The activating receptor NKp65 is selectively expressed by human ILC3 and demarcates ILC3 from mature NK cells.

Innate lymphocytes comprise cytotoxic natural killer (NK) cells and tissue-resident innate lymphoid cells (ILC) that are subgrouped according to their cytokine profiles into group 1 ILC (ILC1), ILC2, and ILC3. However, cell surface receptors unambiguously defining or specifically activating such ILC subsets are scarcely known. Here, we report on the physiologic expression of the human activating C-type lectin-like receptor (CTLR) NKp65, a high-affinity receptor for the CTLR keratinocyte-associated C-type lectin (KACL). Tracking rare NKp65 transcripts in human blood, we identify ILC3 to selectively express NKp65. NKp65 expression not only demarcates "bona fide" ILC3 from likewise RORγt-expressing ILC precursors and lymphoid tissue inducer cells but also from mature NK cells which acquire the NKp65-relative NKp80 during a Notch-dependent differentiation from NKp65+ precursor cells. Hence, ILC3 and NK cells mutually exclusively and interdependently express the genetically coupled sibling receptors NKp65 and NKp80. Much alike NKp80, NKp65 promotes cytotoxicity by innate lymphocytes which may become relevant during pathophysiological reprogramming of ILC3. Altogether, we report the selective expression of the activating immunoreceptor NKp65 by ILC3 demarcating ILC3 from mature NK cells and endowing ILC3 with a dedicated immunosensor for the epidermal immune barrier.

NK-cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK-cell receptors are divided into either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK-cell activation requires stimulation of activating receptors. In viral infections, NK-cell interaction with infected cells can result in activation, memory-like NK-cell differentiation, or NK-cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK-cell receptor expression. In this review, we summarize the current knowledge of NK-cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK-cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK-cell receptor activation gives a promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of the application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK-cell receptor biology, a deeper understanding of NK-cell receptors role in viral infections is still needed.

NK cells to cure cancer.

Natural Killer (NK) cells are innate lymphocytes able to mediate immune-surveillance and clearance of viral infected and tumor-transformed cells. Growing experimental and clinical evidence highlighted a dual role of NK cells either in the control of cancer development/progression or in promoting the onset of immune-suppressant tumor microenvironments. Indeed, several mechanisms of NK cell-mediated tumor escape have been described and these includes cancer-induced aberrant expression of activating and inhibitory receptors (i.e. NK cell immune checkpoints), impairments of NK cell migration to tumor sites and altered NK cell effector-functions. These phenomena highly contribute to tumor progression and metastasis formation. In this review, we discuss the latest insights on those NK cell receptors and related molecules that are currently being implemented in clinics either as possible prognostic factors or therapeutic targets to unleash NK cell anti-tumor effector-functions in vivo. Moreover, we address here the major recent advances in regard to the genetic modification and ex vivo expansion of anti-tumor specific NK cells used in innovative adoptive cellular transfer approaches.

Human NK cells, their receptors and function.

NK cells are cytotoxic components of innate lymphoid cells (ILC) that provide a first line of defense against viral infections and contribute to control tumor growth and metastasis. Their function is finely regulated by an array of HLA-specific and non-HLA-specific inhibitory and activating receptors which allow to discriminate between healthy and altered cells. Human NK cells gained a major attention in recent years because of the important progresses in understanding their biology and of some promising data in tumor therapy. In this review, we will outline well-established issues of human NK cells and discuss some of the open questions, debates, and recent advances regarding their origin, differentiation, and tissue distribution. Newly defined NK cell specializations, including the impact of inhibitory checkpoints on their function, their crosstalk with other cell types, and the remarkable adaptive features acquired in response to certain virus infections will also be discussed.

NK Cells in Chronic Lymphocytic Leukemia and Their Therapeutic Implications.

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.

The Ubiquitin-proteasome pathway regulates Nectin2/CD112 expression and impairs NK cell recognition and killing.

