Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.

Reduced IFNL1 and/or IFNL2, but not IFNL3 is associated with worse outcome in patients with COVID-19.

The recent pandemic was caused by the emergence of a new human pathogen, SARS-CoV-2. While the rapid development of many vaccines provided an end to the immediate crisis, there remains an urgent need to understand more about this new virus and what constitutes a beneficial immune response in terms of successful resolution of infection. Indeed, this is key for development of vaccines that provide long lasting protective immunity. The interferon lambda (IFNL) family of cytokines are produced early in response to infection and are generally considered anti-viral and beneficial. However, data regarding production of IFNL cytokines in COVID-19 patients is highly variable, and generally from underpowered studies. In this study, we measured all three IFNL1, IFNL2 and IFNL3 cytokines in plasma from a well characterised, large COVID-19 cohort (n=399) that included good representation from patients with a more indolent disease progression, and hence a beneficial immune response. While all three cytokines were produced, they differed in both the frequency of expression in patients, and the levels produced. IFNL3 was produced in almost all patients but neither protein level nor IFNL3/IFNL4 SNPs were associated with clinical outcome. In contrast, both IFNL1 and IFNL2 levels were significantly lower, or absent, in plasma of patients that had a more severe disease outcome. These data are consistent with the concept that early IFNL1 and IFNL2 cytokine production is protective against SARS-CoV-2 infection.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Canonical TGF-ß Signaling Pathway Represses Human NK Cell Metabolism.

Cytokines stimulate rapid metabolic changes in human NK cells, including increases in both glycolysis and oxidative phosphorylation pathways. However, how these are subsequently regulated is not known. In this study, we demonstrate that TGF-ß can inhibit many of these metabolic changes, including oxidative phosphorylation, glycolytic capacity, and respiratory capacity. TGF-ß also inhibited cytokine-induced expression of the transferrin nutrient receptor CD71. In contrast to a recent report on murine NK cells, TGF-ß-mediated suppression of these metabolic responses did not involve the inhibition of the metabolic regulator mTORC1. Inhibition of the canonical TGF-ß signaling pathway was able to restore almost all metabolic and functional responses that were inhibited by TGF-ß. These data suggest that pharmacological inhibition of TGF-ß could provide a metabolic advantage to NK cells that is likely to result in improved functional responses. This has important implications for NK cell-based cancer immunotherapies.

The cells that mediate innate immune memory and their functional significance in inflammatory and infectious diseases.

Immunological memory mediated by antigen-specific T and B cells is the foundation of adaptive immunity and is fundamental to the heightened and rapid protective immune response induced by vaccination or following re-infection with the same pathogen. While the innate immune system has classically been considered to be non-specific and devoid of memory, it now appears that it can be trained following exposure to microbes or their products and that this may confer a form of memory on innate immune cells. The evidence for immunological memory outside of T and B cells has been best established for natural killer (NK) cells, where it has been known for decades that NK cells have heighten responses following immunological re-challenge. Furthermore, recent studies have demonstrated that monocyte/macrophages, and probably dendritic cells, can be re-programmed through epigenetic modification, following exposure to pathogens or their products, resulting in heighted responses following a second stimulation. Unlike antigen-specific memory of the adaptive immune system, the second stimulation does not have to be with the same pathogen or antigen. Indirect evidence for this comes from reports on the non-specific beneficial effect of certain live vaccines, such as Bacillus Calmette Guerin (BCG) against unrelated childhood infectious diseases. It also appears that certain pathogen or pathogen-derived molecules can prime immune cells, especially macrophages, to secrete more anti-inflammatory and less pro-inflammatory cyokines, thus opening up the possibility of exploiting innate immune training as a new therapeutic approach for inflammatory diseases.

Metabolic Reprogramming Supports IFN-γ Production by CD56bright NK Cells.

Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56(dim) cells are generally considered more cytotoxic, whereas the CD56(bright) cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2-induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56(bright) cells were more metabolically active compared with CD56(dim) cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56(bright) cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56(dim) cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56(bright) subset of cells. Overall, we have defined CD56(bright) NK cells to be more metabolically active than CD56(dim) cells, which supports their production of large amounts of IFN-γ during an immune response.

Serum Immune System Biomarkers Neopterin and Interleukin-10 Are Strongly Related to Tryptophan Metabolism in Healthy Young Adults.

