ILC2-driven innate immune checkpoint mechanism antagonizes NK cell antimetastatic function in the lung.

Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden. ILC2-driven innate type 2 inflammation is accompanied by profound local suppression of interferon-γ production and cytotoxic function of lung NK cells. ILC2-dependent suppression of NK cells is elaborated via an innate regulatory mechanism, which is reliant on IL-5-induced lung eosinophilia, ultimately limiting the metabolic fitness of NK cells. Therapeutic targeting of IL-33 or IL-5 reversed NK cell suppression and alleviated cancer burden. Thus, we reveal an important function of IL-33 and ILC2s in promoting tumor metastasis via their capacity to suppress innate type 1 immunity.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Group 2 innate lymphoid cells license dendritic cells to potentiate memory TH2 cell responses.

Rapid activation of memory CD4(+) T helper 2 (TH2) cells during allergic inflammation requires their recruitment into the affected tissue. Here we demonstrate that group 2 innate lymphoid (ILC2) cells have a crucial role in memory TH2 cell responses, with targeted depletion of ILC2 cells profoundly impairing TH2 cell localization to the lungs and skin of sensitized mice after allergen re-challenge. ILC2-derived interleukin 13 (IL-13) is critical for eliciting production of the TH2 cell-attracting chemokine CCL17 by IRF4(+)CD11b(+)CD103(-) dendritic cells (DCs). Consequently, the sentinel function of DCs is contingent on ILC2 cells for the generation of an efficient memory TH2 cell response. These results elucidate a key innate mechanism in the regulation of the immune memory response to allergens.

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells.

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7raCre/+Tnfsf4fl/fl mice). Moreover, Il7raCre/+Tnfsf4fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish.

Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation.

Naive CD4(+) T cell differentiation into distinct subsets of T helper (Th) cells is a pivotal process in the initiation of the adaptive immune response. Allergens predominantly stimulate Th2 cells, causing allergic inflammation. However, why allergens induce Th2 cell differentiation is not well understood. Here we show that group 2 innate lymphoid cells (ILC2s) are required to mount a robust Th2 cell response to the protease-allergen papain. Intranasal administration of papain stimulated ILC2s and Th2 cells, causing allergic lung inflammation and elevated immunoglobulin E titers. This process was severely impaired in ILC2-deficient mice. Whereas interleukin-4 (IL-4) was dispensable for papain-induced Th2 cell differentiation, ILC2-derived IL-13 was critical as it promoted migration of activated lung dendritic cells into the draining lymph node where they primed naive T cells to differentiate into Th2 cells. Papain-induced ILC2 activation and Th2 cell differentiation was IL-33-dependent, suggesting a common pathway in the initiation of Th2 cell responses to allergen.

Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation.

Overproduction of cytokines by T helper 2 (Th2) cells in the lung is thought to be a cause of asthma. Here we report that innate lymphocytes termed lung natural helper (LNH) cells are a T cell-independent source of Th2 cell-type cytokines in protease allergen-treated lungs. LNH (Lin(-)Sca-1(+)c-kit(+/lo)CD25(+)CD127(+)) cells, when stimulated by IL-33 plus IL-2, IL-7, or thymic stroma lymphopoietin (TSLP), produced large amounts of IL-5 and IL-13. Intranasal administration of protease allergen papain induced eosinophil infiltration and mucus hyperproduction in the lung of wild-type and Rag1(-/-) mice, but not in Rag2(-/-)Il2rg(-/-) mice that lack LNH cells. LNH cell depletion inhibited papain-induced airway inflammation in Rag1(-/-) mice whereas adoptive transfer of LNH cells enabled Rag2(-/-)Il2rg(-/-) mice to respond to papain. Treatment of lung explants with papain induced IL-33 and TSLP production by stroma cells and IL-5 and IL-13 production by LNH cells. Thus, LNH cells are critical for protease allergen-induced airway inflammation.

Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation.

Natural helper (NH) cells are innate lymphoid cells (ILCs) that produce T helper-2 (Th2)-cell-type cytokines in the lung- and gut-associated lymphoid tissues. Currently, the lineage relationship between NH cells in different tissues and between NH cells and interleukin-22 (IL-22)-producing retinoic-acid-receptor-related orphan receptor (ROR)γt-positive ILCs is unclear. Here, we report that NH cells express RORα, but not RORγt. RORα-deficient, but not RORγt-deficient, mice lacked NH cells in all tissues, whereas all other lymphocytes, including RORγt(+) ILCs, were unaffected. NH-cell-deficient mice generated by RORα-deficient bone-marrow transplantation had normal Th2 cell responses but failed to develop acute lung inflammation in response to protease allergen, thus confirming the essential role of NH cells in allergic lung inflammation. We have also identified RORα-dependent NH cell progenitors in the bone marrow. Thus, all NH cells belong to a unique RORα-dependent cell lineage separate from other lymphoid cell lineages.

Regulation of regulatory T cells in cancer.

The inflammatory response to transformed cells forms the cornerstone of natural or therapeutically induced protective immunity to cancer. Regulatory T (Treg) cells are known for their critical role in suppressing inflammation, and therefore can antagonize effective anti-cancer immune responses. As such, Treg cells can play detrimental roles in tumour progression and in the response to both conventional and immune-based cancer therapies. Recent advances in our understanding of Treg cells reveal complex niche-specific regulatory programmes and functions, which are likely to extrapolate to cancer. The regulation of Treg cells is reliant on upstream cues from haematopoietic and non-immune cells, which dictates their genetic, epigenetic and downstream functional programmes. In this review we will discuss how Treg cells are themselves regulated in normal and transformed tissues, and the implications of this cross talk on tumour growth.

