Cellular ligands of activating NK receptors

Human natural killer (NK) cells are equipped with a series of surface receptors that recognise different cellular ligands on potential target cells. Some of these ligands [e.g. human leukocyte antigen (HLA) class I] prevent an NK-mediated attack by interacting with inhibitory NK receptors (e.g. killer Ig-like receptors). Other ligands interact with activating NK receptors that, once engaged, induce both cytotoxicity and lymphokine release. Tumour transformation (or viral infection) frequently results in downregulation of surface HLA class I molecules together with upregulation or de novo expression of ligands of triggering NK receptors. Thus, transformed cells can become highly susceptible to NK-mediated lysis. However, although NK cells use different means to identify and fight target cells, target cells have various strategies to hide themselves, and disarm or even confuse the immune system.     

Multiple natural killer cell-activating signals are inhibited by major histocompatibility complex class I expression in target cells

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggest that specific properties of the target cell other than the absence of class I expression are necessary to activate NK-mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin-2 activated NK cells, differ in their capacity to kill class I-deficient lymphoblast target cells. In general, class I-deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody-dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK-mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I-mediated inhibition.     

Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines

The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30, 2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity.     

Natural killer lymphocytes: "null cells" no more

Natural Killer (NK) lymphocytes were initially described as potent effector cells that, unlike T lymphocytes, were able to kill targets in the absence of a priori stimulation and without specific recognition mechanisms. Over the past ten years however, it has been clearly demonstrated that NK cell function is regulated by a number of surface receptors that bind specific ligands expressed by target cells. Some of these receptors display inhibitory functions and recognize MHC class I molecules expressed by normal autologous cells that, as a consequence, are spared from indiscriminate NK-mediated killing. Other receptors are involved in NK cell activation against allogeneic cells or cells that, upon viral infection or tumor transformation, down-regulate MHC Class I expression. Altogether these data provide important advances toward the understanding of the complexity of the molecular mechanisms that regulate NK-mediated functions.     

Comparative analysis of NK- or NK-CTL-mediated lysis of immature or mature autologous dendritic cells

Natural killer (NK) cells have been shown to kill efficiently autologous immature dendritic cells (iDC), while sparing those undergone maturation. In this study we investigated the effect of the interaction between autologous DC and NK-cytolytic T lymphocytes (NK-CTL), a subset of HLA-E-restricted CD8(+) T cells that express HLA class I-specific inhibitory NK receptors. Although these cells share with NK cells various phenotypic and functional features (such as the capacity to lyse most allogeneic, NK-susceptible tumor cell lines), different from NK cells, NK-CTL failed to lyse autologous DC. However, after pulsing DC with a cytomegalovirus-derived, HLA-E-binding peptide recognized by NK-CTL, both iDC and mature DC became highly susceptible to lysis. On the other hand,the addition of the peptide resulted in the down-regulation of the NK-mediated lysis of the same autologous iDC. The capability of killing autologous DC, presenting a non-self, HLA-E-binding peptide, may represent a feedback mechanism by which NK-CTL down-regulate HLA-E-restricted responses to certain pathogens.     

Non-MHC-restricted CD4+ T lymphocytes are regulated by HLA-Cw7-mediated inhibition

Natural killer cells (NK cells) represent an important component of innate immunity with the capacity to kill many tumor and virus-infected cells. The discovery of several classes of killer cell inhibitory receptors expressed by NK cells that bind specific MHC class I ligands on target cells provides detailed insight into the regulation of NK cells. Inhibitory receptors deliver negative signals following MHC ligand binding that abrogate cytotoxicity and, thus, determine the specificity of NK effector cell function. Here, we describe a novel subset of human memory CD4⁺ T lymphocytes that display an NK-like pattern of regulation. These CD4⁺ T cells display non-MHC-restricted cytotoxicity that is governed by HLA-Cw7 mediated inhibition. In NK cells, such specificity is associated with expression of the inhibitory receptor p58.2. In contrast, neither p58.2 nor other known inhibitory receptors were detected on these non-MHC-restricted CD4⁺ T cells. This suggests that these cells are regulated by a hitherto unknown inhibitory receptor. The finding that interactions with MHC molecules downregulate the function of these CD4⁺ T cells suggests that these non-MHC-restricted T cells may function to detect and eliminate cells with aberrant MHC expression.     

Surface NK receptors and their ligands on tumor cells

The identification of MHC-class I-specific inhibitory receptors in humans and mice provided a first explanation of why NK cells can kill target cells that have lost or underexpress MHC-class I molecules but spare normal cells. However, the molecular basis of NK-mediated recognition and tumor cell killing revealed a higher degree of complexity. Thus, under pathological conditions, NK cells may express insufficient amounts of triggering receptors and target cells may or may not express ligands for such receptors. Here we briefly illustrate the main NK receptors and their cellular ligands and we delineate the major receptor/ligands interactions leading to NK cell activation and tumor cell lysis.     

Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays

A microelectronic sensor-based platform, the RT-CES (real time electronic sensing) system, is introduced for label free assessment of natural killer (NK) cell-mediated cytotoxic activity. The RT-CES system was used to dynamically and quantitatively monitor NK-mediated cytotoxic activity towards 8 different adherent target cell lines, including cancer cell lines commonly used in laboratories. The cytotoxic activity monitored by RT-CES system was compared with standard techniques such as MTT measurement and shows good correlation and sensitivity. To test the specificity of the assay, pharmacological agents that inhibit NK cell degranulation and cytotoxic activity were employed and were shown to selectively and dose-dependently inhibit NK-mediated cytotoxic activity toward target cells. In summary, the RT-CES system offers fully automated measurement of cytotoxic activity in real time, which enables large-scale screening of chemical compounds or genes responsible for the regulation of NK-mediated cytotoxic activity.     

Histocompatibility antigens and natural killer susceptibility

The central question of the nature of the structure(s) involved in the recognition of targets by natural killer (NK) cells remains unresolved. Although NK-mediated cytotoxicity is not MHC-restricted, it has been suggested that these cells could recognize the targets more effectively in the absence of MHC class I antigens. In this paper we review the contradictory results obtained when studying the NK susceptibility of cell lines which constitutively express different levels of MHC antigens, or which have been induced to express MHC antigens by gene transfection or gamma-interferon treatment. Taken together, the results indicate that MHC antigens play a differential role in NK lysis depending on the nature of the target cells used; MHC class I antigens play a role in the NK resistance of cells from a hematopoietic lineage, but this does not extend to cells from other origins. The data reviewed also support the hypothesis that MHC class I antigens induced NK resistance by interfering with target structures, and that multiple NK molecules are involved in NK-mediated lysis as part of a possible advanced recognition system.     

PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis

The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by "orphan" triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60-65 kDa. Tryptic digestion and mass spectra analysis of purified proteins identified these molecules as PVR and Nectin-2, respectively. PVR-Fc and Nectin-2-Fc chimeric molecules stained COS-7 cells expressing the DNAM-1 activating receptor and conversely, PVR and Nectin-2 CHO-K cell transfectants were stained by DNAM-1-Fc. Thus, both PVR and Nectin-2 represent specific ligands for DNAM-1. Importantly, the specific interaction between DNAM-1 (in NK cells) and PVR or Nectin-2 (in target cells) enhanced the NK-mediated lysis of tumor cells that was downregulated by mAb-mediated masking of the receptor or its ligands.     

