Purpose of review
Inborn errors of human natural killer (NK) cells may affect the development of these cells, their function, or both. There are two broad categories of genetic defects of NK-cell development, depending on whether the deficiency is apparently specific to NK cells or clearly affects multiple hematopoietic lineages. We review here recent progress in the genetic dissection of NK deficiencies (NKDs).
Patients with severe combined immunodeficiencies (SCID) bearing mutations of ADA, AK2, IL2RG and JAK3 genes present NKDs and are prone to a broad range of infections. Patients with GATA2 deficiency are susceptible to both mycobacterial and viral infections and display NKD and a lack of monocytes. Patients with MCM4 deficiency display an apparently selective NKD associated with viral infections, but they also display various non hematopoietic phenotypes, including adrenal insufficiency and growth retardation.
These studies have initiated genetic dissection of the development of human NK cells. Further studies are warranted, including the search for genetic etiologies of NKD in particular. This research may lead to the discovery of molecules specifically controlling the development of NK cells and to improvements in our understanding of the hitherto elusive function of these cells in humans.
Natural Killer cells; Immunodeficiencies; Genetic diseases
Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by a lymphocytic exocrinopathy. However, patients often have evidence of systemic autoimmunity and they are at markedly increased risk for the development of non- Hodgkin’s lymphoma. Similar to other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, though the precise etiology remains uncertain. NCR3/NKp30 is a NK-specific activating receptor regulating the cross-talk between NK and dendritic cells and type II IFN secretion. We performed a case-control study of genetic polymorphisms of the NCR3/NKp30 gene and found that rs11575837 (G>A) residing in the promoter was associated with reduced gene transcription and function as well as protection to pSS. We also demonstrated that circulating levels of NCR3/NKp30 were markedly increased among pSS patients compared with controls and correlated with higher NCR3/NKp30 but not CD16-dependent IFN-γ secretion by NK cells. Excess accumulation of NK cells in minor salivary glands correlated with the severity of the exocrinopathy. B7H6, the ligand of NKp30, was expressed by salivary epithelial cells. These findings suggest that NK cells may promote an NKp30-dependent inflammatory state in salivary glands, and that blockade of the B7H6/NKp30 axis could be clinically relevant in pSS.
Sjögren’s syndrome; autoimmunity; NK cells; innate immunity; NKp30/NCR3
Inflammatory cytokines drive NK cell expansion in the absence of the transcription factor Nfil3, and Nfil3 is dispensable for the maintenance and function of mature NK cells.
Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3−/− mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3−/− NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15–dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells.
Cyclophosphamide is one of several clinically important cancer drugs whose therapeutic efficacy is due in part to their ability to stimulate anti-tumor immune responses. Studying mouse models, we demonstrate that cyclophosphamide alters the composition of microbiota in the small intestine and induces the translocation of selected species of Gram+ bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of “pathogenic” T helper 17 (pTh17) cells and memory Th1 immune responses. Tumor-bearing mice that were germ-free or that had been treated with antibiotics to kill Gram+ bacteria showed a reduction in pTh17 responses and their tumors were resistant to cyclophosphamide. Adoptive transfer of pTh17 cells partially restored the anti-tumor efficacy of cyclophosphamide. These results suggest that the gut microbiota help shape the anticancer immune response.
A major challenge for the immune system is to control pathogens and stressed cells, such as infected or tumors cells, while sparing healthy self-cells. To achieve this tolerance to self, immune cells must recognize and differentiate “self” versus “nonself” and “self” versus “altered self.” In the absence of self-tolerance, cells of the adaptive immune system attack healthy cells and cause autoimmune diseases such as lupus, psoriasis, and type I diabetes. Mechanisms at work to ensure tolerance in the innate immune system are still poorly understood. Natural killer cells are innate immune lymphocytes, which have the capacity to kill cellular targets and produce cytokines without prior specific sensitization. Because of these intrinsic effector capacities, tolerance mechanisms must exist to prevent autoreactivity. Herein, we will review the present knowledge on NK cell tolerance.
