NK Cell Metabolism Research Articles

C/EBPβ-dependent autophagy inhibition hinders NK cell function in cancer

NK cells are endowed with tumor killing ability, nevertheless most cancers impair NK cell functionality, and cell-based therapies have limited efficacy in solid tumors. How cancers render NK cell dysfunctional is unclear, and overcoming resistance is an important immune-therapeutic aim. Here, we identify autophagy as a central regulator of NK cell anti-tumor function. Analysis of differentially expressed genes in tumor-infiltrating versus non-tumor NK cells from our previously published scRNA-seq data of advanced human prostate cancer shows deregulation of the autophagic pathway in tumor-infiltrating NK cells. We confirm this by flow cytometry in patients and in diverse cancer models in mice. We further demonstrate that exposure of NK cells to cancer deregulates the autophagic process, decreases mitochondrial polarization and impairs effector functions. Mechanistically, CCAAT enhancer binding protein beta (C/EBPβ), downstream of CXCL12-CXCR4 interaction, acts as regulator of NK cell metabolism. Accordingly, inhibition of CXCR4 and C/EBPβ restores NK cell fitness. Finally, genetic and pharmacological activation of autophagy improves NK cell effector and cytotoxic functions, which enables tumour control by NK and CAR-NK cells. In conclusion, our study identifies autophagy as an intracellular checkpoint in NK cells and introduces autophagy regulation as an approach to strengthen NK-cell-based immunotherapies.

Metabolic Regulation of Natural Killer Cells

Natural Killer cells, known as NK cells act crucially in protection of anti-tumor and anti-virus activities as innate lymphocytes. Due to its unique characteristics such as recognizing and attacking stressed cells independently, and bridge connection function between innate immunity and adaptive immunity, it possesses profound potential and prospect in immunotherapy. However, due to resistance existing in combination of therapies and undeclared metabolism pathways, its application in clinical trials has been underestimated. Metabolic configurations are adopted to support functions, which is dynamic when immune cells experience the procedure of metabolic reprogramming in responses. In order to support anti-tumor functions, NK cells up regulate both glycolysis and OXPHOS, leading to the alternative of energy metabolism structure and ATP relief upon activation with pro-inflammatory cytokines. After activation, what exactly determines phenotypes and destiny of NK cells is also being considered as the classification standard of receptors distribution is found both vague and not universally applicable. Additionally, author focus on metabolic factors in solid cancer treating, which are highly relative to tumor microenvironment (TME). TME provides many molecules for the prevention of potential effector function of normal cells (especially immune cells) and protection of tumor. For example, substances such as TGF-β are capable to not only downregulate NK cell cytotoxicity and cytokine secretion but also reduce the extent of metabolism and proliferation, and also induce effector NK cells to increase the production of ILC1-like characteristics. This review will discuss NK cells’ metabolic foundation to realize NK cell effector functions and analyze the relationship between phenotypes, NK cell fates and metabolism. Also, basing on impaired functions, immune suppression and resistance of joint therapies, the author will discuss different pathways and possible treating targets and therapies in order to boost cancer treatment progress such as adoptive NK cell therapy, bio- or tri- engager therapy and CAR-NK therapy.

