Innate Lymphoid Cells Subsets Research Articles

Cutting Edge: Salivary Gland NK Cells Develop Independently of Nfil3 in Steady-State

Nfil3 is viewed as an obligate transcription factor for NK cell development. However, mouse CMV (MCMV) infection recently was shown to bypass the requirement for Nfil3 by inducing the appearance of NK cells that express the MCMV-specific receptor Ly49H. Thus, signals transmitted by Ly49H and proinflammatory cytokines are sufficient to promote NK cell differentiation in the absence of Nfil3. In this study, we report that salivary gland (SG) NK cells develop in an Nfil3-independent fashion in the steady-state in the absence of MCMV or any infection. Moreover, we show that SG NK cells have an integrin profile reminiscent of tissue-resident lymphocytes and express TRAIL for killing target cells. These results demonstrate that SG NK cells, although related to conventional NK cells, are a distinct subset of innate lymphoid cells that deviates from the conventional developmental pathway, perhaps under the influence of tissue-specific factors.

Sa1769 TRIF Confers Expansion of Unique Innate Lymphoid Cell Populations During Enteric Infection With Gram-Negative Bacteria

Background: Increasing evidence has highlighted an important contribution of innate lymphoid cells (ILCs) to host defense against enteric pathogens. We have found that mice deficient in TIR-domain-containing adapter-inducing interferon-β (TRIF) (TrifLPS2 mice) have an increased susceptibility to Gram-negative enteropathogens due to defective induction of IFNs in macrophages and natural killer (NK) cells. Since other subsets of ILCs also express IFN-γ, this study examined the role of TRIF signaling in the regulation of IFN-γ expressing ILCs during enteric infection with a Gram-negative enteropathogen Yersinia enterocolitica. Method: WT and TrifLPS2 mice were orally infected with Y. enterocolitica. Four days post infection, IFN-γ expressing ILCs were analyzed in the mesenteric lymph nodes (MLN), Peyer's patches (PPs), and the small intestinal lamina propria by flow cytometry. The single cell suspensions of mononuclear cells were isolated from these organs and incubated with PMA and ionomycin for four hours in the presense of golgiplug. Specific surface markers and intracellular cytokines were stained. IFN-γ expressing ILCs were characterized by NKp46+IL-17-IL-22cells (include NK cells: ILC1), NKp46+IL-22+ cells (IL-22+ILC3), and NKp46-IL-17+ cells (IL-17+ILC3) within IFN-γ+CD3population. The expression of cytokines that are involved in the activation of ILCs (IL-12p35, IL-23p19, and IL-15) and IFN-β were analyzed in the MLN and PPs by real-time PCR. Results: WT mice had significantly greater population of IFN-γ+CD3cells in the MLN and PPs compared to TrifLPS2 mice: the population of these cells in the small intestine was similar between WT and TrifLPS2 mice. In the MLN, ILC1 was dominant in IFN-γ+CD3population, which was significantly greater in WT mice compared to TrifLPS2 mice. Whereas the number of ILC3 (both IL-22+ and IL-17+) in the MLN was similar between WT and TrifLPS2 mice. On the other hand, IL-22+ILC3 was more abundant in the small intestine than other ILCs, which was greater in TrifLPS2 mice compared to WT mice: there were no differences between WT and TrifLPS2 mice in the number of ILC1 or IL-17+ILC3. In the PPs, ILC1 was more abundant than ILC3. IL-22+ILC3 population in the PPs was slightly greater in TrifLPS2 mice than WT mice. Defective expression of IFN-γ, IL-12p35, and IL-15 in the MLN and PPs was found in TrifLPS2 mice suggesting the involvement of these cytokines in observed differences in ILC expansion. Conclusion: Taken together, TRIF signaling regulates IFN-γ expressing ILC1 expansion in the MLN and IL-22+ILC3 expansion in the small intestine and PPs during enteric infection with Y. enterocolitica. Establishing the role of TRIF signaling in intestinal ILC expansion may contribute to the managements of infectious as well as inflammatory diseases in the intestine.

