Family Of Innate Lymphoid Cells Research Articles

Targeting natural killer cells: from basic biology to clinical application in hematologic malignancies.

Natural killer (NK) cell belongs to innate lymphoid cell family that contributes to host immunosurveillance and defense without pre-immunization. Emerging studies have sought to understand the underlying mechanism behind NK cell dysfunction in tumor environments, and provide numerous novel therapeutic targets for tumor treatment. Strategies to enhance functional activities of NK cell have exhibited promising efficacy and favorable tolerance in clinical treatment of tumor patients, such as immune checkpoint blockade (ICB), chimeric antigen receptor NK (CAR-NK) cell, and bi/trispecific killer cell engager (BiKE/TriKE). Immunotherapy targeting NK cell provides remarkable advantages compared to T cell therapy, including a decreased rate of graft versus-host disease (GvHD) and neurotoxicity. Nevertheless, advanced details on how to support the maintenance and function of NK cell to obtain better response rate and longer duration still remain to be elucidated. This review systematically summarizes the profound role of NK cells in tumor development, highlights up-to-date advances and current challenges of therapy targeting NK cell in the clinical treatment of hematologic malignancies.

Innate lymphoid cells: New players in osteoimmunology.

Innate lymphoid cells (ILCs) are the most recently identified immune cell types existing in lymphoid and nonlymphoid organs. Albeit they lack the expression of antigen receptors, ILCs play vital roles in innate immune responses by producing multiple effector cytokines. The ILC family includes conventional natural killer cells and cytokine-producing ILCs, which are divided into group 1, group 2, and group 3 ILCs based on their effector cytokines and developmental requirements. Emerging evidence has indicated that ILCs are essential immune regulators of bone homeostasis, playing a critical role in osteoimmunology. In this mini-review, we discuss recent advances in the understanding of ILC functions in bone homeostasis under physiological and pathological conditions, with an emphasis on the communication between ILCs and bone cells including osteoclasts and osteoblasts, as well as the underlying immunoregulatory networks involving ILC-derived cytokines and growth factors. This review also discusses future research directions and the potential of targeting ILCs for the treatment of inflammation-associated bone disorders.

Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells.

Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) 'helper' ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.

Tumor-associated NK cells facilitate tumor growth via NKp46 in immunocompetent murine hepatocellular carcinoma

Natural killer(NK) cells comprise one subset of the innate lymphoid cells family. Despite reported anti-tumor activity of NK cells, their tangible contribution to tumor control remains controversial. This is due to the incomplete understanding of NK alterations within tumor microenvironment(TME). Here we showed, using murine hepatocellular carcinoma(HCC) model, that early NK cells deletion markedly attenuated tumor growth in a CD8+T cells dependent manner. This effect was accompanied by an enhanced CD8+T cells effector function in tumor rather than circulating blood. Then, we demonstrated that abundant NKp46+ NK subset, but not NKp46− NK, were recruited towards tumor microenvironment during tumor progression. Frequency of intratumor NKP46+ NK cells were inversely related to CD8+T cells activation, and positively correlated with tumor growth. Intratumor NKp46+ NK cells exhibited dysfunction and increased expression of inhibitory receptors, when compared with NKp46− NK cells. Blockade of NK cells-associated NKp46 effectively attenuated HCC growth. Infusion of tumor-derived NKp46+ NK cells markedly enhanced HCC growth in vivo, in contrast to tumor cells inoculation alone. The further mechanistic investigations unveiled that NK cells boosted tumor growth by NKp46-mediated impairment of CD8+T cells effector function.Overall, this work supported a previously unappreciated regulatory property of tumor-associated NK cells in HCC, and NKp46 as a potential target against HCC in clinical setting.

Natural Killer Cells, as the Rising Point in Tissues, Are Forgotten in the Kidney.

Natural killer (NK) cells are members of a rapidly expanding family of innate lymphoid cells (ILCs). NK cells play roles in the spleen, periphery, and in many tissues, such as the liver, uterine, lung, adipose, and so on. While the immunological functions of NK cells are well established in these organs, comparatively little is known about NK cells in the kidney. Our understanding of NK cells is rapidly rising, with more and more studies highlighting the functional significance of NK cells in different types of kidney diseases. Recent progress has been made in translating these findings to clinical diseases that occur in the kidney, with indications of subset-specific roles of NK cells in the kidney. For the development of targeted therapeutics to delay kidney disease progression, a better understanding of the NK cell with respect to the mechanisms of kidney diseases is necessary. In order to promote the targeted treatment ability of NK cells in clinical diseases, in this paper we demonstrate the roles that NK cells play in different organs, especially the functions of NK cells in the kidney.