Abstract A021: Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer

Abstract Tumor cells present constant nutritional demand of macronutrients and micronutrients for their rapid growth needs. Often this nutrient limitation hampers the function of anti-tumor immune cells, which need basal energy levels to execute cytotoxic effects against malignant cells. Hence, we investigated nutrient limitation-induced metabolic alterations in NK cells that regulate the anti-tumor activity of NK cells. Using 2D cultures and PDAC organoid models, we demonstrate that pancreatic tumor cells exhaust vitamin B6 (VB6) in the co-culture milieu, causing reduced killing activity of NK cells. Additionally, we demonstrate that PDAC patients have significantly reduced plasma VB6 as compared to the healthy counterparts. This data aligns with the epidemiological studies showing that VB6 intake reduces the risk of pancreatic cancer incidence. Moreover, we noted a significant reduction in circulating VB6 in the pancreatic tumor-bearing mice as compared to healthy counterparts. Utilizing mass spectrometry-based metabolomics we noted that limitation of the VB6 prevents glycogen breakdown in NK cells. This was validated by follow up electron microscopy-based studies that demonstrated glycogen depots in NK cells that were very prominent upon inhibiting glycogenolysis. Metabolic tracing studies demonstrated that NK cells require VB6 for intracellular glycogen breakdown. Correspondingly, knocking down glycogen phosphorylase, a key enzyme involved in glycogen breakdown abrogated anti-tumoral NK cell function. Notably, supplementation of co-cultures in 2D and organoid models and in vivo orthotopic mouse models, with VB6 restored NK cell function against pancreatic tumor cells. In parallel, we observed dependence of tumor cell metabolism on VB6 for sustaining growth. Accordingly, we observed that tumor cells actively deplete VB6 in tumor microenvironments, as observed by comparing VB6 levels in circulation and in tumor interstitial fluids. Finally, we observed that VB6 supplementation in combination with inhibitors targeting VB6-driven metabolic pathway dependencies in tumor cells effectively enhances NK cell frequency and diminishes tumor burden in vivo. These studies demonstrate a novel role of glycogen breakdown as a critical energy source for activated NK cells and demonstrate, for the first time a key role of glycogenolysis in NK cell cytotoxic activities. Our results expand the understanding of the critical role of micronutrients (vitamin B6) in regulating cancer progression and anti-tumor immunity and open new avenues for developing NK cell-based immunotherapies for PDAC patients. Citation Format: Kamiya Mehla. Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A021.

CD8α identifies human NK cells with altered proliferation, metabolism, and IL-15 signaling

Abstract NK cells are cytotoxic innate lymphoid cells that can differentiate into memory-like (ML) NK cells following IL-12/15/18 stimulation. Previous work identified that CD8α expression on donor ML NK cells was negatively associated with outcome for cellular therapy of AML. However, the role of CD8α on human NK cells remains poorly studied, and murine NK cells are CD8α-precluding study in mice. We found that CD8α-NK cells had greater proliferation and survival in vitro and in vivo. Interestingly, we observed that CD8α expression was dynamic, and a subset of CD8α-NK cells upregulated CD8α (“new” CD8+) after IL-15 stimulation. Notably, the “new” CD8+ NK cells were the most proliferative in vitro and in vivo, and had significantly greater signaling following IL-15 stimulation. Strikingly, we also found that “new” CD8+ NK cells had significantly higher glucose uptake, nutrient receptor expression, and an enhanced capacity for glycolysis and oxidative phosphorylation. These results indicate that recent CD8α acquisition marks the ability of NK cells to respond to IL-15 signals to engage in the robust metabolic activity required for proliferation. We also investigated the role of CD8α in NK cell activation, given that CD8α can bind HLA and associate with Lck. In the absence of CD8α, NK responses (CD107a, IFNg, TNF) following activating receptor ligation were enhanced for several receptors, suggesting an intrinsic role for CD8α in inhibiting NK cell activation. Together, these results identify a novel association of CD8α with treatment outcome for NK cellular therapy and identify a previously unappreciated role of CD8α on human NK cell proliferation, metabolism, and response through activating receptors. Supported by grants from NIAID F30AI16131

MEF2C is a critical regulator of human NK cell metabolism.

