Interferon-producing killer dendritic cells (IKDCs) are a recently described subset of CD11c(lo)B220(+) cells that share phenotypic and functional properties of DCs and natural killer (NK) cells (Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, et al. 2006. Nat. Med. 12:207-213; Taieb, J., N. Chaput, C. Menard, L. Apetoh, E. Ullrich, M. Bonmort, M. Pequignot, N. Casares, M. Terme, C. Flament, et al. 2006. Nat. Med. 12:214-219). IKDC development appears unusual in that cytokines using the interleukin (IL)-2 receptor beta (IL-2Rbeta) chain but not those using the common gamma chain (gamma(c)) are necessary for their generation. By directly comparing Rag2(-/-)gamma(c)(-/y), Rag2(-/-)IL-2Rbeta(-/-), Rag2(-/-)IL-15(-/-), and Rag2(-/-)IL-2(-/-) mice, we demonstrate that IKDC development parallels NK cell development in its strict IL-15 dependence. Moreover, IKDCs uniformly express NK-specific Ncr-1 transcripts (encoding NKp46), whereas NKp46(+) cells are absent in Ncr1(gfp/+)gamma(c)(-/y) mice. Distinguishing features of IKDCs (CD11c(lo)B220(+)MHC-II(+)) were carefully examined on developing NK cells in the bone marrow and on peripheral NK cells. As B220 expression was heterogeneous, defining B220(lo) versus B220(hi) NK1.1(+) NK cells could be considered as arbitrary, and few phenotypic differences were noted between NK1.1(+) NK cells bearing different levels of B220. CD11c expression did not correlate with B220 or major histocompatibility complex (MHC) class II (MHC-II) expression, and most MHC-II(+) NK1.1(+) cells did not express B220 and were thus not IKDCs. Finally, CD11c, MHC-II, and B220 levels were up-regulated on NK1.1(+) cells upon activation in vitro or in vivo in a proliferation-dependent fashion. Our data suggest that the majority of CD11c(lo)B220(+) "IKDC-like" cells represent activated NK cells.

Natural killer dendritic cells (NKDCs) possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT) cells is required for the anti-tumor immune responses that are elicited by α -galactosylceramide ( α -GC) in mice. The rapid and strong expression of interferon- γ by NKDCs after α -GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α -GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α -GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α -GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.

Natural killer (NK) cells are innate immune lymphocytes critical for host defense against viral infection and surveillance against malignant transformation. MicroRNAs (miRNAs) are a family of small, non-coding RNAs that regulate a wide variety of cellular processes. Recent advances have highlighted the importance of miRNA-mediated post-transcriptional regulation in NK cell development, maturation, and function. This review focuses on several facets of this regulatory mechanism in NK cells: (1) the expressed NK cell miRNA transcriptome; (2) the impact of total miRNA deficiency on NK cells; (3) the role of specific miRNAs regulating NK cell development, survival, and maturation; (4) the intrinsic role of miRNAs regulating NK cell function, including cytokine production, proliferation, and cytotoxicity; and (5) the role of NK cell miRNAs in disease. Currently our knowledge of how miRNAs regulate NK cell biology is limited, and thus we also explore key open questions in the field, as well as approaches and techniques to ascertain the role of individual miRNAs as important molecular regulators.

No abstract available

Natural killer (NK) cells, as members of the innate immune system, and natural killer T (NKT) cells, bridging innate and adaptive immunity, play a prominent role in chronic inflammatory diseases and cancerogenesis, yet have scarcely been examined in oral diseases. Therefore, systematic research on the latest literature focusing on NK/NKT cell-mediated mechanisms in periodontal disease, including the time period 1988-2020, was carried out in MEDLINE (PubMed) using a predetermined search strategy, with a final selection of 25 studies. The results showed that NK cells tend to have rather proinflammatory influences via cytokine production, cytotoxic effects, dendritic-cell-crosstalk, and autoimmune reactions, while contrarily, NKT cell-mediated mechanisms were proinflammatory and immunoregulatory, ranging from protective effects via B-cell-regulation, specific antibody production, and the suppression of autoimmunity to destructive effects via cytokine production, dendritic-cell-crosstalk, and T-/B-cell interactions. Since NK cells seem to have a proinflammatory role in periodontitis, further research should focus on the proinflammatory and immunoregulatory properties of NKT cells in order to create, in addition to antibacterial strategies in dental inflammatory disease, novel anti-inflammatory therapeutic approaches modulating host immunity towards dental health.

Canines spontaneously develop many cancers similar to humans - including osteosarcoma, leukemia, and lymphoma - offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3-/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3-/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3-/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3-/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3-/CD21-/CD14-/NKp46-) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3-/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46- subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy.