Embryonic ILC-poiesis across tissues.

The family of innate lymphoid cells (ILCs), consisting of Group 1 ILCs (natural killer cells and ILC1), ILC2, and ILC3, are critical effectors of innate immunity, inflammation, and homeostasis post-natally, but also exert essential functions before birth. Recent studies during critical developmental periods in the embryo have hinted at complex waves of tissue colonization, and highlighted the breadth of multipotent and committed ILC progenitors from both classic fetal hematopoietic organs such as the liver, as well as tissue sites such as the lung, thymus, and intestine. Assessment of the mechanisms driving cell fate and function of the ILC family in the embryo will be vital to the understanding ILC biology throughout fetal life and beyond.

Oxygen regulates ILC3 antigen presentation potential and pregnancy-related hormone actions

Early pregnancy is marked by placentation and embryogenesis, which take place under physiological low oxygen concentrations. This oxygen condition is crucial for many aspects of placentation, trophoblast function, vascularization and immune function. Recently, a new family of innate lymphoid cells has been found to be expressed at the fetomaternal interface. Among these, type 3 innate lymphoid cells (ILC3) are important antigen presenting cells in the context of MHC-II. The expression of MHC-II on ILC3s during pregnancy is reduced. We tested the hypothesis that low oxygen concentrations reduce the potential of ILC3s to present antigens promoting fetal tolerance.Using an in vitro approach, NCR+ ILC3s generated from cord blood stem cell precursors were incubated under different O2 concentrations in the presence or absence of the pregnancy-related hormones hCG and TGF-β1. The expression of MHC-II, accessory molecules and an activation marker were assessed by flow cytometry. We observed that 1% O2 reduced the expression of the MHC-II molecule HLA-DR as compared to 21% O2 and modulated the relative effects of hCG and TGF-β1.Our data indicate that low oxygen concentrations reduce the antigen presentation potential of NCR+ ILC3s and suggest that it may promote fetal tolerance during the first trimester of pregnancy.

Searching for the Elusive Regulatory Innate Lymphoid Cell.

The complex nature of the innate lymphoid cell (ILC) family and wide range of ILC effector functions has been the focus of intense research. In addition to important roles in host defense, ILCs have central roles in maintaining tissue homeostasis and can promote immune tolerance. Alterations within the microenvironment can impart new functions on ILCs, and can even induce conversion to a distinct ILC family member. Complicating current definitions of ILCs are recent findings of distinct regulatory ILC populations that limit inflammatory responses or recruit other immunosuppressive cells such as regulatory T cells. Whether these populations are distinct ILC family members or rather canonical ILCs that exhibit immunoregulatory functions due to microenvironment signals has been the subject of much debate. In this review, we highlight studies identifying regulatory populations of ILCs that span regulatory NK-like cells, regulatory ILCs, and IL-10-producing ILC2s.

Unraveling the Role of Innate Lymphoid Cells in AcuteMyeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive form of cancer found in the blood and bone marrow with poor survival rates. Patients with AML are known to have many defects in their immune system which render immune cells unable to detect and/or kill cancer cells. Natural Killer (NK) cells are innate immune effector cells responsible for surveying the body to eliminate cancer cells as well as alert other immune cells to help clear the cancer cells. NK cells have developmental and functional defects in AML patients. While advances have been made to understand these NK cell defects in the setting of AML, the role of other closely related and recently discovered members of the innate lymphoid cell (ILC) family is much less clear. The ILC family is comprised of NK cells, ILC1s, ILC2s, and ILC3s, and due in part to their recent discovery, non-NK ILCs are just now beginning to be investigated in the setting of AML. By better understanding how AML alters the normal function of these cell types, and how the alteration regulates AML growth, we may be able to target and tailor new forms of therapy for patients.Over the past 50 years, few therapeutic advances have been made in treating acute myeloid leukemia (AML), an aggressive form of blood cancer, despite vast improvements in our ability to classify the disease. Emerging evidence suggests the immune system is important in controlling AML progression and in determining prognosis. Natural killer (NK) cells are important cytotoxic effector cells of the innate lymphoid cell (ILC) family that have been shown to have potent anti-leukemic functions. Recent studies are now revealing impairment or dysregulation of other ILCs in various types of cancers, including AML, which limits the effectiveness of NK cells in controlling cancer progression. NK cell development and function are inhibited in AML patients, which results in worse clinical outcomes; however, the specific roles of other ILC populations in AML are just now beginning to be unraveled. In this review, we summarize what is known about the role of ILC populations in AML.

