Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF-1α.

Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+ ) levels. The HIF-1α/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD+ metabolism.

Escape from NK cell tumor surveillance by NGFR-induced lipid remodeling in melanoma.

Metastatic disease is a major cause of death for patients with melanoma. Melanoma cells can become metastatic not only due to cell-intrinsic plasticity but also due to cancer-induced protumorigenic remodeling of the immune microenvironment. Here, we report that innate immune surveillance by natural killer (NK) cells is bypassed by human melanoma cells expressing the stem cell marker NGFR. Using in vitro and in vivo cytotoxic assays, we show that NGFR protects melanoma cells from NK cell-mediated killing and, furthermore, boosts metastasis formation in a mouse model with adoptively transferred human NK cells. Mechanistically, NGFR leads to down-regulation of NK cell activating ligands and simultaneous up-regulation of the fatty acid stearoyl-coenzyme A desaturase (SCD) in melanoma cells. Notably, pharmacological and small interfering RNA-mediated inhibition of SCD reverted NGFR-induced NK cell evasion in vitro and in vivo. Hence, NGFR orchestrates immune control antagonizing pathways to protect melanoma cells from NK cell clearance, which ultimately favors metastatic disease.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic T cell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for T cell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ T cell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence.

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.

Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors.

Monoclonal antibodies (mAbs) have significantly improved the treatment of certain cancers. However, in general mAbs alone have limited therapeutic activity. One of their main mechanisms of action is to induce antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated by natural killer (NK) cells. Unfortunately, most cancer patients have severe immune dysfunctions affecting NK activity. This can be circumvented by the injection of allogeneic, expanded NK cells, which is safe. Nevertheless, despite their strong cytolytic potential against different tumors, clinical results have been poor. Methods: We combined allogeneic NK cells and mAbs to improve cancer treatment. We generated expanded NK cells (e-NK) with strong in vitro and in vivo ADCC responses against different tumors and using different therapeutic mAbs, namely rituximab, obinutuzumab, daratumumab, cetuximab and trastuzumab. Results: Remarkably, e-NK cells can be stored frozen and, after thawing, armed with mAbs. They mediate ADCC through degranulation-dependent and -independent mechanisms. Furthermore, they overcome certain anti-apoptotic mechanisms found in leukemic cells. Conclusion: We have established a new protocol for activation/expansion of NK cells with high ADCC activity. The use of mAbs in combination with e-NK cells could potentially improve cancer treatment.

Hypoxia, Metabolism and Immune Cell Function.

Hypoxia is a hallmark of inflamed, infected or damaged tissue, and the adaptation to inadequate tissue oxygenation is regulated by hypoxia-inducible factors (HIFs). HIFs are key mediators of the cellular response to hypoxia, but they are also associated with pathological stress such as inflammation, bacteriological infection or cancer. In addition, HIFs are central regulators of many innate and adaptive immunological functions, including migration, antigen presentation, production of cytokines and antimicrobial peptides, phagocytosis as well as cellular metabolic reprogramming. A characteristic feature of immune cells is their ability to infiltrate and operate in tissues with low level of nutrients and oxygen. The objective of this article is to discuss the role of HIFs in the function of innate and adaptive immune cells in hypoxia, with a focus on how hypoxia modulates immunometabolism.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

Boosting the hypoxic response in myeloid cells accelerates resolution of fibrosis and regeneration of the liver in mice.

We have recently shown that targeting Vascular Endothelial Growth Factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis. In a gain of function approach, we wanted to test the impact of VEGF overexpression in myeloid cells on fibrolysis. We observe that genetic inactivation of the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) in myeloid cells, leads to increased VEGF expression and most importantly, accelerated matrix degradation and reduced myofibroblast numbers after CCl4 challenge. This is associated with enhanced expression of MMP-2 and -14 as well as lower expression of TIMP-2 in liver endothelial cells. In addition, we report increased expression of MMP-13 in scar-associated macrophages as well as improved liver regeneration upon ablation of VHL in myeloid cells. Finally, therapeutic infusion of macrophages nulli-zygous for VHL or treated with the pharmacologic hydroxylase inhibitor and HIF-inducer Dimethyloxalylglycine (DMOG) accelerates resolution of fibrosis. Hence, boosting the HIF-VEGF signaling axis in macrophages represents a promising therapeutic avenue for the treatment of liver fibrosis.

Targeting VEGF-A in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia.