Abstract Natural killer (NK) cells are critical for the first line of defense against viral infection and malignancy. While the transcriptional and epigenetic regulation of activated NK cells is well characterized in mice, these processes are not yet fully understood in human NK cells. We performed a targeted CRISPR/Cas9 ribonucleoprotein (RNP) transcription factor screen in mature primary human NK cells to identify putative transcription factors required for optimal effector function. The transcription factor myocyte enhancer factor 2C (MEF2C) emerged as a previously unidentified regulator of NK cell homeostasis, cytokine production, and cytotoxicity. MEF2C-deficient NK cells activated with IL-2 and IL-15 displayed impaired proliferation, degranulation, and production of granzyme B. CRISPR-mediated deletion of MEF2C resulted in disrupted glycolysis and oxidative phosphorylation using both SCENITH and Seahorse extracellular flux analysis. Liquid chromatography/mass spectrometry (LC/MS)-based metabolomics of MEF2C-deficient NK cells revealed reductions in the metabolites glyceraldehyde-3-phosphate and a-ketoglutarate/succinate, accompanied by increased accumulation of lipid metabolism products including phosphorylethanolamine and saturated long-chain fatty acids. In vivo, CRISPR/Cas9 RNP-mediated disruption of MEF2C expression in mouse NK cells resulted in decreased expansion during mouse cytomegalovirus infection. Together, these studies reveal MEF2C as a novel regulator of NK cell effector function through control of multiple critical cell-intrinsic metabolic pathways.

Butyrate limits human natural killer cell effector function

The gut microbiota regulates chronic inflammation and has been implicated in the pathogenesis of a broad spectrum of disease including autoimmunity and cancer. Microbial short-chain fatty acids (SCFAs) e.g., butyrate have demonstrated immunomodulatory effects and are thought to be key mediators of the host-microbiome interaction. Here, we investigated the effect of butyrate on effector functions of blood derived human NK cells stimulated for 18 h with a combination of IL-12/IL-15, a potent mix of cytokines that drive NK cell activation. We show that butyrate has a strong anti-inflammatory effect on NK cells. NK cells cultured in the presence of butyrate expressed lower levels of activating receptors (TRAIL, NKp30, NKp44) and produced lower levels of cytokines (IFNγ, TNF-α, IL-22, granzyme B, granzyme A, perforin) in response to IL-12/IL-15. Butyrate restricted NK cell function by downregulation of mTORC1 activity, c-Myc mRNA expression and metabolism. Using a shotgun proteomic approach, we confirmed the effect of butyrate on NK cell cytokine signaling and metabolism and identified BRD2, MAT2A and EHD1 as downstream mediators of these effects. This insight into the immunomodulatory activity of butyrate on human NK cell function might help to develop new ways to limit NK cell function during chronic inflammation.

Metabolism of NK cells during viral infections.

Cellular metabolism is essential for the correct function of immune system cells, including Natural Killer cells (NK). These cells depend on energy to carry out their effector functions, especially in the early stages of viral infection. NK cells participate in the innate immune response against viruses and tumors. Their main functions are cytotoxicity and cytokine production. Metabolic changes can impact intracellular signals, molecule production, secretion, and cell activation which is essential as the first line of immune defense. Metabolic variations in different immune cells in response to a tumor or pathogen infection have been described; however, little is known about NK cell metabolism in the context of viral infection. This review summarizes the activation-specific metabolic changes in NK cells, the immunometabolism of NK cells during early, late, and chronic antiviral responses, and the metabolic alterations in NK cells in SARS-CoV2 infection. The modulation points of these metabolic routes are also discussed to explore potential new immunotherapies against viral infections.

TGFβ and CIS Inhibition Overcomes NK-cell Suppression to Restore Antitumor Immunity.

Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor β (TGFβ), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFβ signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFβ, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.

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

AbstractNatural 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.

Abstract 1536: Functional characterization of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cell therapy, reveals increased granzyme B and degranulation supporting improved serial killing capacity