Natural killer (NK) cells destroy pathogens and stressed cells; tolerance mechanisms prevent autoreactivity. NK cell self-tolerance relies on both MHC-I-dependent and -independent mechanisms.
Eric Vivier and colleagues discuss recent publications demonstrating that T reg cell restriction of IL-2 availability regulates NK cell function.
In light of their role in the immune response against tumors and viruses, natural killer (NK) cells represent a promising target for immunotherapy. Before this target is reached, the various mechanisms that control NK cell activity must first be identified and understood. In the past decades, studies have identified two critical processes that prevent spontaneous NK cell–mediated autoimmune activation while maximizing the efficiency of these cells during an immune response. First is the education process, whereby NK cells adapt to their environment by sensing ligands for inhibitory and activating receptors. Second is the priming phase of NK cell activation, which arms NK cells with appropriate cytotoxic molecules during inflammation. New studies now indicate that NK cell proliferation, accumulation, and activation are also under the control of regulatory T cells that restrict availability of IL-2 released by activated CD4+ T cells. Together with other recent studies, these data highlight the importance of the adaptive immune system in the regulation of NK cell activity.
The role of the immune response to oncolytic Herpes Simplex viral (oHSV) therapy for glioblastoma is controversial. Within hours of oHSV infection of human or syngeneic glioblastoma in mice, activated natural killer (NK) cells are recruited to the site of infection. This response significantly diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on NK cell natural cytotoxicity receptors (NCR) NKp30 and NKp46, whose ligands were up-regulated in oHSV-infected glioblastoma cells. HSV titers and oHSV efficacy were increased in Ncr1−/− mice and in a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These in vitro and in vivo (mouse) results demonstrate that glioblastoma virotherapy is partly limited by an antiviral NK cell response involving specific NCRs, uncovering novel potential targets to enhance cancer virotherapy.
Herpes simplex virus; gene therapy; oncolytic virus; brain tumor; microglia; macrophages
Although hepatic fibrosis typically follows chronic inflammation, fibrosis will often regress after cessation of liver injury. Here we examined whether liver dendritic cells (DC) play a role in liver fibrosis regression using carbon tetrachloride (CCl4) to induce liver injury. We examined DC dynamics during fibrosis regression and their capacity to modulate liver fibrosis regression upon cessation of injury. We show that conditional DC depletion soon after discontinuation of the liver insult leads to delayed fibrosis regression and reduced clearance of activated hepatic stellate cells, the key fibrogenic cell in liver. Conversely, DC expansion induced either by Flt3L (Fms-like tyrosine kinase-3 ligand) or adoptive transfer of purified DC accelerates liver fibrosis regression. DC modulation of fibrosis was partially dependent on MMP-9, as MMP-9 inhibition abolished Flt3L-mediated effect and the ability of transferred DC to accelerate fibrosis regression. In contrast, transfer of DC from MMP-9 deficient mice failed to improve fibrosis regression.
Altogether, these results suggest that DC increase fibrosis regression, and that the effect is correlated with their production of MMP-9. These results also suggest that Flt3L treatment during fibrosis resolution merits evaluation to accelerate regression of advanced liver fibrosis.
Flt3L; NK cells; MMP-9; collagen; hepatic stellate cells
Cells of the immune system have evolved various molecular mechanisms to sense their environment and react to alterations of self. NK cells are lymphocytes with effector and regulatory functions, which are remarkably adaptable to changes in self. In a study published in this issue of the JCI, Tarek and colleagues report the clinical benefits of manipulating NK cell adaptation to self in an innovative mAb-based therapy against neuroblastoma (NB). This novel therapeutic strategy should stimulate further research on NK cell therapies.
Natural killer cells, as a major source of interferon-γ, contribute to the amplification of the inflammatory response as well as to mortality during severe sepsis in animal models.
We studied the phenotype and functions of circulating NK cells in critically-ill septic patients.
