Natural Killer Cell Effector Functions Research Articles

Glycolysis and Oxidative Phosphorylation Play Critical Roles in Natural Killer Cell Receptor-Mediated Natural Killer Cell Functions.

Natural killer (NK) cells are innate lymphocytes that directly kill tumor and pathogen-infected cells upon activation by cytokines and NK cell receptors (NKRs) without previous sensitization. It is known that cell metabolism affects the differentiation and effector functions of immune cells. For instance, interleukin−2 and interleukin−15 treatment increases glycolysis and oxidative phosphorylation (OXPHOS) in NK cells to support their effector functions. However, little is known about the metabolic reprogramming of human NK cells upon their activation by NKRs. In this study, we investigated the metabolism of NK cells stimulated via NKRs. We found that NK cells upregulated glycolysis and OXPHOS in response to anti-CD16 antibody or NKG2D ligand engagement. Inhibition of either glycolysis or OXPHOS impaired NK cell production of interferon-γ. Interestingly, inhibition of glycolysis but not OXPHOS decreased NK cell killing and dampened NK cell degranulation and Fas ligand expression, suggesting that glycolysis is more critical for NKR-activated cell cytotoxicity. Thus, our study provides insight into understanding the metabolic requirements underlying different effector functions of human NK cells.

Targeting Natural Killer Cells for Tumor Immunotherapy.

Natural killer (NK) cells are important innate cytotoxic lymphocytes with a rapid and efficient capacity to recognize and kill tumor cells. In recent years, adoptive transfer of autologous- or allogeneic-activated NK cells has become a promising cellular therapy for cancer. However, the therapeutic efficiency is encouraging in hematopoietic malignancies, but disappointing in solid tumors, for which the use of NK-cell-based therapies presents considerable challenges. It is difficult for NK cells to traffic to, and infiltrate into, tumor sites. NK cell function, phenotype, activation, and persistence are impaired by the tumor microenvironment, even leading to NK cell dysfunction or exhaustion. Many strategies focusing on improving NK cells' durable persistence, activation, and cytolytic activity, including activation with cytokines or analogs, have been attempted. Modifying them with chimeric antigen receptors further increases the targeting specificity of NK cells. Checkpoint blockades can relieve the exhausted state of NK cells. In this review, we discuss how the cytolytic and effector functions of NK cells are affected by the tumor microenvironment and summarize the various immunotherapeutic strategies based on NK cells. In particular, we discuss recent advances in overcoming the suppressive effect of the tumor microenvironment with the aim of enhancing the clinical outcome in solid tumors treated with NK-cell-based immunotherapy.

Epstein-Barr Virus Peptides Derived from Latent Cycle Proteins Prevent the Inhibition of NKG2A Expressing NK Cells

Natural killer (NK) cells control viral infection through the interaction between inhibitory receptors and human leukocyte antigen (HLA) ligands and bound peptide. NK cells expressing the inhibitory receptor NKG2A/CD94 recognize and respond to autologous B cells latently infected with Epstein–Barr virus (EBV). The mechanism is not yet understood, thus we investigated peptides derived from the seven latent proteins of EBV in the interaction of NKG2A and its ligand HLA-E. Functional analysis demonstrated that EBV peptides can bind to HLA-E and block inhibition of NK cell effector function. Moreover, analysis of DNA from 79 subjects showed sequence variations in the latent protein, LMP1, which alters NK responses to EBV. We provide evidence that peptides derived from EBV latent cycle proteins can impair the recognition of NKG2A despite being presented by HLA-E, resulting in NK cell activation. Thus, blocking of the NKG2A-HLA-E axis with viral peptides may have therapeutic potential.

Mannan-Binding Lectin Regulates Inflammatory Cytokine Production, Proliferation, and Cytotoxicity of Human Peripheral Natural Killer Cells.