Nectin2 is a member of immunoglobulin-like cell adhesion molecules and plays a prominent role in the establishment of adherens and tight junctions. It is also upregulated on the surface of tumor and virus-infected cells where it functions as a ligand for the activating receptor CD226, thus contributing to cytotoxic lymphocyte-mediated recognition and killing of damaged cells. Little is currently known about the regulation of Nectin2 expression and, in particular, whether posttranscriptional and posttranslational mechanisms are involved. Here, we analyzed Nectin2 expression on a panel of human tumor cell lines and primary cultures and we found that Nectin2 is mainly expressed in cytoplasmic pools. Moreover, we demonstrated that ubiquitination of Nectin2 promotes its degradation and is responsible for protein intracellular retention. Indeed, inhibition of the ubiquitin pathway results in increased Nectin2 surface expression and enhances tumor cell susceptibility to NK cell cytotoxicity. Our results demonstrate a previously unknown mechanism of Nectin2 regulation revealing that the ubiquitin pathway represents a potential target of intervention in order to increase susceptibility to NK cell-mediated lysis.

NK Cells in the Tumor Microenvironment.

Natural killer cells are powerful effectors of innate immunity that constitute a first line of defense against cancer. NK cells express an array of germline-encoded receptors which allow them to eliminate transformed cells and spare normal, healthy cells. Owing to their ability to kill circulating tumor cells, NK cells play a major role in the protection against cancer metastases. There is also convincing evidence that NK cells protect against some hematological cancers such as acute myeloid leukemia. However, the importance of NK cells for the control of established solid tumors is rather uncertain. Several mechanisms impede NK cell-mediated elimination of solid tumors, starting with the incapacity of NK cells to infiltrate the core of the tumor. In addition, immune escape mechanisms are at play in both solid and hematological cancers. These include the immunoediting of tumor cells and aberrant chronic inflammation that renders NK cells ineffective. In this chapter, I review the phenotypic characteristics of NK cells within the tumor microenvironment. Furthermore, I describe the mechanisms by which NK cells contribute to antitumor immunity. Finally, I review the different immune-evasion factors that impair NK cell activity against cancer.

CD155: A Multi-Functional Molecule in Tumor Progression.

CD155 is an adhesion molecule belonging to the Nectin/Nectin-like family often overexpressed on tumor cells and involved in many different processes such as cell adhesion, migration and proliferation. In contrast to these pro-tumorigenic functions, CD155 is also a ligand for the activating receptor DNAM-1 expressed on cytotoxic lymphocytes including Natural Killer (NK) cells and involved in anti-tumor immune response. However, during tumor progression inhibitory receptors for CD155 are up-regulated on the surface of effector cells, contributing to an impairment of their cytotoxic capacity. In this review we will focus on the roles of CD155 as a ligand for the activating receptor DNAM-1 regulating immune surveillance against cancer and as pro-oncogenic molecule favoring tumor proliferation, invasion and immune evasion. A deeper understanding of the multiple roles played by CD155 in cancer development contributes to improving anti-tumor strategies aimed to potentiate immune response against cancer.

KIR downregulation by IL-12/15/18 unleashes human NK cells from KIR/HLA-I inhibition and enhances killing of tumor cells.

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition of human NK cells by HLA-I-expressing tumor cells. Here, we show that stimulation of NK cells with IL-12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re-induced already 3 days after re-culture in IL-2. Reduced KIR2DL2/L3 expression on IL-12/15/18-activated NK cells resulted in less inhibition upon antibody-mediated KIR engagement and increased CD16-dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL-12/15/18-stimulated NK cells against tumor cells expressing cognate HLA-I molecules. NK cells pre-activated with IL-12/15/18 were previously shown to exert potent anti-tumor activity and memory-like long-lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL-12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self-HLA-I due to decreased KIR expression, highlighting the potency of IL-12/15/18-activated NK cells for anti-tumor immunotherapy protocols.

T Cells Going Innate.

Natural killer (NK) cell receptors (NKRs) play a crucial role in the homeostasis of antigen-experienced T cells. Indeed, prolonged antigen stimulation may induce changes in the receptor repertoire of T cells to a profile that features NKRs. Chronic antigen exposure, at the same time, has been shown to trigger the loss of costimulatory CD28 molecules with recently reported intensified antigen thresholds of antigen-experienced CD8(+) T cells. In transplantation, NKRs have been shown to assist allograft rejection in a CD28-independent fashion. We discuss here a role for CD28-negative T cells that have acquired the competency of the NKR machinery, potentially promoting allorecognition either through T cell receptor (TCR) crossreactivity or independently from TCR recognition. Collectively, NKRs can bring about innate-like T cells by providing alternative costimulatory pathways that gain relevance in chronic inflammation, potentially leading to resistance to CD28-targeting immunosuppressants.

