Natural killer (NK) cells are endowed with germline-encoded receptors that enable them to detect and kill malignant cells without prior priming. Over the years, overwhelming evidence has identified an essential role for NK cells in tumor immune surveillance. More recently, clinical trials have also highlighted their potential in therapeutic settings. Yet, data show that NK cells can be dysregulated within the tumor microenvironment (TME), rendering them ineffective in eradicating the cancer cells. This has been attributed to immune suppressive factors, including the tumor cells per se, stromal cells, regulatory T cells, and soluble factors such as reactive oxygen species and cytokines. However, the TME also hosts myeloid cells such as dendritic cells, macrophages, neutrophils, and myeloid-derived suppressor cells that influence NK cell function. Although the NK-myeloid cell crosstalk can promote anti-tumor responses, myeloid cells in the TME often dysregulate NK cells via direct cell-to-cell interactions down-regulating key NK cell receptors, depletion of nutrients and growth factors required for NK cell growth, and secretion of metabolites, chemokines and cytokines that ultimately alter NK cell trafficking, survival, and cytotoxicity. Here, we review the complex functions of myeloid-derived cytokines in both supporting and suppressing NK cells in the TME and how NK cell-derived cytokines can influence myeloid subsets. We discuss challenges related to these interactions in unleashing the full potential of endogenous and adoptively infused NK cells. Finally, we present strategies aiming at improving NK cell-based cancer immunotherapies via pathways that are involved in the NK-myeloid cell crosstalk in the TME.

Natural Killer (NK) cells are potent cytotoxic cells belonging to the family of Innate Lymphoid Cells (ILCs). Their most characterized effector functions are directed to the control of aberrant cells in the body, including both transformed and virus-infected cells. NK cell-mediated recognition of abnormal cells primarily occurs through receptor-ligand interactions, involving an array of inhibitory and activating NK receptors and different types of ligands expressed on target cells. While most of the receptors have become known over many years, their respective ligands were only defined later and their impressive complexity has only recently become evident. NKp44, a member of Natural Cytotoxicity Receptors (NCRs), is an activating receptor playing a crucial role in most functions exerted by activated NK cells and also by other NKp44 + immune cells. The large and heterogeneous panel of NKp44 ligands (NKp44L) now includes surface expressed glycoproteins and proteoglycans, nuclear proteins that can be exposed outside the cell, and molecules that can be either released in the extracellular space or carried in extracellular vesicles. Recent findings have extended our knowledge on the nature of NKp44L to soluble plasma glycoproteins, such as secreted growth factors or extracellular matrix (ECM)-derived glycoproteins. NKp44L are induced upon tumor transformation or viral infection but may also be expressed in normal cells and tissues. In addition, NKp44-NKp44L interactions are involved in the crosstalk between NK cells and different innate and adaptive immune cell types. NKp44 expression in different ILCs located in tissues further extends the potential role of NKp44-NKp44L interactions.

The release of soluble ligands of activating Natural Killer (NK) cell receptors may represent a regulatory mechanism of NK cell function both in physiologic and in pathologic conditions. Here, we identified the extracellular matrix protein Nidogen-1 (NID1) as a ligand of NKp44, an important activating receptor expressed by activated NK cells. When released as soluble molecule, NID1 regulates NK cell function by modulating NKp44-induced IFN-γ production or cytotoxicity. In particular, it also modulates IFN-γ production induced by Platelet-Derived Growth Factor (PDGF)-DD following NKp44 engagement. We also show that NID1 may be present at the cell surface. In this form or when bound to a solid support (bNID1), NID1 fails to induce NK cell cytotoxicity or cytokine release. However, analysis by mass spectrometry revealed that exposure to bNID1 can induce in human NK cells relevant changes in the proteomic profiles suggesting an effect on different biological processes.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8 + T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8 + T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

Angiogenesis represents a hallmark of tumor progression in Multiple Myeloma (MM), a still incurable malignancy. Here we analyzed the activity of cytokine-stimulated NK cells against tumor-associated endothelial cells isolated from bone marrow aspirates of MM patients with active disease (MMECs). We show that NK cells activated with optimal doses of IL-15 killed MMECs thanks to the concerted action of multiple activating receptors. In particular, according to the high expression of PVR and Nectin-2 on MMECs, DNAM-1 actively participated in target recognition. Interestingly, in MMECs the surface density of PVR was significantly higher than that detected in endothelium from patients with MM in complete remission or with monoclonal gammopathy of undetermined significance (MGUS). Importantly, IL-27, which unlike IL-15 does not display pro-angiogenic properties, maintained or increased the NK cell functions induced by suboptimal concentrations of IL-15. NK cell properties included killing of MMECs, IFN-γ production as well as a peculiar increase of NKp46 expression on NK cell surface. Finally, IL-27 showed a striking capability of up-regulating the expression of PD-L2 and HLA-I on tumor endothelium, whereas it did not modify that of PD-L1 and HLA-II.Our results suggest that cytokine-activated endogenous or adoptively transferred NK cells might support conventional therapies improving the outcome of MM patients.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-kB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-kB recruitment in response to high LPS doses. Using the transposaseaccessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

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