36]. The extent of RORgt loss was dependent on the organ environment. While only a few cells downregulated the expression of RORgt in the small intestine, the loss of RORgt was more complete in the colon or spleen [25]. IL-2, IL-12, and IL-15 promote this reversible conversion of ILC3s into ILC1s. Conversely, IL-1, IL-23, AhR signaling, and retinoic acid preserve the ILC3 phenotype, or even favor the transformation of IL-7R/+ ILC1s into NKp44+ ILC3s [25,36,74–76]. This enhanced accumulation of ILC3s likely promotes tumorigenesis within different tissue locations [68,69,77]. IL-1b also converts ILC2s into ILC1s as a result of the induction of T-bet- and IL-12Rb2 expression, which can promote the ILC1 phenotype in response to IL-12 [78]. Accordingly, stimulation via the IL-12/IL-12R pathway promoted IFN-g production by blood ILC2s In vitro, and IFN-g+/IL-13+ ILC2s were identified in the intestines of patients with Crohn's disease [79]. In consequence, local IL-12 production by accessory cells might provide antitumor effects by converting immunosuppressive ILC2s into IFN-g producers. ILC1s and NK cells also possess substantial developmental plasticity [80]. There is also evidence that in the lung IL-25-responsive ILC2 populations can differentiate into RORgt+ ILC3-like cells that produce IL-17 [81]. Whether such a functional plasticity occurs in vivo, and in particular in a tumor microenvironment, remains to be investigated.
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Concluding Remarks
Outstanding Questions
Even in our infancy of understanding their development and function, ILCs are revealed to be important mediators in multiple diseases, and the secreted factors they produce remain intriguing targets for therapy. Given the emerging role of ILCs in both tumor immunosurveillance and promotion, and the first reports about alterations in ILC numbers/functions in defined patient populations, there exits an urgent need to investigate therapeutic strategies to modulate ILC responses and provide clinical benefit. Although there is now substantial evidence for their potential involvement in the promotion, maintenance, or elimination of tumors at various anatomic sites, more specific research efforts as well as improved experimental and diagnostic tools will be necessary to specifically address their value as future therapeutic targets (see Outstanding Questions). High similarities between ILCs and their corresponding T helper cell populations in their transcriptional profiles and developmental programs have so far been a challenge for functional in vivo studies in mice. As a consequence, many of the studies described in this review were conducted in the absence of adaptive immune cells, and the role of ILCs in a normal tumor microenvironment remains somewhat enigmatic. The development of specific genetic tools selectively targeting distinct ILC populations without affecting adaptive immunity will certainly support a clearer description of their roles in tumor biology. In addition, currently no markers exist that exclusively define individual ILC subsets, and this prevents routine diagnostic detection of ILCs in patient material. Hence, standardized reagents and protocols for monitoring the presence and function of ILCs in human blood will be needed.
Do ILCs actively migrate to human tumors or are they tissue-resident, as indicated by studies in parabiosis models? If they do not migrate, do they play cancer-related roles only in tissues harboring substantial steady-state ILC numbers?
Acknowledgments This work has received funding from Deutsche Forschungsgemeinschaft (DFG) projects within CRC1181 (A08, C04) and the clinical research unit KFO257 (TP1). Further support was provided by the Interdisciplinary Center for Clinical Research (IZKF) at the University Erlangen-Nuremberg (to S.W.) and by the Staedtler Stiftung.
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What is the precise role of natural cytotoxicity receptors on helper ILC1 and ILC3? What are their ligands and do they allow ILCs to directly sense tumor cells/antigens? Do ILC2s shift immune responses within tumors to establish a tumor-promoting microenvironment, for example by the production of IL-4 and IL-13? Is it possible to develop specific genetic tools to permit selective targeting of distinct ILC populations without affecting adaptive immunity? Can these results from murine models be extrapolated to humans? Does the tumor microenvironment actively regulate the plasticity of ILCs? Can this be employed in future therapeutic strategies?