Human NK cell response to pathogens

NK cells represent important effectors of the innate immunity in the protection of an individual from microbes. During an NK-mediated anti-microbial response, the final fate (survival or death) of a potential infected target cell depends primarily on the type and the number of receptor/ligand interactions occurring at the effector/target immune synapse. The identification of an array of receptors involved in NK cell triggering has been crucial for a better understanding of the NK cell biology. In this context, NCR play a predominant role in NK cell activation during the process of natural cytotoxicity. Regarding the NK-mediated pathogen recognition and NK cell activation, an emerging concept is represented by the involvement of TLRs and activating KIRs.NK cells express certain TLRs in common with other innate cell types. This would mean that specific TLR ligands are able to promote the simultaneous and synergistic stimulation of these innate cells, providing a coordinated mechanism for regulating the initiation and amplification of immune responses.Evidences have been accumulated indicating that viral infections may have a significant impact on NK cell maturation, promoting the expansion of phenotypically and functionally aberrant NK cell subpopulations. For example, during chronic HIV-infection, an abnormal expansion of a dysfunctional CD56neg NK cell subset has been detected that may explain, at least in part, the defective NK cell-mediated antiviral activity. An analogous imbalance of NK cell subsets has been detected in patients receiving HSCT to cure high risk leukemias and experiencing HCMV infection/reactivation. Remarkably, NK cells developing after CMV reactivation may contain “memory-like” or “long-lived” NK cells that could exert a potent anti-leukemia effect.     

NKp46 regulates allergic responses

Natural killer (NK) cells are cytotoxic cells that are able to rapidly kill viruses, tumor cells, parasites, bacteria, and even cells considered “self”. The activity of NK cells is controlled by a fine balance of inhibitory and activating signals mediated by a complex set of different receptors. However, the function of NK cells is not restricted only to the killing of target cells, NK cells also possess other properties such as the secretion of proangiogenic factors during pregnancy. Here, we demonstrate another unique NK‐cell activity, namely the regulation of T‐cell mediated allergic responses, which is dependent on the NK‐cell specific receptor NKp46 (Ncr1 in mice). Using mice in which the Ncr1 gene has been replaced with a green fluorescent protein, we demonstrate reduced delayed‐type hypersensitivity and airway hypersensitivity. Interestingly, we show that this reduction in airway hypersensitivity is due to differences in the stimulation of T cells resulting in an altered cytokine profile.     

NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals

A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.     

Killer immunoglobulin-like receptors

Killer Ig-like receptors (KIRs) are surface inhibitory receptors specific for allelic forms of human leukocyte antigen (HLA) class I molecules, which are expressed by natural killer (NK) cells and a subset of T lymphocytes. Upon engagement with HLA class I molecules, KIRs block NK cell activation and function. Cells lacking HLA class I molecules are promptly killed by NK cells because of the predominant effect of several activating NK receptors. The NK-mediated killing of these cells might represent an important defence mechanism, antagonizing spreading of pathogens and tumours. Evidence has been accumulated that KIR-encoding genes have evolved and diversified rapidly in primates and in humans. Similar to HLA loci, KIR sequences are highly polymorphic and, moreover, KIR haplotypes greatly vary in the number of the type of genes they contain. KIR gene expression is regulated by mechanisms of DNA methylation. As recently shown, the HLA class I regulated control of NK cell function can be exploited in an allogeneic bone marrow transplantation setting to eradicate acute myeloid leukaemias.     

The evolutionary arms race between NK cells and viruses: Who gets the short end of the stick?

NK cells are innate lymphocytes that play a key role in the control of various viral infections. Recent studies indicate that NK cells may acquire some features of adaptive immune cells, including the formation of long‐lived memory cells. A large and growing body of data indicates that NK cells regulate the adaptive immune response as well. The function and the activation status of NK cells are tightly regulated by signals induced by a broad range of inhibitory and activating cell surface receptors and cytokines released by other immune cells. Here, we review the function of mouse NK‐cell receptors involved in virus control and in the regulation of the adaptive immune response. In addition, we discuss viral strategies used to evade NK‐cell‐mediated control during infection. Finally, the role of several activating Ly49 receptors specific for mouse cytomegalovirus (MCMV), as well as some controversial issues in the field, will be discussed.     

Dysregulated cellular functions and cell stress pathways provide critical cues for activating and targeting natural killer cells to transformed and infected cells

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer.