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Review

Human NK cell response to pathogens Mariella Della Chiesa 1 , Emanuela Marcenaro 1 , Simona Sivori 1 , Simona Carlomagno, Silvia Pesce, Alessandro Moretta ∗ DI.ME.S. Dipartimento di Medicina Sperimentale and Centro di Eccellenza per la Ricerca Biomedica, Università di Genova, Genova, Italy

a r t i c l e

i n f o

Keywords: Natural killer cells Toll-like receptors Anti-viral response HIV HCMV BCG

a b s t r a c t NK cells represent important effectors of the innate immunity in the protection of an individual from microbes. During an NK-mediated anti-microbial response, the final fate (survival or death) of a potential infected target cell depends primarily on the type and the number of receptor/ligand interactions occurring at the effector/target immune synapse. The identification of an array of receptors involved in NK cell triggering has been crucial for a better understanding of the NK cell biology. In this context, NCR play a predominant role in NK cell activation during the process of natural cytotoxicity. Regarding the NK-mediated pathogen recognition and NK cell activation, an emerging concept is represented by the involvement of TLRs and activating KIRs. NK cells express certain TLRs in common with other innate cell types. This would mean that specific TLR ligands are able to promote the simultaneous and synergistic stimulation of these innate cells, providing a coordinated mechanism for regulating the initiation and amplification of immune responses. Evidences have been accumulated indicating that viral infections may have a significant impact on NK cell maturation, promoting the expansion of phenotypically and functionally aberrant NK cell subpopulations. For example, during chronic HIV-infection, an abnormal expansion of a dysfunctional CD56neg NK cell subset has been detected that may explain, at least in part, the defective NK cell-mediated antiviral activity. An analogous imbalance of NK cell subsets has been detected in patients receiving HSCT to cure high risk leukemias and experiencing HCMV infection/reactivation. Remarkably, NK cells developing after CMV reactivation may contain “memory-like” or “long-lived” NK cells that could exert a potent anti-leukemia effect. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Innate and adaptive immune responses cooperate to protect the host against microbial infections [1–3]. NK cells are innate immune cells whose function is critical in the first-line of defense against different types of pathogens, including viruses, bacteria and fungi [4,5]. Pathogen recognition by NK cell is mediated by different types

Abbreviations: HIV, human immunodeficiency virus; HCMV, human cytomegalovirus; MCMV, murine cytomegalovirus; HSCT, hematopoietic stem cell transplantation; TLRs, Toll-like receptors; PRR, pattern recognition receptor; PAMPs, pathogen-associated molecular pattern; poly I:C, polyinosinic–polycytidylic acid; NCR, natural cytotoxicity receptor; BCG, bacillus Calmette-Guerin; UCBT, umbilical cord blood transplantation; SOT, solid organ transplantation; PB, peripheral blood. ∗ Corresponding author at: Dipartimento di Medicina Sperimentale, Sezione di Istologia, Via G.B. Marsano 10, 16132 Genova, Italy; Tel.: +39 010 3537868; fax: +39 010 3537576. E-mail address: [email protected] (A. Moretta). 1 These authors equally contributed to this work.

of receptors, including Toll-like receptors (TLRs), that display broad specificities for conserved and invariant features of microorganisms, and a series of activating receptors, able to recognize specific ligands on infected/transformed cells [6,7]. NK cells also express inhibitory receptors that may counteract the function of activating receptors. The expression or the lack of ligands specific for inhibitory or activating NK receptors by target cells is thought to determine the final outcome, i.e. to determine whether a given target cell will be killed or spared by NK cells [8–12]. NK cells can also respond to signals/cytokines derived from accessory cells including IL-12 which is crucial for IFN-␥ production in response to various pathogen-associated TLR agonists [13,14]. Remarkably, several recent studies reported that during infection with some viruses, a progressive, impairment of NK cell function may occur as a consequence of a progressive perturbation of NK cell compartment [15–17]. In this review, we will discuss some of the molecular mechanisms by which NK cells recognize pathogens or virus-infected cells, mediate appropriate innate immune effector responses

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and favor the development of efficacious downstream adaptive immune responses to viral antigens. In this context, the impact of human CMV (HCMV) infection on NK cell development and function will be analyzed in more detail. In addition, we will describe some aberrant redistribution of dysfunctional NK cell subsets, during HIV or HCMV infection/reactivation [17–19].