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with a CISH and TGFBR2 knockout mediated by CRISPR-Cas12a that results in resistance to TGF-β inhibition and increased effector function. EDIT-201 kills tumor cells more efficiently and for a longer duration than control NK cells in the presence of TGF-β. Based on these data, we hypothesized that EDIT-201 has increased serial killing capacity. First, we explored the transcriptional changes contributed by each gene edit with a focus on transcripts critical for NK cell effector function and metabolism using Nanostring analysis. Unedited, mock electroporated, and control edited (targeting a biologically irrelevant site) NK cells were included as controls in addition to CISH and TGFBR2 single and double gene knockout (KO) NK cells to interrogate the potential impact of electroporation and double-stranded DNA breaks on NK cell function. All samples included in the analysis were cultured for 3 days in IL-15 post-electroporation. No significant transcriptional changes were detected in all control conditions, while samples that contained CISH editing clearly upregulated transcripts relevant for NK cell effector function, including contents of cytolytic granules (GZMB, GZMA and GZMH). EDIT-201 expressed an average of 22 fold more GZMB transcript than control NK cells as measured by RT-qPCR in four unique NK cell donors. Furthermore, EDIT-201 showed significantly higher levels of CD107a, a marker of degranulation, after 14hrs of co-culture with SKOV-3 tumor cells, suggesting that EDIT-201 had an increased capacity to degranulate relative to control NK cells. To determine the presence of granzyme proteins within tumor cells following engagement with NK cells, we developed a novel GzmB reporter gene and used lentiviral vectors to introduce this reporter into tumor cell lines. Using SK-OV-3 tumor cells transduced with our reporter, we demonstrated that upon coculture of EDIT-201 with tumor cells, intratumoral GzmB activity was identified 4 hours sooner than when the target cells were cultured with control NK cells. In addition, EDIT-201 affected 80% more SK-OV-3 tumor cells with granzyme B compared to control NK cells over a 20 hour period. Most significantly, our data demonstrated that EDIT-201 not only discharged GzmB more rapidly than control NK cells, but also the amount of GzmB degranulated is greater as well, confirming that enhanced degranulation is a key mechanism by which EDIT-201 has superior functional capacity relative to control NK cells. Together, these data demonstrate one mechanism by which EDIT-201 demonstrates superior cytotoxicity during in vitro killing of SK-OV-3 tumor targets, and continues to support the advancement of EDIT-201 as a potential novel gene edited NK cell-based treatment for cancer. Citation Format: Christopher M. Borges, Shuqi Zhang, Kevin Wasko, Kelly Donahue, Lincy Prem Antony, Jared Nasser, Steven Sexton, Amanda Pfautx, Richard A. Morgan, Karrie K. Wong. Functional characterization of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cell therapy, reveals increased granzyme B and degranulation supporting improved serial killing capacity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1536.

Abstract 1532: EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with CRISPR-Cas12a mediated CISH and TGFBR2 double gene knockout that demonstrated resistance to TGF-β inhibition and increased tumor control in vitro and in vivo. The tumor microenvironment (TME) is known to be nutrient-deprived due to active tumor cell metabolism leading to competition for essential nutrients with infiltrating effector cells, while at the same time being enriched in immunosuppressive metabolites such as lactic acid due to Warburg Metabolism. To explore whether EDIT-201 is functional in such hostile metabolic conditions, we aimed to more accurately model the metabolic microenvironment in a long-term tumor spheroid model. To model this, we generated SK-OV-3 ovarian tumor spheroids in decreasing concentrations of glucose (10-0.5mM) or glutamine (2-0.1mM), two important fuels for NK cell metabolism, as well as increasing concentrations of inhibitory metabolite lactate (0-50mM), or decreasing pH (7.2-6.5). Each of these metabolic conditions are known to suppress effector cell function; we additionally performed spheroid cells co-cultures in the presence of 10ng/mL TGF-β to increase the potential for NK inhibition. We demonstrated that, relative to control NK cells, EDIT-201 demonstrated rapid and sustained tumor killing in all conditions tested including, the absence of critical nutrients. We next created a multifactorial matrix of metabolic conditions where we combined deprivation of multiple nutrients, in the presence of lactate and/or acidic cell culture media. Remarkably, in all combinations tested, we surprisingly found that EDIT-201 continued to demonstrate increased cytotoxicity against SK-OV-3 spheroids relative to control NK cells, suggesting a clear and robust metabolic advantage of EDIT-201 over control NK cells. We additionally observed a corresponding increase in the concentrations of IFN-γ and TNF-α by EDIT-201 in all of these conditions relative to control NK cells. Given that mitochondrial respiration is key to NK cell persistence and function, we next interrogated the mitochondrial function of EDIT-201. We find that EDIT-201 consistently demonstrated greater spare respiratory capacity (SRC) relative to control NK cells, suggesting enhanced mitochondrial reserve as a result of CISHand TGFBR2 knockout. The increase in SRC likely enables EDIT-201 to meet enhanced energy demands necessary to mediate effector function in metabolically challenging conditions, thus sustaining superior cytotoxic capacity and cytokine production. In summary, our data demonstrate enhanced metabolic function of EDIT-201, resulting in superior cytotoxicity in metabolically unfavorable conditions. These data further support the development of EDIT-201 as a novel cell therapy for cancer. Citation Format: Karrie K. Wong, Steven Sexton, Lincy Prem Antony, Kevin Wasko, Jared Nasser, Kelly Donahue, Amanda Pfautz, Shuqi Zhang, Richard A. Morgan, Christopher M. Borges. EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1532.