BACKGROUND: Changes in tryptophan metabolism through the vitamin B-6-dependent kynurenine pathway have been linked to activation of the immune system. OBJECTIVE: We hypothesized that blood concentrations of tryptophan and its catabolites were associated with biomarkers relevant to inflammatory processes in healthy noninflamed subjects. METHODS: Healthy young adults (n = 737) aged 18-28 y without any known diseases or clinical evidence of inflammation provided blood samples for analysis of serum tryptophan/kynurenine metabolites, neopterin, C-reactive protein (CRP), and plasma pyridoxal 5'-phosphate (PLP) with LC-tandem mass spectrometry methodologies. A panel of cytokines was measured in serum by using high-sensitivity ELISA assays. Anthropometric and lifestyle data were collected by questionnaire. Multiple linear regression analysis to determine the effect of measured serum cytokine concentrations as predictors of tryptophan metabolites was performed on inverse normal-rank transformations of the data, adjusted for sex, body mass index, smoking, alcohol intake, and contraceptive use in women. RESULTS: Median serum CRP and neopterin concentrations were well below established clinical cutoffs for inflammation. We observed significant positive associations between serum interleukin-10 (IL-10) and serum kynurenine (P = 0.0002), the kynurenine-to-tryptophan ratio (KTR) (P = 0.003), 3-hydroxykynurenine (P = 0.01), and 3-hydroxyanthranilic acid (P = 0.04). Serum neopterin was positively associated with kynurenine, the KTR (both P < 0.0001), and anthranilic acid (P = 0.004), and was negatively associated with serum tryptophan (P = 0.01) and PLP (P < 0.0001). Serum tumor necrosis factor α was also negatively associated with tryptophan (P < 0.001). CONCLUSIONS: In healthy young adults with no apparent inflammatory conditions, serum tryptophan metabolites are significantly associated with key immune system biomarkers. The observed association between IL-10 and kynurenine is unexpected and suggests that kynurenine-linked mechanisms promoting negative regulation of inflammatory responses are associated with normal immune homeostasis.

mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function.

The mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cellular metabolism and also has fundamental roles in controlling immune responses. Emerging evidence suggests that these two functions of mTORC1 are integrally linked. However, little is known regarding mTORC1 function in controlling the metabolism and function of NK cells, lymphocytes that play key roles in antiviral and antitumor immunity. This study investigated the hypothesis that mTORC1-controlled metabolism underpins normal NK cell proinflammatory function. We demonstrate that mTORC1 is robustly stimulated in NK cells activated in vivo and in vitro. This mTORC1 activity is required for the production of the key NK cell effector molecules IFN-γ, which is important in delivering antimicrobial and immunoregulatory functions, and granzyme B, a critical component of NK cell cytotoxic granules. The data reveal that NK cells undergo dramatic metabolic reprogramming upon activation, upregulating rates of glucose uptake and glycolysis, and that mTORC1 activity is essential for attaining this elevated glycolytic state. Directly limiting the rate of glycolysis is sufficient to inhibit IFN-γ production and granzyme B expression. This study provides the highly novel insight that mTORC1-mediated metabolic reprogramming of NK cells is a prerequisite for the acquisition of normal effector functions.

Innate immune genes synergize to predict increased risk of chronic disease in hepatitis C virus infection.

Hepatitis C is a common infection with significant morbidity and mortality, and only a minority of patients successfully clear the infection. Identification of factors that influence disease progression in HCV infection is difficult owing to the lack of well-defined patient cohorts. However, recent evidence supports a role for the innate immune system in virus clearance. In this study, we investigated innate immune genes for their contribution to disease progression in a unique cohort of well-controlled HCV-infected patients. The Irish cohort of HCV patients is uniquely homogenous; patients were infected with a single genotype of HCV from contaminated anti-D Ig. We genotyped 543 infected patients, including 247 patients who spontaneously resolved infection, for natural killer (NK) cell-associated killer cell Ig-like receptors (KIR) genes and the recently reported IL28B (IFNλ3) SNP. The NK cell gene KIR2DS3 was significantly increased in patients with chronic infection [odds ratio (OR) 1.90, 95% confidence interval (CI) 1.25-2.90, P < 0.002]. The IL28B "T" allele was also significantly increased in chronically infected patients (OR 7.38, 95% CI 4.93-11.07, P < 10(-8)). The presence of both markers synergized to significantly increase the risk of chronic infection over either factor alone (OR 20.11, 95% CI 9.05-44.68, P < 10(-7)). In functional experiments, we found that IL28A significantly inhibited IFN-γ production by NK cells. Thus, we demonstrate a functional link between NK cells and type 3 IFN. Our findings may contribute to the development of a prognostic test for HCV and identify therapeutic strategies for the clinical management of HCV-infected patients.

Common HIV-1 peptide variants mediate differential binding of KIR3DL1 to HLA-Bw4 molecules.