Group 2 innate lymphoid cells in disease.

Group 2 innate lymphoid cells (ILC2) are now recognized as an important innate source of type-2 effector cytokines. Although initially associated with mucosal tissues, it is clear that ILC2 are present in diverse anatomical locations. The function of ILC2 at these sites is equally varied, and although ILC2 represent a relatively minor population, they are fundamentally important regulators of innate and adaptive immune processes. As such, there is much interest to understand the role of ILC2 in diseases with a type-2 inflammatory component. This review explores the known roles of ILC2 in disease, and the diseases that show associations or other strong evidence for the involvement of ILC2.

Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures.

BACKGROUND: Allergic inflammation involves the sensitization of naive CD4(+) T cells to allergens, resulting in a TH2-skewed inflammatory response. Although antigen presentation by dendritic cells to T cells in the lymph node is crucial for TH2 cell development, the innate signals that initiate adaptive type 2 inflammation and the role of group 2 innate lymphoid cells (ILC2s) are poorly understood. OBJECTIVE: We sought to investigate the influence of ILC2s and the route of priming on the development of an adaptive type 2 immune response to lung allergens. METHODS: Wild-type and ILC2-deficient mice were exposed intranasally or systemically to the TH2-inducing antigens house dust mite or ovalbumin in a model of allergic airway inflammation or the TH17-inducing bacterial antigen Saccharopolyspora rectivirgula in a model of hypersensitivity pneumonitis. The formation of an adaptive immune response was evaluated based on serum antibody titers and production of T cell-derived cytokines (IL-4, IL-5, IL-13 and IL-17A). RESULTS: We find that lung ILC2s play a critical role in priming the adaptive type 2 immune response to inhaled allergens, including the recruitment of eosinophils, TH2 cytokine production and serum IgE levels. Surprisingly, systemic priming with ovalbumin, with or without adjuvants, circumvents the requirement for ILC2s in inducing TH2-driven lung inflammation. ILC2s were also found to be dispensable for the sensitization to TH1- or TH17-inducing antigens. CONCLUSION: These data highlight a critical role for ILC2s in the development of adaptive type 2 responses to local, but not systemic, antigen exposure.

Cross-talk between ILC2 and Gata3high Tregs locally constrains adaptive type 2 immunity.

Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia.

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors.

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

Context Dependent Role of Type 2 Innate Lymphoid Cells in Allergic Skin Inflammation.

The discovery of innate lymphoid cells (ILC) has profoundly influenced the understanding of innate and adaptive immune crosstalk in health and disease. ILC and T cells share developmental and functional characteristics such as the lineage-specifying transcription factors and effector cytokines, but importantly ILC do not display rearranged antigen-specific receptors. Similar to T cells ILC are subdivided into 3 different helper-like subtypes, namely ILC1-3, and a killer-like subtype comprising natural killer (NK) cells. Increasing evidence supports the physiological relevance of ILC, e.g., in wound healing and defense against parasites, as well as their pathogenic role in allergy, inflammatory bowel diseases or psoriasis. Group 2 ILC have been attributed to the pathogenesis of allergic diseases like asthma and atopic dermatitis. Other inflammatory skin diseases such as allergic contact dermatitis are profoundly shaped by inflammatory NK cells. This article reviews the role of ILC in allergic skin diseases with a major focus on ILC2. While group 2 ILC are suggested to contribute to the pathogenesis of type 2 dominated inflammation as seen in atopic dermatitis, we have shown that lack of ILC2 in type 1 dominated contact hypersensitivity results in enhanced inflammation, suggesting a regulatory role of ILC2 in this context. We provide a concept of how ILC2 may influence context dependent the mutual counterbalance between type I and type II immune responses in allergic skin diseases.

Group 2 innate lymphocytes at the interface between innate and adaptive immunity.

Group 2 innate lymphoid cells (ILC2) are innate immune cells that respond rapidly to their environment through soluble inflammatory mediators and cell-to-cell interactions. As tissue-resident sentinels, ILC2 help orchestrate localized type 2 immune responses. These ILC2-driven type 2 responses are now recognized in diverse immune processes, different anatomical locations, and homeostatic or pathological settings. ILC2-derived cytokines and cell surface signaling molecules function as key regulators of innate and adaptive immunity. Conversely, ILC2 are governed by their environment. As such, ILC2 form an important nexus of the immune system and may present an attractive target for immune modulation in disease.

Lack of Type 2 Innate Lymphoid Cells Promotes a Type I-Driven Enhanced Immune Response in Contact Hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity, are T-cell-mediated inflammatory skin diseases that require activation of the innate immune system. Here we investigate the role of innate lymphoid cells (ILCs) during the elicitation phase of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity using EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice. Ear swelling responses, cutaneous ILC numbers, and cytokine production were determined at different time points. Functional analyses were performed in a CD90.1/.2 congenic adoptive transfer model that allowed selective antibody-mediated depletion of ILCs before hapten challenge, and in Rorasg/floxIl7rCre/+ mice, which lack ILC2. Hapten challenge induced early increases of natural killer cells in skin and ear draining lymph nodes corresponding to the peak ear swelling response. In contrast, ILC1, 2, and 3 showed a delayed increase in numbers corresponding to the contact hypersensitivity resolution phase. Hapten challenge induced increased marker cytokines in all ILC subtypes and an activated phenotype in ILC2. Depletion of all ILC resulted in a significantly enhanced ear swelling response. Similarly, ILC2-deficient mice (Rorasg/floxIl7rCre/+) displayed increased ear swelling responses on hapten challenge, suggesting that ILC2 act as negative regulators in the type 1-dominated immune response of contact hypersensitivity.