CMV drives the expansion of highly functional memory T cells expressing NK-cell receptors in renal transplant recipients.

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post-transplant. We describe how active and latent CMV affect T-cell subsets in RTRs who are stable on maintenance therapy. T-cell responses to CMV were assessed in RTRs (n = 54) >2 years post-transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8+ T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T-cell (TEM ), terminally differentiated T-cell (TEMRA ) and CD57+ TEMRA cell populations. Expression of NK-cell receptors, LIR-1 and KLRG1 on CD4+ and CD8+ CD57+ TEM and TEMRA cells correlated with elevated interferon-γ and cytotoxic responses to anti-CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8+ T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) -1 peptides. The data show that latent and active CMV infection can alter T-cell subsets in RTRs many years after transplantation, and up-regulate T-cell expression of NK-cell receptors. This may enhance effector responses of CD4+ and CD8+ T cells against CMV.

Higher expression of inhibitory CD158b and CD158e NK cell receptor and age predicts treatment response in children with chronic hepatitis C.

Treatment with pegylated interferon-α and ribavirin (PEG-IFN/RBV) is the only choice for chronic hepatitis C (CHC) in children. Natural killer (NK) cells were described to play a vital role in CHC. The aim of this study was to analyze the expression of peripheral blood NK cell receptors in their relation to PEG-IFN/RBV treatment response. Study included 26 children with CHC-13 boys, age range 13.42 ± 3.28 years. Blood for biochemical, virological and cytometric testing was taken for evaluation prior to the antiviral treatment. NK cell receptors were detected by flow cytometry and the results were presented as proportion of cells and mean fluorescence intensity (MFI). Therapy consisted of PEG-IFNα-2b (60 µg/m2 s.c 1×/week) and RBV (15 mg/kg p.o. daily). Treatment duration was response-related and varied from 12 to 72 weeks. Rapid virological response (RVR) was evaluated in the 4th week and sustained virological response (SVR) 6 months after completion of the therapy. RVR children were younger (11.67 ± 3.74 vs 15.35 ± 2.42; p = 0.001) and displayed higher CD158b (3.58 ± 0.16 vs 3.45 ± 0.13; p = 0.038) and CD158e expression (4.33 ± 0.21 vs 4.03 ± 0.16; p = 0.039). Density of CD158b (logMFI = 3.68 ± 0.22 vs 3.36 ± 0.16; p = 0.036) and CD158e expression was significantly higher (4.37 ± 0.14 vs 4.12 ± 0.21; p = 0.046) and NKG2D expression significantly lower (97.50 ± 3.46 vs 94.92 ± 5.93; p = 0.049) in SVR children. SVR children were also significantly younger (12.40 ± 3.66 vs 15.13 ± 2.83; p = 0.003). Significance of the age of patients, and expression of CD158b and CD158e were confirmed in univariate and multivariate analysis. Age of patients is negatively related to RVR and SVR. NK cell phenotype with higher expression density of CD158b and CD158e receptor was a positive predictor of SVR.

Natural killer cells in the treatment of high-risk acute leukaemia.

Several studies have shown that in patients with acute leukaemia given allogeneic haematopoietic stem cell transplantation (allo-HSCT) large part of the therapeutic effect lies on the anti-tumour effect displayed by cells of both adaptive and innate immunity. This evidence has also opened new scenarios for the treatment of patients with other haematological malignancies/solid tumours. In particular, donor-derived natural killer (NK) cells play a crucial role in the eradication of cancer cells in patients given an allograft from an HLA-haploidentical relative, especially when there is a killer inhibitory-receptor (KIR)-KIR ligand mismatched in the donor-recipient direction. Alloreactive donor-derived NK cells have been also demonstrated to kill recipient antigen-presenting cells and cytotoxic T lymphocytes, thus preventing graft-versus-host disease (GvHD) and graft rejection and to largely contribute to the defence against cytomegalovirus infection in the early post-transplant period. Several clinical studies have recently focused also on the influence of NK-cell activating receptors on the outcome of allo-HSCT recipients; in particular, B/x haplotype donors offer clinical advantages compared with A/A donors, even when the donor is an HLA-identical volunteer. Altogether, these data have provided the rationale for implementing phase I/II clinical trials based on adoptive infusion of either selected or ex vivo activated NK cells from an HLA-mismatched donor. This review summarizes the biological and clinical data on the role played by NK cells in patients with high-risk acute leukaemia, focusing also on the still unsolved issues and the future perspectives related to the approaches of adoptive NK cell therapy.