Blood samples were taken <48 hours after admission from 42 ICU patients with severe sepsis (n = 15) or septic shock (n = 14) (Sepsis group), non-septic SIRS (n = 13) (SIRS group), as well as 21 healthy controls. The immuno-phenotype and functions of NK cells were studied by flow cytometry.
The absolute number of peripheral blood CD3–CD56+ NK cells was similarly reduced in all groups of ICU patients, but with a normal percentage of NK cells. When NK cell cytotoxicity was evaluated with degranulation assays (CD107 expression), no difference was observed between Sepsis patients and healthy controls. Under antibody-dependent cell cytotoxicity (ADCC) conditions, SIRS patients exhibited increased CD107 surface expression on NK cells (62.9[61.3–70]%) compared to healthy controls (43.5[32.1–53.1]%) or Sepsis patients (49.2[37.3–62.9]%) (p = 0.002). Compared to healthy (10.2[6.3–13.1]%), reduced interferon-γ production by NK cells (K562 stimulation) was observed in Sepsis group (6.2[2.2–9.9]%, p<0.01), and especially in patients with septic shock. Conversely, SIRS patients exhibited increased interferon-γ production (42.9[30.1–54.7]%) compared to Sepsis patients (18.4[11.7–35.7]%, p<0.01) or healthy controls (26.8[19.3–44.9]%, p = 0.09) in ADCC condition.
Extensive monitoring of the NK-cell phenotype and function in critically-ill septic patients revealed early decreased NK-cell function with impaired interferon-γ production. These results may aid future NK-based immuno-interventions.
Neutropenia in mice and humans results in the generation of NK cells with an immature and hyporesponsive phenotype.
Natural killer (NK) cells are bone marrow (BM)–derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases.
The risk of influenza infection depends on biological characteristics, individual or collective behaviors and the environmental context. The Cohorts for Pandemic Influenza (CoPanFlu) France study was set up in 2009 after the identification of the novel swine-origin A/H1N1 pandemic influenza virus. This cohort of 601 households (1450 subjects) representative for the general population aims at using an integrative approach to study the risk and characteristics of influenza infection as a complex combination of data collected from questionnaires regarding sociodemographic, medical, behavioral characteristics of subjects and indoor environment, using biological samples or environmental databases.
Households were included between December 2009 and July 2010. The design of this study relies on systematic follow-up visits between influenza seasons and additional visits during influenza seasons, when an influenza-like illness is detected in a household via an active surveillance system. During systematic visits, a nurse collects individual and environmental data on questionnaires and obtains blood samples from all members of the household. When an influenza-like-illness is detected, a nurse visits the household three times during the 12 following days, and collects data on questionnaires regarding exposure and symptoms, and biological samples (including nasal swabs) from all subjects in the household. The end of the follow-up period is expected in fall 2012.
The large amount of data collected throughout the follow-up will permit a multidisciplinary study of influenza infections. Additional data is being collected and analyzed in this ongoing cohort. The longitudinal analysis of these households will permit integrative analyses of complex phenomena such as individual, collective and environmental risk factors of infection, routes of transmission, or determinants of the immune response to infection or vaccination.
Influenza a virus H1N1 subtype; Cohort study; Risk factors; France
Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56dim NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients’ fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients’ growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56dim subset in patients was associated with a lower rate of NK CD56bright cell proliferation, and the maturation of NK CD56bright cells toward an NK CD56dim phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to the association of an infectious agent with lymphocyte cytotoxicity defects, either of congenital genetic origin in children or presumably acquired in adults. In HLH patients, an excess of lymphocyte or macrophage cytokines, such as IFN-γ and TNFα is present in serum. In animal models of the disease, IFN-γ and TNF-α have been shown to play a central pathogenic role. In humans, unusually high concentrations of IL-18, an inducer of IFN-γ, and TNF-α have been reported, and are associated with an imbalance between IL-18 and its natural inhibitor IL-18 binding protein (IL-18BP) resulting in an excess of free IL-18. Here we studied whether IL-18BP could reduce disease severity in an animal model of HLH. Mouse cytomegalovirus infection in perforin-1 knock-out mice induced a lethal condition similar to human HLH characterized by cytopenia with marked inflammatory lesions in the liver and spleen as well as the presence of hemophagocytosis in bone marrow. IL-18BP treatment decreased hemophagocytosis and reversed liver as well as spleen damage. IL-18BP treatment also reduced both IFN-γ and TNF-α production by CD8+ T and NK cells, as well as Fas ligand expression on NK cell surface. These data suggest that IL-18BP is beneficial in an animal model of HLH and in combination with anti-infectious therapy may be a promising strategy to treat HLH patients.