2. NK and TLRs 2.1. TLR-mediated recognition of pathogens by NK cells TLRs are germ-line encoded pattern-recognition receptors (PRRs) that are essential for the recognition of invading pathogens, triggering of innate responses and shaping of subsequent adaptive immune responses [20,21]. Currently, at least 11 mammalian TLRs have been identified, but only nine have been well characterized. Each TLR has a broad specificity for conserved molecular structures that are unique to microorganisms. These targets are referred to as pathogen associated molecular patterns (PAMPs), although they may actually be components of both pathogenic and non-pathogenic microorganisms. PAMPs include: lipopolysaccharide (LPS), an essential structure of the outer membrane of Gram-negative bacteria, which is recognized by TLR4; bacterial lipoproteins and lipoteichoic acids, recognized by TLR2; flagellin, a bacterial protein recognized by TLR5; microbial unmethylated CpG DNA motifs, recognized by TLR9; double-stranded RNA (dsRNA), recognized by TLR3; and single-stranded RNA (ssRNA), recognized by TLR7/TLR8 [20]. TLRs are expressed on various cells of the innate immunity, including NK cells [1], monocytes/macrophages [22], neutrophils [22], basophils [23], eosinophils [24], myeloid dendritic cells (DCs) [25], plasmacytoid DCs (pDCs) [25] and mast cells [6,26]. TLR expression is dependent on the cell type and in most instances, a given cell type expresses a small number of TLR. Moreover, TLRs differ in their signal transduction pathways. Certain TLRs (TLR1,2,4,5,6) are expressed on the cell surface, whereas others (TLR3,7,8,9) are localized in intracellular compartments (i.e. endosomes). Therefore, their ligands require internalization to generate signals [21]. Upon recognition of PAMPs, TLRs initiate a signaling cascade resulting in activation of tissue-resident innate cells (such as DCs, macrophages and mast cells) and in chemokine secretion. In turn, these factors recruit circulating leukocytes to the site of infection which further limit the spread of invading pathogens [1,21]. Among the recruited cells, an important role is played by NK cells, that, once reached the inflammatory sites, require activation in order to carry out their function [27]. NK cell activation occurs through different mechanisms, including engagement of different triggering NK cell receptors or recognition of appropriate ligands on transformed cells, and/or stimulation via TLRs [1]. NK cells express different functional TLRs, independent of their state of activation, including TLR2 [28,29], TLR3 [30,31], TLR5 [32], TLR7/8 [33,34] and TLR9 [30]. Thus, responses to TLR ligands occur in both fresh and IL-2activated NK cells and, in most instances, are sharply increased in the presence of inflammatory cytokines, primarily IL-12. TLRs can activate NK cell function either directly or in cooperation with accessory cells in a cytokine or cell-to-cell contact-dependent manner [13] (Fig. 1). Because certain TLRs are shared by different innate cells that participate to the early phases of an immune response, certain PAMPs are able to promote the simultaneous and synergistic stimulation of these cells. For example, since both NK cells and DCs express TLR3, the engagement of such receptor may represent a crucial step for promoting the cross-talk between these cells in peripheral tissues. In particular, in the presence of

IL-12 (released by DCs upon TLR3 engagement), NK cells respond via TLR3 to dsRNA by increasing their anti-tumor/anti-viral cytotoxicity and acquire also the ability to kill immature DCs (iDCs). This activity favors the selective survival of mature DCs (NK cellmediated ‘editing’ of DCs) [30]. Moreover, upon TLR stimulation, NK cells greatly increase their capability of secreting pro-inflammatory cytokines (such as TNF-␣ and IFN-␥), which promote further DC maturation and subsequent induction of Th1 responses in lymph nodes [35–37]. Remarkably, NK cell populations, derived from different donors may respond to TLR3 stimulation with different efficiency. Such heterogeneous response to poly I:C, a classic TLR3 ligand, has been explained with the observation that the percentage of NK cells expressing high levels of TLR3 mRNA transcripts may vary in different donors. Moreover, the analysis of NK cell clones revealed that heterogeneity exists not only among different donors, but also among NK cell clones derived from the same individual [38]. It has also been shown that NK cell clones expressing low levels of Natural Cytotoxicity Receptors [39–41](NCR dull phenotype) that are poorly cytotoxic, can up-regulate their cytolitic activity upon TLR3 engagement provided that they express high levels of TLR3 mRNA transcript. This capability of responding to TLR3 ligands may be critical in the context of some pathologic conditions, including HIV infection [42] or acute myeloid leukemia (AML) [43], in which down-regulation of NCR expression may represent a mechanism by which virus-infected or tumor cells escape NK-mediated lysis. Notably, NK cells also express functional TLR9, which are present in pDCs, but not in conventional DCs. Thus, stimuli acting on TLR9 are able to simultaneously activate both NK cells and pDCs [44]. In particular, IFN-␣, released by pDCs upon TLR9 engagement, may play an important role in supporting the activation of TLR9responsive NK cells [45]. Moreover, the exposure of NK cells to IL-12 (produced by DCs) amplifies the response and further potentiates NK-mediated induction of IFN-␣ release by pDCs [46]. Also TLR2 is expressed by NK cells and other innate cells (including monocytes/macrophages and DCs). This TLR binds to products of bacterial origin. In particular, a direct involvement of TLR2 in the recognition of Mycobacterium tuberculosis by NK cells has been clearly demonstrated [29,47] (see Section 2.2). Flagellin the TLR5 ligand, can directly act on NK cells by inducing the secretion of IFN-␥ and ␣-defensins that contribute, respectively, to activate accessory cells (e.g., macrophages) and to exert an anti-microbial effect. In turn, accessory cells, activated by PAMPs and/or cytokines present in the inflammatory microenvironment, can release other cytokines (e.g., IL-12 and IL-2), which may modulate PAMP-mediated activation of NK cell effector functions [32]. Finally, Hart et al. demonstrated that human NK cells may also express functional TLR7 and TLR8 [33]. In this regard, Alter et al. showed that NK cells may be significantly activated by the TLR7/8 ligand uridine-rich ssRNA derived from HIV. The functional activation of NK cells is strictly dependent on the direct contact of NK cells with pDCs or CD14+ monocytes resulting in increased IFN-␥ secretion [34]. All these data indicate that, although in some cases NK cells can be directly activated by some TLR agonists, both the cross-talk with other innate cells (in particular DCs) and the cytokine milieu may play a crucial role in the activation of their effector function. Thus, NK cells, in addition to a direct TLR-engagement, require appropriate costimulatory signals, to elicit an optimal response, capable of modulating downstream adaptive immune responses [6,48] (Fig. 1). 2.2. Direct recognition of BCG by human NK cells A number of experimental evidences indicate that NK cells are the main responsible of the anti-tumor responses induced

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Fig. 1. TLR-mediated activation of human NK cells. NK cells express different functional TLRs: TLR2 recognizes bacterial lipoproteins and lipoteichoic acids is present, for example, in BCG cell wall; TLR3 recognizes viral dsRNA; TLR5 binds flagellin; TLR7/8 recognize viral ssRNA and TLR9 microbial unmethylated CpG DNA motifs. Since both NK cells and DCs express TLR2 and TLR3, the engagement of these receptors by their specific ligands may represent a crucial step to promote the cross-talk between these two cell types. In most cases, IL-12 (released by DCs after TLR2 or TLR3 engagement), leads to optimal NK cell activation in response to TLR2 and TLR3 specific ligands. Stimuli acting on TLR7 or TLR9 are able to simultaneously activate both NK cells and pDCs, sharing these TLRs. In particular, IFN-␣, released by pDCs upon TLR engagement, may support the activation of TLR-responsive NK cells. Thus, although NK cells can be directly activated by some TLR agonists, the cross-talk with other innate cells (in particular DCs and pDCs) and the cytokine milieu play a crucial role in the activation of their multiple effector functions as explained in the figure box.

by Mycobacterium bovis (bacille Calmette-Guérin, BCG) in the treatment of superficial bladder carcinoma [49–52], as well as in innate immune protection from tubercolosis [53,54]. However, the molecular mechanisms of interaction between human NK cells and mycobacteria have yet to be fully clarified. Some recent studies would indicate that BCG, through the engagement of TLR2, can directly induce the acquisition of potent effector functions by NK cells [29,47,55,56]. These include the ability to potentiate the cytolytic activity against both tumor cells and iDCs, and to release proinflammatory cytokines (including IFN-␥ and TNF-␣), that, in turn, mediate the maturation of DCs, thus favoring the induction of adaptive Th1 immune responses. Remarkably, different from other TLR-mediated signaling, the release of cytokines by NK cells does not strictly require the presence of IL-12 [29,56]. Notably, in response to BCG stimulation, both CD56bright and CD56dim NK cell subsets release IFN-␥ [29]. In this context, however, some authors suggest that the CD56bright subset is primarily involved in the release of IFN-␥, in response to BCG [56]. Accordingly, a recent report would support the crucial role of IFN-␥ produced by CD56bright NK cells in mycobacterial infection and in BCG immunotherapy of bladder cancer [57]. Recently, Esin et al. have shown that different components abundant in mycobacterium tuberculosis cell wall may directly interact with the NKp44 NCR [58] and TLR2 [47]. However, only the interaction via TLR2 promotes cell activation and IFN-␥ production by resting NK cells, whereas NKp44, expressed upon TLR2-mediated NK cell activation, may promote later signal resulting in prolonged NK cell activation [47].

All these findings suggest that a direct interaction with extracellular mycobacteria may represent an efficient stimulus to induce NK cell activation and cytokine secretion by these innate cells [29,55] (Fig. 1). 2.3. KIR3DL2 as chaperon for TLR9 ligands Quite surprisingly, recent data have shown that KIR3DL2 [59] plays a direct role in the events that allow NK cell responsiveness to CpG-ODNs, i.e. the ligands of TLR9 receptor. In particular, it has been demonstrated that KIR3DL2 works as a sensor for microbial products and as a chaperon for TLR9 ligands [60]. KIR3DL2, expressed at the NK cell surface, can bind CpG-ODNs and shuttle them to early endosomes where TLR9 translocates upon CpG-ODNs cell stimulation. Following CpG-ODNs binding, TLR9 leads to NK cell activation and cytokine secretion (Fig. 1). Also other members of the KIR family (KIR3DL1, KIR3DS1, KIR2DL4) are able to bind CpG-ODNs, however, functional assays confirmed that IFN-␥ production in response to CpG-ODNs, is mostly confined to NK cells expressing KIR3DL2+ . This suggests that the other CpG-ODN-binding KIRs do not mediate efficient CpGODNs shuttling to TLR9-rich intracellular compartments [61]. KIR3DL2 is known as an HLA-specific receptor that binds HLAA*03 and A*11 allotypes. However, it is characterized by low inhibitory capacity and its interaction with HLA ligands is highly dependent on the peptide(s) bound to HLA-A molecules [62]. Together with KIR2DL4 and KIR3DL3, KIR3DL2 is a framework gene which is present in all KIR haplotypes [63]. The capability of KIR3DL2 described above may provide novel important clue to understand the driving force leading to the conservation of

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the KIR3DL2-encoding gene in all haplotypes, in spite of the low frequency of HLA-A3 or -A11 alleles in humans [1]. Thus, the need of a prompt NK-mediated reaction to microbial products may represent an important factor of selective genetic pressure [60]. 2.4. Correlation between TLRs and anti-HIV/HCMV responses HCMV and HIV can activate the innate immune system through both TLR-dependent and -independent pathways [19,64]. The precise role of TLR in early recognition and control of HIV and HCMV by NK cells or other immune cells, has not been fully elucidated, even though different innate immune receptors, including TLR2, TLR3, TLR7, TLR8 and TLR9, have been reported to play a role in innate responses against these viruses. In particular, HIV, similar to other ssRNA viruses, was described to trigger TLR7/TLR8 expressed on pDC/DCs and induce the release of proinflammatory cytokines capable of promoting the activation of other innate cells, including NK cells [65–67]. In addition, it has been possible to demonstrate a direct binding of gp120 to TLR9 on pDCs. This interaction could suppress pDC activation, preventing the TLR9-mediated production of proinflammatory cytokines and the pDC-induced stimulation of NK cytotoxicity [68]. The HIV-mediated DCs triggering results in production of different cytokines at different time intervals after acute infection [69]. Remarkably, the early cytokine storm preceding the peak of viremia includes cytokines capable of increasing the NK cell function. It has been reported that gB and gH glycoproteins of HCMV, an enveloped dsDNA virus, can stimulate TLR2-expressing fibroblasts [70]. Along this line, a recent study shows that NK cells can directly recognize HCMV virions through TLR2. Upon TLR2 engagement, NK cells become activated and produce IFN-␥ [64]. In mice, TLR2 and TLR9 have been shown to participate in the recognition of viral particles (including envelope glycoproteins and viral DNA, respectively) [71–74], while TLR3 and TLR7 could be involved in sensing CMV-derived products during the infection cycle [72,74]. Similarly, TLR9 is likely to be involved in sensing HCMV as suggested by epidemiological and genetic studies showing that specific single nucleotide polymorphism in TLR9 gene is highly predictive of susceptibility to HCMV infection [75]. 3. NK cells in HIV and HCMV infections 3.1. NK cell responses in HIV infection HIV has developed multiple strategies to evade detection by NK cells, strongly suggesting that these cells exert a pressure on the virus [4,19,76,77]. However, also the opposite may be true, since several functional defects of NK cell have been observed in HIVinfected patients, particularly in active and advanced disease stages [15]. A number of studies indicate that HIV uses the Nef gene to evade innate and adaptive immune responses. Indeed, Nef may downregulate the expression of the dominant T-cell receptor ligands HLA-A and -B molecules at the surface of infected cells thus preventing their recognition by T cells. On the other hand, HLA-C, the dominant ligand of inhibitory KIR2D is not affected preventing their killing by NK cells [78,79]. In addition, Nef may prevent both the expression of some NKG2D ligands (including MIC-A, ULBP-1, and -2) and the DNAM-1 ligand PVR on the surface of infected cells, thus impairing, at least in part, the NK-mediated cytotoxicity [80]. It has also been suggested that particular KIR/HLA combinations may impact the outcome of HIV-infection [81–83]. In particular, individuals expressing the activating KIR3DS1 allele, in

conjunction with its putative HLA class I ligand, were characterized by a slower disease progression as compared to controls [81]. In addition, KIR3DS1+ NK cells have been reported to exert their anti-viral activity preferentially against HIV-infected target cells expressing its putative ligand [84]. Moreover, different data indicated that given amino acid changes in the peptide associated with HLA-class I molecules could abrogate binding of inhibitory KIRs to their HLA class I ligands, thus allowing target cell lysis [14,85–89]. On the other hand, certain viral peptides could favor binding of an activating KIR to its HLA ligand, thus increasing NK-cell cytotoxicity [76]. 3.2. Presence of dysfunctional CD56neg NK cell subset in HIV infection Several studies support the notion that NK cells, in most infected patients, are unable to control the progression of HIV infection. This is associated with the expansion of dysfunctional NK cell subpopulations that are virtually absent in uninfected/aviremic donors [19,42]. Peripheral blood (PB) NK cells include two main subsets based on the expression of the CD56 molecule: the minor NK cell subpopulation characterized by a CD56bright phenotype and the major subset expressing a CD56dim phenotype [90]. CD56dim cells are strongly cytotoxic and can produce high levels of proinflammatory and antiviral cytokines following stimulation via activating NK receptors [27,91–93]. Both chronic and active phases of HIV infection are associated with decreased proportions of CD56dim /CD16+ NK cells and with the emergence of an aberrant CD56neg/CD16+ NK cell subset [16,42,94–96]. This unusual CD56neg NK cell subset displays phenotypic perturbations, including down-regulation of the major activating NK receptors (e.g. NCRs), associated with relevant functional abnormalities [42,97]. These cells appear to exert a limited ability to control opportunistic infections and tumors occuring at late stages of HIV infection. They also display a reduced secretion of cytokines such as IFN-␥, TNF-␣, and GM-CSF. The expression of inhibitory NK receptors (including KIRs) was either normal or increased in NK cells of viremic patients. As a consequence, these receptors could mediate an even greater inhibition of cytolytic function. Several studies reported in the course of HIV infection, abnormalities in the interaction between NK cells and DCs, occurring in peripheral tissue at inflammation sites [3,48,98–100]. This led to an impaired activation of NK cell and to a deficient killing of iDCs by NK cells. These defects prevent a correct DC-mediated priming of autologous naive CD4+ T cells thus compromising downstream antigen-specific, adaptive immune responses [101]. In line with the observation that only high levels of viral replication during chronic HIV infection correlate with the appearance of functionally impaired CD56neg NK cells, suppression of viral load by antiretroviral therapy resulted in progressive restoration of CD56 expression in PB NK. Recent studies have shown that a decreased expression of the cellular marker Siglec-7 occurred at the initial stages of HIV infection preceding down-regulation of CD56 [102]. In addition to abnormalities in the levels of expression of NK cell receptors in chronic viremic HIV-infected individuals, another finding was the decrease of NKG2A+ NK cells and a dramatic expansion of NKG2C+ NK cells leading to a low NKG2A/C+ NK cell ratio. In these patients changes in the expression of NKG2C were shown to be linked to a concomitant infection/reactivation with HCMV rather than to the HIV infection alone. Indeed, the fraction of NK cells expressing NKG2C in HCMV seronegative donors was low or undetectable, regardless of their HIV status [18,103,104] (Fig. 2).

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Fig. 2. Virus-induced shaping of human NK cell phenotype and function. (Left) HCMV infection promotes differentiation of functional NK cells, characterized by a mature phenotype (CD56dim CD16bright NKG2A− KIR+ NKG2C+ ). In HCMV-infected HSCT recipients, Siglec-7 is sharply down-regulated on this mature NK cell subset. Similarly CD56dim Siglec-7− NK cells are also observed already in early stages of HIV infection. In the absence of NKG2C (i.e. in NK cells derived from NKG2C−/− subjects), the expression of activating KIRs by HCMV-induced NK cells could play a role in driving their differentiation and in killing infected targets (e.g. fibroblasts). (Right) In both chronically HIV-infected subjects and HCMV-infected HSCT recipients, aberrant NK cells lacking both CD56 and Siglec-7 surface expression can develop. These NK cells are characterized by a mature phenotype, but display impaired effector function.

3.3. NK cell responses in HCMV infection HCMV affects most humans (50–100% depending on geographical location and socioeconomic conditions) [105] and can persist lifelong after primary infection [106]. In immunocompetent individuals, HCMV infection is usually asymptomatic, but it may become cause of life threatening complications in primary or acquired immunodeficiencies and in immunosuppressed patients such as transplanted recipients. During the HCMV-host interplay, NK cells and T cells, which are primarily involved in controlling HCMV infection [107], undergo a persistent reconfiguration of their receptor repertoire. In particular, it has been reported that HCMV is capable of inducing an expansion of NK cells expressing the activating receptor CD94/NKG2C. Remarkably, this expanded NKG2C+ NK cell subset is characterized by a mature phenotype, mostly NKG2A− and KIR+ [103,108]. In most cases HCMV-induced NKG2C+ NK cells express self-reactive KIRs, i.e. KIR specific for self HLA class I molecules [109]. Increased proportions of KIR+ NKG2A− NKG2C+ NK cells have also been described in subjects affected by HIV [18,104], Chikungunya virus [110], Hantavirus [111] or HBV/HCV [112] and EBV [113] infections. However, it is conceivable that induction of such NK cell phenotype in these patients may actually be related to HCMV co-infection and reactivation. It is also possible that HCMV infection may prime NK cells inducing the differentiation of mature KIR+ NKG2C+ NK cells that could undergo expansion in response to a subsequent virus infection, such as Hantavirus [111], EBV [113] or HBV [112]. The preferential proliferation of NKG2C+ NK cells in response to HCMV-infected fibroblasts demonstrated that this expansion is HCMV-specific [103]. It is possible that NKG2C+ NK cells recognize HLA-E molecules upregulated in certain HCMV-infected targets including fibroblasts. Remarkably, the signal peptide of the HCMV UL40 protein stabilizes HLA-E expression on HCMV-infected fibroblasts, while other HCMV-derived peptides (including US2, US3, US6, US10 and US11) dampen the surface expression of classical HLA class I molecules [114]. Thus, it is conceivable that, upon the interaction with HCMV-infected cells, the expansion of mature NK cells expressing inhibitory KIRs specific for self HLA-class I could be favored by the lack of inhibitory interactions with clas-

sical HLA class I molecules. The stabilized surface expression of HLA-E molecules in HCMV-infected cells, while favoring the expansion of NKG2C+ NK cells, would also inhibit cells expressing the HLA-E-specific NKG2A receptor. However, we cannot exclude that surface molecules, other than HLA-E, expressed by HCMV-infected targets may be responsible for the expansion of NKG2C+ NK cells (Fig. 2). In this context the HCMV derived glycoprotein UL-18, the viral ligand for the inhibitory receptor LIR-1/ILT-2, has been shown to directly bind to the CD94/NKG2C heterodimer as well [115]. This molecular interaction could play a role in triggering LIR-1− NKG2C+ NK cells [116]. Therefore, NKG2C+ NK cells may contribute to the control of HCMV-infected cells upon recognition of HLA-E [117] or of still undefined ligands expressed by infected targets [103]. In contrast with this interpretation, HCMV infected DCs down-regulate both HLA-I and HLA-E, become susceptible to killing by KIR+ NK cells and are poorly susceptible to NKG2C+ NK cells [118,119]. 3.4. NK cell maturation is skewed by HCMV infection toward highly differentiated stages in HSCT recipients: emergence of “memory-like” NK cells? The imprinting on the NK cell phenotype induced by HCMV infection is more evident when T-cell immunity is impaired in the infected host, as in HIV-infected patients [18,104], congenitally immunodeficient individuals [120,121] and patients undergoing HSCT. In this context, recent studies [122,123] have shown that, in umbilical cord blood transplant (UCBT) recipients HCMV infection/reactivation can promote a rapid development of highly differentiated NK cells characterized by the NKG2A− KIR+ NKG2C+ CD57+ CD16+ Siglec-7− signature. These NK cells were highly cytolytic and produced cytokines. However, in some HCMV-reactivating patients a unusual and hypofunctional CD56negCD16+ Siglec-7− NK cell subset could be detected [122]. This subset was reminiscent of that previously described in viremic HIV-infected patients undergoing HCMV reactivation [15]. Importantly, the acquisition of CD57 (a marker of terminal NK cell differentiation) [124,125] by some NKG2C+ KIR+ NK cells [122,123] further confirmed the primary role of HCMV infection

