Restriction of Glycolysis Increases Serial Killing Capacity of Natural Killer Cells.

Tumor cells rely heavily on glycolysis to meet their high metabolic demands. While this results in nutrient deprivation within the tumor microenvironment and has negative effects on infiltrating immune cells such as natural killer (NK) cells, it also creates a potential target for cancer therapies. Here we use Glupin, an inhibitor of glucose transporters, to study the effect of limited glucose uptake on NK cells and their anti-tumor functions. Glupin treatment effectively inhibited glucose uptake and restricted glycolysis in NK cells. However, acute treatment had no negative effect on NK cell cytotoxicity or cytokine production. Long-term restriction of glucose uptake via Glupin treatment only delayed NK cell proliferation, as they could switch to glutaminolysis as an alternative energy source. While IFN-γ production was partially impaired, long-term Glupin treatment had no negative effect on degranulation. Interestingly, the serial killing activity of NK cells was even slightly enhanced, possibly due to changes in NAD metabolism. This demonstrates that NK cell cytotoxicity is remarkably robust and insensitive to metabolic disturbances, which makes cellular metabolism an attractive target for immune-mediated tumor therapies.

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

Analyzing the activity of the proteases granzyme B and caspase-8 inside living cells using fluorescence localization reporters.

Natural Killer (NK) cells are innate lymphocytes that are important for early immune reactions against viral infections and cancer. Their cytotoxic activity is mediated by the release of perforin and granzymes or by engaging death receptors on the surface of their target cells. Here we provide a protocol for the use of fluorescence localization reporters to measure the activity of granzyme B or caspase-8 activity inside living target cells. This method can be used to investigate how these two killing pathways are used by NK cells. By modifying the modular structure of the reporters, they can be adapted to study other cytotoxic effector cells or signaling pathways, where proteases play an important role.

Inhibition of Glucose Uptake Blocks Proliferation but Not Cytotoxic Activity of NK Cells.

Tumor cells often have very high energy demands. Inhibition of glucose uptake is therefore a possible approach to limit the proliferation and survival of transformed cells. However, immune cells also require energy to initiate and to maintain anti-tumor immune reactions. Here, we investigate the effect of Glutor, an inhibitor of glucose transporters, on the function of human Natural Killer (NK) cells, which are important for the immunosurveillance of cancer. Glutor treatment effectively inhibits glycolysis in NK cells. However, acute treatment with the inhibitor has no effect on NK cell effector functions. Prolonged inhibition of glucose uptake by Glutor prevents the proliferation of NK cells, increases their pro-inflammatory regulatory function and reduces the stimulation-dependent production of IFN-γ. Interestingly, even after prolonged Glutor treatment NK cell cytotoxicity and serial killing activity were still intact, demonstrating that cytotoxic NK cell effector functions are remarkably robust against metabolic disturbances.

Impact of N6-methyladenosine (m6A) modification on immunity.

N6-methyl-adenosine (m6A) is the most prevalent modification on mRNAs and long noncoding RNAs (lnRNAs) in higher eukaryotes. Modulation of m6A relies on m6A writers, erasers and readers. m6A modification contributes to diverse fundamental biological functions at the molecular, cellular, and physiological levels. The dysregulation of m6A modification has been implicated in various human diseases. Thus, m6A modification has now become a research hotspot for its potential therapeutic applications in the treatment of various cancers and diseases. The immune system is essential to provide defense against infections and cancers. This review summarizes the current knowledge about the roles of m6A in regulating immune cell functions and immune responses. Video abstract.

Distinct distribution patterns of exercise-induced natural killer cell mobilization into the circulation and tumor tissue of patients with prostate cancer.

The mobilization and activation of natural killer (NK) cells have been proposed as key mechanisms promoting anti-oncogenic effects of physical exercise. Although mouse models have proven that physical exercise recruits NK cells to tumor tissue and inhibits tumor growth, this preclinical finding has not been transferred to the clinical setting yet. In this first-in-human study, we found that physical exercise mobilizes and redistributes NK cells, especially those with a cytotoxic phenotype, in line with preclinical models. However, physical exercise did not increase NK cell tumor infiltrates. Future studies should carefully distinguish between acute and chronic exercise modalities and should be encouraged to investigate more immune-responsive tumor entities.

Human NK cells responses are enhanced by CD56 engagement.

