PD-1 regulates ILC3-driven intestinal immunity and homeostasis.

Interleukin-(IL) 22 production by intestinal group 3 innate lymphoid cells (ILC3) is critical to maintain gut homeostasis. However, IL-22 needs to be tightly controlled; reduced IL-22 expression is associated with intestinal epithelial barrier defect while its overexpression promotes tumor development. Here, using a single-cell ribonucleic acid sequencing approach, we identified a core set of genes associated with increased IL-22 production by ILC3. Among these genes, programmed cell death 1 (PD-1), extensively studied in the context of cancer and chronic infection, was constitutively expressed on a subset of ILC3. These cells, found in the crypt of the small intestine and colon, displayed superior capacity to produce IL-22. PD-1 expression on ILC3 was dependent on the microbiota and was induced during inflammation in response to IL-23 but, conversely, was reduced in the presence of Notch ligand. PD-1+ ILC3 exhibited distinct metabolic activity with increased glycolytic, lipid, and polyamine synthesis associated with augmented proliferation compared with their PD-1- counterparts. Further, PD-1+ ILC3 showed increased expression of mitochondrial antioxidant proteins which enable the cells to maintain their levels of reactive oxygen species. Loss of PD-1 signaling in ILC3 led to reduced IL-22 production in a cell-intrinsic manner. During inflammation, PD-1 expression was increased on natural cytotoxicity receptor (NCR)- ILC3 while deficiency in PD-1 expression resulted in increased susceptibility to experimental colitis and failure to maintain gut barrier integrity. Collectively, our findings uncover a new function of the PD-1 and highlight the role of PD-1 signaling in the maintenance of gut homeostasis mediated by ILC3 in mice.

A tuft cell - ILC2 signaling circuit provides therapeutic targets to inhibit gastric metaplasia and tumor development.

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest a role for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing an opportunity to therapeutically inhibit early-stage gastric cancer through repurposing antibody-mediated therapies.

Innate lymphoid cells: More than just immune cells.

Since their discovery, innate lymphoid cells (ILCs) have been described as the innate counterpart of the T cells. Indeed, ILCs and T cells share many features including their common progenitors, transcriptional regulation, and effector cytokine secretion. Several studies have shown complementary and redundant roles for ILCs and T cells, leaving open questions regarding why these cells would have been evolutionarily conserved. It has become apparent in the last decade that ILCs, and rare immune cells more generally, that reside in non-lymphoid tissue have non-canonical functions for immune cells that contribute to tissue homeostasis and function. Viewed through this lens, ILCs would not be just the innate counterpart of T cells, but instead act as a link between sensory cells that monitor any changes in the environment that are not necessarily pathogenic and instruct effector cells that act to maintain body homeostasis. As these non-canonical functions of immune cells are operating in absence of pathogenic signals, it opens great avenues of research for immunologists that they now need to identify the physiological cues that regulate these cells and how the process confers a finer level of control and a greater flexibility that enables the organism to adapt to changing environmental conditions. In the review, we highlight how ILCs participate in the physiologic function of the tissue in which they reside and how physiological cues, in particular neural inputs control their homeostatic activity.

A protocol to isolate bone marrow innate lymphoid cells for alymphoid mouse reconstitution.

Innate lymphoid cells (ILCs) and adaptive T cells remain a challenge to study because of a significant overlap in their transcriptomic profiles. Here, we describe the adoptive transfer of ILC progenitors into mice genetically deficient in innate and adaptive immune cells to allow detailed study of the development and function of ILCs and gene regulation in an in vivo setting. For complete details on the use and execution of this protocol, please refer to Jacquelot et al. (2021) and Seillet et al. (2016).

Innate lymphoid cells and cancer.

The innate lymphoid cell (ILC) family is composed of natural killer (NK) cells, ILC1, ILC2 and ILC3, which participate in immune responses to virus, bacteria, parasites and transformed cells. ILC1, ILC2 and ILC3 subsets are mostly tissue-resident, and are profoundly imprinted by their organ of residence. They exhibit pleiotropic effects, driving seemingly paradoxical responses such as tissue repair and, alternatively, immunopathology toward allergens and promotion of tumorigenesis. Despite this, a trickle of studies now suggests that non-NK ILCs may not be overwhelmingly tumorigenic and could potentially be harnessed to drive anti-tumor responses. Here, we examine the pleiotropic behavior of ILCs in cancer and begin to unravel the gap in our knowledge that exposes a new horizon for thinking about modifying ILCs and targeting them for immunotherapy.