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in driving NK cell maturation and acquisition of effector function. High proportions of NKG2C+ CD57+ NK cells have also been detected in healthy HCMV seropositive individuals [108,126] and in recipients of solid organ transplantation (SOT) [127]. Notably, this NK cell subset displays a poor natural cytotoxicity, however, these cells can efficiently kill HCMV-infected targets, in the presence of anti-HCMV antibodies because they maintain the ability to respond to signaling through CD16 cross-linking [126]. The high proportions of HCMV-induced, NKG2C+ KIR+ NK cells persisted over one year after HSCT [122,123]. This expanded NKG2C+ NK cell subset is reminiscent of a population of Ly49H+ NK cells found in MCMV-infected mice that is responsible for the recovery from the disease through the induction of a memory response [128]. Importantly, in mice, the Ly49H receptor has been shown to bind the m157 protein, expressed by MCMV-infected cells [128]. On the other hand, in humans, as discussed above, the nature of the putative viral ligands recognized by NKG2C has yet to be identified. However, it has been shown that NKG2C+ NK cells, transplanted from a seropositive donor, undergo expansion both in recipients experiencing HCMV reactivation and in seropositive recipients with detectable viremia. Moreover, such expanded, NKG2C+ NK cells had an increased capacity of producing IFN-␥, as compared to those isolated from seronegative recipients [129]. This suggests that “primed” NKG2C+ NK cells exposed to the same viral antigens in seropositive recipients may exert a more efficient anti-viral activity by producing higher amounts of cytokines. Although this is not a classic recall response against HCMV-infected targets, it represents an example of “memory-like” response, possibly contributing to the control of HCMV reactivation in HCMV+ recipients. Of note, recent studies indicated that NK cells exposed to multiple cytokines can acquire a “memory-like” phenotype and release higher amounts of IFN-␥ following restimulation with cytokines or NK-susceptible target cells [130,131]. Despite these observations, NKG2C cannot be considered a univocal marker of “memory-like” NK cells. Indeed, although NKG2C expression appears to be a hallmark of HCMV-induced NK cell expansions [103], recent reports suggest that also other NK receptors might be involved in driving HCMV-induced NK cell differentiation and contribute to shape the NK cell receptor repertoire. In this context, recent data have shown that HCMV infection can drive NK cell maturation even in patients receiving UCBT from donors carrying a homozygous deletion of the NKG2C gene. In these patients, HCMV infection induced a rapid expansion of mature NK cells (NKG2A− NKG2C− KIR+ ) expressing activating KIRs that could efficiently trigger NK cells. In the absence of NKG2C, it is conceivable that activating KIRs may play a role in the HCMV-driven NK cell maturation [138], possibly contributing to the control of infections. In this context, in a cohort of HCMV seropositive healthy individuals not carrying the NKG2C deletion, the expansion of NKG2A− NKG2C− NK cells expressing activating KIRs [109], has been recently described. Along this line, a number of other studies suggested that the presence of activating KIRs correlates with protection against viral infections. A reduced risk of HCMV reactivation has been reported in SOT recipients expressing two or more activating KIRs [132,133], as well as in patients given HSCT from donors expressing two or more activating KIRs [134]. However, the nature of the putative viral ligands recognized by the activating KIRs has yet to be identified. Thus, both the mechanisms promoting HCMV-driven maturation and the anti-HCMV activity mediated by this subset remain to be clarified. Moreover, it cannot be excluded that HCMV infection may determine a continuous replenishment of new mature NK cells rather than the expansion of long-living NK cells [17]. Remarkably, a sharp down-regulation of the surface receptor Siglec-7 has been detected in NK cells maturing in UCBT recipients

reactivating HCMV, also in cases lacking NKG2C [122,138]. These findings suggest that the loss of Siglec-7 may be a typical feature of HCMV infection (Fig. 2). 3.5. HCMV infection could favor an anti-leukemic effect in HSCT recipients Recent studies suggested a correlation between early HCMV reactivation and reduction of leukemia relapses after allogenic HSCT in adult patients [135,136]. Accordingly, HCMV infection/reactivation would be beneficial rather than detrimental in HSCT recipients. The HCMV-induced rapid maturation of functional NK cells could favor an NK cell-mediated anti-leukemic activity in the case of a KIR-mismatched haploidentical HSCT. In this transplantation setting, the rapid emergence of large proportions of KIR+ NKG2A− NK cells would coincide with the appearance of alloreactive NK cells, capable of efficiently killing leukemic blasts [137]. However, the positive effect of HCMV infection [135] may not necessarily be related to NK cells but rather depend on a direct cytotoxic virus-mediated lysis of HCMV-infected leukemic blasts. In addition, HCMV infection may induce the expression of ligands recognized by activating NK receptors (e.g. NCR) and down-regulate HLA-class I molecules on leukemic blasts [135] that become highly susceptible to NK cell-mediated lysis even in case of a KIR-matched HSCT. Indeed, in this case, mature and functional KIR+ NK cells expanded upon HCMV infection could efficiently kill leukemic blasts lacking HLA-I. In addition, NKG2C+ NK cells could contribute to kill leukemic blasts expressing HLA-E or still unknown NKG2C ligands. As discussed above, although this is an intriguing possibility, the actual role of NKG2C+ NK cells in protecting from leukemia relapses has still to be proven. 4. Conclusions During the past two decades, the central role played by NK cells in immune responses against different pathogens has been progressively elucidated. In early studies, NK cells were shown to provide innate defenses against viral infections. More recently, it became clear that NK cells are also capable of recognizing and providing efficient responses against other microorganisms different from viruses (e.g. BCG, fungi). In particular, recognition of pathogens through TLRs has been shown to represent a crucial event in NK-mediated anti-microbial responses. The ability of NK cells to respond directly or indirectly to different pathogens offered a clue to understand the importance of multi-directional interactions among innate cells, showing that innate responses could not only limit the spreading of pathogens, but also influence downstream adaptive immune responses. A remarkable finding was the recent demonstration that NK cell development and function can be deeply influenced by certain viral infections, including primarily HIV and HCMV infections. The imprinting induced by HCMV infection on the NK cell receptor repertoire, as revealed by the HSCT setting, has also suggested that NK cells may keep memory of past infections, thus sharing features with adaptive immune cells. Whether virus-induced skewing of NK cell differentiation and generation of “memorylike” NK cells could be beneficial in pathologic conditions requires further investigation. In addition, both HIV and HCMV infection may induce the emergence of aberrant/hypofunctional NK cell subsets, thus compromising the efficacy of NK-mediated anti-viral responses. In conclusion, harnessing the ability of NK cells to sense and respond to pathogens and their developmental plasticity in the course of viral infections still represents an important field of investigation to design new NK-cell based therapies.

