Natural Killer Cell Effector Functions Research Articles

T cell receptor engineering of primary NK cells to therapeutically target tumors and tumor immune evasion

BackgroundT cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can negatively impact the success...

Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious disorder of unknown aetiology. While the pathomechanism of ME/CFS remains elusive, reduced natural killer (NK) cell cytotoxic function is a consistent immunological feature. NK cell effector functions rely on long-term sustained calcium (Ca2+) influx. In recent years evidence of transient receptor potential melastatin 3 (TRPM3) dysfunction supports the hypothesis that ME/CFS is potentially an ion channel disorder. Specifically, reports of single nucleotide polymorphisms, low surface expression and impaired function of TRPM3 have been reported in NK cells of ME/CFS patients. It has been reported that mu (µ)-opioid receptor (µOR) agonists, known collectively as opioids, inhibit TRPM3. Naltrexone hydrochloride (NTX), a µOR antagonist, negates the inhibitory action of µOR on TRPM3 function. Importantly, it has recently been reported that NTX restores impaired TRPM3 function in NK cells of ME/CFS patients.MethodsLive cell immunofluorescent imaging was used to measure TRPM3-dependent Ca2+ influx in NK cells isolated from n = 10 ME/CFS patients and n = 10 age- and sex-matched healthy controls (HC) following modulation with TRPM3-agonist, pregnenolone sulfate (PregS) and TRPM3-antaognist, ononetin. The effect of overnight (24 h) NTX in vitro treatment on TRPM3-dependent Ca2+ influx was determined.ResultsThe amplitude (p < 0.0001) and half-time of Ca2+ response (p < 0.0001) was significantly reduced at baseline in NK cells of ME/CFS patients compared with HC. Overnight treatment of NK cells with NTX significantly improved TRPM3-dependent Ca2+ influx in ME/CFS patients. Specifically, there was no significance between HC and ME/CFS patients for half-time response, and the amplitude of Ca2+ influx was significantly increased in ME/CFS patients (p < 0.0001).ConclusionTRPM3-dependent Ca2+ influx was restored in ME/CFS patients following overnight treatment of isolated NK cells with NTX in vitro. Collectively, these findings validate that TRPM3 loss of function results in altered Ca2+ influx supporting the growing evidence that ME/CFS is a TRP ion channel disorder and that NTX provides a potential therapeutic intervention for ME/CFS.

Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing.

Genome engineered natural killer (NK) cell therapies are emerging as a promising cancer immunotherapy platform with potential advantages and remaining uncertainties. Feeder cells induce activation and proliferation of NK cells via cell surface receptor-ligand interactions, supported by cytokines. Feeder cell expanded NK cell products have supported several NK cell adoptive transfer clinical trials over the past decade. Genome engineered NK cell therapies, including CAR-NK cells, seek to combine innate and alloreactive NK cell anti-tumor activity with antigen specific targeting or additional modifications aimed at improving NK cell persistence, homing or effector function. The profound activating and expansion stimulus provided by feeder cells is integral to current applications of clinical-scale genome engineering approaches in donor-derived, primary NK cells. Herein we explore the complex interactions that exist between feeder cells and both viral and emerging non-viral genome editing technologies in NK cell engineering. We focus on two established clinical-grade feeder systems; Epstein-Barr virus transformed lymphoblastoid cell lines and genetically engineered K562.mbIL21.4-1BBL feeder cells.

Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells

Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

An HLA-I signature favouring KIR-educated Natural Killer cells mediates immune control of HIV in children and contrasts with the HLA-B-restricted CD8+ T-cell-mediated immune control in adults.

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children.

A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability.Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence.CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity.The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time.In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. DisclosuresAllen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Deletion of CISH and TGFβR2 in iPSC-Derived NK Cells Promotes High Cytotoxicity and Enhances In Vivo Tumor Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells.Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity.We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing.In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. DisclosuresGerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Key Activating and Inhibitory Ligands Involved in the Mobilization of Natural Killer Cells for Cancer Immunotherapies.

Natural killer (NK) cells are the most potent arm of the innate immune system and play an important role in immunity, alloimmunity, autoimmunity, and cancer. NK cells recognize “altered-self” cells due to oncogenic transformation or stress due to viral infection and target to kill them. The effector functions of NK cells depend on the interaction of the activating and inhibitory receptors on their surface with their cognate ligand expressed on the target cells. These activating and inhibitory receptors interact with major histocompatibility complex I (MHC I) expressed on the target cells and make decisions to mount an immune response. NK cell immune response includes cytolytic activity and secretion of cytokines to help with the ongoing immune response. The advancement of our knowledge on the expression of inhibitory and activating molecules led us to exploit these molecules in the treatment of cancer. This review discusses the importance of activating and inhibitory receptors on NK cells and their clinical importance in cancer immunotherapy.