Natural killer (NK) cells represent the founding members of innate lymphoid cells (ILC) and play critical roles in inflammation and the immune response. NK cell effector functions are regulated and fine-tuned by various immune modulators. Mannan (or mannose)-binding lectin (MBL), a soluble C-type lectin, is traditionally recognized as an initiator of the complement pathway. Recently, it is also considered as an immunomodulator by its interaction with kinds of immune cells. However, the effect of MBL on NK cell function remains unexplored. In this study, we found that human plasma MBL could interact directly with peripheral NK cells partially via its collagen-like region (CLR). This MBL binding markedly suppressed the interleukin-2- (IL-2-) induced inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) production but increased the IL-10 production in NK cells. In addition, the expression of activation surface markers such as CD25 and CD69 declined after MBL treatment. Also, MBL impaired the proliferation and lymphokine-activated killing (LAK) of NK cells. Moreover, we demonstrated that MBL inhibited IL-2-induced signal transducers and activators of transcription 5 (STAT5) activation in NK cells. In conclusion, we have uncovered a far unknown regulatory role of MBL on NK cells, a new clue that could be important in the immunomodulatory networks of immune responses.

Immunomodulation of NK Cell Activity.

Natural killer (NK) cells can kill virus-infected cells and tumor cells without prior sensitization and secrete numerous cytokines and chemokines that modulate the activity of different cells of the immune system. The recognition of target cells is mediated by germ line-encoded receptors, and the activity of NK cells can be further regulated by soluble factors such as cytokines and Toll-like receptor ligands. Thus, NK cells display an exciting potential as a powerful immunotherapeutic tool against malignant diseases, and different strategies are being tested aiming to overcome tumor-induced NK cell suppression and restore NK-cell mediated antitumor activity. This section describes different flow cytometry-based protocols to study NK cell effector functions, which can be used to evaluate the immunomodulatory ability of different therapeutic compounds.

The IRE1-XBP1s Pathway Impairment Underpins NK Cell Dysfunction in Hodgkin Lymphoma, That Is Partly Restored By PD-1 Blockade

Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Restoring Innate and Adaptive Immune Repertoire in Multiple Myeloma for Therapeutic Application

Despite advances and improvements in survival, majority of multiple myeloma (MM) patients ultimately relapses. Extensive analysis on the properties of tumor cells has provided interesting insights into the disease biology allowing for the identification of novel targets and development of related therapeutics. However, the key microenvironmental influences, especially immune microenvironment, that drive the disease and impact outcome remain to be fully characterized. We have now performed both single cell RNA-sequencing and high-dimensional CyTOF analysis of both peripheral blood (PBMC) and bone marrow mononuclear cells (BMMC) from MM patients and healthy donors (HD) and evaluated both phenotypic and functional state of the MM immune repertoire. In addition to humoral immunodeficiency impacting aberrations in B-cell subsets (B1a, B1b, B2 and Breg cells) and the disfunction in the adaptive immune system including an increase in the immunosuppressive cells (Tregs or myeloid-derived suppressor cells), a significant impairment of the innate immunity was identified in MM patients with a progressive decline in functional state of natural killer (NK) cells. Additionally, a lower expression of the activating receptors NKG2D and NKp46 as opposed to a higher expression of certain inhibitory KIR receptors was observed in NK cells from MM patients compared to HD. This immunosuppressive microenvironment allows tumor immune escape and ultimately myeloma cell growth. To overcome some of the immune dysfunction observed in MM, we have evaluated the role of IL15, a key player in both innate and adaptive immunity, with antitumor activity via enhancing both NK and memory T cell functions. Using extensive flow cytometry-based analysis, we evaluated impact of recombinant IL15 on PBMC from HD and MM patients at different stages of disease. Treatment with recombinant IL15 rescued the immune effector cell decline observed in MM patients. Specifically, a 2.9-fold-increase in CD8+ CD45RO+ CCR7- effector memory T cells and a 1.5-fold-increase in NK populations in both HD and MM PBMC was observed. For a subset of MM patients, we have also confirmed these positive effects on BMMC. Importantly, cytotoxicity tests revealed an improvement in NK cell effector functions leading to increased tumor cell recognition and killing, while we did not observe any direct effect of IL15 on growth and viability of MM cells. Integration of IL15 in the immunotherapeutic arsenal is limited by the unfavorable pharmacokinetic properties. Therefore, for clinical application and to restore both innate and adaptive immunity, we evaluated impact of NKTR-255, a polymer-conjugated IL15 receptor agonist designed to engage the IL-15 pathway to stimulate and expand natural killer (NK) cells and promote the survival and expansion of central memory CD8+ T cells without inducing suppressive regulatory T cells. Treatment with NKTR-255 enhanced the number and function of both NK and CD8+ effector memory T cell populations in PBMC from HD and MM patients in a dose dependent manner. Interestingly, the natural killer T (NKT) cells, heterogeneous group of T cells that share properties of both T cells and NK cells with important role in MM, were also increased in number by NKTR-255. To evaluate if NKTR-255 increases NK recognition of MM cells, cytotoxicity tests were performed using several MM cell lines and primary MM cells as targets, and both NK cells purified from PBMC of HD and MM patients as effectors. NKTR-255 was able to revert the inhibitory status of NK cells from MM patients (via induction of NKG2D expression), and significantly increase NK susceptibility of the MM cells in a dose dependent manner. Taken together, our data suggest a significant impact of NKTR-255 on the activation of effector cell function to efficiently target MM cells. This study has important translational implications and highlights the importance of restoring the balance between innate and adaptive immunity in MM. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Munshi:Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy.