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Review
Friend or Foe? The Ambiguous Role of Innate Lymphoid Cells in Cancer Development Jochen Mattner1 and Stefan Wirtz2,* The development of immunotherapies represents a major advance towards the effective eradication of malignant tumors. So far, therapeutic approaches have largely focused on T lymphocytes, but the innate arm of the immune system might be similarly important. Innate lymphoid cells (ILCs) are rapidly-responding cells that are functionally analogous to diverse T cell subsets. In recent years these cells have attracted enormous attention owing to their pleiotropic effects in early host defense to infection and organ pathologies. ILCs might also represent promising targets in the context of cancer therapy because they are an innate immune cell population endowed with potent immunomodulatory properties. In this review we discuss the impact of the three ILC subsets and the signature cytokines they release on cancer development and tumor growth. ILCs in the Tumor Microenvironment Most types of malignancies are characterized by substantial immune cell infiltrations, and complex mutual interactions between innate/adaptive immune cells and tumor cells are believed to profoundly affect tumor development and the success of therapeutic strategies. While the presence of some immune cells such as cytotoxic T cells is important for limiting tumor growth, the occurrence of others, including tumor-associated macrophages, correlates with invasiveness, metastasis, and poor prognosis [1]. Although many studies have focused on the interaction between immune cells and the tumor microenvironment, the role of ILCs remains largely unexplored. ILCs represent a heterogeneous group of developmentally related cells that in adults derive from shared lymphoid precursor populations including the common lymphoid progenitor (CLP) in the bone marrow, but typically lack recombination-activating gene (Rag)dependent rearranged antigen receptors [2]. Based on functional criteria, cytokine production characteristics, and transcription factor expression profiles, they were recently categorized into three major subgroups (ILC1, ILC2, and ILC3) that strikingly resemble the corresponding T helper cell subsets (Th1, Th2, and Th17) [3] (Table 1). The main biological function of ILCs is likely to center on their capacity to rapidly respond to environmental and inflammatory signals by the secretion of cytokines implicated in tissue repair and immune defense mechanisms. In addition, ILCs may substantially contribute to T cell polarization and effector functions by secretion of regulatory cytokines, antigen presentation, or direct cellular interactions [4]. Hypoxia and the acid milieu in the tumor microenvironment favor necrotic cell death and an abundance of immunostimulatory danger signals [5]. Given their activation characteristics and their importance in the context of tissue damage and repair, ILCs may be important players in tumor-associated host responses, and could represent interesting target cell populations for
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Trends ILCs are categorized into three major subpopulations (ILC1s, ILC2s, ILC3s) that have key and non-redundant functions during infections as well as in the pathogenesis of inflammatory and allergic diseases. Recent evidence suggests that ILC subsets may have important roles both in tumor immunosurveillance and cancer promotion. ILC1s include conventional NK and lin CD127+ cells that characteristically express IFN-g and the transcription factor T-bet. They most likely inhibit malignant transformation and tumor growth. ILC2s abundantly secrete the type 2 cytokines IL-5 and IL-13, and are strongly implicated with a microenvironment promoting tumor growth and blocking antitumor immunity. ILC3s display dependency on RORgt and secrete IL-17 and IL-22. While in the case of inflammation-driven cancers there is evidence for a tumor-promoting role of ILC3, other studies have indicated direct antitumor functions.
1 Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany 2 Department of Medicine 1, FriedrichAlexander University, Erlangen, Germany
*Correspondence: [email protected] (S. Wirtz).
http://dx.doi.org/10.1016/j.it.2016.10.004 © 2016 Elsevier Ltd. All rights reserved.
