Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations.

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

Decidual natural-killer-cell interaction with trophoblast: cytolysis or cytokine production?

At the implantation site, the uterine mucosa (decidua) is infiltrated by large numbers of natural killer (NK) cells. These NK cells are in close contact with the invading fetal trophoblast and we have proposed that they might be the effector cells that control the implantation of the allogeneic placenta. Recent characterization of NK cell receptors and their HLA class I ligands has suggested potential mechanisms by which NK cells might interact with trophoblast. However, what happens as a result of this interaction is not clear. The traditional method for investigating NK cell function in vitro is the protection from lysis of target cells by expression of HLA class I antigens. This might not be an accurate reflection of what happens in vivo. Another function of NK cells is the production of cytokines on contact with target cells. This could be an important outcome of the interaction between decidual NK cells and trophoblast. Decidual NK cells are known to produce a variety of cytokines; trophoblast cells express receptors for many of these cytokines, indicating that they can potentially respond. In this way, decidual NK cells have a significant influence on trophoblast behaviour during implantation.     

Tumor-induced apoptosis of human IL-2-activated NK cells: role of natural cytotoxicity receptors

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46. Indeed, the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis, and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA, FasL protein synthesis, and release. In turn, FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly, NK cell apoptosis, but not killing of tumor target cells, is inhibited by cyclosporin A, suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity, but also surface receptors involved in NK cell suicide.     

Inhibition of human NK cell-mediated killing by CD1 molecules

It is now well established that NK cells recognize classical and nonclassical MHC class I molecules and that such recognition typically results in the inhibition of target cell lysis. Given the known structural similarities between MHC class I and non-MHC-encoded CD1 molecules, we investigated the possibility that human CD1a, -b, and -c proteins might also function as specific target structures for NK cell receptors. Here we report that expression of CD1a, -b, or -c can partially inhibits target cell lysis by freshly isolated human NK cells and cultured NK lines. The inhibitory effects of CD1 molecules on NK cell could be shown upon expression of individual CD1 proteins in transfected NK-sensitive target cells, and these effects could be reversed by incubation of the target cells with mAbs specific for the expressed form of CD1. Inhibitory effects of CD1 expression on NK-mediated lysis could also be shown for cultured human dendritic cells, which represent a cell type that prominently expresses the various CD1 proteins in vivo. In addition, the bacterial glycolipid Ags known to be bound and presented by CD1 proteins could significantly augment the observed inhibitory effects on target cell lysis by NK cells.     

Targeted cellular therapy with natural killer cells.

Natural killer (NK) cells are believed to be important contributors to a patient's immune armamentarium to fight cancer. However, cancer patients have reportedly defective NK cells and the malignant target frequently has developed mechanisms to escape detection of NK cells. Our research is aimed at overcoming this NK cell paralysis through three different approaches: instead of using autologous NK cells for adoptive immunotherapy, allogeneic NK cells are used that are not inhibited by self histocompatibility antigens. Further, NK cells, selected for a high affinity Fc receptor can, together with monoclonal antibodies, kill targets through ADCC irrespective of any inhibitory receptors. Finally, the genetic engineering of NK cells to express chimeric antigen receptors recognizing antigens on tumor target can overcome inhibitory mechanism and effectively lyse tumor cells.     

The activating NKG2D ligand MHC class I-related chain A transfers from target cells to NK cells in a manner that allows functional consequences

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.     

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activating receptors and their ligands

Natural killer (NK) cell activation is strictly regulated to ensure that healthy cells are preserved, but tumour-transformed or virus-infected cells are recognized and eliminated. To carry out this selective killing, NK cells have an ample repertoire of receptors on their surface. Signalling by inhibitory and activating receptors by interaction with their ligands will determine whether the NK cell becomes activated and kills the target cell. Here, we show reduced expression of NKp46, NKp30, DNAM-1, CD244 and CD94/NKG2C activating receptors on NK cells from acute myeloid leukaemia patients. This reduction may be induced by chronic exposure to their ligands on leukaemic blasts. The analysis of ligands for NK cell-activating receptors showed that leukaemic blasts from the majority of patients express ligands for NK cell-activating receptors. DNAM-1 ligands are frequently expressed on blasts, whereas the expression of the NKG2D ligand MICA/B is found in half of the patients and CD48, a ligand for CD244, in only one-fourth of the patients. The decreased expression of NK cell-activating receptors and/or the heterogeneous expression of ligands for major receptors on leukaemic blasts can lead to an inadequate tumour immunosurveillance by NK cells. A better knowledge of the activating receptor repertoire on NK cells and their putative ligands on blasts together with the possibility to modulate their expression will open new possibilities for the use of NK cells in immunotherapy against leukaemia.     