Expression of Nutrient Transporters on NK Cells during Murine Cytomegalovirus Infection is MyD88-Dependent

Abstract Natural killer (NK) cells are the predominant innate lymphocytes that provide the first line of defence without prior sensitization. In the inflammatory milieu, NK cells modify their metabolism to cope with the high energy demand required to support their proliferation, activation, and functional plasticity. This metabolic reprogramming is usually accompanied by the upregulated expression of nutrient transporters on the cell surface, leading to increased nutrient uptake needed for robust proliferation. The members of interleukin-1 family of inflammatory cytokines are critical in activating NK cells during infection; however, their function in NK cell metabolism is not fully elucidated. Previously, IL-18 has been shown to upregulate the expression of solute carrier transmembrane proteins and thereby induces a robust metabolic boost in NK cells. However, we demonstrated that IL-18 signaling is dispensable during viral infection in vivo whereas the expression of nutrient transporters on NK cells is primarily regulated by MyD88-pathway since NK cells from Myd88−/− mice displayed significantly reduced expression of nutrient receptors. Moreover, we identified that IL-33, another cytokine employing MyD88 signaling, can induce the expression of nutrient receptor but requires a sequential exposure to IL-12. Furthermore, signaling through NK cells activating receptor, Ly49H, can also promote the expression of nutrient transporters. In summary, our findings revealed multiple pathways that induce the expression of nutrient transporters on NK cells while highlighting the imperative role of MyD88 in NK cell metabolism during infection.

Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment

NK cells are central to anti-tumor immunity and recently showed efficacy for treating hematologic malignancies. However, their dysfunction in the hostile tumor microenvironment remains a pivotal barrier for cancer immunotherapies against solid tumors. Using cancer patient samples and proteomics, we found that human NK cell dysfunction in the tumor microenvironment is due to suppression of glucose metabolism via lipid peroxidation-associated oxidative stress. Activation of the Nrf2 antioxidant pathway restored NK cell metabolism and function and resulted in greater anti-tumor activity invivo. Strikingly, expanded NK cells reprogrammed with complete metabolic substrate flexibility not only sustained metabolic fitness but paradoxically augmented their tumor killing in the tumor microenvironment and in response to nutrient deprivation. Our results uncover that metabolic flexibility enables a cytotoxic immune cell to exploit the metabolic hostility of tumors for their advantage, addressing a critical hurdle for cancer immunotherapy.

TGFβ drives NK cell metabolic dysfunction in human metastatic breast cancer

BackgroundNatural 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...

Dysregulation of Metabolic Pathways in Circulating Natural Killer Cells Isolated from Inflammatory Bowel Disease Patients.