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Table 1. Markers Commonly Used for Identification of Mouse and Human ILC Subsetsa Subset
Mouse
Human
cNK
Eomes, T-bet, NK1.1, NKp46, CD49b, IFN-g, TNF-/
Eomes, T-bet, NKp46, CD56, KIR, CD94, IFN-g, TNF-/
Helper ILC1
T-bet, NKp46, NK1.1, CD90, CD127, IFN-g, TNF-/
T-bet, NKp46, CD56+/ , NK1.1, CD90, CD117, CD127+/ , CD161, IFN-g, TNF-/
ILC2
Gata3, ROR/, IL-33R, IL-17RB, TSLPR, CD25, CD90, CD117+/ , CD127, SCA-1, ICOS, KLRG1, IL-4+/ , IL-5, IL-9, IL-13
Gata3, IL-33R, IL-17RB, TSLPR, NKp30+/ , CRTH2, CD25, CD117+/ , CD127, ICOS
NCR ILC3
RORgt, AhR, CCR6+/ , CD4+/ , CD25, CD90, CD117, CD127, IL-17, IL-22
RORgt, AhR, CCR6, CD117, CD127, CD161, IL-17+/ , IL-22+/
NCR+ ILC3
RORgt, T-bet, AhR, NKp46, CD90, CD117, CD127, IL-22
RORgt, Ahr, NKp30, NKp44, NKp46, CD117, CD127, CD161, IL-22, TNF-/
ILC1
ILC3
a
+ / indicates variability of expression in different subsets. Abbreviations: AhR, aryl hydrocarbon receptor; CRTH2, T helper 2 chemoattractant receptor; Gata3, GATA-binding protein 3; ICOS, inducible T cell costimulatory; KIR, killer cell immunoglobulin-like receptor; KLRG1, killer cell lectin-like receptor subfamily G member 1; NCR, natural cytotoxicity receptor; ROR/, RAR-related orphan receptor /; RORgt, RAR-related orphan receptor g; SCA-1, stem cell antigen-1; TSLPR, thymic stromal lymphopoietin receptor.
future therapeutic interventions. Indeed, the potent anticancer properties of natural killer (NK) cells, a well-known cytotoxic cell type that is nowadays assigned to the ILC1 family, are well established [6]. By contrast, the roles of the non-cytotoxic so-called helper ILC populations remain poorly understood. In the following we discuss how activation of these different ILC classes within tumors may provide signals modulating the tumor microenvironment and how the individual cytokine responses of the distinct ILC populations may overall impact on tumorigenesis (Figure 1).
Group 1 ILCs All members of the group 1 ILC family constitutively express the transcription factor T-bet, respond to interleukin-12 (IL-12) and release Th1 effector cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-/ [7,8]. Based on their developmental needs and their expression profiles for IL-7, IL-15, T-bet, and eomesodermin (Eomes), group 1 ILCs are currently subdivided into three subsets [2]: NK cells [9], CD127low CD103+ intraepithelial ILC1s [7], and CD127high ILC1s [8,9]. While the pivotal role of NK cells in cancer immunosurveillance and therapy is well established (reviewed in [6]), direct evidence for the involvement of helper ILC1s in tumor immunity is sparse. Nonetheless, a recent study reported cell transformation-induced expansion of an NK1.1+ CD49a+ CD103+ ILC1-like cell population which lysed tumor cells in a granzyme B- and TRAIL-dependent manner in an oncogene-induced cancer model [10]. Because the tumor also induced cytotoxic T cells, further studies will be necessary to dissect the impact of the different immune cell populations on cancer immunosurveillance. It is important to note here that the role of ILCs has generally been evaluated by comparing Rag-deficient mice, which lack adaptive cells but have ILCs, to Il2rg-deficient mice, which lack both populations. The indirect evidence for the antitumor functions of ILC1s includes their responsiveness to IL-12 and their identification as a cellular source for the effector cytokines IFN-g and TNF-/. IL-12, for example, which has emerged as one of the most potent antitumor cytokines [11],
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ILC1
ILC2
ILC3
IL-12
IL-33, IL-25, TSLP
IL-1β, IL-23
helper ILC1
NK IL-12R
NCR
IL-12R
NCR- ILC3 NCR
IL-1R
IL-23R
IL-25R
GATA3 RORα
T-bet IL-7Rα
IFN-γ, TNF-α, perforin, granzymes
IFN-γ, TNF-α
An-tumorigenic: • M1 macrophages, DC ↑ • CTL responses ↑ • Angen presentaon ↑ • Tumor cell proliferaon ↓ • Tumor cell apoptosis ↑ Pro-tumorigenic: • Angiogenesis ↑ (TNF) • EMT, Metastasis ↑ (TNF)
IL-1R
IL-23R CCR6
IL-33R T-bet
NCR+ ILC3
NCR
ICOS RORγt Ahr IL-7Rα
IL-13, IL-5, IL-9, IL-4, Areg
An-tumorigenic: • Eosinophilia ↑ (IL-5) Pro-tumorigenic: • M2 macrophages, MDSC ↑ (IL-13, IL-4) • Type 2 polarizaon of T cells (IL-4) • Treg ↑ (Areg) • Tumor cell proliferaon ↑ (Areg, IL-9) • Tissue remodeling ↑ (IL-13)
RORγt Ahr IL-7Rα
IL-17, IL-22, LT
IL-7Rα
IL-22, LT
An-tumorigenic: • TLS ↑ (LT) • Leukocyte invasion ↑ (NCRs) Pro-tumorigenic: • Tumor proliferaon ↑ (IL-22, IL-17) • Apoptosis ↓ (IL-22, IL-17)