NK Cell Metabolism and Tumor Microenvironment.

Natural Killer (NK) cells are characterized by their potential to kill tumor cells by different means without previous sensitization and have, therefore, become a valuable tool in cancer immunotherapy. However, their efficacy against solid tumors is still poor and further studies are required to improve it. One of the major restrictions for NK cell activity is the immunosuppressive tumor microenvironment (TME). There, tumor and other immune cells create the appropriate conditions for tumor proliferation while, among others, preventing NK cell activation. Furthermore, NK cell metabolism is impaired in the TME, presumably due to nutrient and oxygen deprivation, and the higher concentration of tumor-derived metabolic end products, such as lactate. This metabolic restriction of NK cells limits their effector functions, and it could represent a potential target to focus on to improve the efficacy of NK cell-based therapies against solid tumors. In this review, we discuss the potential effect of TME into NK cell metabolism and its influence in NK cell effector functions.

The Food Additive tBHQ Impairs NK Cell Cytotoxicity Against Influenza Infection (FS12-02-19)

ObjectivesWe examined the effects of the food preservative, tert-Butylhydroquinone (tBHQ), on natural killer (NK) cells when infected with influenza in C57Bl/6 mice. Previously, we found tBHQ to negatively impact splenic NK cell effector function in vitro. Therefore, we hypothesized that the consumption of tBHQ would impair the NK cell response against a primary influenza virus infection. MethodsFemale, C57Bl/6 mice were fed either a diet containing 0.0014% tBHQ or a control diet two weeks prior to infection. Mice were instilled intranasally with 21.78 HAU of influenza A/PR/8/34 (H1N1) and were monitored for two days. Body weight was recorded as a measurement for infection. After day two of infection, lungs were collected and processed for analysis by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and total antioxidant capacity (TAC). ResultsThe NK cell activation markers, CD25 and CD69, were not affected by tBHQ consumption. Additionally, tBHQ did not have an effect on maturation of NK cells or interferon (IFN)γ production. Notably, NK cell expression of granzyme B was significantly decreased with tBHQ in infected mice. Furthermore, mice on the tBHQ diet showed decreased TAC after two days of infection compared to mice on the control diet, suggesting increased oxidative stress in the tBHQ group. ConclusionstBHQ had no effect on NK cell activation, maturation, and IFNγ production after two days of influenza infection. However, decreased expression of granzyme B by NK cells suggests tBHQ may impair NK cell cytoxicity during the infection. Moreover, the decrease in TAC suggests increased oxidative stress in the tBHQ-treated mice. Funding SourcesThis study was funded by National Institute of Environmental Health Sciences (ES024966).

