Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a+Eomes+ subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a+Eomes+ NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.

Toxoplasma gondii Causes Adverse Pregnancy Outcomes by Damaging Uterine Tissue-Resident NK Cells That Secrete Growth-Promoting Factors.

Vertical transmission of the intracellular parasite, Toxoplasma gondii can lead to adverse pregnancy outcomes especially when infection occurs in early pregnancy. Decidual natural killer (dNK) cells accumulate at the maternal-fetal interface in large numbers during early pregnancy. Their nutritional roles during infection with T. gondii remain poorly defined. In the present study, we demonstrated that a functional deficiency of the uterine tissue-resident NK (trNK) cells, a subset of dNK cells, contributes to the adverse pregnancy outcomes induced by T. gondii in early pregnancy. Adverse pregnancy outcomes could be ameliorated by adoptive transfer of trNK cells. Moreover, fetal growth restriction could be improved after supplementation of growth-promoting factors. In addition to the widely recognized disturbance of the immune balance at the interface between the mother and the fetus, our study reveals a novel mechanism in T. gondii that contributes to the adverse pregnancy outcomes.

Tissue-resident CD69+ CXCR6+ Natural Killer cells with exhausted phenotype accumulate in human non-small cell lung cancer.

Natural killer (NK) cells with tissue-residency features (trNK cells) are a new subpopulation of NK cells, which plays an important role in tissue homeostasis. However, the characteristics of trNK cells in the tumor microenvironment (TME) of human cancers remain unclear. Using multicolor flow cytometry, we investigated the quantity, phenotype, and function of trNK cells in biospecimens freshly resected from 60 non-small cell lung cancer (NSCLC) patients. We successfully identified a new CD69+ CXCR6+ trNK subset with an immunomodulatory-like and exhausted phenotype, specifically accumulated in the TME of NSCLC. In vitro experiments showed that CD69+ CXCR6+ trNK cells more readily secreted IFN-γ and TNF-α spontaneously. Furthermore, the production of IFN-γ and TNF-α by tumor-infiltrating CD69+ CXCR6+ trNK cells was not induced by their reactivation in vitro, which is analogous to T-cell exhaustion. Finally, we demonstrated that the dysfunction of CD69+ CXCR6+ trNK cells could be partly ameliorated by PD-1 and CTLA-4 blockade. In summary, we identified a new dysfunctional CD69+ CXCR6+ trNK cell subset that specifically accumulates in the TME of NSCLC. Our findings suggest that CD69+ CXCR6+ trNK cells are a promising target for immune checkpoint inhibitors in the treatment of NSCLC.

Diversity of tissue-resident NK cells.

Although natural killer (NK) cells were initially named for their spontaneous tumor-killing capacity, their concept has been greatly expanded with more than 40 years of extensive investigation. Currently, NK cells are known as a heterogeneous population of innate lymphoid cell (ILC) family, consisting of different subsets with unique phenotypic and functional features. Recent studies have shown that tissue-resident NK (trNK) cells, which are distinct from conventional NK (cNK) cells, preferentially distribute in non-lymphoid tissues, such as the liver, uterus, salivary gland, and adipose. In this review, we provide a comprehensive overview of the current knowledge about the phenotype, function and development of trNK cells across different tissues and describe the similarities and differences between diverse trNK cells and cNK cells, with a particular focus on the tissue-specific characteristics of different trNK cells.

NK cell subsets in autoimmune diseases.

Natural killer (NK) cells are lymphocytes of the innate immune system. They not only exert cell-mediated cytotoxicity against tumor cells or infected cells, but also play regulatory role through promoting or suppressing functions of other immune cells by secretion of cytokines and chemokines. However, overactivation or dysfunction of NK cells may be associated with pathogenesis of some diseases. NK cells are found to act as a two edged weapon and play opposite roles with both regulatory and inducer activity in autoimmune diseases. Though the precise mechanisms for the opposite effects of NK cells has not been fully elucidated, the importance of NK cells in autoimmune diseases might be associated with different NK cell subsets, different tissue microenvironment and different stages of corresponding diseases. The local tissue microenvironment, unique cellular interactions and different stages of corresponding diseases shape the properties and function of NK cells. In this review, we focus on recent research on the features and function of different NK cell subsets, particularly tissue-resident NK cells in different tissues, and their potential role in autoimmune diseases.