Figure 1. Potential Functions of Innate Lymphoid Cell Subsets in Cancer. Except for NK cells, precise functional data demonstrating a direct involvement of the ILC subsets in cancer is limited. However, the studies available so far indicate that depending on the environmental context all subsets may have pro- and antitumorigenic roles. Helper ILC1s secrete IFN-g and TNF-/ that could orchestrate several antitumorigenic events. However, the role of TNF in particular is possibly ambiguous. ILC2 may promote the establishment of a pro-tumorigenic microenvironment via a variety of secreted factors. They may also have the capacity to support the development of infiltrating eosinophils attacking tumor cells. The release of IL-17 and IL-22 by the NCR and NCR+ (NKp46 in mice; NKp30, NKp44 and NKp46 in humans) ILC3 subsets might promote the growth of tumors. By contrast, via NCR-based interactions with malignant cells or their potential to drive the generation of TLS, they possibly also promote antitumor immunity. Abbreviations: Ahr, aryl hydrocarbon receptor; Areg, amphiregulin; CTL, cytotoxic T cell; DC, dendritic cell; EMT, epithelial–mesenchymal transition; IFN, interferon; ILC, innate lymphoid cell, LT, lymphotoxin; MDSC, myeloid-derived suppressor cell; NCR, natural cytotoxicity receptor; NK, natural killer; TNF, tumor necrosis factor; TLS, tertiary lymphoid structure; Treg, regulatory T cell; TSLP, thymic stromal lymphopoietin.
prevents melanoma growth only in ILC-containing hosts [12,13]. The IL-12-responsive and tumor-repressive cell population in this model was later identified as an ILC3 subset carrying the natural cytotoxicity receptor NKp46 [14]. Although presently used for the treatment of malignancies in clinical settings, IFN-g plays a more ambiguous role in tumor immunity [15]. Thus, IFN-g inhibits tumor cell proliferation [16] and angiogenesis [17], promotes the differentiation of Th1 cells [18,19], and boosts the response of macrophages, NK cells, and cytotoxic T lymphocytes (CTLs) against tumor tissues [20,21]. The upregulation of major histocompatibility complex (MHC) class I molecules on tumor cells by IFNg contributes thereby to the antitumor effects of this pleiotropic cytokine [22]. In addition, IFN-g can promote tumor cell apoptosis [23]. The pivotal role of IFN-g in the elimination of malignant cells was also confirmed in animals deficient for the receptor for this cytokine [24]. However, ILCs also promote inflammation in several mouse models and patient populations [2], and IFN-g released within these inflammatory conditions might even perpetuate tumor growth and/or malignant transformation. Furthermore, IFN-g is known to enhance cellular proliferation and might hamper the CTL- and/or NK cell-mediated lysis of tumor cells [15]. Subsets other than ILC1s might contribute to these adverse effects of IFN-g because, for example, RORgt natural
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cytotoxicity receptor (NCR)+ lymphoid tissue inducer cells (LTi) cells have been reported as an alternative cellular source for IFN-g which induced colitis [25]. TNF-/ is also involved in tumor immunity because it interferes with angiogenesis, cellular growth, and cell migration [26]. On the one hand, TNF-/ stimulates strong antitumor responses owing to the promotion of macrophage and dendritic cell (DC) recruitment and the generation of CTLs [26,27]. Because it selectively destroys tumor vasculature and induces apoptosis and necrosis of cancer cells, TNF-/ may even exert direct antitumor effects [28], and thus may serve as pivotal regulator of immune-mediated cancer cell destruction [29,30]. On the other hand, TNF-/ induces tumor formation, spread, and growth because of its versatile impact on the expression of angiogenic and growth factors, cytokines, adhesion receptors, and proteases [31,32]. Similarly to IFN-g, the proinflammatory functions of TNF-/ may favor tumorigenesis in some settings. Hence, blockade of TNF-/ in colitis-associated tumor and skin cancer models prevented tumor formation [33,34]. In summary, it appears that ILC1s primarily inhibit malignant transformation and tumor growth despite the ambiguous effects of their two signature cytokines, IFN-g and TNF-/, in tumor immunity. However, the protective function of ILC1s might be hampered in cancer patients. For example, patients with acute myeloid leukemia revealed shifts in the composition of ILC subsets and a reduced capacity of ILC1s to secrete IFN-g and TNF-/ compared to controls [35]. Bidirectional plasticity between ILC family members, which affects tumor immunosurveillance, has been also described in the gut in response to various environmental changes [36].