Comprehensive Genetic Analysis Revealed Myeloid/Natural Killer (NK) Cell Precursor Acute Leukemia As a Novel Distinctive Leukemia Entity

Introduction: Myeloid/Natural killer (NK) cell precursor acute leukemia (MNKPL) is a rare hematologic malignancy prevalent in East Asia. MNKPL is characterized by extramedullary involvement, immature lymphoblastoid morphology without myeloperoxidase (MPO) reactivity, the CD7+/CD33+/CD34+/CD56+/HLA−DR+ phenotype. MNKPL is classified as mixed phenotype acute leukemia, and not otherwise specified rare types (MPAL NOS rare types) in WHO classification. However, its characteristic clinical feature and undetermined genetic feature suggests that MNPKL leaves open the possibility of a new independent disease concept. Here, we report clinical features and genetic alterations in patients with MNKPL. Methods: The Leukemia and Lymphoma Committee of the Japanese Society of Pediatric Hematology and Oncology (JSPHO) sent out questionnaires to 110 JSPHO affiliated hospitals and collected cases of MNPKL diagnosed during the period 2000-2013. Besides, the cases published as literature were recruited. The data of clinical features, cell surface antigen profiling, overall survival (OS), and event-free survival (EFS) defined as relapse or death were also collected as a secondary survey. The protocol of this retrospective study was approved by the review boards of JSPHO and Ehime Prefectural Central Hospital. Comprehensive genetic analysis including 13 whole-exome sequences (WES), 2 target sequence, 6 RNA sequence (RNA-seq), and 8 DNA methylation analysis was performed. We also performed single-cell RNA-seq using 1 sample of MNKPL patients and a normal bone marrow sample as the reference. The research protocol was approved by the review board of TMDU. Results: Sixteen children or young adults (< 39 years old) and 2 older adults with MNKPL were identified. The median age of MNKPL patients was 11 (0.5-75) years old. There are 12 males and 6 females. The extramedullary involvement was observed in 7 patients. Complete remission after induction therapy was achieved in 8/14 (57%) patients treated with acute myeloid leukemia (AML) type chemotherapy and 2/4 (50%) patients treated with acute lymphoblastic leukemia (ALL)/non-Hodgkin lymphoma type chemotherapy, respectively. Fifteen patients underwent hematopoietic cell transplantation (HCT). The median follow-up period was 3.8 (0.1-16.0) years. 5-year OS and 5-year EFS was 49.5% and 40.7%, respectively. In genetic analysis, median 388 somatic mutations in MNKPL were identified by WES. The recurrent mutations were observed in NOTCH1 (n=5), MAML3 (n=4), NRAS, MAP3K4, RECQL4, CREBBP, ASXL2, and KMT2D (n=3, respectively), and MAML2, MAP3K1, FLT3, CARD11, MSH4, FANCI, WT1, ZNF384, and ERG (n=2, respectively). The distinct expression pattern, higher expression of RUNX3 and NOTCH1, and lower expression of BCL11B were identified in MNKPL samples which were compared to MPAL, AML, and T cell ALL in RNA-seq. The distinct methylation profile, hypomethylation of RUNX3 regulatory region, and hypermethylation of BCL11B regulatory region were identified in DNA methylation analysis. Single-cell RNA-seq analysis also showed distinct 4 subsets of MNKPL. Discussion and Conclusions: NK cells are the founding member of a family of innate lymphoid cells (ILC). Genetic abnormality of NOTCH1 pathway is a hallmark of MNPKL. RUNX3 is required for NK cell survival and proliferation in response to IL-15 signaling. RUNX3 high expression and hypomethylation of RUNX3 regulatory region also characterize MNKPL. Currently, MNKPL is classified as MPAL NOS, our genetic analysis revealed that MNKPL is a distinct group from MPAL. The prognosis of MNKPL was not satisfactory even though HCT was performed. The development of new therapeutic approaches based on these genetic analyses is highly expected. Disclosures Saito: Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

Recent Advances in the Role of Natural Killer Cells in Acute Kidney Injury.

Growing evidence is revealing a central role for natural killer (NK) cells, cytotoxic cells belonging to the broad family of innate lymphoid cells (ILCs), in acute and chronic forms of renal disease. NK cell effector functions include both the recognition and elimination of virus-infected and tumor cells and the capability of sensing pathogens through Toll-like receptor (TLR) engagement. Notably, they also display immune regulatory properties, exerted thanks to their ability to secrete cytokines/chemokines and to establish interactions with different innate and adaptive immune cells. Therefore, because of their multiple functions, NK cells may have a major pathogenic role in acute kidney injury (AKI), and a better understanding of the molecular mechanisms driving NK cell activation in AKI and their downstream interactions with intrinsic renal cells and infiltrating immune cells could help to identify new potential biomarkers and to select clinically valuable novel therapeutic targets. In this review, we discuss the current literature regarding the potential involvement of NK cells in AKI.

Protocols for Innate Lymphoid Cell Phenotypic and Functional Characterization: An Overview.

The innate immune system forms a first line of defense against infections and is an essential component in immune defense prior to the generation of sustained adaptive immune responses. Until recently this innate response was believed to be predominantly elicited by cells of myeloid origin. The last 10years have seen the discovery of an extensive family of innate lymphoid cells (ILCs) characterized by the ability to express cytokines, traditionally associated with activated T effector cells, and lack of expression of antigen receptors.ILCs are enriched at barrier surfaces and rapidly respond to alarmins within the tissue microenvironment. ILCs lack recombinant activating gene (RAG)-dependent rearranged receptors, lack myeloid and dendritic cell lineage defining factors, and possess lymphoid morphology. The first characterized innate lymphoid cells were natural killer cells (NK) cells and lymphoid tissue inducer (LTi) cells. NK cells, defined in 1975), mediate important early antigen independent immune responses. By contrast, LTi cells, defined in 1997, are vital for formation of lymph nodes during embryogenesis. In the past decade, research on ILCs has defined them as important regulators of barrier immunity. However, ILC phenotype and functional characterization is complex and requires highly specific protocols for delineating ILC specific function from other well-characterized immune cells. Within this book, each chapter provides an in-depth protocol that will expand on techniques used by laboratories to study ILC development, characterization and function in mice and humans.

Transcriptional regulation of natural killer cell development and maturation.

Natural killer cells are lymphocytes that respond rapidly to intracellular pathogens or cancer/stressed cells by producing pro-inflammatory cytokines or chemokines and by killing target cells through direct cytolysis. NK cells are distinct from B and T lymphocytes in that they become activated through a series of broadly expressed germ line encoded activating and inhibitory receptors or through the actions of inflammatory cytokines. They are the founding member of the innate lymphoid cell family, which mirror the functions of T lymphocytes, with NK cells being the innate counterpart to CD8 T lymphocytes. Despite the functional relationship between NK cells and CD8 T cells, the mechanisms controlling their specification, differentiation and maturation are distinct, with NK cells emerging from multipotent lymphoid progenitors in the bone marrow under the control of a unique transcriptional program. Over the past few years, substantial progress has been made in understanding the developmental pathways and the factors involved in generating mature and functional NK cells. NK cells have immense therapeutic potential and understanding how to acquire large numbers of functional cells and how to endow them with potent activity to control hematopoietic and non-hematopoietic malignancies and autoimmunity is a major clinical goal. In this review, we examine basic aspects of conventional NK cell development in mice and humans and discuss multiple transcription factors that are known to guide the development of these cells.

The Interplay Between Innate Lymphoid Cells and the Tumor Microenvironment.

The multifaceted roles of Innate Lymphoid Cells (ILC) have been widely interrogated in tumor immunity. Whereas, Natural Killer (NK) cells possess undisputable tumor-suppressive properties across multiple types of cancer, the other ILC family members can either promote or inhibit tumor growth depending on the environmental conditions. The differential effects of ILCs on tumor outcome have been attributed to the high degree of heterogeneity and plasticity within the ILC family members. However, it is now becoming clear that ILCs responses are shaped by their dynamic crosstalk with the different components of the tumor microenvironment (TME). In this review, we will give insights into the molecular and cellular players of the ILCs-TME interactions and we will discuss how we can use this knowledge to successfully harness the activity of ILCs for anticancer therapies.