IL-18; IL-18 binding protein; natural killer cells; hemophagocytic lymphohistiocytosis; macrophage activation syndrome
Understanding Natural Killer (NK) cell anatomical distribution is key to dissect the role of these unconventional lymphocytes in physiological and disease conditions. In mouse, NK cells have been detected in various lymphoid and non-lymphoid organs, while in humans the current knowledge of NK cell distribution at steady state is mainly restricted to lymphoid tissues. The translation to humans of findings obtained in mice is facilitated by the identification of NK cell markers conserved between these two species. The Natural Cytotoxicity Receptor (NCR) NKp46 is a marker of the NK cell lineage evolutionary conserved in mammals. In mice, NKp46 is also present on rare T cell subsets and on a subset of gut Innate Lymphoid Cells (ILCs) expressing the retinoic acid receptor-related orphan receptor γt (RORγt) transcription factor. Here, we documented the distribution and the phenotype of human NKp46+ cells in lymphoid and non-lymphoid tissues isolated from healthy donors. Human NKp46+ cells were found in splenic red pulp, in lymph nodes, in lungs, and gut lamina propria, thus mirroring mouse NKp46+ cell distribution. We also identified a novel cell subset of CD56dimNKp46low cells that includes RORγt+ ILCs with a lineage−CD94−CD117brightCD127bright phenotype. The use of NKp46 thus contributes to establish the basis for analyzing quantitative and qualitative changes of NK cell and ILC subsets in human diseases.
NKp46; human NK cells; tissue distribution; phenotype; expression profiling
Severe sepsis and septic shock are still deadly conditions urging to develop novel therapies. A better understanding of the complex modifications of the immune system of septic patients is needed for the development of innovative immunointerventions. Natural killer (NK) cells are characterized as CD3−NKp46+CD56+ cells that can be cytotoxic and/or produce high amounts of cytokines such as IFN-γ. NK cells are also engaged in crosstalks with other immune cells, such as dendritic cells, macrophages, and neutrophils. During the early stage of septic shock, NK cells may play a key role in the promotion of the systemic inflammation, as suggested in mice models. Alternatively, at a later stage, NK cells-acquired dysfunction could favor nosocomial infections and mortality. Standardized biological tools defining patients' NK cell status during the different stages of sepsis are mandatory to guide potential immuno-interventions. Herein, we review the potential role of NK cells during severe sepsis and septic shock.
Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity endowed with constitutive cytolytic functions. More recently, a more nuanced view of NK cells has emerged. NK cells are now recognized to express a repertoire of activating and inhibitory receptors that is calibrated to ensure self-tolerance while allowing efficacy against assaults such as viral infection and tumor development. Moreover, NK cells do not react in an invariant manner but rather adapt to their environment. Finally, recent studies have unveiled that NK cells can also mount a form of antigen-specific immunologic memory. NK cells thus exert sophisticated biological functions that are attributes of both innate and adaptive immunity, blurring the functional borders between these two arms of the immune response.