Group 2 ILCs ILC2s share several developmental and transcriptional signatures with Th2 cells, suggesting a role in type 2 immune responses. Upon activation by cytokines such as IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), ILC2 cells produce large amounts of IL-5 and IL-13, and have been demonstrated to be a dominant innate source of these cytokines. By virtue of their strategic location and relative abundance in tissues harboring barrier functions such as gut, lung and skin, they have been shown to vitally contribute to early phases of host protection against helminths and viral infections [37]. In addition, ILC2s are enriched in visceral adipose tissue and fatassociated lymphoid clusters, and contribute prominently to steady-state metabolic homeostasis, for example by sustaining the pools of eosinophils and M2 macrophages [38–40]. Given that type 2-related immune responses and the presence of high levels of type 2 cytokines are strongly associated with a microenvironment promoting tumor growth and blocking antitumor immunity, ILC2 may most likely be considered as a somewhat pro-carcinogenic ILC subtype. Indeed, although virtually no robust mouse functional data about their precise role during carcinogenesis exist, and their accumulation in patients has only been reported in gastric cancer [41], this notion is clearly supported by evidence obtained in studies addressing their secreted downstream effector molecules or factors that trigger ILC2 proliferation and activation. Several studies have implicated IL-33, an important activator of ILC2, in the formation of tumors and metastasis [42–44]. Although several IL-33R+ cell types represent potential targets of the tumor-promoting properties of IL-33, the induction of IL-13 production seems to be particularly important in some settings. IL-13 via activation of signal transducer and activator of transcription (STAT) 6 signaling is an important mediator of macrophage polarization towards an immunosuppressive M2 phenotype. Similarly, IL-13 promotes functions of tumor-associated macrophages (TAM) that display similar characteristics to M2 macrophages and provide an immunosuppressive microenvironment for tumor growth [1]. Furthermore, IL-13 has been shown to be directly involved in the differentiation and activation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immune cells supporting tumor growth by multiple mechanisms including induction of T cell suppression via TGF-b and pro-angiogenesis
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via production of vascular endothelial growth factor (VEGF) [45]. Moreover, IL-13 production by ILC2 drives the development of tissue fibrosis in liver and lung [46,47]. Because such dysregulation of extracellular matrix homeostasis commonly culminates in cancer, the ILC2/IL-13 axis might contribute to the initiation of cancers linked to extensive tissue remodeling. By contrast, ILC2-derived IL-13 may also have direct pro-carcinogenic effects on tumor cells. Notably, IL-13 employs the IL-4 receptor / (IL-4Ra) subunit for signaling, and numerous studies implicate IL-4R activation in tumorigenesis. IL-4Ra seems to be particularly overexpressed by many epithelial cancers, and IL-4R signaling may support the proliferation and migration of epithelial cancer cells. Moreover, IL-4R activation has been shown to support tumor growth in vivo by mediating resistance to apoptosis [48]. Additional evidence for a potential role of ILC2-derived IL-13 in the context of cancer formation comes from in vivo studies of the pathogenesis of biliary atresia that demonstrated ILC2-dependent cholangiocyte hyperplasia [49]. In some settings, however, ILC2s have also been described as a key source of the Th2 master regulator IL-4, which shares with IL-13 the IL-4Ra chain for signaling [50,51]. Thus, IL-4 secretion by intratumoral ILC2 or other mechanisms of ILC2/T cell interactions may promote tumor growth and metastasis. Additional studies will be necessary to