Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration

Abstract

Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues.

Innate Cytokine Induced Early Release of IFNγ and CC Chemokines from Hypoxic Human NK Cells Is Independent of Glucose

“…Likewise, neither for normoxic nor hypoxic NK cells, glucose deprivation affected the release of CCL3/4/5 ( Figure 9D-F), TNFα ( Figure 9I) and VEGF ( Figure 9J) and levels of intracellular perforin and granzyme B ( Figure 9G,H). The influence of hypoxia on the levels of secreted IFNγ, TNFα, CCL3, CCL5 and VEGF observed here was consistent with reported results obtained with human NK cells stimulated exclusively with IL-12/IL-18 for 20 h under normoxia and hypoxia [64]. Overall, the simultaneous independence from glucose and oxygen of the early cytokine response and of the maintenance of cytotoxic granule proteins in primed human NK cells in response to secondary stimulation shows that these cellular processes require little if any active energy production.…”

Unleashing Natural Killer Cells in the Tumor Microenvironment–The Next Generation of Immunotherapy?

“…NK cells also undergo functional exhaustion when exposed to the conditions prevalent in the TME. These phenotypes in NK cells are induced by high expression of checkpoint ligands on tumors (such as Programmed death-ligand 1 (PD-L1) and HLA-E) (69,70), inhibitory cytokines and inhibitory soluble factors (such as TGF-β and IL-10) (71-73), hypoxia (74,75), exposure to tumor suppressor cells (i.e., Tregs, tumor associated macrophages, and MDSCs) (76)(77)(78), and sustained chronic activation (such as sustained activation through the activating NKG2D receptor) (79,80).…”

Immunometabolic Responses of Natural Killer Cells to Inhibitory Tumor Microenvironment Checkpoints

“…The responses of NK cells to hypoxic environments have been discussed previously, yet despite the available knowledge, the specific role of hypoxia on NK cells is still not fully clear. Hypoxia was shown to affect functions of NK cells by impairing their cytotoxicity [134][135][136] and altering their metabolic signatures [137]. Expression of activating receptors NKp46, NKp30, NKp44, and NKG2D [138] as well as perforin and granzyme B [138] are all downregulated on NK cells in hypoxia.…”

“…Cytokines have been shown to strongly affect NK cell metabolism [155]. Among them, IL-15 is the most widely studied [156] PD-1 Impairs lytic granule polarization; likely contributes to impaired NK anti-tumor immunity; in T cells PD-1 blocks glycolysis and glutaminolysis [47,49,54] Tumor microenvironment Adenosine Impairs cytotoxicity, glycolysis and alters mitochondrial respiration [86] TGF-β Inhibits mTOR in NK cells; impairs oxidative phosphorylation, glycolytic capacity and capacity and expression of CD71 [13,131] Hypoxia Impairs function and metabolism of NK cells; effects of NK cells are condition specific and somewhat inconclusive [137,140] Lactate Impairs NK cell activation, function and IFN-γ production [149][150][151] Despite the growing body of work that continues to uncover new mechanisms which tumors use to evade NK cell-mediated recognition and killing our understanding of NK cell metabolism in pathological settings lacks significant knowledge. For example, many of the studies on NK cell metabolism are carried out in ex vivo environments not representative of TME conditions, precluding accurate mechanistic insights to be elucidated.…”