Allotransplantation of natural killer (NK) cells has been shown to be a key factor in the control and cure of at least some hematologic diseases, such as acute myeloid leukemia or pediatric acute lymphocytic leukemia. These results support the idea that stimulation of NK cells could be an important therapeutic tool in many diseases, and several such approaches are now in clinical trials, sometimes with conflicting results. In parallel, recent advances in the understanding of the molecular mechanisms governing NK-cell maturation and activity show that NK-cell effector functions are controlled by complex mechanisms that must be taken into account for optimal design of therapeutic protocols. We review here innovative protocols based on allotransplantation, use of NK-cell therapies, and use of newly available drug candidates targeting NK-cell receptors, in the light of fundamental new data on NK-cell biology.
Inhibitory receptors that engage self-MHC class I molecules enable NK cells to detect disease-associated loss of MHC class I on surrounding cells. Previous studies showed that some NK cells lack all receptors for self-MHC class I, yet fail to exhibit autoimmunity because they are generally hyporesponsive to stimulation. We asked whether NK cells exist in only two states, responsive and hyporesponsive, corresponding to cells that express or fail to express inhibitory receptors for self-MHC class I. The alternative model is that NK cells vary continuously in their responsiveness, based on variations in the number of different inhibitory and stimulatory receptors they express, which is known to vary. Here we show in the murine system that NK cell responsiveness increases quantitatively with each added self MHC-specific inhibitory receptor. Genetic analysis demonstrated that interactions of each of the receptors with self-MHC class I were necessary to observe augmented responsiveness. These findings suggest that NK cell responsiveness is comparable to a rheostat: it is tuned to an optimal set point depending on the inhibitory and stimulatory interactions encountered in the normal environment, so as to ensure self-tolerance and yet optimize sensitivity to changes in normal cells.
Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.
Fc receptor-like 6 (FCRL6), the most recently characterized member of the FCRL family, is a cell surface glycoprotein with tyrosine-based regulatory potential. An extensive survey of human hematopoietic tissues disclosed that FCRL6 expression by NK and T cell subpopulations increases as a function of differentiation and is remarkably restricted to mature lymphocytes with cytotoxic capability. In particular, FCRL6 distinguishes perforin-expressing CD56dim NK cells, Vδ1+ and Vδ2+ γδ T cells, effector and effector memory CD8+ T cells, and rare cytotoxic CD4+ T cells in adult tissues. Analysis of this receptor in B cell chronic lymphocytic leukemia (CLL) was also performed. FCRL6 was found to mark significantly expanded populations of cytotoxic CD8+ T, CD4+ T, and NK cells in patients with CLL. Despite sequence homology with the known Fc receptors for IgG and IgE, FCRL6 did not bind immunoglobulin. Although FCRL6 can be tyrosine-phosphorylated, its antibody-mediated ligation was unable to influence cellular activation. Collectively these results demonstrate that FCRL6 is a distinct indicator of cytotoxic effector lymphocytes that is upregulated in diseases characterized by chronic immune stimulation.
Human NK cells; Human cytotoxic T cells; Fc Receptor-like; Chronic lymphocytic leukemia
Lymphoid tissue-inducer cells are hematopoietic cells essential for the organogenesis of several lymphoid structures during both fetal and adult life, whereas natural killer cells are key effector lymphocytes of the innate immune system. A series of recent reports has identified RORγt+NKp46+ interleukin-22-producing cells in gut and tonsils that share features with both lymphoid tissue-inducer cells and natural killer cells and that may be involved in mucosal immunity and homeostasis.
Multinucleated giant cells, formed by fusion of macrophages, are a hallmark of granulomatous inflammation. With a genetic approach, we show that signaling through the adaptor protein DAP12 (DNAX activating protein of 12 kD), its associated receptor triggering receptor expressed by myeloid cells 2 (TREM-2), and the downstream protein tyrosine kinase Syk is required for the cytokine-induced formation of giant cells and that overexpression of DAP12 potentiates macrophage fusion. We also present evidence that DAP12 is a general macrophage fusion regulator and is involved in modulating the expression of several macrophage-associated genes, including those encoding known mediators of macrophage fusion, such as DC-STAMP and Cadherin 1. Thus, DAP12 is involved in programming of macrophages through the regulation of gene and protein expression to induce a fusion-competent state.