High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Innate Lymphoid Cells: 10 Years On.

Innate lymphoid cells (ILCs) are lymphocytes that do not express the type of diversified antigen receptors expressed on T cells and B cells. ILCs are largely tissue-resident cells and are deeply integrated into the fabric of tissues. The discovery and investigation of ILCs over the past decade has changed our perception of immune regulation and how the immune system contributes to the maintenance of tissue homeostasis. We now know that cytokine-producing ILCs contribute to multiple immune pathways by, for example, sustaining appropriate immune responses to commensals and pathogens at mucosal barriers, potentiating adaptive immunity, and regulating tissue inflammation. Critically, the biology of ILCs also extends beyond classical immunology to metabolic homeostasis, tissue remodeling, and dialog with the nervous system. The last 10 years have also contributed to our greater understanding of the transcriptional networks that regulate lymphocyte commitment and delineation. This, in conjunction with the recent advances in our understanding of the influence of local tissue microenvironments on the plasticity and function of ILCs, has led to a re-evaluation of their existing categorization. In this review, we distill the advances in ILC biology over the past decade to refine the nomenclature of ILCs and highlight the importance of ILCs in tissue homeostasis, morphogenesis, metabolism, repair, and regeneration.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Innate Lymphoid Cell-Epithelial Cell Modules Sustain Intestinal Homeostasis.

The intestines have the essential but challenging mission of absorbing nutrients, restricting damage from food-derived toxins, promoting colonization by symbionts, and expelling pathogens. These processes are often incompatible with each other and must therefore be prioritized in view of the most crucial contemporary needs of the host. Recent work has shown that tissue-resident innate lymphoid cells (ILCs) constitute a central sensory module allowing adaptation of intestinal organ function to changing environmental input. Here, we propose a conceptual framework positing that the various types of ILC act in distinct modules with intestinal epithelial cells, collectively safeguarding organ function. Such homeostasis-promoting circuitry has high potential to be plumbed for new therapeutic approaches to the treatment of immune-mediated inflammatory diseases.

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues.

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-ß receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection.

The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were 'preferentially' expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons.

Antigen-presenting innate lymphoid cells orchestrate neuroinflammation.

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases1-6, but the pathways that control these responses remain unclear. Here we define a population of inflammatory group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen-presenting cells that restimulate myelin-specific T cells, and are increased in individuals with multiple sclerosis. Notably, antigen presentation by inflammatory ILC3s is required to promote T cell responses in the CNS and the development of multiple-sclerosis-like disease in mouse models. By contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but instead limit autoimmune T cell responses and prevent multiple-sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define a population of inflammatory ILC3s that is essential for directly promoting T-cell-dependent neuroinflammation in the CNS and reveal the potential of harnessing peripheral tissue-resident ILC3s for the prevention of autoimmune disease.

Untimely TGFß responses in COVID-19 limit antiviral functions of NK cells.

SARS-CoV-2 is a single-stranded RNA virus that causes COVID-19. Given its acute and often self-limiting course, it is likely that components of the innate immune system play a central part in controlling virus replication and determining clinical outcome. Natural killer (NK) cells are innate lymphocytes with notable activity against a broad range of viruses, including RNA viruses1,2. NK cell function may be altered during COVID-19 despite increased representation of NK cells with an activated and adaptive phenotype3,4. Here we show that a decline in viral load in COVID-19 correlates with NK cell status and that NK cells can control SARS-CoV-2 replication by recognizing infected target cells. In severe COVID-19, NK cells show defects in virus control, cytokine production and cell-mediated cytotoxicity despite high expression of cytotoxic effector molecules. Single-cell RNA sequencing of NK cells over the time course of the COVID-19 disease spectrum reveals a distinct gene expression signature. Transcriptional networks of interferon-driven NK cell activation are superimposed by a dominant transforming growth factor-ß (TGFß) response signature, with reduced expression of genes related to cell-cell adhesion, granule exocytosis and cell-mediated cytotoxicity. In severe COVID-19, serum levels of TGFß peak during the first two weeks of infection, and serum obtained from these patients severely inhibits NK cell function in a TGFß-dependent manner. Our data reveal that an untimely production of TGFß is a hallmark of severe COVID-19 and may inhibit NK cell function and early control of the virus.

Regulation of innate immune system function by the microbiome: Consequences for tumor immunity and cancer immunotherapy.

Innate effector cells are immune cells endowed with host protective features and cytotoxic functions. By sensing the tissue environment, innate cells have an important role in regulating the transition from homeostasis to inflammation and the establishment of pathological states, including the onset and development of cancer. The tumor microenvironment induces molecular and functional modifications in innate cells, dampening their capability to initiate and sustain anti-tumor immune responses. Emerging studies clearly showed a contribution of the microbiota in modulating the functions of innate cells in cancer. Commensal microorganisms can not only directly interact with innate cells in the tumor microenvironment but can also exert immunomodulatory features from non-tumor sites through the release of microbial products. The microbiota can mediate the priming of innate cells at mucosal tissues and determine the strength of immune responses mediated by such cells when they migrate to non-mucosal tissues, having an impact on cancer. Finally, several evidences reported a strong contribution of the microbiota in promoting innate immune responses during anti-cancer therapies leading to enhanced therapeutic efficacy. In this review, we considered the current knowledge on the role of the microbiota in shaping host innate immune responses in cancer.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.

The ILC World Revisited.

Immunology has recently witnessed several new developments in understanding the biology of innate lymphocytes. In particular, the discovery of innate lymphoid cells (ILCs) has opened entirely new avenues for research. The exciting new developments in this rapidly expanding field were the focus of the 2ndEMBO Conference on Innate Lymphoid Cells, which took place from November 30 to December 2, 2016 in Berlin, Germany. Here, we summarize the key new developments reported at the conference.

Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

Transcription factor EBF1 is essential for the maintenance of B cell identity and prevention of alternative fates in committed cells.

The transcription factors EBF1 and Pax5 have been linked to activation of the B cell lineage program and irreversible loss of alternative lineage potential (commitment), respectively. Here we conditionally deleted Ebf1 in committed pro-B cells after transfer into alymphoid mice. We found that those cells converted into innate lymphoid cells (ILCs) and T cells with variable-diversity-joining (VDJ) rearrangements of loci encoding both B cell and T cell antigen receptors. As intermediates in lineage conversion, Ebf1-deficient CD19(+) cells expressing Pax5 and transcriptional regulators of the ILC and T cell fates were detectable. In particular, genes encoding the transcription factors Id2 and TCF-1 were bound and repressed by EBF1. Thus, both EBF1 and Pax5 are required for B lineage commitment by repressing distinct and common determinants of alternative cell fates.

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria.

Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohn's disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1ß dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn's disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.

B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen.

Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell-independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell-helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.