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed.

A Coevolutionary Arms Race between Hosts and Viruses Drives Polymorphism and Polygenicity of NK Cell Receptors.

Natural killer cell receptors (NKRs) monitor the expression of major histocompatibility class I (MHC-I) and stress molecules to detect unhealthy tissue, such as infected or tumor cells. The NKR gene family shows a remarkable genetic diversity, containing several genes encoding receptors with activating and inhibiting signaling, and varying in gene content and allelic polymorphism. The expansion of the NKR genes is species-specific, with different species evolving alternative expanded NKR genes, which encode structurally different proteins, yet perform comparable functions. So far, the biological function of this expansion within the NKR cluster has remained poorly understood. To study the evolution of NKRs, we have developed an agent-based model implementing a coevolutionary scenario between hosts and herpes-like viruses that are able to evade the immune response by downregulating the expression of MHC-I on the cell surface. We show that hosts evolve specific inhibitory NKRs, specialized to particular MHC-I alleles in the population. Viruses in our simulations readily evolve proteins mimicking the MHC molecules of their host, even in the absence of MHC-I downregulation. As a result, the NKR locus becomes polygenic and polymorphic, encoding both specific inhibiting and activating receptors to optimally protect the hosts from coevolving viruses.

Functional characterization of a conserved pair of NKR-P1 receptors expressed by NK cells and T lymphocytes in liver and gut.

Natural killer cell receptor protein 1 (NKR-P1) molecules are C-type lectin-like receptors modulating cellular responses toward target cells expressing C-type lectin-like related (Clr) molecules. Although the function of the prototypic rat NKR-P1A receptor and its inhibitory counterpart NKR-P1B are known, little is known about NKR-P1F and NKR-P1G apart from their promiscuity for Clr ligands. Here we generated mAbs against both receptors for phenotypic and functional analyses in rat tissues. NKR-P1F induced redirected lysis and robust Ca(2+) signaling in NK cells, which were prevented by simultaneous engagement of NKR-P1G. NKR-P1G also inhibited NK-cell lysis of Clr transfectants. NKR-P1F was expressed by most NK cells and NKR-P1A(+) T cells in all tissues analyzed, and by many NKR-P1A(-) intestinal T cells, while NKR-P1G was expressed by subsets of these cells with highest prevalence in gut and liver. In the intraepithelial compartment, the proportion of NKR-P1A(+) and NKR-P1F(+) cells was high at birth and thereafter declined, while NKR-P1B(+) and NKR-P1G(+) cells increased with age. Expression levels were also modulated by cytokines, with an increase of NKR-P1B and NKR-P1G induced by inflammatory cytokines, and a reduction of NKR-P1A by TGF-ß. The physiological impact of NKR-P1 receptors might thus be dependent on age, tissue, and inflammatory status.

The evolution of natural killer cell receptors.

Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Human NK cells and cancer.

The long story of NK cells started about 50 y ago with the first demonstration of a natural cytotoxic activity within an undefined subset of circulating leukocytes, has involved an ever-growing number of researchers, fascinated by the apparently easy-to-reach aim of getting a "universal anti-tumor immune tool". In fact, in spite of the impressive progress obtained in the first decades, these cells proved far more complex than expected and, paradoxically, the accumulating findings have continuously moved forward the attainment of a complete control of their function for immunotherapy. The refined studies of these latter years have indicated that NK cells can epigenetically calibrate their functional potential, in response to specific environmental contexts, giving rise to extraordinarily variegated subpopulations, comprehensive of memory-like cells, tissue-resident cells, or cells in various differentiation stages, or distinct functional states. In addition, NK cells can adapt their activity in response to a complex body of signals, spanning from the interaction with either suppressive or stimulating cells (myeloid-derived suppressor cells or dendritic cells, respectively) to the engagement of various receptors (specific for immune checkpoints, cytokines, tumor/viral ligands, or mediating antibody-dependent cell-mediated cytotoxicity). According to this picture, the idea of an easy and generalized exploitation of NK cells is changing, and the way is opening toward new carefully designed, combined and personalized therapeutic strategies, also based on the use of genetically modified NK cells and stimuli capable of strengthening and redirecting their effector functions against cancer.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.