Background and AimsInflammatory bowel diseases [IBD], comprising Crohn’s disease [CD] and ulcerative colitis [UC], are chronic conditions characterized by severe dysregulation of innate and adaptive immunity resulting in the destruction of the intestinal mucosa. Natural killer [NK] cells play a pivotal role in the dynamic interaction between the innate and adaptive immune response. There is an increasing appreciation for the key role immunometabolism plays in the regulation of NK cell function, yet little remains known about the metabolic profile, cytokine secretion, and killing capacity of human NK cells during active IBD.MethodsPeripheral blood mononuclear cells were isolated from peripheral blood of patients with moderate to severely active IBD and healthy controls. NK cells were stained with a combination of cell surface receptors, intracellular cytokines, and proteins and analyzed by flow cytometry. For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed. The metabolic profile was analyzed by an extracellular flux analyzer.ResultsNK cells from IBD patients produce large quantities of pro-inflammatory cytokines, IL-17A and TNF-α ex vivo, but have limited killing capability. Furthermore, patient NK cells have reduced mitochondrial mass and oxidative phosphorylation. mTORC1, an important cell and metabolic regulator, demonstrated limited activity in both freshly isolated cells and cytokine-stimulated cells.ConclusionsOur results demonstrate that circulating NK cells of IBD patients have an unbalanced metabolic profile, with faulty mitochondria and reduced capacity to kill. These aberrations in NK cell metabolism may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients.

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.

Tgfβ-Imprinting Decrease CD38 Expression and Lead to Metabolic Reprogramming on Primary NK Cell

Transforming growth factor-β (TGF-β) plays an essential role in regulating immune responses through its immunossupressive effect on adaptive and innate immune cells. In addition, we and others have shown that alterations in cell-intrinsic metabolism profiles can significantly impact the function of immune cells. Previously, our group demonstrated that TGFβ-imprinting (TGFβi) decreases NK cell sensitivity to TGFβ through SMAD3 suppression and enhances a pro-inflammatory phenotype with hypersecretion of IFN-γ, TNF-α, and GM-CSF, which appeared to be mediated by an epigenetic mechanism. To evaluate this process, we conducted RNA-seq and ATAC-seq to evaluate the impact of chromosomal remodeling on gene expression. In addition to confirming low SMAD3 transcription, this data showed that TGFβi NK cells have decreased CD38 expression, which we confirmed at the protein level. Since CD38 is an ectoenzyme that regulates nicotinamide adenine dinucleotide (NAD+), a critical component of OXPHOS in both T and NK cells, we examined the effect of TGFβi on NK cell metabolism. TGFβi primary NK cells were generated from peripheral blood of healthy donors as previously described, and using a mitochondrial stress test assay, we observed higher oxygen consumption rates (OCR). However, in the glycolysis stress test assay we observed comparable extracellular acidification rates (ECAR) between Standard expanded and TGFbi NK cells, resulting in higher OCR/ECAR ratios in TGFbi NK cells. Since TGFβ has been shown to induce fragmented mitochondria, and inhibition of mitochondrial fragmentation improved mitochondrial metabolism, we evaluated subcellular structure of TGFβi NK cellsby transmission electron microscopy (TEM). We found no difference in mitochondrial fragmentation between STD and TGFβi NK cells. Together, these results show that TGFβi induces epigenetic reprograming of NK cells that results in increased OXPHOS through CD38 suppression. Moreover, the decreases CD38 expression in TGFβi NK cells could be a promising non-genetic alternative to generating CD38-negative NK cells to avoid fratricide in combination with DARA Figure Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Scaffolding LSD1 Inhibitors Impair NK Cell Metabolism and Cytotoxic Function Through Depletion of Glutathione.

