In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus-infected or transformed cells, however it is unknownwhether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia-inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK-cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL-2 (or other activating cytokines, including IL-15, IL-12, and IL-21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc-γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody-dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody-based immunotherapy of cancer. Keywords: ADCC · Hypoxia · NK cells · Tumor Escape · Tumor MicroenvironmentAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionAs a component of innate immunity, natural killer (NK) cells play an important role in the control of virus infections and in cancer immune surveillance [1][2][3][4][5]. They can kill a wide range of cells that, Correspondence: Prof. Lorenzo Moretta e-mail: lorenzomoretta@ospedale-gaslini.ge.it upon neoplastic transformation or virus infection, have downregulated major histocompatibility complex class I surface expression.To recognize their targets, NK cells use a complex array of activating receptors and/or coreceptors. These mainly include the natural cytotoxicity receptors (NCRs, i.e. NKp46, NKp30, and * These authors contributed equally to this work. * * These authors share senior authorship. [5,[11][12][13][14], there are still many obstacles for the effective use of these cells in immunotherapy. Both tumors and viruses have developed different escape mechanisms to avoid NK-cell immunosurveillance. For example, certain viruses can shape the expression profile of various NK-receptor ligands in infected cells [15]. Similarly, tumor cells may shed from the surface certain NKG2D ligands thus avoiding NK-cell-mediated attack [16]. In addition, several lines of evidence indicate that the tumor microenvironment may impact the real ability of NK cells to clear pathologic cells [17][18][19][20][21][22]. Indeed, while cytokines such as IL-2, IL-15, IL-12, and IL-21 can enhance NK-cell function, other factors induced at the tumor site, such as IDO, PGE 2 , and TGF-β, or even the direct interaction with tumor cells or tumor-associated stromal cells, may impai...

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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.

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The identification of COVID-19 patients with high-risk of severe disease is a challenge in routine care. We performed cell phenotypic, serum and RNA-seq gene expression analyses in severe hospitalized patients (n = 61). Relative to healthy donors, results showed abnormalities of 27 cell populations and an elevation of 42 cytokines, neutrophil chemo-attractants, and inflammatory components in patients. Supervised and unsupervised analyses revealed a high abundance of CD177 , a specific neutrophil activation marker, contributing to the clustering of severe patients. Gene abundance correlated with high serum levels of CD177 in severe patients. Higher levels were confirmed in a second cohort and in ICU than non-ICU patients (P< 0.001). Longitudinal measurements discriminated between patients with the worst prognosis, leading to death, and those who recovered (P = 0.01). These results highlight neutrophil activation as a hallmark of severe disease and CD177 assessment as a reliable prognostic marker for routine care.