Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44

Background

The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer.

Methodology/Principal Findings

Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G₂/M phase.

Conclusion/Significance

These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.     

Novel Interaction between Proliferating Cell Nuclear Antigen and HLA I on the Surface of Tumor Cells Inhibits NK Cell Function through NKp44

NK cell function is closely regulated by numerous inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface PCNA. Other reports have pointed to potential associations between NKp44 and HLA I molecules, as well as HLA I and Damage Associated Molecular Pattern molecules (DAMPs) on the surface of tumor cells. In this report, we have identified novel interaction between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK cell cytotoxicity. We further postulate NCR ligands are composed of DAMP molecules localized to the cell surface, colocalizing with HLA I, and potentially heparin sulfate proteoglycans.     

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.     

Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44

NK cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only natural cytotoxicity receptor that is expressed exclusively by primate NK cells, yet its cellular ligands remain largely unknown. Proliferating cell nuclear Ag (PCNA) is overexpressed in cancer cells. In this study, we show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through NKp44/ITIM. The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.     

Generation and Preclinical Characterization of an NKp80-Fc Fusion Protein for Redirected Cytolysis of Natural Killer (NK) Cells against Leukemia

The capacity of natural killer (NK) cells to mediate Fc receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for the NK-activating receptor NKp80, is frequently highly expressed on leukemia cells, the lack of therapeutic AICL-specific antibodies limits clinical application. Here we explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-Fc fusion proteins. The NKp80-Fc fusion protein we generated bound specifically to leukemia cells in an AICL-specific manner. Cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced the ADCC effect of NK cells. NKp80-Fc not only promoted NK-mediated leukemia cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in the late stage. The bifunctional NKp80-Fc could redirect NK cells toward leukemia cells and triggered NK cell killing in vitro. Moreover, NKp80-Fc enhanced the lysis of NK cells against tumors in leukemia xenograft non-obese diabetic/severe combined immunodeficiency mice. Taken together, our results demonstrate that NKp80-Fc potently amplifies NK cell anti-leukemia effects in vitro and in vivo through induction of the NK cell ADCC effect. This method could potentially be useful for molecular targeted therapy, and the fusion proteins may be a promising drug for immunotherapy of leukemia.     

Tumour‐experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)‐γ secretion on engagement of the natural‐killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell‐mediated anti‐tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co‐culture with some melanomas, activated CD4⁺ T cells promoted degranulation, and NKG2D‐ and NKp46‐dependent IFN‐γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour‐cell surface (trogocytosis). This effect was observed in CD4⁺, CD8⁺ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain‐related protein A on co‐culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.     

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46⁺ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46⁺ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46⁺ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46⁺ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46⁺ ILC in the regulation of mucosal CD8 T cell responses.     

Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.     

Differentiation of natural killer cells from induced pluripotent stem cells under defined,serum- and feeder-free conditions

Background aimsTraditionally, natural killer (NK) cells are sourced from the peripheral blood of donors―a laborious and highly donor-specific process. Processes for generating NK cells from induced pluripotent stem cells (iPSCs) have demonstrated that it is possible to successfully generate renewable alloreactive NK cells that are not only functional in vivo but can also be genetically engineered for enhanced function. However, poor standardization and cumbersome differentiation procedures suggest that further improvements in the control of the differentiation process are necessary.MethodsHere the authors evaluated the potential of differentiating NK cells from centrally authenticated iPSCs under entirely chemically defined and serum-free conditions as well as their immunotherapeutic potential, after expansion in feeder-free media, against solid tumors targets. To address limitations of current differentiation approaches, the authors did not utilize feeder or stromal cell layers, TrypLE adaptation or peripheral blood during the differentiation process. The authors also evaluated the feasibility of utilizing centrally authenticated iPSC lines, thus circumventing protocol- and donor-induced variability associated with reprogramming approaches, and characterized these iPSC-NK cells in terms of cytotoxicity, cytokine production and degranulation potential against solid tumor cell lines and patient-derived targets.ResultsDifferentiation of iPSCs generated NK cells that were predominantly CD56⁺/CD16⁺/CD3⁻ and expressed NK activation markers NKG2D, NKp30, NKp44, NKp46 and DNAM-1. These iPSC-NK cells mediated effector functions, including cytotoxicity, degranulation and IFN-γ production, in response to solid tumor targets, including patient-derived cancer cells, and could be cryopreserved and expanded in culture.ConclusionsThe ability to produce NK cells under defined conditions and the functional responses elicited by these iPSC-NK cells suggest that they could represent promising effectors in clinical adoptive transfer settings as a renewable source of donor-independent NK cells for immunotherapy of solid tumors.     