Cytomegalovirus Infection Drives Avidity Selection of Natural Killer Cells

The process of affinity maturation, whereby T and Bcells bearing antigen receptors with optimal affinity to the relevant antigen undergo preferential expansion, is a key feature of adaptive immunity. Natural killer (NK) cells are innate lymphocytes capable of "adaptive" responses after cytomegalovirus (CMV) infection. However, whether NK cells are similarly selected on the basis of their avidity for cognate ligand is unknown. Here, we showed that NK cells with the highest avidity for the mouse CMV glycoprotein m157 were preferentially selected to expand and comprise the memory NK cell pool, whereas low-avidity NK cells possessed greater capacity for interferon-γ (IFN-γ) production. Moreover, we provide evidence for avidity selection occurring in human NK cells during human CMV infection. These results delineate how heterogeneity in NK cell avidity diversifies NK cell effector function during antiviral immunity, and how avidity selection might serve to produce the most potent memory NK cells.

Cytomegalovirus infection drives avidity selection of natural killer cells

Abstract Adaptive lymphocyte clones with the greatest receptor affinity tend to dominate primary and secondary immune responses. Strength of receptor signal and receptor abundance, along with competition for antigen and cytokines, drive preferential outgrowth of certain clones, shaping the effector and memory pool in selection processes known as affinity and avidity maturation. Natural killer (NK) cells are innate lymphocytes capable of “adaptive” responses following infectious challenges. However, whether NK cells undergo a similar selection process on the basis of their avidity for cognate ligand is not known. Here, we show that NK cells with a broad range of avidities for the mouse cytomegalovirus (MCMV) glycoprotein m157 are initially recruited after virus infection, but those with highest avidity are selected to undergo the greatest clonal expansion to comprise the memory NK cell population. Furthermore, pre-established levels of Ly49H receptor expression within the Ly49H+ NK cell pool dictated the functional contribution of a given NK cell during MCMV infection, with lower avidity NK cells possessing greater capacity for IFN-γ production, and higher avidity NK cells possessing greater capacity for cytotoxicity and adaptive responses. Moreover, we provide evidence for avidity selection also occurring in human NK cells during human CMV (HCMV) infection. These results provide a mechanistic understanding of how heterogeneity in NK cell avidity underlies the diversification of NK cell effector function during a primary antiviral immune response, and how the process of avidity selection may serve to produce the most potent memory NK cell pool.

Kctd9 Deficiency Impairs Natural Killer Cell Development and Effector Function.

We previously showed that potassium channel tetramerization domain containing 9 (KCTD9) is aberrantly expressed in natural killer (NK) cells in patients with hepatitis B virus-associated acute-on-chronic liver failure and mice with experimental fulminant hepatitis. However, the mechanism underlying the regulation of NK cell function and fulminant hepatitis progression by KCTD9 is unknown. Here, we investigated the role of Kctd9 in regulation of early development, maturation, and function of NK cells using Kctd9-knockout mice. Compared to wild-type mice, Kctd9-deficient mice exhibited impaired NK cell lineage commitment, as evidenced by selective reduction in the refined NK progenitors, and incomplete NK cell maturation, as manifested by a higher proportion of CD11b− NK cells and a lower percentage of CD11b+ NK cells with high proliferative potential. Moreover, Kctd9-depleted NK cells displayed insufficient IFN-γ production, degranulation, and granzyme B production in response to cytokine stimulation, and attenuated cytotoxicity to tumor cells in vitro. The defect in NK cells was further supported by ameliorated liver damage and improved survival in Kctd9-deficient mice following murine hepatitis virus strain-3 (MHV-3) infection, which otherwise leads to immune-mediated fulminant hepatitis, a phenotype homologous to that caused by NK cell depletion in wild-type mice. Further investigation to identify the underlying mechanism revealed that Kctd9 deficiency hindered the expression of transcription factors, including Ets1, Nfil3, Eomes, and Id2 in NK cells. Collectively, our data reveal that Kctd9 acts as a novel regulator for NK cell commitment, maturation, and effector function.

Novel Approach to Cell Surface Discrimination Between KIR2DL1 Subtypes and KIR2DS1 Identifies Hierarchies in NK Repertoire, Education, and Tolerance.

Cumulative activating and inhibitory receptor signaling controls the functional output of individual natural killer (NK) cells. Investigation of how competing signals impact response, however, has been hampered by the lack of available antibodies capable of distinguishing inhibitory and activating receptors with highly homologous ectodomains. Utilizing a novel combination of monoclonal antibodies with specificity for discrete inhibitory KIR2DL1 and activating KIR2DS1 allotypes found among 230 healthy donors, we investigated allele-specific receptor expression and function driven by KIR2DL1 and KIR2DS1 alleles. We found that co-expression of the HLA-C2 ligand diminishes KIR2DL1, but not KIR2DS1, cell surface staining, but does not impact the respective frequencies of KIR2DL1- and KIR2DS1-expressing cells within the NK repertoire. We can distinguish by flow cytometry NK cell populations expressing the most common KIR2DL1-C245 allotypes from those expressing the most common KIR2DL1-R245 allotypes, and we show that the informative differential binding anti-KIR2DL1/S1 clone 1127B is determined by amino acid residue T154. Although both KIR2DL1-C245 and KIR2DL1-R245 subtypes can be co-expressed in the same cell, NK cells preferentially express one or the other. Cells expressing KIR2DL1-C245 exhibited a lower KIR2DL1 cell surface density and lower missing-self reactivity in comparison to cells expressing KIR2DL1-R245. We found no difference, however, in sensitivity to inhibition or cell surface stability between the two KIR2DL1 isoforms, and both demonstrated similar expansion among NKG2C+ KIR2DL1+ NK cells in HCMV-seropositive healthy individuals. In addition to cell surface density of KIR2DL1, copy number of cognate HLA-C2 hierarchically impacted the effector capacity of both KIR2DL1+ cells and the tolerization of KIR2DS1+ NK cells. HLA-C2 tolerization of KIR2DS1+ NK cells could be overridden, however, by education via co-expressed self-specific inhibitory receptors, such as the heterodimer CD94/NKG2A. Our results demonstrate that effector function of NK cells expressing KIR2DL1 or KIR2DS1 is highly influenced by genetic variability and is calibrated by co-expression of additional NK receptors and cognate HLA-C2 ligands. These findings define the molecular conditions under which NK cells are activated or inhibited, potentially informing selection of donors for adoptive NK therapies.

Dissecting the Role of ArhGAP12 in Natural Killer Cell‐Mediated Cytotoxicity

The immune system consists of two broad branches: innate and adaptive. In the innate branch of the immune system, one will find natural killer (NK) cells, which are responsible for recognizing and killing virally infected or cancerous cells within the human body. Numerous immunodeficiency syndromes arise from mutations in genes that encode proteins essential to NK cells including those involved in NK cell development and effector functions. Among the most important NK cell effector functions is cell‐mediated cytotoxicity. Our lab has been interested in understanding the molecular machinery that regulates this process, in particular those regulated by the small GTP‐binding proteins of the Rho family. With this in mind, the ArhGAP12 protein, which functions as a GTPase activating protein for members of the Rho family, was tested in NK cells to see if it had an effect on cytotoxicity. Significantly, knockdown of ArhGAP12 in NK cells resulted in diminished NK cell‐mediated cytotoxicity. In addition, we found that ArhGAP12 is tyrosine phosphorylated following stimulation through the NKG2D+2B4 activating receptors, indicating that it is linked to this important signaling pathway. As ArhGAP12 is a multi‐modular signaling protein containing a src‐homology 3 (SH3), WW, pleckstrin homology (PH), and GAP domains, we performed proteomics to identify potential interacting partners using the SH3 domain as bait. We found that ArhGAP12 interacts strongly with multiple regulators of F‐actin dynamics including WAVE2, WASP, Evl and VASP. Taken together, these results suggest that ArhGAP12 is linked to activating receptors on NK cells, associates with F‐actin regulators, and is involved in NK cell‐mediated cytotoxicity.Support or Funding InformationMayo Clinic Graduate School of Biomedical Sciences SURF Program and the R01 grant AI120949 to D.D.B.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IL-27 promotes NK cell effector functions via Maf-Nrf2 pathway during influenza infection

Influenza virus targets epithelial cells in the upper respiratory tract. Natural Killer (NK) cell-mediated early innate defense responses to influenza infection include the killing of infected epithelial cells and generation of anti-viral cytokines including interferon gamma (IFN-γ). To date, it is unclear how the underlying cytokine milieu during infection regulates NK cell effector functions. Our data show during influenza infection myeloid cell-derived IL-27 regulates the early-phase effector functions of NK cells in the bronchioalveolar and lung tissue. Lack of IL-27R (Il27ra−/−) or IL-27 (Ebi3−/−) resulted in impaired NK cell effector functions including the generation of anti-viral IFN-γ responses. We identify CD27+CD11b+ NK cells as the primary subset that expresses IL-27R, which predominantly produces IFN-γ within the upper respiratory tract of the infected mice. IL-27 alone was incapable of altering the effector functions of NK cells. However, IL-27 sensitizes NK cells to augment both in vitro and in vivo responses mediated via the NKG2D receptor. This ‘priming’ function of IL-27 is mediated partly via transcriptional pathways regulated by Mafs and Nrf2 transcriptionally regulating TFAM and CPT1. Our data for the first time establishes a novel role for IL-27 in regulating early-phase effector functions of NK cells during influenza infection.

Viral Infection of Human Natural Killer Cells.

Natural killer (NK) cells are essential in the early immune response against viral infections, in particular through clearance of virus-infected cells. In return, viruses have evolved multiple mechanisms to evade NK cell-mediated viral clearance. Several unrelated viruses, including influenza virus, respiratory syncytial virus, and human immunodeficiency virus, can directly interfere with NK cell functioning through infection of these cells. Viral infection can lead to immune suppression, either by downregulation of the cytotoxic function or by triggering apoptosis, leading to depletion of NK cells. In contrast, some viruses induce proliferation or changes in the morphology of NK cells. In this review article, we provide a comprehensive overview of the viruses that have been reported to infect NK cells, we discuss their mechanisms of entry, and describe the interference with NK cell effector function and phenotype. Finally, we discuss the contribution of virus-infected NK cells to viral load. The development of specific therapeutics, such as viral entry inhibitors, could benefit from an enhanced understanding of viral infection of NK cells, opening up possibilities for the prevention of NK cell infection.

Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

Upon inflammation, natural killer (NK) cells undergo metabolic changes to support their high energy demand for effector function and proliferation. The metabolic changes are usually accompanied by an increase in the expression of nutrient transporters, leading to increased nutrient uptake. Among various cytokines inducing NK cell proliferation, the mechanisms underlying the effect of interleukin (IL)-18 in promoting NK cell proliferation are not completely understood. Here, we demonstrate that IL-18 is a potent cytokine that can enhance the expression of the nutrient transporter CD98/LAT1 for amino acids independently of the mTORC1 pathway and thereby induce a dramatic metabolic change associated with increased proliferation of NK cells. Notably, treatment of IL-18-stimulated NK cells with leucine activates the metabolic sensor mTORC1, indicating that the high expression of amino acid transporters induces amino acid-driven mTORC1 activation. Inhibition of the amino acid transporter CD98/LAT1 abrogated the leucine-driven mTORC1 activation and reduced NK cell effector function. Taken together, our study identified a novel role of IL-18 in up-regulating nutrient transporters on NK cells and thereby inducing metabolic changes, including the mTORC1 activation by amino acids.

Immunometabolism and natural killer cell responses.

Natural killer (NK) cells are lymphocytes with important roles in innate and adaptive immune responses to tumours and viral infection. However, in certain chronic diseases, including obesity and cancer, NK cell functional responses are impaired. Recently, research has highlighted the importance of NK cell metabolism in facilitating robust NK cell effector functions. This Review describes our current understanding of mouse and human NK cell metabolism and the key signalling pathways that mediate metabolic responses in NK cells. Furthermore, it explores howdefects in metabolism can contribute to the generation of dysfunctional NK cells in chronic disease. Finally, the potential for new therapeutic strategies targeting cellular metabolism isdiscussed.

Abstract A065: Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells

Abstract Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library. The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockout clones were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse cancer types, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T-cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagström, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.

NK cells specifically TCR-dressed to kill cancer cells.

BackgroundAdoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome.MethodsWe herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells.FindingsThis is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells.InterpretationThese results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution.FundThis work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.