Natural Killer (NK) cells are important innate lymphocytes for effective immune responses against intracellular pathogens and tumors. CD56 is a well-known marker for human NK cells, but there is very limited information about a functional role of this surface receptor. Here, we show that engagement of CD56 can induce NK cell activation resulting in degranulation, IFN-γ secretion and morphological changes, making CD56 a potential co-activating receptor in NK cells. Interestingly, this effect was only observed in cytokine pre-activated and not in freshly isolated human NK cells, demonstrating that NK cell reactivity upon CD56 engagement was dependent on cytokine stimulation. Inhibition of Syk, PI3K, Erk, and src-family-kinases impaired CD56-mediated NK cell stimulation. Finally, we used CRISPR/Cas9 to delete CD56 from primary human NK cells. While this abolished the stimulatory effect of CD56 on pre-activated NK cells, the cytotoxic activity of NK cells against several tumor target cells was not affected by the absence of CD56. This demonstrates that the stimulating effect of CD56 on pre-activated NK cells does not have a major impact on their cytotoxic activity, but it may contribute to the function of CD56 as a fungal recognition receptor and in the NK cell developmental synapse.

Human ILC3 Exert TRAIL-Mediated Cytotoxicity Towards Cancer Cells.

Group 3 helper Innate Lymphoid Cells (ILC3s) are cytokine-producing lymphocytes that respond to stress signals released during disturbed tissue homeostasis and infection. Upon activation, ILC3s secrete IL-22 and IL-17, and orchestrate immune responses against extracellular pathogens. Their role in cancer remains poorly explored. To determine their anti-cancer effector potential, we co-cultured cytokine-activated human ILC3s with cancer cells of different origins. ILC3s were able to directly respond to tumor cells, resulting in enhanced IFN-γ production. Upon tumor cell encounter, ILC3s maintained expression of the transcription factor RORγt, indicating that ILC3s preserved their identity. ILC3s were able to directly kill both hepatocellular carcinoma and melanoma tumor cells expressing cell-death receptor TRAILR2, through the activation of Caspase-8 in target cells. Moreover, liver-derived cytokine-activated ILC3s also expressed TRAIL and were able to eliminate hepatoblastoma cells. Together, our data reveal that ILC3s can participate in anti-tumor immune response through direct recognition of tumor cells resulting in IFN-γ release and TRAIL-dependent cytotoxicity. Thus, ILC3s might be ancillary players of anti-tumor immunity in tissues, acting as primary responders against transformed or metastasizing cells, which might be further exploited for therapies against cancer.

Activating Natural Killer Cell Receptors, Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein.

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.

Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression.

Mouse models of non-alcoholic fatty liver disease (NAFLD) are required to define therapeutic targets, but detailed time-resolved studies to establish a sequence of events are lacking. Here, we fed male C57Bl/6N mice a Western or standard diet over 48 weeks. Multiscale time-resolved characterization was performed using RNA-seq, histopathology, immunohistochemistry, intravital imaging, and blood chemistry; the results were compared to human disease. Acetaminophen toxicity and ammonia metabolism were additionally analyzed as functional readouts. We identified a sequence of eight key events: formation of lipid droplets; inflammatory foci; lipogranulomas; zonal reorganization; cell death and replacement proliferation; ductular reaction; fibrogenesis; and hepatocellular cancer. Functional changes included resistance to acetaminophen and altered nitrogen metabolism. The transcriptomic landscape was characterized by two large clusters of monotonously increasing or decreasing genes, and a smaller number of 'rest-and-jump genes' that initially remained unaltered but became differentially expressed only at week 12 or later. Approximately 30% of the genes altered in human NAFLD are also altered in the present mouse model and an increasing overlap with genes altered in human HCC occurred at weeks 30-48. In conclusion, the observed sequence of events recapitulates many features of human disease and offers a basis for the identification of therapeutic targets.

Results of the Optimune trial: A randomized controlled trial evaluating a novel Internet intervention for breast cancer survivors.