Metabolites from commensal microbes are closely associated with maintaining mucosal homeostasis through modulation of innate lymphoid cells in Peyer’s patch (MUC4P.849)

Abstract It was characterized that the innate lymphoid cells (ILCs) are a group of immune cells closely associated with immune functions such as lymphoid organogenesis, tissue remodeling, antimicrobial immunity, and inflammation through cytokine-mediated cell regulation. ILC subsets can be categorized into three groups based on cytokine-secreting patterns. Group 1 ILCs secrete IFN-γ. Group 2 produces type 2 cytokines dependent on GATA-3. Group 3 represents all RORγt+ ILC subtypes secreting IL-17 and/or IL-22. In mucosal immunity, RORγt+ ILCs play an essential role in early protection against enteric pathogen infections, although its regulation by mucosal environment is unknown. In this study, we suggest that butyrate, one of commensal microbe-derived metabolites, as a modulator of PP ILCs, because it is known that butyrate treatment closely associated with the protection of bacterial infection or DSS-induced colitis. In order to understand the mechanism underlying the proposed role of butyrate, we monitored the expression pattern of each transcription factors and cytokines in PP ILCs after oral administration of sodium butyrate in SPF and antibiotics-treated mice. To understand the regulating activity of PP ILCs induced by butyrate in mucosal immunity, we try to understand the regulation of DSS colitis in the same mice. Collectively, this study suggests that regulation of ILCs by butyrate in gastrointestinal tract was closely associated with maintaining mucosal homeostasis.

Characterization of Innate Lymphoid Cells in Human Skin and Blood Demonstrates Increase of NKp44+ ILC3 in Psoriasis

Innate lymphoid cells (ILC) are increasingly appreciated as key regulators of tissue immunity. However, their role in human tissue homeostasis and disease remains to be fully elucidated. Here we characterise the ILC in human skin from healthy individuals and from the inflammatory skin disease psoriasis. We show that a substantial proportion of IL-17A and IL-22 producing cells in skin and blood of normal individuals and psoriasis patients are CD3 negative innate lymphocytes. Deep immunophenotyping of human ILC subsets showed a statistically significant increase in the frequency of circulating NKp44+ ILC3 in blood of psoriasis patients compared to healthy individuals or atopic dermatitis patients. More than 50% of circulating NKp44+ ILC3 expressed cutaneous lymphocyte-associated antigen indicating their potential for skin homing. Analysis of skin tissue revealed a significantly increased frequency of total ILC in skin compared to blood. Moreover the frequency of NKp44+ ILC3 was significantly increased in non-lesional psoriatic skin compared to normal skin. A detailed time course of a psoriasis patient treated with anti-TNF showed a close association between therapeutic response, decrease in inflammatory skin lesions, and decrease of circulating NKp44+ ILC3. Overall, data from this initial observational study suggest a potential role for NKp44+ ILC3 in psoriasis pathogenesis.

The Transcription Factor GATA3 Is Critical for the Development of All IL-7Rα-Expressing Innate Lymphoid Cells

Innate lymphoid cells (ILCs) are critical in innate immune responses to pathogens and lymphoid organ development. Similar to CD4(+) T helper (Th) cell subsets, ILC subsets positive for interleukin-7 receptor α (IL-7Rα) produce distinct sets of effector cytokines. However, the molecular control of IL-7Rα(+) ILC development and maintenance is unclear. Here, we report that GATA3 was indispensable for the development of all IL-7Rα(+) ILC subsets and T cells but was not required for the development of classical natural killer cells. Conditionally Gata3-deficient mice had no lymph nodes and were susceptible to Citrobactor rodentium infection. After the ILCs had fully developed, GATA3 remained important for the maintenance and functions of ILC2s. Genome-wide gene expression analyses indicated that GATA3 regulated a similar set of cytokines and receptors in Th2 cells and ILC2s, but not in ILC3s. Thus, GATA3 plays parallel roles in regulating the development and functions of CD4(+) T cells and IL-7Rα(+) ILCs.

A committed precursor to innate lymphoid cells

Innate lymphoid cells (ILC) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers.1–9 Their diversity and similarities with previously characterized NK cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 based solely on cytokine properties,10 but their developmental pathways and lineage relationships remain elusive. Using lineage tracing and transfer studies, we identified and characterized a novel subset of lymphoid precursors in fetal liver and adult bone marrow that transiently expressed high amounts of PLZF, a transcription factor previously associated with NKT cell development.11,12 PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+DX5− ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of Id2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages.13 These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILC, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

Chapter One - Group 2 Innate Lymphoid Cells in the Lung

As the first line of defense, innate immunity plays an important role in protecting the host against pathogens. Innate lymphoid cells (ILCs) are emerging as important effector cells in the innate immune system and the cell type that regulate immune and tissue homeostases. Group 2 ILCs (ILC2s) are a subset of ILCs and are characterized by their capacity to produce large quantities of type 2 cytokines and certain tissue growth factors. In animal models, lung ILC2s are involved in allergic airway inflammation induced by exposure to allergens even in the absence of CD4(+) T cells and are likely responsible for tissue repair and recovery after respiratory virus infection. ILC2s are also identified in various organs in humans, and the numbers are increased in mucosal tissues from patients with allergic disorders. Further investigations of this novel cell type will provide major conceptual advances in our understanding of the mechanisms of asthma and allergic diseases.