Methods for Isolating and Defining Single-Cell Transcriptomes of Tissue-Resident Human NK Cells.

Natural killer (NK) cells are innate lymphocytes that control tumors and microbial infections. Human NK cells are transcriptomically and phenotypically heterogeneous. The site where NK cells develop and reside determines their phenotype and effector functions. Our current knowledge about human NK cells is primarily from blood- and bone marrow-derived NK cells. The major limitation in formulating organ-specific clinical therapy is the knowledge gap on how tissue-resident NK cells develop, home, and function. Thus, it is crucial to define the transcriptomic profiles and the transcriptional regulation of tissue-resident NK cells. The major challenges in studying tissue-resident NK cells include their total number and the complexity of the tissue. Additionally, during isolation, keeping them viable and naïve without activation are challenging tasks. Here, we provide methods for isolating and performing transcriptomic analyses of NK cells at the individual cell level. Single-cell RNA sequencing provides a higher resolution of cellular heterogeneity and a better understanding of cell-cell interactions within the microenvironment. Using these methods, we can efficiently identify distinct populations of NK cells in tissues and define their unique transcriptomic profiles.

Human-Induced CD49a+ NK Cells Promote Fetal Growth.

CD49a+ natural killer (NK) cells play a critical role in promoting fetal development and maintaining immune tolerance at the maternal-fetal interface during the early stages of pregnancy. However, given their residency in human tissue, thorough studies and clinical applications are difficult to perform. It is still unclear as to how functional human CD49a+ NK cells can be induced to benefit pregnancy outcomes. In this study, we established three no-feeder cell induction systems to induce human CD49a+ NK cells from umbilical cord blood hematopoietic stem cells (HSCs), bone marrow HSCs, and peripheral blood NK cells in vitro. These induced NK cells (iNKs) from three cell induction systems display high levels of CD49a, CD9, CD39, CD151 expression, low levels of CD16 expression, and no obvious cytotoxic capability. They are phenotypically and functionally similar to decidual NK cells. Furthermore, these iNKs display a high expression of growth-promoting factors and proangiogenic factors and can promote fetal growth and improve uterine artery blood flow in a murine pregnancy model in vivo. This research demonstrates the ability of human-induced CD49a+ NK cells to promote fetal growth via three cell induction systems, which could eventually be used to treat patients experiencing adverse pregnancy outcomes.

Plasticity of NK cells in Cancer.

Natural killer (NK) cells are crucial to various facets of human immunity and function through direct cytotoxicity or via orchestration of the broader immune response. NK cells exist across a wide range of functional and phenotypic identities. Murine and human studies have revealed that NK cells possess substantial plasticity and can alter their function and phenotype in response to external signals. NK cells also play a critical role in tumor immunity and form the basis for many emerging immunotherapeutic approaches. NK cells can directly target and lyse malignant cells with their inherent cytotoxic capabilities. In addition to direct targeting of malignant cells, certain subsets of NK cells can mediate antibody-dependent cellular cytotoxicity (ADCC) which is integral to some forms of immune checkpoint-blockade immunotherapy. Another important feature of various NK cell subsets is to co-ordinate anti-tumor immune responses by recruiting adaptive and innate leukocytes. However, given the diverse range of NK cell identities it is unsurprising that both pro-tumoral and anti-tumoral NK cell subsets have been described. Here, NK cell subsets have been shown to promote angiogenesis, drive inflammation and immune evasion in the tumor microenvironment. To date, the signals that drive tumor-infiltrating NK cells towards the acquisition of a pro- or anti-tumoral function are poorly understood. The notion of tumor microenvironment-driven NK cell plasticity has substantial implications for the development of NK-based immunotherapeutics. This review will highlight the current knowledge of NK cell plasticity pertaining to the tumor microenvironment. Additionally, this review will pose critical and relevant questions that need to be addressed by the field in coming years.