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94⁺ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94⁺ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL⁺ CD56^dim NK cells, in contrast to the efficient responses by CD56^bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94⁺ KIR2DL^- NK cells may be uniquely beneficial.     

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.     

The role of Ia antigens in the activation of T cells by concanavalin A: an evidence for the species restriction between T cells and accessory cells

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.     

“Harpoon” Model for Cell–Cell Adhesion and Recognition of Target Cells by the Natural Killer Cells

The mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition or NK-sensitive target cells (TCs) by NK cells of NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of “non-NK receptors” and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.); (2) the presence of multiple disorders in the organization of “extracellular matrix-surface membrane-submembrane cytoskeleton” assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can “harpoon” the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this “harpoon” model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.     

Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy

Human NK cells lyse Ab-coated target cells through the process of Ab-dependent cellular cytotoxicity (ADCC). Improving ADCC responses is desirable because it is thought to be an important antitumor mechanism for some Abs. NK cell inhibitory receptors, such as killer cell Ig-like receptors, engage with MHC class I molecules on self-cells to block NK cell activation. Accordingly, we enhanced ADCC responses by blocking NK cell inhibitory receptors, thus perturbing induction of the self-recognition signal. In a cell line model of anti-lymphoma therapy, the combination of rituximab with an Ab that blocks inhibitory self-recognition yielded increased NK cell-mediated target cell lysis when compared with rituximab alone. To validate this proof-of-concept, we then used a more representative approach in which an individual's fresh primary NK cells encountered autologous, EBV-transformed B cells. In this system, rituximab and a combination of Abs that block NK cell inhibitory receptors yielded improved NK cell-mediated lysis over rituximab alone. The results show, for the first time, that disruption of inhibitory self-recognition can efficiently promote ADCC in a human model, applying an autologous system in which physiologic checkpoints are in place. This method provides an alternative approach to potentiate the therapeutic benefit of antitumor Abs that mediate ADCC.     

Intercellular transfer of carcinoembryonic antigen from tumor cells to NK cells

The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.     

Surrogate receptor-mediated cellular cytotoxicity. A method for "custom designing" killer cells of desired target specificity

Palmitate-derivatized antibody molecules can function as surrogate receptors when incorporated into the plasma membranes of nylon wool non-adherent spleen cells. Surrogate receptor molecules are attached to the membranes by insertion of the palmitate hydrocarbon chains into the phospholipid bilayer. This mode of attachment precludes interactions between surrogate receptors and intracellular and intramembranous structures. Despite these limitations, surrogate receptors consisting of either palmitate-derivatized intact antibody molecules or their corresponding F(ab')2 fragments specific for Ag on syngeneic B lymphocytes or hapten-modified EL-4 lymphoma cells can direct cell-mediated cytotoxic activity against the appropriate target. Treatment of the surrogate receptor-decorated effector cell populations with anti-asialo GM1 plus C eliminated the observed target cell lysis, suggesting a role for NK cells in this cytolytic process. The efficiency of this surrogate receptor-mediated cellular cytotoxicity parallels that of natural receptor-mediated target cell lysis and is not limited by inherent resistance of a target cell to NK cell-mediated cytotoxicity. Application of this technology to study the requirements for productive lytic interactions between effector and target cells may provide valuable insights into the mechanism of cell-mediated cytotoxicity. Furthermore, these results provide the rationale for future studies designed to evaluate the ability of surrogate receptors to focus cytotoxic cell activity onto a specified target in situ, in an attempted elimination of diseased cells.     

Natural killer cells: detectors of stress

Natural killer (NK) cells are a key component of the innate immune system, as they are able to detect microbe-infected cells, tumors as well as allogeneic cells, without specific sensitization. NK cell effector functions (cytotoxicity, cytokine secretion) are regulated by a wide array of inhibitory and activating receptors. MHC class I molecules are the ligands of most inhibitory receptors, while activating receptors recognize either pathogen-encoded molecules, or self-proteins whose expression is up-regulated upon microbial infection or tumor development. Upon integration of these negative and positive signals, Natural Killer cells can discriminate between healthy "self" (tolerance) and autologous cells undergoing different types of cellular stress or allogeneic cells (immunosurveillance). The knowledge of the different mechanisms of target cell recognition is thus crucial to dissect NK cell involvement in homeostatic and disease conditions as well as to develop novel alternative therapeutic approaches based on NK cell manipulation.     

Activating Ly-49 receptors regulate LFA-1-mediated adhesion by NK cells

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors, and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions, yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore, we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1, and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors, leading to target cell death.