Sense and immuno-sensibility: innate lymphoid cell niches and circuits.

Tissue-resident lymphocytes that lack expression of rearranged antigen receptors and are lineage negative for classical T and B cell markers are collectively known as innate lymphoid cells (ILCs). The ILC family is remarkably heterogeneous and exhibits plasticity; however, mature ILCs can be grouped based on their steady state expression of distinct surface receptors and transcription factors as well as production of signature cytokines following activation. The study of ILC subsets in mouse and human tissues has revealed that the elicitation and magnitude of their effector functions are determined by a combination of extrinsic cues specific to the niches in which they reside. In this short review, we will summarize some recent findings related to tissue-specific signals that govern ILC responses and localization.

The interaction of intestinal microbiota and innate lymphoid cells in health and disease throughout life.

SummaryImmunity is shaped by commensal microbiota. From early life onwards, microbes colonize mucosal surfaces of the body and thereby trigger the establishment of immune homeostasis and defense mechanisms. Recent evidence reveals that the family of innate lymphoid cells (ILCs), which are mainly located in mucosal tissues, are essential in the maintenance of barrier functions as well as in the initiation of an appropriate immune response upon pathogenic infection. In this review, we summarize recent insights on the functional interaction of microbiota and ILCs at steady‐state and throughout life. Furthermore, we will discuss the interplay of ILCs and the microbiota in mucosal infections focusing on intestinal immunity.

Rapid isolation of mouse ILCs from murine intestinal tissues.

Tissue-resident immune cells like innate lymphoid cells (ILCs) are regulators of local immunity and tissue homeostasis. Similar to Natural Killer (NK) cells, ILCs express germline-encoded natural cytotoxicity receptors (NCRs) that facilitate the rapid execution of effector functions. Recent advances using transgenic animal models have further uncovered the developmental, transcriptional, epigenetic, and functional differences between members of the ILC family. Isolation of ILCs, which are particularly enriched in non-lymphoid tissues, can often be challenging and time consuming. Here, we provide a simple and rapid protocol for the isolation of NK cells and ILCs from murine intestinal tissues. This protocol is suitable for Fluorescence Activated Cell Sorting (FACS) and intracellular analysis of cytokine and transcription factor expression using flow and mass cytometry.

Roles of innate lymphoid cells in tumor immunity and their clinical significance

Innate lymphoid cells (ILCs) are a recently characterized family of immune cells that have critical roles in innate immunity, immune regulation, maintenance of tissue homeostasis, and tissue repair and remodeling. Besides the conventional innate lymphocytes including NK cells and lymphoid tissue-inducer cells, the ILC family can be categorized into three groups, ILC1s, ILC2s and ILC3s. These non-cytotoxic ILC subsets have been identified to confer a diverse array of functions in oncogenesis and metastasis, immune surveillance, and antitumor immunity. In this review, we summarized the emerging findings in recent years regarding the roles of ILCs in immuno-oncology, and highlighted their potentials in immunotherapeutic approaches to tumors. Key words: Innate lymphoid cell; Immuno-oncology; Immunomodulation

Regulating the development of pulmonary Group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s) are members of the family of innate lymphoid cells and are innately committed to type 2 immune responses. In the lungs, ILC2s are the predominant population of innate lymphoid cells (ILCs) and their development is orchestrated by several different transcription factors ensuring lineage commitment by intrinsic regulation. ILC2s are present in the lungs from the foetal period onwards and are thus exposed to extrinsic regulation due to the airways' continuous morphological changes upon birth. In this review, we will briefly summarise the dependence of ILC2s on transcription factors and discuss recently described characteristics and function of early life ILC2s in the lungs.

Function of innate lymphoid cells in the immune-related disorders.

Innate lymphoid cells (ILCs) are a recently described group of innate immune cells that mirror the characteristics of CD4+ T cell subsets. Based on their transcriptional factor and cytokine profile, ILCs family is divided into main subgroups-ILC1s, ILC2s, and ILC3s. Recently, one new subpopulation of ILCs with immunosuppressive characteristics has been described and named as regulatory ILCs. Various roles of ILCs have been confirmed including the role during the response to microbial signals, the role in inflammation and process of tissue repair. Function of ILCs is mediated through the cytokines production and direct cell-to-cell contact. This article summarizes in detail, the relationship between the ILCs and various immune-related disorders.