Natural Killer Cells and Type 1 Innate Lymphoid Cells in Hepatocellular Carcinoma: Current Knowledge and Future Perspectives.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) are specific innate lymphoid cell subsets that are key for the detection and elimination of pathogens and cancer cells. In liver, while they share a number of characteristics, they differ in many features. These include their developmental pathways, tissue distribution, phenotype and functions. NK cells and ILC1 contribute to organ homeostasis through the production of key cytokines and chemokines and the elimination of potential harmful bacteria and viruses. In addition, they are equipped with a wide range of receptors, allowing them to detect "stressed cells' such as cancer cells. Our understanding of the role of innate lymphoid cells in hepatocellular carcinoma (HCC) is growing owing to the development of mouse models, the progress in immunotherapeutic treatment and the recent use of scRNA sequencing analyses. In this review, we summarize the current understanding of NK cells and ILC1 in hepatocellular carcinoma and discuss future strategies to take advantage of these innate immune cells in anti-tumor immunity. Immunotherapies hold great promise in HCC, and a better understanding of the role and function of NK cells and ILC1 in liver cancer could pave the way for new NK cell and/or ILC1-targeted treatment.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Neuroimmune Interactions and Rhythmic Regulation of Innate Lymphoid Cells.

The Earth's rotation around its axis, is one of the parameters that never changed since life emerged. Therefore, most of the organisms from the cyanobacteria to humans have conserved natural oscillations to regulate their physiology. These daily oscillations define the circadian rhythms that set the biological clock for almost all physiological processes of an organism. They allow the organisms to anticipate and respond behaviorally and physiologically to changes imposed by the day/night cycle. As other physiological systems, the immune system is also regulated by circadian rhythms and while diurnal variation in host immune responses to lethal infection have been observed for many decades, the underlying mechanisms that affect immune function and health have only just started to emerge. These oscillations are generated by the central clock in our brain, but neuroendocrine signals allow the synchronization of the clocks in peripheral tissues. In this review, we discuss how the neuroimmune interactions create a rhythmic activity of the innate lymphoid cells. We highlight how the disruption of these rhythmic regulations of immune cells can disturb homeostasis and lead to the development of chronic inflammation in murine models.

Natural killers or ILC1s? That is the question.

Group 1 innate lymphoid cells (ILCs) comprise the natural killer (NK) cells and ILC1s. Both cells co-exist in peripheral tissues and despite effort to characterize the molecular identity and developmental pathways of ILC1s, their relationship with NK cells remains elusive. ILC1s and NK cells share many common features and analysis of ILC1s in tissues revealed a great heterogeneity and distinct transcriptional requirement of each ILC1 subsets complexifying the organization of this group. Here, we discuss whether ILC1 and NK cells can be considered as distinct lineages based on their origin, location, phenotype or transcriptional regulation. Discrimination of NK cells and ILC1s represent an important challenge to unravel the individual functions of these cells during infection and tumor immunosurveillance.

Tissue-resident lymphocytes: weaponized sentinels at barrier surfaces.

Tissue-resident immune cells stably localize in tissues largely independent of the circulatory system. While initial studies have focused on the recognition of CD8 + tissue-resident memory T (CD8 T RM) cells, it is now clear that numerous cell types such as CD4 + T cells, gd T cells, innate lymphoid cells and mucosal-associated invariant T (MAIT) cells form stable populations in tissues. They are enriched at the barrier surfaces and within non-lymphoid compartments. They provide an extensive immune network capable of sensing local perturbations of the body's homeostasis. This positioning enables immune cells to positively influence immune protection against infection and cancer but paradoxically also augment autoimmunity, allergy and chronic inflammatory diseases. Here, we highlight the recent studies across multiple lymphoid immune cell types that have emerged on this research topic and extend our understanding of this important cellular network. In addition, we highlight the areas that remain gaps in our knowledge of the regulation of these cells and how a deeper understanding may result in new ways to 'target' these cells to influence disease outcome and treatments.

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Physiological Regulation of Innate Lymphoid Cells.

Discovery of innate lymphoid cells (ILCs) have provoked a paradigm shift in our understanding of the immune protection. Their constitutive presence and activity at the body's barrier surfaces ensure the maintenance of the tissue homeostasis and immune protection. This complex family has distinct and non-redundant functions that can have both beneficial and detrimental effects on disease outcome. The capacity of ILCs to perform their function effectively relies on their ability to sense and integrate intrinsic and extrinsic signals. Recent studies have shown that ILCs are not only sensitive to pathogen-derived stimuli but are also very well equipped to sense host-derived signals such as neuropeptides, hormones, and metabolites. The integration of these signals represents a complex and constant cross-talk between the immune system and the physiological systems of the body, including the nervous, endocrine, digestive, and reproductive systems. The physiological regulation of ILCs constitutes an important step in our understanding of the events leading to the protective and pathological properties of these cells. This review summarizes the recent advances in the understanding of the regulation of ILCs by physiological signals and their consequences on the maintenance of tissue homeostasis.

Assessment of Gene Function of Mouse Innate Lymphoid Cells for In Vivo Analysis Using Retroviral Transduction.

Retroviral transduction is commonly used to modulate gene expression and is a powerful approach to understand the role of a gene using gain- or loss-of-function strategies. Retroviral vectors can stably integrate non-viral genes into host genomes, providing long-term modulation of gene expression in infected cells and their progeny. Here we describe the generation of retroviral supernatants and the steps to efficiently transduce genes in innate lymphoid cell (ILC) progenitors for subsequent analysis of ILC populations in vivo.

Sensing of physiological regulators by innate lymphoid cells.

Maintenance of homeostasis and immune protection rely  on the coordinated action of different physiological systems. Bidirectional communication between the immune system and physiological systems is required to sense and restore any disruption of equilibrium. Recent transcriptomic analyses of innate lymphoid cells (ILCs) from different tissues have revealed that ILCs express a large array of receptors involved in the recognition of neuropeptides, hormones and metabolic signals. ILCs rapidly secrete effector cytokines that are central in the development and activation of early immune responses, but they also constitutively secrete mediators that are important for tissue homeostasis. To achieve these functions effectively, ILCs integrate intrinsic and extrinsic signals that modulate their constitutive and induced activity. Disruption of the regulation of ILCs by physiological regulators leads to altered immune responses with harmful consequences for the organism. An understanding of these complex interactions between the immune system and physiological mediators is crucial to decipher the events leading to the protective versus pathological effects of these cells.

Constitutive overexpression of TNF in BPSM1 mice causes iBALT and bone marrow nodular lymphocytic hyperplasia.

BPSM1 (Bone phenotype spontaneous mutant 1) mice develop severe polyarthritis and heart valve disease as a result of a spontaneous mutation in the Tnf gene. In these mice, the insertion of a retrotransposon in the 3' untranslated region of Tnf causes a large increase in the expression of the cytokine. We have found that these mice also develop inducible bronchus-associated lymphoid tissue (iBALT), as well as nodular lymphoid hyperplasia (NLH) in the bone marrow. Loss of TNFR1 prevents the development of both types of follicles, but deficiency of TNFR1 in the hematopoietic compartment only prevents the iBALT and not the NLH phenotype. We show that the development of arthritis and heart valve disease does not depend on the presence of the tertiary lymphoid tissues. Interestingly, while loss of IL-17 or IL-23 limits iBALT and NLH development to some extent, it has no effect on polyarthritis or heart valve disease in BPSM1 mice.

NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology.

OBJECTIVES: NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. METHODS: Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. RESULTS: The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1ß production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1ß as a pivotal cytokine. CONCLUSIONS: NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1ß over-production.

Innate Lymphoid Cells in Colorectal Cancers: A Double-Edged Sword.

The immune system plays a fundamental role at mucosal barriers in maintaining tissue homeostasis. This is particularly true for the gut where cells are flooded with microbial-derived signals and antigens, which constantly challenge the integrity of the intestinal barrier. Multiple immune cell populations equipped with both pro- and anti-inflammatory functions reside in the gut tissue and these cells tightly regulate intestinal health and functions. Dysregulation of this finely tuned system can progressively lead to autoimmune disease and inflammation-driven carcinogenesis. Over the last decade, the contribution of the adaptive immune system in controlling colorectal cancer has been studied in detail, but the role of the innate system, particularly innate lymphoid cells (ILCs), have been largely overlooked. By sensing their microenvironment, ILCs are essential in supporting gut epithelium repair and controling bacterial- and helminth-mediated intestinal infections, highlighting their important role in maintaining tissue integrity. Accumulating evidence also suggests that they may play an important role in carcinogenesis including intestinal cancers. In this review, we will explore the current knowledge about the pro- and anti-tumor functions of ILCs in colorectal cancer.

Shaping Innate Lymphoid Cell Diversity.

Innate lymphoid cells (ILCs) are a key cell type that are enriched at mucosal surfaces and within tissues. Our understanding of these cells is growing rapidly. Paradoxically, these cells play a role in maintaining tissue integrity but they also function as key drivers of allergy and inflammation. We present here the most recent understanding of how genomics has provided significant insight into how ILCs are generated and the enormous heterogeneity present within the canonical subsets. This has allowed the generation of a detailed blueprint for ILCs to become highly sensitive and adaptive sensors of environmental changes and therefore exquisitely equipped to protect immune surfaces.