Phenotypic and Functional Plasticity of CXCR6+ Peripheral Blood NK Cells.

Human NK cells are comprised of phenotypic subsets, whose potentially unique functions remain largely unexplored. C-X-C-motif-chemokine-receptor-6 (CXCR6) + NK cells have been identified as phenotypically immature tissue-resident NK cells in mice and humans. A small fraction of peripheral blood (PB)-NK cells also expresses CXCR6. However, prior reports about their phenotypic and functional plasticity are conflicting. In this study, we isolated, expanded, and phenotypically and functionally evaluated CXCR6+ and CXCR6- PB-NK cells, and contrasted results to bulk liver and spleen NK cells. We found that CXCR6+ and CXCR6- PB-NK cells preserved their distinct phenotypic profiles throughout 14 days of in vitro expansion ("day 14"), after which phenotypically immature CXCR6+ PB-NK cells became functionally equivalent to CXCR6- PB-NK cells. Despite a consistent reduction in CD16 expression and enhanced expression of the transcription factor Eomesodermin (Eomes), day 14 CXCR6+ PB-NK cells had superior antibody-dependent cellular cytotoxicity (ADCC) compared to CXCR6- PB-NK cells. Further, bulk liver NK cells responded to IL-15, but not IL-2 stimulation, with STAT-5 phosphorylation. In contrast, bulk splenic and PB-NK cells robustly responded to both cytokines. Our findings may allow for the selection of superior NK cell subsets for infusion products increasingly used to treat human diseases.

Uterine Natural Killer Cell Heterogeneity: Lessons From Mouse Models.

Natural killer (NK) cells are the most abundant lymphocytes at the maternal-fetal interface. Epidemiological data implicate NK cells in human pregnancy outcomes. Discoveries using mouse NK cells have guided subsequent advances in human NK cell biology. However, it remains challenging to identify mouse and human uterine NK (uNK) cell function(s) because of the dynamic changes in the systemic-endocrinological and local uterine structural microenvironments during pregnancy. This review discusses functional similarities and differences between mouse and human NK cells at the maternal-fetal interface.

Tissue-resident NK cells and other innate lymphoid cells.

Natural killer (NK) cells are important innate effectors for their defense against pathogens and tumors without the need of prior sensitization. Along with the growing understanding of basic NK cell biology, it has been widely accepted that NK cells are a heterogeneous population of innate lymphoid cell (ILC) family. Apart from the conventional NK cell (cNK) subset that circulates throughout the body, some non-lymphoid tissues contain tissue-resident NK (trNK) cell subsets, and the composition of NK cell subsets varies greatly with different locations. Except for cNK cells, other ILCs are known as tissue-resident cells. In this review, we summarize the unique properties of trNK cells, discuss their lineage relationship with other ILCs, and highlight recent advances in our understanding of the functions of trNK cells and other ILCs.

Transcriptional Regulation of Mouse Tissue-Resident Natural Killer Cell Development.

Natural killer (NK) cells are cytotoxic innate lymphocytes that are well-known for their ability to kill infected or malignant cells. Beyond their roles in tumor surveillance and anti-pathogen defense, more recent studies have highlighted key roles for NK cells in a broad range of biological processes, including metabolic homeostasis, immunomodulation of T cells, contact hypersensitivity, and pregnancy. Consistent with the breadth and diversity of these functions, it is now appreciated that NK cells are a heterogeneous population, comprised of specialized and sometimes tissue-specific subsets with distinct phenotypes and effector functions. Indeed, in addition to the conventional NK cells (cNKs) that are abundant and have been well-studied in the blood and spleen, distinct subsets of tissue-resident NK cells (trNKs) and "helper" Group 1 innate lymphoid cells (ILC1s) have now been described in multiple organs and tissues, including the liver, uterus, thymus, adipose tissue, and skin, among others. The cNK, trNK, and/or helper ILC1 populations that co-exist in these various tissues exhibit both common and distinct developmental requirements, suggesting that a combination of lineage-, subset-, and tissue-specific differentiation processes may contribute to the unique functional properties of these various populations. Here, we provide an overview of the transcriptional regulatory pathways known to instruct the development and differentiation of cNK, trNK, and helper ILC1 populations in specific tissues in mice.