Cell therapies such as chimeric-antigen receptor (CAR) T-cells and NK cells are cutting-edge methods for treating cancer and other diseases. There is high interest in optimizing drug treatment regimens to best work together with emerging cell therapies, such as targeting epigenetic enzymes to stimulate recognition of tumor cells by immune cells. Herein, we uncover new mechanisms of the histone demethylase LSD1, and various inhibitors targeting unique domains of LSD1, in the function of NK cells grown for cell therapy. Catalytic inhibitors (tranylcypromine and the structural derivatives GSK LSD1 and RN-1) can irreversibly block the demethylase activity of LSD1, while scaffolding inhibitors (SP-2509 and clinical successor SP-2577, also known as seclidemstat) disrupt epigenetic complexes that include LSD1. Relevant combinations of LSD1 inhibitors with cell therapy infusions and immune checkpoint blockade have shown efficacy in pre-clinical solid tumor models, reinforcing a need to understand how these drugs would impact T- and NK cells. We find that scaffolding LSD1 inhibitors potently reduce oxidative phosphorylation and glycolysis of NK cells, and higher doses induce mitochondrial reactive oxygen species and depletion of the antioxidant glutathione. These effects are unique to scaffolding inhibitors compared to catalytic, to NK cells compared to T-cells, and importantly, can fully ablate the lytic capacity of NK cells. Supplementation with biologically achievable levels of glutathione rescues NK cell cytolytic function but not NK cell metabolism. Our results suggest glutathione supplementation may reverse NK cell activity suppression in patients treated with seclidemstat.

Correction: Differential Fuel Requirements of Human NK Cells and Human CD8 T Cells: Glutamine Regulates Glucose Uptake in Strongly Activated CD8 T Cells.

CD8 T cells and NK cells are the two major cytotoxic lymphocytes that carry out cell-mediated immunity and regulate other immune responses. However, we do not completely understand human CD8 T cell and NK cell metabolic requirements and they have not been compared in the same experiments. We activated human CD8 T cells by two anti–CD3/CD28 mAb methods, and we stimulated both CD8 T cells and NK cells with IL-12/IL-18. When glucose (Glc) could not be used, human CD8 T cells either died or became hypofunctional, depending upon the anti–CD3/CD28 activation method. In contrast, Glc starvation did not decrease the percentage of IL-12/IL-18–stimulated human NK cells that made IFN-γ. NK cells were relatively fuel resilient and used Glc, glutamine (Gln), fatty acid, or acetate to power IFN-γ expression. Surprisingly, strongly activated human CD8 T cells required Gln for glycolysis and Glc uptake. We showed that human CD8 T cells regulate Glc uptake by a novel mechanism related to the TXNIP pleiotropic protein. These conditions may be relevant to septic patients who have high blood Glc but low Gln. Under the conditions tested, Gln did not change human NK cell TXNIP expression. Our experiments reveal fundamental differences in human CD8 T cell and NK cell metabolism and the fuels needed for IFN-γ production.

Differential Fuel Requirements of Human NK Cells and Human CD8 T Cells: Glutamine Regulates Glucose Uptake in Strongly Activated CD8 T Cells.

CD8 T cells and NK cells are the two major cytotoxic lymphocytes that carry out cell-mediated immunity and regulate other immune responses. However, we do not completely understand human CD8 T cell and NK cell metabolic requirements and they have not been compared in the same experiments. We activated human CD8 T cells by two anti–CD3/CD28 mAb methods, and we stimulated both CD8 T cells and NK cells with IL-12/IL-18. When glucose (Glc) could not be used, human CD8 T cells either died or became hypofunctional, depending upon the anti–CD3/CD28 activation method. In contrast, Glc starvation did not decrease the percentage of IL-12/IL-18–stimulated human NK cells that made IFN-γ. NK cells were relatively fuel resilient and used Glc, glutamine (Gln), fatty acid, or acetate to power IFN-γ expression. Surprisingly, strongly activated human CD8 T cells required Gln for glycolysis and Glc uptake. We showed that human CD8 T cells regulate Glc uptake by a novel mechanism related to the TXNIP pleiotropic protein. These conditions may be relevant to septic patients who have high blood Glc but low Gln. Under the conditions tested, Gln did not change human NK cell TXNIP expression. Our experiments reveal fundamental differences in human CD8 T cell and NK cell metabolism and the fuels needed for IFN-γ production.