INTRODUCTION: After the acute treatment phase, breast cancer patients often experience low quality of life and impaired mental health, which could potentially be improved by offering cognitive behavioural therapy (CBT) and addressing exercise and dietary habits. However, CBT and other behavioural interventions are rarely available beyond the acute treatment phase. Internet-based interventions could bridge such treatment gaps, given their flexibility and scalability. In this randomized controlled trial (RCT), we investigated the effects of such an intervention ("Optimune") over three months. METHODS: This RCT included 363 female breast cancer survivors (age range = 30-70), recruited from the community, who had completed the active treatment phase. Inclusion criteria were: breast cancer diagnosis less than 5 years ago and acute treatment completion at least 1 month ago. Participants were randomly assigned to (1) an intervention group (n = 181), in which they received care as usual (CAU) plus 12-month access to Optimune immediately after randomization, or (2) a control group (n = 182), in which they received CAU and Optimune after a delay of 3 months. Primary endpoints were quality of life (QoL), physical activity, and dietary habits at three months. We hypothesized that intervention group participants would report better QoL, more physical activity, and improved dietary habits after 3 months. RESULTS: Intention-to-treat (ITT) analyses revealed significant effects on QoL (d = 0.27, 95% CI: 0.07-0.48) and dietary habits (d = 0.36, 95% CI: 0.15-0.56), but the effect on physical exercise was not significant (d = 0.30; 95% CI: 0.10-0.51). DISCUSSION: These findings suggest the effectiveness of Optimune, a new CBT-based Internet intervention for breast cancer survivors, in facilitating improvements in quality of life and dietary habits. Efforts to disseminate this intervention more broadly may be warranted. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03643640. Registered August 23rd 2018, https://clinicaltrials.gov/ct2/show/NCT03643640.

Regulation of natural killer cell activity by glucocorticoids, serotonin, dopamine, and epinephrine.

The immune system and the nervous system are highly complex organs composed of various different cells that must interact with each other for proper function of the system. This communication can be mediated by soluble factors. The factors released by the nervous system (neurotransmitters) differ from those released by the immune system (cytokines). Nevertheless, the nervous and immune systems can influence each other's activity because immune cells express neurotransmitter receptors, and neurons express cytokine receptors. Moreover, immune cells can synthesize and release neurotransmitters themselves, thus using neurotransmitter-mediated pathways via autocrine and paracrine mechanisms. Natural killer (NK) cells are innate lymphocytes that are important for early and effective immune reactions against infections and cancer. Many studies have shown the strong influence of stress and the nervous system on NK cell activity. This phenomenon may be one reason why chronic stress leads to a higher incidence of infections and cancer. Here, we review the effects of neuroendocrine factors on the different activities of NK cells. Understanding the effects of neuroendocrine factors on NK cell activities during physiological and pathophysiological conditions may result in novel therapeutic strategies to enhance NK cell functions against tumors.

Protocol for the Optimune trial: a randomized controlled trial evaluating a novel Internet intervention for breast cancer survivors.

INTRODUCTION: Depression and fatigue are common in breast cancer survivors, and their presence is associated with personal suffering and worse prognosis. While many women receive short-term psychological support in the acute treatment phase, this is rarely available in subsequent phases. Internet interventions for breast cancer survivors could provide additional psychological support, as they are easily accessible and may be effective. However, no trial has yet examined the effectiveness of an Internet intervention that provides cognitive behavioural therapy techniques plus lifestyle advice for this population. This trial aims to test whether Optimune, a novel Internet intervention we developed for that purpose, leads to improvements in quality of life and relevant lifestyle habits over the course of 3 to 6 months. METHODS: This randomized controlled trial (RCT) will include 360 female breast cancer survivors who have completed the active tumour eradication phase. Participants will be recruited from various settings, including web-based advertisements and Internet forums in German-speaking countries. The main inclusion criteria are a breast cancer diagnosis less than 5 years ago and completion of acute treatment at least 1 month ago, as verified by discharge letter from an oncology treatment centre. Participants will be randomly assigned to either (1) a control group, in which they receive care as usual (CAU) and are given access to Optimune after a delay of 3 months (CAU/wait list control), or (2) a treatment group that may also use CAU and will receive 12-month access to Optimune immediately after randomization. The three primary endpoints are quality of life, physical activity and diet quality, assessed with the World Health Organization Quality of Life Questionnaire, the International Physical Activity Questionnaire and the Food Quality Questionnaire, at 3 months post-baseline; secondary outcomes include cancer-related fatigue, emotional stress, depression, anxiety, fear of progression, insomnia, usefulness of the programme and negative treatment effects. Online assessments are conducted at baseline (T0), 3 months (T1) and 6 months (T2). DISCUSSION: Results of this RCT are expected to extend the body of knowledge with regard to the effectiveness of CBT-based Internet interventions for female breast cancer survivors. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03643640. Registered on 23 August 2018.

NK cell detachment from target cells is regulated by successful cytotoxicity and influences cytokine production.