Epidemiological studies have shown the protective effect of KIR3DL1/HLA-Bw4 genotypes in human immunodeficiency virus type 1 (HIV-1) infection; however, the functional correlates for the protective effect remain unknown. We investigated whether human leukocyte antigen (HLA)-Bw4-presented HIV-1 peptides could affect the interaction between the inhibitory natural killer (NK) cell receptor KIR3DL1 and its ligand HLA-Bw4. Distinct HIV-1 epitopes differentially modulated the binding of KIR3DL1 to HLA-Bw4. Furthermore, cytotoxic T lymphocyte (CTL) escape mutations within the immunodominant HLA-B57 (Bw4)-restricted Gag epitope TSTLQEQIGW abrogated KIR3DL1 binding to HLA-B57, suggesting that sensing of CTL escape variants by NK cells can contribute to the protective effect of the KIR3DL1/HLA-Bw4 compound genotype.

Heightened metabolic responses in NK cells from patients with neuroblastoma suggests increased potential for immunotherapy.

High risk neuroblastoma is responsible for 15% of deaths in pediatric cancer patients. The introduction of anti-GD2 immunotherapy has significantly improved outcomes but there is still only approximately a 50% 5 year event-free-survival for these children and improvements in treatments are urgently required. Anti-GD2 immunotherapy uses the patients' own immune system to kill cancer cells. In particular, Natural Killer (NK) cells kill antibody coated tumor cells by a process called antibody dependent cellular cytotoxicity (ADCC). However, our previous work has highlighted metabolic exhaustion of NK cells in circulating blood of adult cancer patients, identifying this as a potential therapeutic target. In this study, we investigated circulating NK cells in patients newly diagnosed with neuroblastoma. We found evidence of activation of NK cells in vivo by the cancer itself. While some evidence of NK cell dysfunction was observed in terms of IFNγ production, most results indicated that the NK cell compartment remained relatively intact. In fact, some aspects of metabolic and functional activities were actually increased in patients compared to controls. Glycolytic responses, which we show are crucial for ADCC, were actually enhanced in patients and CD16, the NK cell receptor that mediates ADCC, was also expressed at high levels in some patients. Overall, the data suggest that patient NK cells could be harvested at diagnosis for subsequent beneficial autologous use during immunotherapy. Enhancing glycolytic capacity of cell therapies could also be a strategic goal of future cell therapies for patients with neuroblastoma and indeed other cancers.

NK cells and psoriasis.

Psoriasis is a chronic condition of the skin characterised by distinctive scaly plaques. The immune system is now thought to play a major role in the development and pathogenesis of psoriasis with immune cells and cytokines influencing keratinocyte function. Keratinocytes in turn, can activate and recruit immune cells leading to a positive feedback loop in disease. Natural Killer (NK) cells are lymphocytes that are best known for killing virally infected and cancer cells. However, evidence is emerging to support a role for NK cells in psoriasis. NK cells are found in the inflammatory infiltrate in psoriatic skin lesions. They can produce a range of inflammatory cytokines, many of which are important in the pathogenesis of psoriasis. Recent genetic studies have identified a range of potential molecules relating to NK cell biology that are known to be important in psoriasis. This paper will discuss the evidence, both cellular and genetic, for NK cell involvement in psoriasis.

Cytokine-induced natural killer cell training is dependent on cellular metabolism and is defective in obesity.

Natural killer (NK) cells are a population of innate immune cells that can rapidly kill cancer cells and produce cytokines such as interferon-γ. A key feature of NK cells is their ability to respond without prior sensitization; however, it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be trained to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study, we investigated if cellular metabolism is required during the initial period of NK cell cytokine training and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and oxidative phosphorylation during the initial cytokine activation period is essential for NK cell training, as is the metabolic signaling factor Srebp. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

TGFß drives NK cell metabolic dysfunction in human metastatic breast cancer.

BACKGROUND: Natural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output. METHODS: Single-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controls RESULTS: In addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-ß (TGFß) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFß processing pathway, was increased on NK cells from some patients. Blocking the GARP-TGFß axis recapitulated the effects of TGFß neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time. CONCLUSIONS: TGFß contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFß and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

Specific human cytomegalovirus signature detected in NK cell metabolic changes post vaccination.

Effective vaccines for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) remain a significant challenge for these infectious diseases. Given that the innate immune response is key to controlling the scale and nature of developing adaptive immune responses, targeting natural killer (NK) cells that can promote a T-helper type 1 (Th1)-type immune response through the production of interferon-γ (IFNγ) remains an untapped strategic target for improved vaccination approaches. Here, we investigate metabolic and functional responses of NK cells to simian adenovirus prime and MVA boost vaccination in a cohort of healthy volunteers receiving a dual HCV-HIV-1 vaccine. Early and late timepoints demonstrated metabolic changes that contributed to the sustained proliferation of all NK cells. However, a strong impact of human cytomegalovirus (HCMV) on some metabolic and functional responses in NK cells was observed in HCMV seropositive participants. These changes were not restricted to molecularly defined adaptive NK cells; indeed, canonical NK cells that produced most IFNγ in response to vaccination were equally impacted in individuals with latent HCMV. In summary, NK cells undergo metabolic changes in response to vaccination, and understanding these in the context of HCMV is an important step towards rational vaccine design against a range of human viral pathogens.