Transcription factors controlling innate lymphoid cell fate decisions.

The mucosal epithelium is in direct contact with symbiotic and pathogenic microorganisms. Therefore, the mucosal surface is the principal portal of entry for invading pathogens and immune cells accumulated in the intestine to prevent infections. In addition to these conventional immune system functions, it has become clear that immune cells during steady-state continuously integrate microbial and nutrient-derived signals from the environment to support organ homeostasis. A major role in both processes is played by a recently discovered group of lymphocytes referred to as innate lymphoid cells (ILCs) Innate lymphoid cells (ILCs) that are specifically enriched at mucosal surfaces but are rather rare in secondary lymphoid organs. In analogy to the dichotomy between CD8 and CD4 T cells, we propose to classify ILCs into interleukin-7 receptor α-negative cytotoxic ILCs and IL-7Rα(+) helper-like ILCs. Dysregulated immune responses triggered by the various ILC subsets have been linked to inflammatory diseases such as inflammatory bowel disease, atopic dermatitis and airway hyperresponsiveness. Here, we will review recent progress in determining the transcriptional and developmental programs that control ILC fate decisions.

Therapeutic opportunities of the IL-22–IL-22R1 system

Interleukin-22 (IL-22) is a key effector molecule that is produced by activated T cells, including T helper 22 (TH22) cells, TH17 cells and TH1 cells, as well as subsets of innate lymphoid cells. Although IL-22 can act synergistically with IL-17 or tumour necrosis factor, some important functions of IL-22 are unique to this cytokine. Data obtained over the past few years indicate that the IL-22-IL-22 receptor subunit 1 (IL-22R1) system has a high potential clinical relevance in psoriasis, ulcerative colitis, graft-versus-host disease, certain infections and tumours, as well as in liver and pancreas damage. This Review highlights current knowledge of the biology of the IL-22-IL-22R1 system, its role in inflammation, tissue protection, regeneration and antimicrobial defence, as well as the positive and potentially negative consequences of its therapeutic modulation.

Natural cytotoxicity receptors and their ligands

Natural killer (NK) cells are innate lymphoid cells (ILCs) that participate to the clearance of pathogen-infected cells and tumour cells. NK cells and subsets of ILCs express the natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 at their surface. NCRs have been shown to recognize a broad spectrum of ligands ranging from viral-, parasite- and bacterial-derived ligands to cellular ligands; however, the full identification of NCR ligands remains to be performed and will undoubtedly contribute to a better understanding of NK cell and ILC biology.

Open questions: a few that need answers in immunology

The cells of the immune system fall into two broad categories - innate and adaptive - with the cells of the innate immune system providing frontline defense by means of invariant receptors to conserved microbial components, and the B and T lymphocytes of the adaptive immune system, with highly variable somatically diversified receptors, providing reinforcement after selective proliferation in response to specific antigen. While this principle still holds, the ‘old’ view of these systems as developmentally separate and functionally distinct has been eroded, most recently and dramatically by the discovery of a whole new group of innate lymphoid cell subsets. Another development has been the increasing emphasis on studying immune responses in vivo in interaction with stromal components that are beginning to be seen as essential contributors to the development of immune responses, rather than just as matrix or scaffold components in lymphoid organs or non-lymphoid peripheral tissues. A major focus now is on immune defense at epithelial barriers and homeostatic coexistence with a healthy microbiota, as it is becoming increasingly clear that intestinal dysbiosis strongly influences immune responses even at distal sites. However, whether dysbiosis is a cause or consequence of dysregulated inflammatory responses remains to be unraveled. I outline below some of the open questions raised by these developments.

Taming the Beast within: Regulation of Innate Lymphoid Cell Homeostasis and Function

Although substantial parallels have been made between transcription factor regulation of cytokine production by innate lymphoid cell (ILC) and Th cell subsets, we are still learning how ILC subsets are regulated during immune responses. Critical factors that promote ILC development and stimulate their effector functions have been identified, but mechanisms that control their homeostasis and downregulate their cytokine secretion remain poorly understood. In this review, we consider some of the potential positive and negative regulators of ILC homeostasis and function in physiological and pathological conditions.