When we can understand what natural killer (NK) cells recognize during an encounter with an infectious pathogen or a tumor cell, and when we can understand how the NK cell responds to that encounter, we can then begin to understand the role of NK cells in human health and how to improve upon their role for the prevention and treatment of human disease. In the quest to understand how these cells function in antiviral and antitumoral immunity, there have been previously described mechanisms established for NK cells to participate in clearing viral infections and tumors, including classical NK cell antibody dependent cellular cytotoxicity (ADCC) as well as recognition and elimination of transformed malignant cells through direct ligand interactions. However, it is now clear that there are additional mechanisms by which NK cells can participate in these critical immune tasks. Here we review two recently described types of NK cell recognition and response: the first is to primary infection with herpes virus, recognized and responded to by non-specific Fc bridged cellular cytotoxicity (FcBCC), and the second describes a novel phenotypic and functional response when a subset of NK cells recognize myeloid leukemia.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

Natural killer (NK) cells acquire effector function through a licensing process and exert anti-leukemia/tumor effect. However, there is no means to promote a licensing effect of allogeneic NK cells other than cytomegalovirus reactivation-induced licensing in allogeneic hematopoietic stem cell transplantation in human. In mice, a licensing process is mediated by Ly49 receptors which recognize self-major histocompatibility complex class I. The distribution of four Ly49 receptors showed similar pattern in congenic mice, B10, B10.BR, and B10.D2, which have B10 background. Forty Gy-irradiated 2 × 10(6) B10.D2 cells including splenocytes, peripheral blood mononuclear cells in untreated mice, or granulocyte colony-stimulating factor treated mice were injected intraperitoneally into B10 mice. We found that murine NK cells were effectively licensed by intraperitoneal injection of donor neutrophils with its corresponding NK receptor ligand in B10 mice as a recipient and B10.D2 as a donor. Mechanistic studies revealed that NK cells showed the upregulation of intracellular interferon-γ and CD107a expression as markers of NK cell activation. Moreover, enriched neutrophils enhanced licensing effect of NK cells; meanwhile, licensing effect was diminished by depletion of neutrophils. Collectively, injection of neutrophils induced NK cell licensing (activation) via NK receptor ligand interaction.

In an adaptive immune response, naive T cells proliferate during infection and generate long-lived memory cells that undergo secondary expansion after a repeat encounter with the same pathogen. Although natural killer (NK) cells have traditionally been classified as cells of the innate immune system, they share many similarities with cytotoxic T lymphocytes. We use a mouse model of cytomegalovirus infection to show that, like T cells, NK cells bearing the virus-specific Ly49H receptor proliferate 100-fold in the spleen and 1,000-fold in the liver after infection. After a contraction phase, Ly49H-positive NK cells reside in lymphoid and non-lymphoid organs for several months. These self-renewing 'memory' NK cells rapidly degranulate and produce cytokines on reactivation. Adoptive transfer of these NK cells into naive animals followed by viral challenge results in a robust secondary expansion and protective immunity. These findings reveal properties of NK cells that were previously attributed only to cells of the adaptive immune system.

Tbet-deficient mice have reduced natural killer (NK) cells in blood and spleen, but increased NK cells in bone marrow and lymph nodes, a phenotype that is thought to be the result of defective migration. Here, we revisit the role of Tbet in NK cell bone marrow egress. We definitively show that the accumulation of NK cells in the bone marrow of Tbet-deficient Tbx21-/- animals occurs because of a migration defect and identify a module of genes, co-ordinated by Tbet, which affects the localization of NK cells in the bone marrow. Cxcr6 is approximately 125-fold underexpressed in Tbx21-/- , compared with wild-type, immature NK cells. Immature NK cells accumulate in the bone marrow of CXCR6-deficient mice, and CXCR6-deficient progenitors are less able to reconstitute the peripheral NK cell compartment than their wild-type counterparts. However, the CXCR6 phenotype is largely confined to immature NK cells, whereas the Tbet phenotype is present in both immature and mature NK cells, suggesting that genes identified as being more differentially expressed in mature NK cells, such as S1pr5, Cx3cr1, Sell and Cd69, may be the major drivers of the phenotype.

Therapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities.

Natural killer (NK) cells are key players in the innate immune response. They kill virus-infected cells and are crucial for the induction of adaptive immune responses. Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes deadly T cell lymphomas in chickens. Host resistance to MDV is associated with differences in NK cell responses; however, the exact role of NK cells in the control of MDV remains unknown. In this study, we assessed if MDV can infect NK cells and alter their activation. Surprisingly, we could demonstrate that primary chicken NK cells are very efficiently infected with very virulent RB-1B MDV and the live-attenuated CVI988 vaccine. Flow cytometry analysis revealed that both RB-1B and CVI988 enhance NK cell degranulation and increase interferon gamma (IFNγ) production in vitro. In addition, we could show that the MDV Eco Q-encoded oncogene (meq) contributes to the induction of NK cell activation using meq knockout viruses. Taken together, our data revealed for the first time that NK cells are efficiently infectable with MDV and that this oncogenic alphaherpesvirus enhances NK cell degranulation and increased IFNγ production in vitro.

The failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression.