Natural killer (NK) cells participate in early immune defenses against pathogens and tumors and play a major role as immune effector and regulatory cells. The NK cell-mediated elimination of an infected or cancerous cell is a highly regulated process that requires the formation of a cell contact, the establishment of an immunological synapse and the polarization and release of lytic granules. Additionally, the detachment of NK cells from target cells is important for NK cells to bind and kill other cells in a process called serial killing. However, very little is known about this detachment process. Here, we show that NK detachment is directly connected to the successful killing of a target cell. The inhibition of killing due to reduced NK cell cytotoxicity or increased target cell resistance results in defective detachment and prolonged contact times. This effect leads to sustained Ca2+ flux in NK cells and the hypersecretion of proinflammatory cytokines. Linking defective cytotoxicity with enhanced cytokine secretion via reduced detachment may explain inflammatory pathologies in several diseases.

Activation of natural killer cells by rituximab in granulomatosis with polyangiitis.

OBJECTIVE: In the last few years, anti-CD20 antibody rituximab profoundly changed the therapeutic landscape of granulomatosis with polyangiitis (GPA). Here, we investigated whether natural killer (NK) cells may play a role in rituximab's mechanism of action in GPA. METHODS: B cell depletion, NK cell degranulation, and the expression of CD69 and CD16 on NK cells were measured in a series of in vitro experiments using peripheral blood mononuclear cells (PBMCs). In vivo activation of NK cells was investigated in patients receiving rituximab infusions. Cells were analyzed by seven-color flow cytometry. RESULTS: NK cells from GPA patients were activated by immobilized rituximab. Also soluble rituximab activated NK cells, provided that B cells were present. NK cells degranulated and expressed the activation marker CD69 while CD16 expression was decreased. This activation of NK cells by soluble rituximab was accompanied by a reduction of B cells. The next-generation anti-CD20 antibody obinutuzumab showed stronger effects compared to rituximab on both the reduction of B cells and the activation of NK cells. Finally, we found that rituximab led to the activation of NK cells in vivo, provided that B cells were not depleted due to prior rituximab infusions. CONCLUSION: B cell-bound rituximab activates NK cells in GPA. While NK cells therefore participate in rituximab's mechanism of action in humans, their potential may be more efficiently exploited, e.g., by Fc engineering of therapeutic antibodies.

NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing.

NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor-mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor-mediated cytotoxicity are differentially regulated during NK cell serial killing.

Mechanisms of natural killer cell-mediated cellular cytotoxicity.

Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the immune system to combat viral infections and cancer. Cytotoxic T cells and natural killer (NK) cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of NK cells. NK cells can kill virally infected of transformed cells via the directed release of lytic granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a highly regulated process to prevent damage during the synthesis of these cytotoxic molecules. Additionally, NK cells have developed several strategies to protect themselves from the cytotoxic activity of granular content upon degranulation. While granule-mediated apoptosis is a fast process, death receptor-mediated cytotoxicity requires more time. Current data suggest that these 2 cytotoxic mechanisms are regulated during the serial killing activity of NK cells. As many modern approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unraveling these pathways will be important to further progress these therapeutic strategies.

Circulating growth/differentiation factor 15 is associated with human CD56bright natural killer cell dysfunction and nosocomial infection in severe systemic inflammation.

BACKGROUND: Systemic inflammation induced by sterile or infectious insults is associated with an enhanced susceptibility to life-threatening opportunistic, mostly bacterial, infections due to unknown pathogenesis. Natural killer (NK) cells contribute to the defence against bacterial infections through the release of Interferon (IFN) γ in response to Interleukin (IL) 12. Considering the relevance of NK cells in the immune defence we investigated whether the function of NK cells is disturbed in patients suffering from serious systemic inflammation. METHODS: NK cells from severely injured patients were analysed from the first day after the initial inflammatory insult until the day of discharge in terms of IL-12 receptor signalling and IFN-γ synthesis. FINDINGS: During systemic inflammation, the expression of the IL-12 receptor ß2 chain, phosphorylation of signal transducer and activation 4, and IFN-γ production on/in NK cells was impaired upon exposure to Staphylococcus aureus. The profound suppression of NK cells developed within 24 h after the initial insult and persisted for several weeks. NK cells displayed signs of exhaustion. Extrinsic changes were mediated by the early and long-lasting presence of growth/differentiation factor (GDF) 15 in the circulation that signalled through the transforming growth factor ß receptor I and activated Smad1/5. Moreover, the concentration of GDF-15 in the serum inversely correlated with the IL-12 receptor ß2 expression on NK cells and was enhanced in patients who later acquired septic complications. INTERPRETATION: GDF-15 is associated with the development of NK cell dysfunction during systemic inflammation and might represent a novel target to prevent nosocomial infections. FUND: The study was supported by the Department of Orthopaedics and Trauma Surgery, University Hospital Essen.

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.