Chemotherapy remains a mainstay of cancer treatment but its use is often limited by the development of adverse reactions. Severe loss of body weight (cachexia) is a frequent cause of death in cancer patients and is exacerbated by chemotherapy. We show that genetic inactivation of vascular endothelial growth factor (VEGF)-A in myeloid cells prevents chemotherapy-induced cachexia by inhibiting skeletal muscle loss and the lipolysis of white adipose tissue. It also improves clearance of senescent tumour cells by natural killer cells and inhibits tumour regrowth after chemotherapy. The effects depend on the chemoattractant chemerin, which is released by the tumour endothelium in response to chemotherapy. The findings define chemerin as a critical mediator of the immune response, as well as an important inhibitor of cancer cachexia. Targeting myeloid cell-derived VEGF signalling should impede the lipolysis and weight loss that is frequently associated with chemotherapy, thereby substantially improving the therapeutic outcome.

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity.

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56bright, NK cells. Most NK cells in healthy human donors are CD45RA+CD45RO-. The CD45RA-RO+ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RAdimRO- (CD45RAdim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RAdim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RAdim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.

Identification of Anti-tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With Hematological Cancers.

Natural killer (NK) cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56(dim)CD16(+) NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46), natural-killer group 2, member D (NKG2D) and killer inhibitory receptors (KIRs) and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA(+)RO(+) phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA(+)RO(-) phenotype similar to naïve T cells. In summary, we show that CD45RA(+)RO(+) cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias.

The presence of wild type p53 in hematological cancers improves the efficacy of combinational therapy targeting metabolism.

Manipulation of metabolic pathways in hematological cancers has therapeutic potential. Here, we determined the molecular mechanism of action of the metabolic modulator dichloroacetate (DCA) in leukemic cells. We found that DCA induces the AMP-activated protein kinase (AMPK)/p53 pathway with increased efficacy in tumors expressing wild type (wt p53). Clinically relevant, low concentrations of doxorubicin synergize in vitro and in vivo with DCA to further enhance p53 activation and to block tumor progression. Leukemia cell lines and primary leukemic cells containing mutant p53 are resistant to the above-described combination approach. However, DCA synergized with the Hsp90 inhibitor 17-AAG to specifically eliminate these cells. Our studies strongly indicate that depending on the p53 status, different combination therapies would provide better treatment with decreased side effects in hematological cancers.

All-trans retinoic acid (ATRA) induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity.

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model.

Chemical metabolic inhibitors for the treatment of blood-borne cancers.

Tumor cells, including leukemic cells, remodel their bioenergetic system in favor of aerobic glycolysis. This process is called "the Warburg effect" and offers an attractive pharmacological target to preferentially eliminate malignant cells. In addition, recent results show that metabolic changes can be linked to tumor immune evasion. Mouse models demonstrate the importance of this metabolic remodeling in leukemogenesis. Some leukemias, although treatable, remain incurable and resistance to chemotherapy produces an elevated percentage of relapse in most leukemia cases. Several groups have targeted the specific metabolism of leukemia cells in preclinical and clinical studies to improve the prognosis of these patients, i.e. using L-asparaginase to treat pediatric acute lymphocytic leukemia (ALL). Additional metabolic drugs that are currently being used to treat other diseases or tumors could also be exploited for leukemia, based on preclinical studies. Finally, we discuss the potential use of several metabolic drugs in combination therapies, including immunomodulatory drugs (IMiDs) or immune cell-based therapies, to increase their efficacy and reduce side effects in the treatment of hematological cancers.

From tumor cell metabolism to tumor immune escape.

Tumorigenesis implies adaptation of tumor cells to an adverse environment. First, developing tumors must acquire nutrients to ensure their rapid growth. Second, they must escape the attack from the host immune system. Recent studies suggest that these phenomena could be related and that tumor cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy. This specific metabolism helps tumor cells to avoid the immune attack from the host by blocking or avoiding the immune attack. By changing their metabolism, tumor cells produce or sequester a variety of amino acids, lipids and chemical compounds that directly alter immune function therefore promoting immune evasion. A second group of metabolism-related modification targets the major histocompatibility complex-I (MHC-I) and related molecules. Tumor MHC-I presents tumor-associated antigens (TAAs) to cytotoxic T-cells (CTLs) and hence, sensitizes cancer cells to the cytolytic actions of the anti-tumor adaptive immune response. Blocking tumor mitochondrial activity decreases expression of MHC-I molecules at the tumor cell surface. And peroxynitrite (PNT), produced by tumor-infiltrating myeloid cells, chemically modifies MHC-I avoiding TAA expression in the plasma membrane. These evidences on the role of tumor cell metabolism on tumor immune escape open the possibility of combining drugs designed to control tumor cell metabolism with new procedures of anti-tumor immunotherapy. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.