Human CD56br natural killer (NK) cells represent a small subset of CD56+ NK cells in circulation and are largely tissue-resident. The frequency and number of CD56br NK cells in blood has been shown to increase following administration of low-dose IL-2 (LD-IL2), a therapy aimed to specifically expand CD4+ regulatory T cells (Tregs). Given the potential clinical application of LD-IL-2 immunotherapy across several immune diseases, including the autoimmune disease type 1 diabetes, a better understanding of the functional consequences of this expansion is urgently needed. In this study, we developed an in vitro co-culture assay with activated CD4+ T cells to measure NK cell killing efficiency. We show that CD56br and CD56dim NK cells show similar efficiency at killing activated CD4+ conventional T (Tconv) and Treg cell subsets. However, in contrast to CD56dim cells, CD56br NK cells preferentially target highly proliferative cells. We hypothesize that CD56br NK cells have an immunoregulatory role through the elimination of proliferating autoreactive CD4+ Tconv cells that have escaped Treg suppression. These results have implications for the interpretation of current and future trials of LD-IL-2 by providing evidence for a new, possibly beneficial immunomodulatory mechanism of LD-IL-2-expanded CD56br NK cells.

Natural killer T (NKT) cells are αβ T cell receptor (TCR) expressing innate-like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I-like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi-invariant Vα14-Jα18 (mouse, Vα24-Jα18 in humans) TCR reactive to the prototypic ligand α-galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d-presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18-/- MHCII-/- mice that harbour type II but not type I NKT cells, and CD1d-/- MHCII-/- mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4- CD8- (double negative, DN) TCRβ+ cells expressing NKG2D or NKG2A/C/E in Jα18-/- MHCII-/- mice compared with CD1d-/- MHCII-/- mice, suggesting that 30%-50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+ , while the DN cells were CD49b+ and CD62L+ . Both subsets expressed the NKT cell-associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN-γ, but rarely IL-4 and IL-17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1-like cytokine production.

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.

To investigate the changes in the maternal immune system at term pregnancy, we studied the expression of natural cytotoxicity receptors (NCRs) and the cytokine production of NK cells in term placenta decidua and peripheral blood.

Antiviral T cells are thought to regulate whether hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections result in viral control, asymptomatic persistence or severe disease, although the reasons for these different outcomes remain unclear. Recent genetic evidence, however, has indicated a correlation between certain natural killer (NK)-cell receptors and progression of both HIV and HCV infection, implying that NK cells have a role in these T-cell-associated diseases. Although direct NK-cell-mediated lysis of virus-infected cells may contribute to antiviral defence during some virus infections--especially murine cytomegalovirus (MCMV) infections in mice and perhaps HIV in humans--NK cells have also been suspected of having immunoregulatory functions. For instance, NK cells may indirectly regulate T-cell responses by lysing MCMV-infected antigen-presenting cells. In contrast to MCMV, lymphocytic choriomeningitis virus (LCMV) infection in mice seems to be resistant to any direct antiviral effects of NK cells. Here we examine the roles of NK cells in regulating T-cell-dependent viral persistence and immunopathology in mice infected with LCMV, an established model for HIV and HCV infections in humans. We describe a three-way interaction, whereby activated NK cells cytolytically eliminate activated CD4 T cells that affect CD8 T-cell function and exhaustion. At high virus doses, NK cells prevented fatal pathology while enabling T-cell exhaustion and viral persistence, but at medium doses NK cells paradoxically facilitated lethal T-cell-mediated pathology. Thus, NK cells can act as rheostats, regulating CD4 T-cell-mediated support for the antiviral CD8 T cells that control viral pathogenesis and persistence.

The multiple myeloma (MM) landscape has changed in the last few years, but most patients eventually relapse because current treatment modalities do not target clonogenic stem cells, which are drug-resistant and can self-renew. We hypothesized that side population (SP) cells represent myeloma clonogenic stem cells and, searching for new treatment strategies, analyzed the anti-myeloma activity of natural killer (NK) cells against clonogenic cells. Activated and expanded NK cells (NKAE) products were obtained by co-culturing NK cells from MM patients with K562-mb15-41BBL cell line and characterized by flow cytometry. Functional experiments against MM cells were performed by Eu-TDA release assays and methylcellulose clonogenic assays. Side population was detected by Dye Cycle Violet labeling and then characterized by flow cytometry and RNA-Seq. Self-renewal capacity was tested by clonogenic assays. Sorting of both kind of cells was performed for time-lapse microscopy experiments. SP cells exhibited self-renewal potential and overexpressed genes involved in stem cell metabolism. NK cells from MM patients exhibited dysregulation and had lower anti-tumor potential against clonogenic cells than healthy donors' NK cells. Patients' NK cells were activated and expanded. These cells recovered cytotoxic activity and could specifically destroy clonogenic myeloma cells. They also had a highly cytotoxic phenotype expressing NKG2D receptor. Blocking NKG2D receptor decreased NK cell activity against clonogenic myeloma cells, and activated NK cells were able to destroy SP cells, which expressed NKG2D ligands. SP cells could represent the stem cell compartment in MM. This is the first report describing NK cell activity against myeloma clonogenic cells.