Expression Regulation and Function of T-Bet in NK Cells.

Natural killer (NK) cells are cytotoxic innate lymphocytes that play an important role in immune surveillance. The development, maturation and effector functions of NK cells are orchestrated by the T-box transcription factor T-bet, whose expression is induced by cytokines such as IFN-γ, IL-12, IL-15 and IL-21 through the respective cytokine receptors and downstream JAK/STATs or PI3K-AKT-mTORC1 signaling pathways. In this review, we aim to discuss the expression and regulation of T-bet in NK cells, the role of T-bet in mouse NK cell development, maturation, and function, as well as the role of T-bet in acute, chronic infection, inflammation, autoimmune diseases and tumors.

P-006: Expanded natural killer cells with daratumumab, lenalidomide and dexamethasone combination potentiates antimyeloma activity in a myeloma xenograft model

<h3>Background</h3> Advances in the treatment of multiple myeloma (MM) significantly improved overall survival of patients. Nonetheless, MM still remains an incurable disease. This emphasizes urgent need for novel combination therapies for the treatment of MM. Recently, natural killer (NK) cells are becoming an attractive and safe tool in the field of immunotherapy. <h3>Methods</h3> Here, we investigated how daratumumab, lenalidomide, and dexamethasone (DRd) treatment potentiate antitumor effects of NK cells in a MM xenograft mouse model. NK cells were expanded and activated using K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells of MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNK), Rd, Rd + eNK, DRd, and DRd + eNK. DRd treatment strongly enhanced the cytotoxicity of eNK by upregulating NK cell's activation ligands and effector function. <h3>Results</h3> DRd combination with eNK significantly prolonged mouse survival and reduced MM progression and serum M-protein level. Moreover, DRd treatment significantly increased eNK persistence and homing into MM sites. <h3>Conclusions</h3> Our findings suggest that DRd treatment augments the anti-myeloma effects of ex vivo expanded and activated NK cells by modulating immune responses in MM-bearing mice.

Natural killer (NK) cell-based immunotherapies and the many faces of NK cell memory: A look into how nanoparticles enhance NK cell activity.

Natural killer (NK) cells are lymphocytes able to exert potent antitumor and antiviral functions by different means. Besides their classification as innate lymphoid cells (ILCs), NK cells exhibit memory-like and memory responses after cytokine preactivation, viral infections and hapten exposure. Multiple NK cell-based immunotherapies have been developed and are currently being tested, including the possibility to translate the NK cell memory responses into the clinic. Nevertheless, still there is a need to improve these therapies, especially for the treatment of solid tumors, and nanotechnology represents an attractive option to increase NK cell effector functions against transformed cells. In this article, we review the basis of NK cell activity, the diversity of the NK cell memory responses and the current NK cell-based immunotherapies that are being used in the clinic. Furthermore, we take a look into nanotechnology-based strategies targeting NK cells to modulate their responses for effective immunotherapy.

Peptide Adjuvant to Invigorate Cytolytic Activity of NK Cells in an Obese Mouse Cancer Model.

Cancer patients who are overweight compared to those with normal body weight have obesity-associated alterations of natural killer (NK) cells, characterized by poor cytotoxicity, slow proliferation, and inadequate anti-cancer activity. Concomitantly, prohibitin overexpressed by cancer cells elevates glucose metabolism, rendering the tumor microenvironment (TME) more tumor-favorable, and leading to malfunction of immune cells present in the TME. These changes cause vicious cycles of tumor growth. Adoptive immunotherapy has emerged as a promising option for cancer patients; however, obesity-related alterations in the TME allow the tumor to bypass immune surveillance and to down-regulate the activity of adoptively transferred NK cells. We hypothesized that inhibiting the prohibitin signaling pathway in an obese model would reduce glucose metabolism of cancer cells, thereby changing the TME to a pro-immune microenvironment and restoring the cytolytic activity of NK cells. Priming tumor cells with an inhibitory the prohibitin-binding peptide (PBP) enhances cytokine secretion and augments the cytolytic activity of adoptively transferred NK cells. NK cells harvested from the PBP-primed tumors exhibit multiple markers associated with the effector function of active NK cells. Our findings suggest that PBP has the potential as an adjuvant to enhance the cytolytic activity of adoptively transferred NK cells in cancer patients with obesity.

HIF1α is required for NK cell metabolic adaptation during virus infection.

Natural killer (NK) cells are essential for early protection against virus infection and must metabolically adapt to the energy demands of activation. Here, we found upregulation of the metabolic adaptor hypoxia-inducible factor-1α (HIF1α) is a feature of mouse NK cells during murine cytomegalovirus (MCMV) infection in vivo. HIF1α-deficient NK cells failed to control viral load, causing increased morbidity. No defects were found in effector functions of HIF1αKO NK cells; however, their numbers were significantly reduced. Loss of HIF1α did not affect NK cell proliferation during in vivo infection and in vitro cytokine stimulation. Instead, we found that HIF1α-deficient NK cells showed increased expression of the pro-apoptotic protein Bim and glucose metabolism was impaired during cytokine stimulation in vitro. Similarly, during MCMV infection HIF1α-deficient NK cells upregulated Bim and had increased caspase activity. Thus, NK cells require HIF1α-dependent metabolic functions to repress Bim expression and sustain cell numbers for an optimal virus response.

The Role of microRNAs in NK Cell Development and Function.

The clinical use of natural killer (NK) cells is at the forefront of cellular therapy. NK cells possess exceptional antitumor cytotoxic potentials and can generate significant levels of proinflammatory cytokines. Multiple genetic manipulations are being tested to augment the anti-tumor functions of NK cells. One such method involves identifying and altering microRNAs (miRNAs) that play essential roles in the development and effector functions of NK cells. Unique miRNAs can bind and inactivate mRNAs that code for cytotoxic proteins. MicroRNAs, such as the members of the Mirc11 cistron, downmodulate ubiquitin ligases that are central to the activation of the obligatory transcription factors responsible for the production of inflammatory cytokines. These studies reveal potential opportunities to post-translationally enhance the effector functions of human NK cells while reducing unwanted outcomes. Here, we summarize the recent advances made on miRNAs in murine and human NK cells and their relevance to NK cell development and functions.

Leveraging NKG2D Ligands in Immuno-Oncology.

Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.

Natural killer cell effector functions in antiviral defense.

Natural killer (NK) cells are innate lymphoid cells involved in the control of tumors and viral infections. They provide protection by producing cytokines and by directly lysing target cells. Both effector mechanisms have been identified to contribute to viral control, depending on the context of infection. Activation of NK cells depends on the integration of signals received by cytokine receptors and activation and inhibitory receptors recognizing ligands expressed by virus-infected cells. While the control of viral infections by NK cells is well established, the signals perceived by NK cells and how these signals integrate to mediate optimal viral control have been focus of ongoing research. Here, we discuss the current knowledge on NK cell activation and integration of signals that lead to interferon gamma production and cytotoxicity in viral infections. We review NK cell interactions with viruses, with particular focus on murine cytomegalovirus studies, which helped elucidate crucial aspects of antiviral NK cell immunity.

Inhibitory axes impacting on the activity and fate of Innate Lymphoid Cells

In neoplastic patients, an effective immune response ideally should be achieved by the coordinated action of different immune cells with tumor-suppressive functions. These include the more cytolytic members of the Innate Lymphoid Cells (ILCs) family represented by the Natural Killer (NK) cells, whose activities in cancer patients, however, can be hampered by several inhibitory signals. These are generated by membrane-bound and soluble molecules that, interacting with specific inhibitory receptors, create inhibitory axes impacting the NK cell differentiation and effector functions. These breaks, which now represent major immunotherapeutic targets, may be sensitive to interferon (IFN)-γ, whose source, in vivo, is represented by different cell types including the NK and ILC1. Since also ILCs can express receptors of the inhibitory axes like PD-1 and TIGIT, their therapeutic blockade might further amplify the IFN-γ release that, as an unwanted side effect, would promote the onset of NK cell-resistant tumor variants (NKRTV) expressing ligands involved in inhibitory axes. These variants might also arise from the activity of other cytokines such as IL-27, which can increase the expression of HLA class I and PD-Ls in different cell types, including tumor cells. Besides the amplification of membrane-bound inhibitory axes, tumors can reduce the number of infiltrating cytolytic ILCs, promote the recruitment of poorly cytolytic NK cell subsets, and manipulate to their advantage the infiltrating immune cells, which acquire tumor-promoting activities. This occurs thanks to the production of soluble factors including TGF-β1 and IL-18 that, alone or in combination, modify the activating and chemokine receptor repertoire of NK cells, and induce the ILCs differentiation towards cells ineffective in fighting cancer or, even worse, with tumor-promoting functions.The present review aims to present and discuss major inhibitory axes impacting on ILCs functions, migration, and differentiation with a major focus on tumor context.

Natural Killer Cells Are Present in Rag1\u2212/\u2212 Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke

Rag1−/− mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1−/− mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1−/− and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1−/− NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1−/− were comparable in number and function to those in WT mice. Rag1−/− mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1−/− controls. Our results indicate that NK cells from Rag1−/− mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.