Metabolic but not transcriptional regulation by PKM2 is important for natural killer cell responses.

Natural Killer (NK) cells have an important role in immune responses to viruses and tumours. Integrating changes in signal transduction pathways and cellular metabolism is essential for effective NK cells responses. The glycolytic enzyme Pyruvate Kinase Muscle 2 (PKM2) has described roles in regulating glycolytic flux and signal transduction, particularly gene transcription. While PKM2 expression is robustly induced in activated NK cells, mice lacking PKM2 in NK cells showed no defect in NK cell metabolism, transcription or antiviral responses to MCMV infection. NK cell metabolism was maintained due to compensatory PKM1 expression in PKM2-null NK cells. To further investigate the role of PKM2, we used TEPP-46, which increases PKM2 catalytic activity while inhibiting any PKM2 signalling functions. NK cells activated with TEPP-46 had reduced effector function due to TEPP-46-induced increases in oxidative stress. Overall, PKM2-regulated glycolytic metabolism and redox status, not transcriptional control, facilitate optimal NK cells responses.

NK cell metabolism.

Natural Killer (NK) cells are important antiviral and anticancer effector cells. They have excellent potential for immunotherapy although impaired functions during cancer limit their effectiveness. The discovery that cellular metabolism can impact on and regulate immune functions has led to an explosion of articles in this new area of immunometabolism. Metabolism has recently been shown to impact both murine and human NK cell biology. This review is targeted for newcomers to the field; it will introduce basic concepts in the area of immunometabolism including key aspects of glucose metabolism and mitochondrial function. It will review our current understanding of how metabolism of NK cells is differentially impacted in a variety of important situations. This is a rapidly expanding and exciting area of research that holds great potential for improving NK cell-based immunotherapies.

NK Cell Metabolism and TGFß - Implications for Immunotherapy.

NK cells are innate lymphocytes which play an essential role in protection against cancer and viral infection. Their functions are dictated by many factors including the receptors they express, cytokines they respond to and changes in the external environment. These cell processes are regulated within NK cells at many levels including genetic, epigenetic and expression (RNA and protein) levels. The last decade has revealed cellular metabolism as another level of immune regulation. Specific immune cells adopt metabolic configurations that support their functions, and this is a dynamic process with cells undergoing metabolic reprogramming during the course of an immune response. Upon activation with pro-inflammatory cytokines, NK cells upregulate both glycolysis and oxphos metabolic pathways and this supports their anti-cancer functions. Perturbation of these pathways inhibits NK cell effector functions. Anti-inflammatory cytokines such as TGFß can inhibit metabolic changes and reduce functional outputs. Although a lot remains to be learned, our knowledge of potential molecular mechanisms involved is growing quickly. This review will discuss our current knowledge on the role of TGFß in regulating NK cell metabolism and will draw on a wider knowledge base regarding TGFß regulation of cellular metabolic pathways, in order to highlight potential ways in which TGFß might be targeted to contribute to the exciting progress that is being made in terms of adoptive NK cell therapies for cancer.

Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses.

Natural Killer (NK) cells are lymphocytes with an important role in anti-tumour responses. NK cells bridge the innate and adaptive arms of the immune system; they are primed for immediate anti-tumour function but can also have prolonged actions alongside the adaptive T cell response. However, the key signals and cellular processes that are required for extended NK cell responses are not fully known. Herein we show that murine NK cell interaction with tumour cells induces the expression of CD25, the high affinity IL2 receptor, rendering these NK cells highly sensitive to the T cell-derived cytokine IL2. In response to IL2, CD25high NK cells show robust increases in metabolic signalling pathways (mTORC1, cMyc), nutrient transporter expression (CD71, CD98), cellular growth and in NK cell effector functions (IFNγ, granzyme B). Specific ligation of an individual activating NK cell receptor, NK1.1, showed similar increases in CD25 expression and IL2-induced responses. NK cell receptor ligation and IL2 collaborate to induce mTORC1/cMyc signalling leading to high rates of glycolysis and oxidative phosphorylation (OXPHOS) and prolonged NK cell survival. Disrupting mTORC1 and cMyc signalling in CD25high tumour interacting NK cells prevents IL2-induced cell growth and function and compromises NK cell viability. This study reveals that tumour cell interactions and T cell-derived IL2 cooperate to promote robust and prolonged NK cell anti-tumour metabolic responses.