investigate these seemingly diverse and complementary functions of IL-4 and IL-13 cytokines during carcinogenesis. While the IL-4/IL-13 release by ILC2s rather supports the idea of a pro-carcinogenic role of these cells, some data indicate that ILC2-derived IL-5 might protect from cancer development. In the lung, tumor cells increased the local production of IL-5 by ILC2 in the B16F10 melanoma model, and this resulted in the recruitment and activation of eosinophils. Likewise, IL-5 signaling deficiency and neutralization of IL-5 led to decreased eosinophilia and increased lung tumor metastasis [52]. Eosinophilia is a common feature of many solid tumors and is associated with a better prognosis [53]. Given the pivotal role of IL-5 production by ILC2 for the homeostasis and migration of eosinophils, and potentially their cytotoxic responses [40], this axis may play an important physiological role in tumor biology, although this hypothesis remains to be formally tested. Among the factors secreted abundantly by ILC2, the cytokine IL-9 recently gained attention in the context of tumor biology. This cytokine has functional importance as a growth factor for ILC2 and is a survival factor for several other cell types [37]. So far, the cancer-related functions of IL-9 have been exclusively related to tumor-infiltrating Th9 cells [54]. However, studies with IL-9 fatereporter mice established that ILC2 are a major source of IL-9 in vivo, suggesting that the ILC2/ IL-9 axis may also account for at least some of the various in vivo functions of this cytokine [55]. In a melanoma model, IL9R deficiency or IL-9 neutralization led to increased tumor growth, while IL-9 treatment reduced metastasis [56,57]. Mechanistically, IL-9 may indirectly support the tumor-specific CTL response by enhancing the cross-presentation capacity of infiltrating DC [58]. Conversely, in some hematopoietic malignancies, high expression of IL-9 is associated with poor prognosis, indicating that its growth factor and anti-apoptotic properties on transformed cells promote tumorigenesis. Indeed, transgenic IL-9 overexpression in mice provoked the generation of lymphomas, and IL-9 production was associated with Hodgkin disease and T cell transformation in humans [59]. Epidermal growth factor receptor (EGFR) antagonists are used for treatment of some types of epithelial-derived metastatic cancer [60]. Interestingly, it was recently demonstrated that ILC2s are a significant source of the EGFR ligand amphiregulin (AREG) during polarized type 2 responses. While ILC2-derived AREG supported the proliferation and repair of the pulmonary epithelium after virus infection-induced injury [61], it has also been reported to endorse the growth of lung tumor cells and to provide resistance to apoptosis [62]. In addition, AREG production by ILC2s might limit antitumor immunity by stimulating regulatory T cells supporting the establishment of an immune-suppressive tumor microenvironment [63].
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Finally, a subset of human ILC2s were shown to express the NCR receptor NKp30 and respond to stimulation with the tumor-associated cognate ligand B7-H6. In vitro studies indicated, furthermore, that B7-H6-expressing tumor cell lines provoked cytokine secretion by ILC2, suggesting that ILC2 may directly interact via NKp30 with malignant or stressed host cells [64]. Collectively, ILC2 are associated with stress responses and tissue remodeling, and overall the evidence suggests that their signature cytokines are implicated in a tumor-promoting microenvironment in several malignancies. Given their relative abundance in barrier tissues, these potential pro-cancerogenic functions may be particularly relevant in the context of tumors of gut, skin, and the lung. Thus, functional studies addressing the precise role of ILC2s within the tumor microenvironment and novel options to modulate their effector functions in a way that can be used to treat cancers are urgently needed.