Tumor-released Galectin-3, a Soluble Inhibitory Ligand of Human NKp30, Plays an Important Role in Tumor Escape from NK Cell Attack

Human Galectin-3 (Gal-3), a β-galactoside-binding protein expressed by tumor cells, has been reported to act as an immune regulator in antitumor T cells. However, its effect on natural killer (NK) cells is elusive. Using a recombinant human NK cell-activating receptor, NKp30 fusion protein (NKp30-Fc), we found that soluble NKp30-Fc could immunoprecipitate Galectin-3. The direct interaction between NKp30 and Galectin-3 was further confirmed using surface plasmon resonance experiments. Because Galectin-3 was mainly released from tumor cells in a soluble form in our study, the binding assay was performed to show that soluble Galectin-3 specifically bound to NK cells and NKp30 on the surface of the NK cells. Functionally, when soluble Galectin-3 was added to the NK-tumor cell coculture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by preincubation of soluble Galectin-3 with NKp30-Fc fusion protein or the addition of anti-Gal-3 antibody alone. Moreover, genetic down-regulation of Galectin-3 (shGal-3) resulted in tumor cells being more sensitive to NK cell lysis, and, reversely, Galectin-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft non-obese diabetic/severe combined immunodeficiency mice after NK cell adoptive immunotherapy, indicating that Galectin-3 strongly antagonizes human NK cell attack against tumors in vivo. These findings indicate that Galectin-3 may function as an immune regulator to inhibit NK cell function against tumors, therefore providing a new therapeutic target for tumor treatment.     

NKp46

Combating diseases such as cancer and virus infection is one of the major goals of the next millennium. Remarkably, our immune system has developed a unique cell type called Natural Killer (NK) cells, able to kill cancer and virus-infected cells. The nature of the lysis/killer receptors expressed on NK cells, mediating this killing, and their ligands, is only little understood. Four lysis receptors for human NK cells were recently identified, including the NKp30, NKp44, NKp46 and CD16, all of which are capable of mediating direct killing of virus-infected and tumor cells. Among these, the NKp46 receptor is considered to be the major lysis receptor for NK cells. The viral hemagglutinin protein was recently identified as the ligand for the NKp46 receptor. However, the cellular (non-viral) ligands recognized by all of the lysis receptors (NKp30, NKp44, NKp46 and CD16) are currently unknown.     

Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

HIV gp41 Engages gC1qR on CD4+ T Cells to Induce the Expression of an NK Ligand through the PIP3/H2O2 Pathway

CD4⁺ T cell loss is central to HIV pathogenesis. In the initial weeks post-infection, the great majority of dying cells are uninfected CD4⁺ T cells. We previously showed that the 3S motif of HIV-1 gp41 induces surface expression of NKp44L, a cellular ligand for an activating NK receptor, on uninfected bystander CD4⁺ T cells, rendering them susceptible to autologous NK killing. However, the mechanism of the 3S mediated NKp44L surface expression on CD4⁺ T cells remains unknown. Here, using immunoprecipitation, ELISA and blocking antibodies, we demonstrate that the 3S motif of HIV-1 gp41 binds to gC1qR on CD4⁺ T cells. We also show that the 3S peptide and two endogenous gC1qR ligands, C1q and HK, each trigger the translocation of pre-existing NKp44L molecules through a signaling cascade that involves sequential activation of PI3K, NADPH oxidase and p190 RhoGAP, and TC10 inactivation. The involvement of PI3K and NADPH oxidase derives from 2D PAGE experiments and the use of PIP3 and H2O2 as well as small molecule inhibitors to respectively induce and inhibit NKp44L surface expression. Using plasmid encoding wild type or mutated form of p190 RhoGAP, we show that 3S mediated NKp44L surface expression on CD4⁺ T cells is dependent on p190 RhoGAP. Finally, the role of TC10 in NKp44L surface induction was demonstrated by measuring Rho protein activity following 3S stimulation and using RNA interference. Thus, our results identify gC1qR as a new receptor of HIV-gp41 and demonstrate the signaling cascade it triggers. These findings identify potential mechanisms that new therapeutic strategies could use to prevent the CD4⁺ T cell depletion during HIV infection and provide further evidence of a detrimental role played by NK cells in CD4⁺ T cell depletion during HIV-1 infection.     

Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan

NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30-dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the nature of the cellular ligand for this important NK cell activation receptor.     

Harnessing soluble NK cell killer receptors for the generation of novel cancer immune therapy

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.     

Novel xeno-free and serum-free culturing condition to improve piggyBac transposon-based CD19 chimeric antigen receptor T-cell production and characteristics

Background aimsChimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production.MethodsPiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity.ResultsXeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay.ConclusionsOur study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion.     

Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). (51)Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.     

Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

Background

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.

Methodology/Principal Findings

We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D^high or NK30^high donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.

Conclusions/Significance

NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities.