Statins inhibit NK‐cell cytotoxicity by interfering with LFA‐1‐mediated conjugate formation

Inhibitors of the 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, commonly referred to as statins, are inhibitors of cholesterol biosynthesis. They are broadly used for treating hypercholesterolemia and for prevention of cardio‐ and cerebrovascular diseases. Recent publications show that statins also act as immunomodulatory drugs. Here, we show that lipophilic statins inhibit NK‐cell degranulation and cytotoxicity. This effect was reversible by addition of substrates of isoprenylation, but not by addition of cholesterol. In NK‐target cell conjugates intracellular Ca²⁺ flux was unaffected by statin treatment. However, statins strongly reduced the amount of conjugate formation between NK and target cells. This inhibition was paralleled by a statin‐dependent inhibition of LFA‐1‐mediated adhesion and a reduction of NK‐cell polarization. This demonstrates that statins impair the formation of effector–target cell conjugates resulting in the disruption of early signaling and the loss of NK‐cell cytotoxicity.     

Adiponectin modulates NK‐cell function

Adiponectin (APN) has been shown to exert antiinflammatory effects in various disease models but little is known concerning its regulation of NK‐cell function. Here, we show that the majority of human CD56^dim NK cells express surface Adiponectin receptor (AdipoR) 1 and 2 while most CD56^high NK cells are AdipoR‐negative. Toll‐like receptor (TLR) ligand‐induced IFN‐γ production was diminished by APN while it had no influence on NK‐cell cytotoxicity. In contrast only a small subpopulation of murine NK cells expresses surface AdipoRs, but about 90% store them intracellularly. APN‐deficient knockout (KO) mice had elevated frequencies of NK cells. However, cytotoxic degranulation of NK cells was decreased in APN knockout (APN‐KO) animals. Accordingly, frequencies of CD11b^highCD27^high and CD94^high effector NK cells and expression of NKG2D were lower in APN‐KO mice. Upon CVB3 infection NK‐cell function was restored in APN‐KO mice. Our data suggest that in addition to its antiinflammatory effects APN also influences the numerical and differentiation status of NK cells, which may further impact the outcome of immune‐mediated diseases in APN‐KO mice.     

TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals

Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T‐cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK‐cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN‐γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK‐cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.     

Ligand dimensions are important in controlling NK‐cell responses

Size‐dependent protein segregation at the cell–cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK‐cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL‐2 stimulated C57BL/6 mouse‐derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK‐cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H‐2K^b, a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H‐2K^b resulted in decreased inhibition of both lysis and IFN‐γ production by NK cells. These results establish that small ligand dimensions are important for both NK‐cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.     

Novel molecular mechanism for generating NK‐cell fitness and memory

The mammalian immune system has been traditionally subdivided into two compartments known as the innate and the adaptive. T cells and B cells, which rearrange their antigen‐receptor genes using the RAG recombinase, comprise the adaptive arm of immunity. Meanwhile, every other white blood cell has been grouped together under the broad umbrella of innate immunity, including NK cells. NK cells are considered innate lymphocytes because of their rapid responses to stressed cells and their ability to develop without receptor gene rearrangement (i.e. in RAG‐deficient mice). However, new findings implicate a critical function for RAG proteins during NK‐cell ontogeny, and suggest a novel mechanism by which controlled DNA breaks during NK‐cell development dictate the fitness, function, and longevity of these cells. This review highlights recent work describing how DNA break events can impact cellular differentiation and fitness in a variety of cell types and settings.     

Effect of tumor cells and tumor microenvironment on NK‐cell function

The ability of tumors to manage an immune‐mediated attack has been recently included in the “next generation” of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross‐interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK‐cell responses to tumors. Tumor cells, tumor‐associated fibroblasts and tumor‐induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK‐cell activation pathways or the complex receptor array that regulate NK‐cell activation and antitumor activity. Thus, the definition of tumor microenvironment‐related immunosuppressive factors, along with the identification of new classes of tissue‐residing NK‐like innate lymphoid cells, represent key issues to design effective NK‐cell‐based therapies of solid tumors.     

Tissue‐resident and memory properties of human T‐cell and NK‐cell subsets

Efficient immune responses to invading pathogens are the result of the complex but coordinated synergy between a variety of cell types from both the innate and adaptive arms of the immune system. While adaptive and innate immune responses are highly complementary, some cells types within these two systems perform similar functions, underscoring the need for redundancy and increased flexibility. In this review, we will discuss the striking shared features of immunological memory and tissue residency recently discovered between T cells, a component of the adaptive immune system, and natural killer (NK) cells, members generally assigned to the innate compartment. Specifically, we will focus on the T‐cell and NK‐cell diversity at the single‐cell level, on the discrete function of specific subsets, and on their anatomical location. Finally, we will discuss the implication of such diversity in the generation of long‐term memory.     

At diagnosis,diffuse large B‐cell lymphoma patients show impaired rituximab‐mediated NK‐cell cytotoxicity

Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of non‐Hodgkin's lymphoma in adults. It is generally treated by a combination of chemotherapy and CD20‐specific mAbs, such as rituximab, which act, at least partially, by activating antibody‐dependent cell‐mediated cytotoxicity (ADCC). ADCC involves NK cells, particularly the CD56^dim NK‐cell subset expressing CD16, the low affinity Fcγ receptor. Here, we show that CD16 expression levels are decreased in a cohort of 36 newly diagnosed DLBCL patients compared with those in 20 healthy controls (HCs). CD137, a co‐stimulatory molecule expressed on activated NK cells, was also expressed at lower levels in patients compared with controls. Cells sampled from our cohort also showed severely reduced degranulation activity when challenged with rituximab‐coated tumor cells, which could not be corrected by stimulation with high doses of IL‐2. These results suggest that rituximab‐induced NK‐cell ADCC could be defective in some DLBCL patients at diagnosis. These patients should be closely monitored and attempts made to improve their NK‐cell function.     

Activated NKT cells imprint NK‐cell differentiation,functionality and education

NK cells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NK cells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross‐talk between NKT cells and NK cells needs to be better understood. Our results provide strong evidence for NKT cell‐induced effects on key biological features of NK cells. NKT‐cell activation results in the generation of highly active CD27^high NK cells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT‐cell stimulation induced the generation of highly functional educated and uneducated NK cells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT‐NK cell axis that provide important hints for the manipulation of NK cells in clinical settings.     

Overexpression of CD85j in TNBC patients inhibits Cetuximab‐mediated NK‐cell ADCC but can be restored with CD85j functional blockade

Clinical studies suggest that triple negative breast cancer (TNBC) patients with epidermal growth factor receptor (EGFR)‐expressing tumors could benefit from therapy with Cetuximab, which targets EGFR. NK cells are the primary effectors of antibody (Ab)‐dependent cell‐mediated cytotoxicity (ADCC) and thus play a role in Ab‐based therapies. We have previously described diminished levels of Cetuximab‐mediated ADCC in vitro in patients with advanced breast cancer. Here, we investigated the potential causes of this NK‐cell functional deficiency. We characterized NK‐cell activating/inhibitory receptors in the peripheral blood of breast cancer patients and found CD85j inhibitory receptor overexpression. The capacity of NK cells to perform Cetuximab‐triggered ADCC against TNBC cells correlated inversely with CD85j expression, even in the presence of the stimulatory cytokines IL‐2 or IL‐15. Hence, patients expressing high levels of CD85j had an impaired ability to lyse TNBC cells in the presence of Cetuximab. We also found that CD85j overexpression was associated with HLA‐I and soluble HLA‐G expression by tumors. A CD85j functional blockade with a CD85j antagonist Ab restored ADCC levels in breast cancer patients and reverted this negative effect. Our data suggest that strategies that overcome the hurdles of immune activation could improve Cetuximab clinical efficacy.     

Autologous and allogeneic HLA KIR ligand environments and activating KIR control KIR NK‐cell functions

NK‐cell function is regulated by a balance between inhibitory and activating killer cell immunoglobulin‐like receptors (KIR) that specifically recognize HLA class I molecules. Using KIR‐specific mAb to discriminate between KIR2DS1 and KIR2DL1 receptors, we show that KIR2DS1⁺ NK cells are C2‐alloreactive only from C2^? individuals. Moreover, using an in vitro model of NK‐cell expansion, we show here that the frequency of KIR2DL1⁺ NK cells is significantly higher in the absence of C2 ligand on stimulator EBV‐B cells than in its presence. This observation was made regardless of the presence or absence of the autologous C2 ligand, suggesting that the C2^? EBV‐B stimulator cells used in this in vitro model could activate unlicensed KIR2DL1⁺ NK cells. In the case of KIR2DL1⁺/S1⁺ genotyped individuals, KIR2DS1⁺ NK‐cell frequency was increased after stimulation with C2⁺ compared with C2^? stimulator B cells, but only from C2^? individuals. Altogether, these data highlight the C2 alloreactivity of KIR2DS1⁺ NK cells that is only observed in C2^? individuals. These results provide new insights into the way in which NK KIR cell expansion might be regulated in an allogeneic environment.     

Slp‐76 is a critical determinant of NK‐cell mediated recognition of missing‐self targets

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing‐self” recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl‐N‐nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of “missing‐self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp‐76—a critical adapter molecule downstream of ITAM‐containing surface receptors. The Slp‐76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T‐cell development/function, a marked defect in NK cell mediated elimination of β2‐microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp‐76 to control NK‐cell receptor expression and maturation; however, activation of Slp‐76^ace/ace NK cells through ITAM‐containing NK‐cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK‐β2M^?/? target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp‐76^ace/ace NK cells. Overall these studies establish Slp‐76 as a critical determinant of NK‐cell development and NK cell mediated elimination of missing‐self target cells in mice.     

B‐cell co‐receptor CD72 is expressed on NK cells and inhibits IFN‐γ production but not cytotoxicity

NK cells have two main functions, namely cell‐mediated cytotoxicity and production of cytokines. Multiple inhibitory receptors that regulate NK‐cell cytotoxicity have been characterized whereas little is known about receptors regulating cytokine production. Here we report that CD72, which is considered to be an important co‐receptor regulating B‐cell activation, is also expressed on mouse NK cells. NK cells expressing high levels of CD72, upon stimulation with IL‐12 and IL‐18 or target cells, produce significantly less IFN‐γ than those expressing low levels of CD72, whereas both subsets are equally cytotoxic. Ectopic expression of CD72 in the murine NK‐cell line KY2 inhibits cytokine‐induced IFN‐γ production, and the inhibitory effect is diminished by mutations in the inhibitory motifs in the intracellular domain or replacement of the extracellular domain of CD72. Thus, CD72 is an inhibitory receptor on NK cells regulating cytokine production.     

Adaptive reconfiguration of the human NK‐cell compartment in response to cytomegalovirus: A different perspective of the host‐pathogen interaction

As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C⁺ NK cells reported in other infectious diseases is systematically associated with HCMV co‐infection. The functionally mature NKG2C^bright NK‐cell subset expanding in HCMV⁺ individuals displays inhibitory Ig‐like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK‐cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T‐cell‐mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H⁺ NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV‐infected cells may drive the expansion of the corresponding NK‐cell subset. Yet, the precise role of NKG2C⁺ cells in the control of HCMV infection, the molecular mechanisms underlying the NK‐cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues.     

Murine CXCR3+CD27bright NK cells resemble the human CD56bright NK‐cell population

Human NK cells can be subdivided into CD56^dim and CD56^bright NK cells, which exhibit different phenotypical and functional characteristics. As murine NK cells lack CD56 or a distinct correlate, direct comparative studies of NK cells in mice and humans are limited. Although CD27 is currently proposed as a feasible subset marker in mice, we assume that the usage of this marker alone is insufficient. We rather investigated the expression of the chemokine receptor CXCR3 for its suitability for distinguishing murine NK‐cell subsets with simultaneous consideration of CD27. Compared with CXCR3^? NK cells, exerting stronger cytotoxic capability, CXCR3⁺ NK cells displayed an activated phenotype with a lower expression of Ly49 receptors, corresponding to human CD56^bright NK cells. Also in common with human CD56^bright NK cells, murine CXCR3⁺ NK cells exhibit prolific expansion as well as robust IFN‐γ, TNF‐α and MIP‐1α production. We additionally demonstrated changes in both CXCR3 and CD27 expression upon NK‐cell activation. In summary, CXCR3 serves as an additional applicable marker for improved discrimination of functionally distinct murine NK‐cell subsets that comply with those in humans.     

microRNA management of NK‐cell developmental and functional programs

NK cells are innate lymphoid cells that are critical for host defense against infection, and mediate anti‐tumor responses. MicroRNAs (miRNAs) are a large family of small noncoding RNAs that target the 3′ untranslated region (UTR) of mRNAs, thereby attenuating protein translation. The expression of miRNAs within human peripheral blood and mouse splenic NK cells has been cataloged, with the majority of the miRNA sequence pool represented in the top 60 most abundantly expressed miRNAs. Global miRNA deficiency within NK cells has confirmed their critical role in NK‐cell biology, including defects in NK‐cell development and altered functionality. Studies using gain‐ and loss‐of‐function of individual miRNAs in NK cells have demonstrated the role of specific miRNAs in regulating NK‐cell development, maturation, and activation. miRNAs also regulate fundamental NK‐cell processes including cytokine production, cytotoxicity, and proliferation. This review provides an update on the intrinsic miRNA regulation of NK cells, including miRNA expression profiles, as well as their impact on NK‐cell biology. Additional profiling is needed to better understand miRNA expression within NK‐cell developmental intermediates, subsets, tissues, and in the setting of disease. Furthermore, key open questions in the field as well as technical challenges in the study of miRNAs in NK cells are highlighted.     

Hypoxia downregulates the expression of activating receptors involved in NK‐cell‐mediated target cell killing without affecting ADCC

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 unknown whether 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.     

NK cell‐extrinsic IL‐18 signaling is required for efficient NK‐cell activation by vaccinia virus

NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR‐dependent and ‐independent pathways, as well as the NKG2D activating receptor that recognizes host stress‐induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study using C57BL/6 mice, we first showed that IL‐18 is critical for NK‐cell activation and viral clearance. We then demonstrated that IL‐18 signaling on both NK cells and DCs is required for efficient NK‐cell activation upon VV infection in vitro. We further showed in vivo that efficient NK‐cell activation in response to VV is dependent on DCs and IL‐18 signaling in non‐NK cells, suggesting an essential role for NK cell‐extrinsic IL‐18 signaling in NK‐cell activation. Mechanistically, IL‐18 signaling in DCs promotes expression of Rae‐1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell‐extrinsic IL‐18 signaling in NK‐cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK‐cell‐based therapies for viral infections and cancer.