Group 3 ILCs ILC3s are a heterogeneous lymphocyte population depending on the retinoic acid receptor (RAR)-related orphan receptor (RORgt) and are predominantly locating in mucosal and mucosaassociated lymphoid tissues (MALT). In mice, expression of the chemokine receptor CCR6 and the NCR NKp46 discriminates between two prototypic ILC3 subsets which both produce IL-22. In humans, most NKp46+ ILC3 co-express NKp44, another NCR member, which is not conserved in rodents. The CCR6+ NKp46 ILC3 population consists of LTi cells that regulate the development of lymph nodes, Peyer's patches, and other organized lymphoid structures such as intestinal isolated lymphoid follicles (ILFs) and cryptopatches [65]. Conversely, NKp46+ ILC3 cells express and developmentally require the transcription factor T-bet and share with ILC1 the capacity to produce IFN-g [66]. In the steady-state, these NKp46+T-bet+ ILC3s are predominantly located in intestinal lamina propria and the skin. While ILC3s play pivotal roles during immunity against extracellular bacteria and fungi, as well as in epithelial tissue repair and homeostasis, dysregulated ILC responses have been implicated in chronic inflammation of skin, lung, and gut. These proinflammatory effects of ILC3 were primarily linked to the action of IL-23, a potent inducer of their IL-17 and IL-22 production [67]. In human colorectal cancer, elevated expression of IL-23, IL-23 receptor, and IL-17A has been correlated with progression to fatal metastatic disease [68]. In addition, IL-23 levels are increased in several solid human cancers, suggesting that IL-23 links tumorpromoting chronic inflammation to a microenvironment providing insufficient immunosurveillance. Given the ample evidence of a pro-tumorigenic role for IL-23-related signaling pathways in colorectal cancer, ILC3 may also play a direct role in carcinogenesis. Accordingly, minicircle-based overexpression of IL-23 was sufficient to rapidly induce duodenal adenoma formation in wild-type and Rag1 / mice, whereas Rag1 / mice lacking the common g-chain and thus ILCs proved to be resistant. IL-17 production by ILCs was important in this model because Rag1 / Il17 / double-deficient mice were also resistant [69]. Further studies will need to address whether ILC3 activation by endogenous IL-23 is sufficient to contribute to intestinal tumor formation. In a model of bacterial inflammationinduced colorectal cancer in Rag / mice, ILC3 were essential for the formation of invasive colonic tumors. However, in this case IL-22 activation of epithelial cells via STAT3 signaling, rather than the function of IL-17, was reported to be crucial for the phenotype [70]. This study also demonstrated the presence of IL-22-expressing cells within human colorectal tumors, and these comprise CD3+ T cells and to a lesser degree lineage-negative cells, most likely ILC3s. Thus, while ILC3-derived IL-22 is important for epithelial regeneration and tissue repair after injury, its proliferative and anti-apoptotic capacities may support malignant transformation in chronic inflammatory diseases. However, whether IL-22 release by ILC3s supports colorectal tumor growth in non-lymphopenic mice was not addressed in this study. Interestingly, the pro-carcinogenic role IL-22 was also established in a colitis-associated
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colon cancer model, where deficiency in IL-22 bp, a neutralizing soluble IL-22 receptor released by colonic dendritic cells, was associated with increased tumorigenesis [71]. Although there is a wealth of evidence supporting a tumor-promoting role for ILC3s in the case of cancers related to chronic inflammation, a recent study also indicated direct antitumor functions of human NCR+ ILC3s. In this report, increased frequencies of this ILC3 subset, but not of NCR ILC3, ILC2, or ILC1 cells, were present in lungs of patients with non-small cell lung cancer. These cells were located near intratumoral tertiary lymphoid structures and had the capability to secrete patterns of proinflammatory cytokines and chemotactic factors that suggest an LTi-like involvement in the formation of protective tertiary lymphoid structures. This active role in immunosurveillance may be linked to their potential ability to recognize transformed cells via the NKp44 receptor [72]. Further evidence for an antitumor activity of ILC3 was provided in mice injected with IL-12-overexpressing B16F10 melanoma cells [14]. Here, NKp46+, RORgt fate-mapped ILC3, but not T cells or NK cells, were crucial for IL-12-dependent suppression of tumor growth. Notably, tumor suppression seemed to be independent of the ILC3 effector cytokines IL-17 and IL-22 and, moreover, a non-redundant role of IFN-g production was not observed. Conversely, this study indicated that ILC3 may favor immune cell recruitment by inducing the upregulation of adhesion molecules in the tumor microvasculature. More recently, CD90+ NK1.1 RORgt+ ILCs were reported to be responsible for the effects of a combinational therapy in a melanoma model in lymphopenic Rag1 / mice [73]. In summary, although some data point to an important role of ILC3s during tumor development, their ambiguous functions in humans are incompletely understood. However, therapeutic targeting of ILC3 may be beneficial in the setting of inflammation-driven cancers.