Corrigendum: Transcriptional Regulation of Mouse Tissue-Resident Natural Killer Cell Development.

[This corrects the article DOI: 10.3389/fimmu.2020.00309.].

NK Cells in the Human Lungs.

The lung offers one of the largest exchange surfaces of the individual with the elements of the environment. As a place of important interactions between self and non-self, the lung is richly endowed in various immune cells. As such, lung natural killer (NK) cells play major effector and immunoregulatory roles to ensure self-integrity. A better understanding of their abilities in health and diseases has been made possible over the past decade thanks to tremendous discoveries in humans and animals. By precisely distinguishing the different NK cell subsets and dissecting the ontogeny and differentiation of NK cells, both blood and tissue-resident NK populations now appear to be much more pleiotropic than previously thought. In light of these recent findings in healthy individuals, this review describes the different lung NK cell populations quantitatively, qualitatively, phenotypically, and functionally. Their identification, immunological diversity, and adaptive capacities are also addressed. For each of these elements, the impact of the mutual interactions of lung NK cells with environmental and microenvironmental factors are questioned in terms of functionality, competence, and adaptive capacities. As pulmonary diseases are major causes of morbidity and mortality worldwide, special attention is also given to the involvement of lung NK cells in various diseases, including infectious, inflammatory, autoimmune, and neoplastic lung diseases. In addition to providing a comprehensive overview of lung NK cell biology, this review also provides insight into the potential of NK cell immunotherapy and the development of targeted biologics.

Tissue-resident lymphocytes: from adaptive to innate immunity.

Efficient immune responses against invading pathogens often involve coordination between cells from both the innate and adaptive immune systems. For multiple decades, it has been believed that CD8+ memory T cells and natural killer (NK) cells constantly and uniformly recirculate. Only recently was the existence of noncirculating memory T and NK cells that remain resident in the peripheral tissues, termed tissue-resident memory T (TRM) cells and tissue-resident NK (trNK) cells, observed in various organs owing to improved techniques. TRM cells populate a wide range of peripheral organs, including the skin, sensory ganglia, gut, lungs, brain, salivary glands, female reproductive tract, and others. Recent findings have demonstrated the existence of TRM in the secondary lymphoid organs (SLOs) as well, leading to revision of the classic theory that they exist only in peripheral organs. trNK cells have been identified in the uterus, skin, kidney, adipose tissue, and salivary glands. These tissue-resident lymphocytes do not recirculate in the blood or lymphatic system and often adopt a unique phenotype that is distinct from those of circulating immune cells. In this review, we will discuss the recent findings on the tissue residency of both innate and adaptive lymphocytes, with a particular focus on CD8+ memory T cells, and describe some advances regarding unconventional T cells (invariant NKT cells, mucosal-associated invariant T cells (MAIT), and γδ T cells) and the emerging family of trNK cells. Specifically, we will focus on the phenotypes and functions of these subsets and discuss their implications in anti-viral and anti-tumor immunity.

Cytokine-Based Generation of CD49a+Eomes-/+ Natural Killer Cell Subsets.