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Conflict-of-interest disclosure Moretta A. is a founder and shareholder of Innate-Pharma (Marseille, France). The remaining authors declare no competing financial interests. Acknowledgements Supported by grants awarded by Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) - Special Project 5 × 1000 n. 9962 (A.M.); PRIN 2010 (A.M.); Progetto di Ricerca Fondazione Carige 2013 (E.M.); Progetto Ricerca Ateneo 2012 (M.D.C.). References [1] Marcenaro E, Carlomagno S, Pesce S, Moretta A, Sivori S. Bridging innate NK cell functions with adaptive immunity. Advances in Experimental Medicine and Biology 2011;780:45–55. [2] Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nature Immunology 2008;9:503–10. [3] Moretta A. Natural killer cells and dendritic cells: rendezvous in abused tissues. Nature Reviews Immunology 2002;2:957–64. [4] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nature Reviews Immunology 2001;1:41–9. [5] Bar E, Whitney PG, Moor K, Reis ESC, Leibundgut-Landmann S. IL-17 regulates systemic fungal immunity by controlling the functional competence of NK cells. Immunity 2014;40:117–27. [6] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nature Immunology 2004;5:987–95. [7] Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007;449:819–26. [8] Lanier LL. NK cell receptors. Annual Review of Immunology 1998;16:359–93. [9] Long EO. Regulation of immune responses through inhibitory receptors. Annual Review of Immunology 1999;17:875–904. [10] Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, et al. Receptors for HLA class-I molecules in human natural killer cells. Annual Review of Immunology 1996;14:619–48. [11] Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annual Review of Immunology 2001;19:197–223. [12] Long EO, Kim HS, Liu D, Peterson ME, Rajagopalan S. Controlling natural killer cell responses: integration of signals for activation and inhibition. Annual Review of Immunology 2013;31:227–58. [13] Della Chiesa M, Sivori S, Castriconi R, Marcenaro E, Moretta A. Pathogeninduced private conversations between natural killer and dendritic cells. Trends in Microbiology 2005;13:128–36. [14] Fadda L, Borhis G, Ahmed P, Cheent K, Pageon SV, Cazaly A, et al. Peptide antagonism as a mechanism for NK cell activation. Proceedings of the National Academy of Sciences of the United States of America 2010;107:10160–5. [15] Brunetta E, Hudspeth KL, Mavilio D. Pathologic natural killer cell subset redistribution in HIV-1 infection: new insights in pathophysiology and clinical outcomes. Journal of Leukocyte Biology 2010;88:1119–30. [16] Alter G, Teigen N, Davis BT, Addo MM, Suscovich TJ, Waring MT, et al. Sequential deregulation of NK cell subset distribution and function starting in acute HIV-1 infection. Blood 2005;106:3366–9. [17] Della Chiesa M, Falco M, Muccio L, Bertaina A, Locatelli F, Moretta A. Impact of HCMV infection on NK cell development and function after HSCT. Frontiers in Immunology 2013;4:458. [18] Brunetta E, Fogli M, Varchetta S, Bozzo L, Hudspeth KL, Marcenaro E, et al. Chronic HIV-1 viremia reverses NKG2A/NKG2C ratio on natural killer cells in patients with human cytomegalovirus co-infection. AIDS 2010;24:27–34. [19] Carrington M, Alter G. Innate immune control of HIV. Cold Spring Harbor Perspectives in Medicine 2012;2. [20] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. [21] Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Immunology 2010;11:373–84. [22] Sabroe I, Jones EC, Usher LR, Whyte MKB, Dower SK. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. Journal of Immunology 2002;168:4701–10. [23] Marone G, Genovese A, Granata F, Forte V, Detoraki A, de Paulis A, et al. Pharmacological modulation of human mast cells and basophils. Clinical and Experimental Allergy 2002;32:1682–9. [24] Bjerke T, Gaustadnes M, Nielsen S, Nielsen LP, Schiotz PO, Rudiger N, et al. Human blood eosinophils produce and secrete interleukin 4. Respiratory Medicine 1996;90:271–7. [25] Jarrossay D, Napolitani G, Colonna M, Sallusto F, Lanzavecchia A. Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. European Journal of Immunology 2001;31:3388–93.

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Please cite this article in press as: Della Chiesa M, et al. Human NK cell response to pathogens. Semin Immunol (2014), http://dx.doi.org/10.1016/j.smim.2014.02.001