Y

R

H

MSD

a

KNTAHHB

1

hcd[

ccPaup

If

h1

ARTICLE IN PRESSG ModelSMIM-991; No. of Pages 9

Seminars in Immunology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Immunology

j ourna l ho me page: www.elsev ier .com/ locate /ysmim

eview

uman NK cell response to pathogens

ariella Della Chiesa1, Emanuela Marcenaro1, Simona Sivori1, Simona Carlomagno,ilvia Pesce, Alessandro Moretta ∗

I.ME.S. Dipartimento di Medicina Sperimentale and Centro di Eccellenza per la Ricerca Biomedica, Università di Genova, Genova, Italy

r t i c l e i n f o

eywords:atural killer cellsoll-like receptorsnti-viral responseIVCMVCG

a b s t r a c t

NK cells represent important effectors of the innate immunity in the protection of an individual frommicrobes. During an NK-mediated anti-microbial response, the final fate (survival or death) of a poten-tial infected target cell depends primarily on the type and the number of receptor/ligand interactionsoccurring at the effector/target immune synapse. The identification of an array of receptors involved inNK cell triggering has been crucial for a better understanding of the NK cell biology. In this context, NCRplay a predominant role in NK cell activation during the process of natural cytotoxicity. Regarding theNK-mediated pathogen recognition and NK cell activation, an emerging concept is represented by theinvolvement of TLRs and activating KIRs.

NK cells express certain TLRs in common with other innate cell types. This would mean that specific TLRligands are able to promote the simultaneous and synergistic stimulation of these innate cells, providinga 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 NKcell maturation, promoting the expansion of phenotypically and functionally aberrant NK cell subpopu-lations. 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 antivi-ral activity. An analogous imbalance of NK cell subsets has been detected in patients receiving HSCT tocure high risk leukemias and experiencing HCMV infection/reactivation. Remarkably, NK cells develop-ing after CMV reactivation may contain “memory-like” or “long-lived” NK cells that could exert a potentanti-leukemia effect.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Innate and adaptive immune responses cooperate to protect theost against microbial infections [1–3]. NK cells are innate immune

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

ells whose function is critical in the first-line of defense againstifferent types of pathogens, including viruses, bacteria and fungi4,5]. Pathogen recognition by NK cell is mediated by different types

Abbreviations: HIV, human immunodeficiency virus; HCMV, humanytomegalovirus; MCMV, murine cytomegalovirus; HSCT, hematopoietic stemell transplantation; TLRs, Toll-like receptors; PRR, pattern recognition receptor;AMPs, pathogen-associated molecular pattern; poly I:C, polyinosinic–polycytidyliccid; NCR, natural cytotoxicity receptor; BCG, bacillus Calmette-Guerin; UCBT,mbilical cord blood transplantation; SOT, solid organ transplantation; PB,eripheral blood.∗ Corresponding author at: Dipartimento di Medicina Sperimentale, Sezione di

stologia, Via G.B. Marsano 10, 16132 Genova, Italy; Tel.: +39 010 3537868;ax: +39 010 3537576.

E-mail address: alemoret@unige.it (A. Moretta).1 These authors equally contributed to this work.

ttp://dx.doi.org/10.1016/j.smim.2014.02.001044-5323/© 2014 Elsevier Ltd. All rights reserved.

of receptors, including Toll-like receptors (TLRs), that display broadspecificities for conserved and invariant features of microorgan-isms, and a series of activating receptors, able to recognize specificligands on infected/transformed cells [6,7]. NK cells also expressinhibitory receptors that may counteract the function of activat-ing receptors. The expression or the lack of ligands specific forinhibitory or activating NK receptors by target cells is thought todetermine the final outcome, i.e. to determine whether a giventarget cell will be killed or spared by NK cells [8–12].

NK cells can also respond to signals/cytokines derived fromaccessory cells including IL-12 which is crucial for IFN-� productionin response to various pathogen-associated TLR agonists [13,14].

Remarkably, several recent studies reported that during infec-tion with some viruses, a progressive, impairment of NK cellfunction may occur as a consequence of a progressive perturbation

an NK cell response to pathogens. Semin Immunol (2014),

of NK cell compartment [15–17].In this review, we will discuss some of the molecular mech-

anisms by which NK cells recognize pathogens or virus-infectedcells, mediate appropriate innate immune effector responses

ING ModelY

2 s in Im

aihtsi

2

2

(ttTtsraacGlbDrb

i[[eaT((ss

rmtwtroeccfi[aican

pPtart

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

nd favor the development of efficacious downstream adaptivemmune responses to viral antigens. In this context, the impact ofuman CMV (HCMV) infection on NK cell development and func-ion will be analyzed in more detail. In addition, we will describeome aberrant redistribution of dysfunctional NK cell subsets, dur-ng HIV or HCMV infection/reactivation [17–19].

. NK and TLRs

.1. TLR-mediated recognition of pathogens by NK cells

TLRs are germ-line encoded pattern-recognition receptorsPRRs) that are essential for the recognition of invading pathogens,riggering of innate responses and shaping of subsequent adap-ive immune responses [20,21]. Currently, at least 11 mammalianLRs have been identified, but only nine have been well charac-erized. Each TLR has a broad specificity for conserved moleculartructures that are unique to microorganisms. These targets areeferred to as pathogen associated molecular patterns (PAMPs),lthough they may actually be components of both pathogenicnd non-pathogenic microorganisms. PAMPs include: lipopolysac-haride (LPS), an essential structure of the outer membrane ofram-negative bacteria, which is recognized by TLR4; bacterial

ipoproteins and lipoteichoic acids, recognized by TLR2; flagellin, aacterial protein recognized by TLR5; microbial unmethylated CpGNA motifs, recognized by TLR9; double-stranded RNA (dsRNA),

ecognized by TLR3; and single-stranded RNA (ssRNA), recognizedy TLR7/TLR8 [20].

TLRs are expressed on various cells of the innate immunity,ncluding NK cells [1], monocytes/macrophages [22], neutrophils22], basophils [23], eosinophils [24], myeloid dendritic cells (DCs)25], plasmacytoid DCs (pDCs) [25] and mast cells [6,26]. TLRxpression is dependent on the cell type and in most instances,

given cell type expresses a small number of TLR. Moreover,LRs differ in their signal transduction pathways. Certain TLRsTLR1,2,4,5,6) are expressed on the cell surface, whereas othersTLR3,7,8,9) are localized in intracellular compartments (i.e. endo-omes). Therefore, their ligands require internalization to generateignals [21].

Upon recognition of PAMPs, TLRs initiate a signaling cascadeesulting in activation of tissue-resident innate cells (such as DCs,acrophages and mast cells) and in chemokine secretion. In turn,

hese factors recruit circulating leukocytes to the site of infectionhich further limit the spread of invading pathogens [1,21]. Among

he recruited cells, an important role is played by NK cells, that, onceeached the inflammatory sites, require activation in order to carryut their function [27]. NK cell activation occurs through differ-nt mechanisms, including engagement of different triggering NKell receptors or recognition of appropriate ligands on transformedells, and/or stimulation via TLRs [1]. NK cells express differentunctional TLRs, independent of their state of activation, includ-ng TLR2 [28,29], TLR3 [30,31], TLR5 [32], TLR7/8 [33,34] and TLR930]. Thus, responses to TLR ligands occur in both fresh and IL-2-ctivated NK cells and, in most instances, are sharply increasedn the presence of inflammatory cytokines, primarily IL-12. TLRsan activate NK cell function either directly or in cooperation withccessory cells in a cytokine or cell-to-cell contact-dependent man-er [13] (Fig. 1).