Recent studies have identified CD49a+Eomes- and CD49a+Eomes+ subsets of tissue-resident NK (trNK) cells in different organs of the mouse. However, the characteristics of CD49a+Eomes-/+ NK cell development and the regulation of Eomes expression in NK cells remain unclear. Here, we established an in vitro cytokine-based feeder-free system in which bone marrow progenitor cells differentiate into CD49a+ NK cells. IL-15 was identified as being the key cytokine in this system that supported the development and maintenance of CD49a+ NK cells. The CD49a+ NK cells generated were Eomes-CD49b- and shared the same phenotype as hepatic trNK cells. IL-4 induced the expression of Eomes in generated NK cells and converted them into CD49a+Eomes+ cells, which were phenotypically and functionally similar to uterine trNK cells. Moreover, the IL-4/STAT6 axis was identified as being important in the generation of CD49a+Eomes+ induced NK cells. Collectively, these studies describe an approach to generate CD49a+Eomes-/+ subsets of NK cells and demonstrate important roles for IL-15 and IL-4 in the differentiation of these cells. These findings have potential for developmental research underlying the generation of different subsets of NK cells and the application of adoptive NK cell transfer therapies.

IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction.

NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6+CD69+ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies.

Tissue-resident natural killer cells in the livers.

Nature killer (NK) cells are important lymphocytes of the innate immune system, well known for their pivotal roles in immune surveillance and defense against tumor and virus-infected cells. Current studies have revealed that NK cells are not a homogeneous population, but instead consist of distinct subsets with diverse characteristics. As an organ with predominant innate immunity, the liver is enriched with NK cells, among which two distinct NK cell subsets have recently been identified: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Liver trNK cells are markedly different from cNK cells in many aspects, representing a new lineage of innate lymphoid cell (ILC) family. Here, we summarize the phenotypic and functional features of liver trNK cells, and review current knowledge regarding developmental pathway of liver trNK cells. We also overview recent advances in human liver trNK cells and discuss the striking shared hallmarks of trNK cells in different tissues.

Unique Eomes(+) NK Cell Subsets Are Present in Uterus and Decidua During Early Pregnancy.

Decidual and uterine natural killer (NK) cells have been shown to contribute to the successful pregnancy both in humans and mice. NK cells represent "cytotoxic" group 1 innate lymphoid cells (ILCs) and are distinct from the recently described "helper" ILC1. Here, we show that both in humans and mice the majority of group 1 ILC in endometrium/uterus and decidua express Eomesodermin (Eomes), thus suggesting that they are developmentally related to conventional NK cells. However, they differ from peripheral NK cells. In humans, Eomes(+) decidual NK (dNK) cells express CD49a and other markers of tissue residency, including CD103, integrin ß7, CD9, and CD69. The expression of CD103 allows the identification of different subsets of IFNγ-producing Eomes(+) NK cells. We show that TGFß can sustain/induce CD103 and CD9 expression in dNK cells and decidual CD34-derived NK cells, indicating that the decidual microenvironment can instruct the phenotype of Eomes(+) NK cells. In murine decidua and uterus, Eomes(+) cells include CD49a(-)CD49b(+) conventional NK cells and CD49a(+) cells. Notably, Eomes(+)CD49a(+) cells are absent in spleen and liver. Decidual and uterine Eomes(+)CD49a(+) cells can be dissected in two peculiar cell subsets according to CD49b expression. CD49a(+)CD49b ((-)) and CD49a(+)CD49b(+) cells are enriched in immature CD11b(low)CD27(high) cells, while CD49a(-)CD49b(+) cells contain higher percentages of mature CD11b(high)CD27(low) cells, both in uterus and decidua. Moreover, Eomes(+)CD49a(+)CD49b(-) cells decrease during gestation, thus suggesting that this peculiar subset may be required in early pregnancy rather than on later phases. Conversely, a minor Eomes(-)CD49a(+) ILC1 population present in decidua and uterus increases during pregnancy. CD49b(-)Eomes(±) cells produce mainly TNF, while CD49a(-)CD49b(+) conventional NK cells and CD49a(+)CD49b(+) cells produce both IFNγ and TNF. Thus, human and murine decidua contains unique subsets of group 1 ILCs, including Eomes(+) and Eomes(-) cells, with peculiar phenotypic and functional features. Our study contributes to re-examination of the complexity of uterine and decidual ILC subsets in humans and mice and highlights the role of the decidual microenvironment in shaping the features of these cells.