The Plasticity of ILCs May Impact on their Role in Tumor Immunity Diverse signals within tumors (e.g., cytokines, danger signals, metabolic signals) can shape the functional polarization of immune cells. In the context of cancer, this plasticity towards cells with either anti-inflammatory or tumor-promoting abilities might be of particular importance for antitumor immunity. Similarly to some of their T cell counterparts (e.g., Th17 and Treg cells), ILC subsets display the noteworthy capacity to convert into one another within distinct cytokine and environmental milieus and tissue locations. In particular, plasticity has been reported between group 1 and group 3 ILCs upon engagement of distinctive signals in mice and humans. The numbers of CD127low ILC1s, for example, increased in inflamed intestinal tissues of patients with Crohn's disease [36]. These ILC1s originated from ILC3s upon downregulation of RORgt and concomitant upregulation of T-bet expression and production of IFN-g [8,9,25,36]. The extent of RORgt loss was dependent on the organ environment. While only a few cells downregulated the expression of RORgt in the small intestine, the loss of RORgt was more complete in the colon or spleen [25]. IL-2, IL-12, and IL-15 promote this reversible conversion of ILC3s into ILC1s. Conversely, IL-1, IL-23, AhR signaling, and retinoic acid preserve the ILC3 phenotype, or even favor the transformation of IL-7R/+ ILC1s into NKp44+ ILC3s [25,36,74–76]. This enhanced accumulation of ILC3s likely promotes tumorigenesis within different tissue locations [68,69,77]. IL-1b also converts ILC2s into ILC1s as a result of the induction of T-bet- and IL-12Rb2 expression, which can promote the ILC1 phenotype in response to IL-12 [78]. Accordingly, stimulation via the IL-12/IL-12R pathway promoted IFN-g production by blood ILC2s In vitro, and IFN-g+/IL-13+ ILC2s were identified in the intestines of patients with Crohn's disease [79]. In consequence, local IL-12 production by accessory cells might provide antitumor effects by converting immunosuppressive ILC2s into IFN-g producers. ILC1s and NK cells also possess substantial developmental plasticity [80]. There is also evidence that in the lung IL-25-responsive ILC2 populations can differentiate into RORgt+ ILC3-like cells that produce IL-17 [81]. Whether such a functional plasticity occurs in vivo, and in particular in a tumor microenvironment, remains to be investigated.
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Concluding Remarks
Outstanding Questions
Even in our infancy of understanding their development and function, ILCs are revealed to be important mediators in multiple diseases, and the secreted factors they produce remain intriguing targets for therapy. Given the emerging role of ILCs in both tumor immunosurveillance and promotion, and the first reports about alterations in ILC numbers/functions in defined patient populations, there exits an urgent need to investigate therapeutic strategies to modulate ILC responses and provide clinical benefit. Although there is now substantial evidence for their potential involvement in the promotion, maintenance, or elimination of tumors at various anatomic sites, more specific research efforts as well as improved experimental and diagnostic tools will be necessary to specifically address their value as future therapeutic targets (see Outstanding Questions). High similarities between ILCs and their corresponding T helper cell populations in their transcriptional profiles and developmental programs have so far been a challenge for functional in vivo studies in mice. As a consequence, many of the studies described in this review were conducted in the absence of adaptive immune cells, and the role of ILCs in a normal tumor microenvironment remains somewhat enigmatic. The development of specific genetic tools selectively targeting distinct ILC populations without affecting adaptive immunity will certainly support a clearer description of their roles in tumor biology. In addition, currently no markers exist that exclusively define individual ILC subsets, and this prevents routine diagnostic detection of ILCs in patient material. Hence, standardized reagents and protocols for monitoring the presence and function of ILCs in human blood will be needed.
Do ILCs actively migrate to human tumors or are they tissue-resident, as indicated by studies in parabiosis models? If they do not migrate, do they play cancer-related roles only in tissues harboring substantial steady-state ILC numbers?
Acknowledgments This work has received funding from Deutsche Forschungsgemeinschaft (DFG) projects within CRC1181 (A08, C04) and the clinical research unit KFO257 (TP1). Further support was provided by the Interdisciplinary Center for Clinical Research (IZKF) at the University Erlangen-Nuremberg (to S.W.) and by the Staedtler Stiftung.
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