TCF-1 Controls ILC2 and NKp46+RORγt+ Innate Lymphocyte Differentiation and Protection in Intestinal Inflammation

Innate lymphocyte populations play a central role in conferring protective immunity at the mucosal frontier. In this study, we demonstrate that T cell factor 1 (TCF-1; encoded by Tcf7), a transcription factor also important for NK and T cell differentiation, is expressed by multiple innate lymphoid cell (ILC) subsets, including GATA3(+) nuocytes (ILC2) and NKp46(+) ILCs (ILC3), which confer protection against lung and intestinal inflammation. TCF-1 was intrinsically required for the differentiation of both ILC2 and NKp46(+) ILC3. Loss of TCF-1 expression impaired the capacity of these ILC subsets to produce IL-5, IL-13, and IL-22 and resulted in crippled responses to intestinal infection with Citrobacter rodentium. Furthermore, a reduction in T-bet expression required for Notch-2-dependent development of NKp46(+) ILC3 showed a dose-dependent reduction in TCF-1 expression. Collectively, our findings demonstrate an essential requirement for TCF-1 in ILC2 differentiation and reveal a link among Tcf7, Notch, and Tbx21 in NKp46(+) ILC3 development.

Innate lymphoid cells in the defense against infections

Barrier surfaces are under constant attack by potentially dangerous microbes. Interestingly, mucosal tissues contain a large number of innate lymphocytes now collectively referred to as innate lymphoid cells (ILCs). Different groups of ILCs are being distinguished, each of which produce an array of cytokines strikingly resembling the profile of the various T helper cell effector subsets. Over the last couple of years, evidence has been emerging that the various ILC subsets play important roles in immune defense against mucosal infections. In this review, I will introduce the various groups of ILCs and then focus on their roles for immunity to mucosal infections.

Critical Role of p38 and GATA3 in Natural Helper Cell Function

Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.

SP0114 Innate lymphoid cells and their interaction with stroma in tertiary lymphoid tissue formation

Ectopic or tertiary lymphoid tissues (TLTs) are frequently associated with sites of chronic inflammation and are thought to contribute to pathological responses. They typically contain various hematopoietic cell types including...

Cutaneous immuno-surveillance and regulation of inflammation by group 2 innate lymphoid cells (P6374)

Abstract Type 2 immunity is critical for defense against cutaneous infections, but also underlies the development of allergic skin diseases. We report the identification in normal murine dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depends on IL-7 and constitutively produces IL-13. Intravital multiphoton microscopy revealed that dermal ILC2 specifically interact with mast cells, whose function is suppressed by IL-13. Treatment of Rag1−/− mice with IL-2 led to a marked expansion of activated, IL-5-producing dermal ILC2, resulting in the development of spontaneous dermatitis characterized by eosinophil infiltrate and activated mast cells. Our data uncover a novel interactive pathway between two innate immune cell populations implicated in type 2 immunity, and demonstrate that ILC2 exhibit both pro- and anti-inflammatory properties.

Cutaneous immunosurveillance and regulation of inflammation by group 2 innate lymphoid cells

Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.

Intraepithelial Type 1 Innate Lymphoid Cells Are a Unique Subset of IL-12- and IL-15-Responsive IFN-γ-Producing Cells

Mucosal innate lymphoid cell (ILC) subsets promote immune responses to pathogens by producing distinct signature cytokines in response to changes in the cytokine microenvironment. We previously identified human ILC3 distinguished by interleukin-22 (IL-22) secretion. Here we characterized a human ILC1 subset that produced interferon-γ (IFN-γ) in response to IL-12 and IL-15 and had a unique integrin profile, intraepithelial location, hallmarks of TGF-β imprinting, and a memory-activated phenotype. Because tissue-resident memory CD8(+) Tcells share this profile, intraepithelial ILC1 may be their innate counterparts. In mice, intraepithelial ILC1 were distinguished by CD160 expression and required Nfil3- and Tbx21-encoded transcription factors for development, but not IL-15 receptor-α, indicating that intraepithelial ILC1 are distinct from conventional NK cells. Intraepithelial ILC1 were amplified in Crohn's disease patients and contributed to pathology in theanti-CD40-induced colitis model in mice. Thus, intraepithelial ILC1 may initiate IFN-γ responses against pathogens but contribute to pathology when dysregulated.

Th1‐ and Th2‐like subsets of innate lymphoid cells

Innate lymphoid cells (ILCs) constitute a family of effectors in innate immunity and regulators of tissue remodeling that have a cytokine and transcription factor expression pattern that parallels that of the T-helper (Th) cell family. Here, we discuss how ILCs can be categorized and summarize the current knowledge of Th1- and Th2-like ILCs with regard to the molecular mechanisms of development, effector functions, and their interplay with other cell types.