Because certain TLRs are shared by different innate cells thatarticipate to the early phases of an immune response, certainAMPs are able to promote the simultaneous and synergis-

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

ic stimulation of these cells. For example, since both NK cellsnd DCs express TLR3, the engagement of such receptor mayepresent a crucial step for promoting the cross-talk betweenhese cells in peripheral tissues. In particular, in the presence of

PRESSmunology xxx (2014) xxx–xxx

IL-12 (released by DCs upon TLR3 engagement), NK cells respondvia TLR3 to dsRNA by increasing their anti-tumor/anti-viral cyto-toxicity and acquire also the ability to kill immature DCs (iDCs).This activity favors the selective survival of mature DCs (NK cell-mediated ‘editing’ of DCs) [30]. Moreover, upon TLR stimulation, NKcells greatly increase their capability of secreting pro-inflammatorycytokines (such as TNF-� and IFN-�), which promote further DCmaturation and subsequent induction of Th1 responses in lymphnodes [35–37].

Remarkably, NK cell populations, derived from different donorsmay respond to TLR3 stimulation with different efficiency. Suchheterogeneous response to poly I:C, a classic TLR3 ligand, has beenexplained with the observation that the percentage of NK cellsexpressing high levels of TLR3 mRNA transcripts may vary in dif-ferent donors. Moreover, the analysis of NK cell clones revealedthat heterogeneity exists not only among different donors, but alsoamong NK cell clones derived from the same individual [38]. Ithas also been shown that NK cell clones expressing low levels ofNatural Cytotoxicity Receptors [39–41](NCR dull phenotype) thatare poorly cytotoxic, can up-regulate their cytolitic activity uponTLR3 engagement provided that they express high levels of TLR3mRNA transcript. This capability of responding to TLR3 ligands maybe critical in the context of some pathologic conditions, includingHIV infection [42] or acute myeloid leukemia (AML) [43], in whichdown-regulation of NCR expression may represent a mechanismby which virus-infected or tumor cells escape NK-mediated lysis.

Notably, NK cells also express functional TLR9, which are presentin pDCs, but not in conventional DCs. Thus, stimuli acting on TLR9are 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 TLR9-responsive NK cells [45]. Moreover, the exposure of NK cells to IL-12(produced by DCs) amplifies the response and further potentiatesNK-mediated induction of IFN-� release by pDCs [46].

Also TLR2 is expressed by NK cells and other innate cells (includ-ing monocytes/macrophages and DCs). This TLR binds to productsof bacterial origin. In particular, a direct involvement of TLR2 in therecognition of Mycobacterium tuberculosis by NK cells has beenclearly demonstrated [29,47] (see Section 2.2).

Flagellin the TLR5 ligand, can directly act on NK cells by inducingthe secretion of IFN-� and �-defensins that contribute, respec-tively, to activate accessory cells (e.g., macrophages) and to exertan anti-microbial effect. In turn, accessory cells, activated byPAMPs and/or cytokines present in the inflammatory microen-vironment, can release other cytokines (e.g., IL-12 and IL-2),which may modulate PAMP-mediated activation of NK cell effectorfunctions [32].

Finally, Hart et al. demonstrated that human NK cells may alsoexpress functional TLR7 and TLR8 [33]. In this regard, Alter et al.showed that NK cells may be significantly activated by the TLR7/8ligand uridine-rich ssRNA derived from HIV. The functional activa-tion of NK cells is strictly dependent on the direct contact of NKcells with pDCs or CD14+ monocytes resulting in increased IFN-�secretion [34].

All these data indicate that, although in some cases NK cellscan be directly activated by some TLR agonists, both the cross-talkwith other innate cells (in particular DCs) and the cytokine milieumay play a crucial role in the activation of their effector function.Thus, NK cells, in addition to a direct TLR-engagement, requireappropriate costimulatory signals, to elicit an optimal response,capable of modulating downstream adaptive immune responses[6,48] (Fig. 1).

an NK cell response to pathogens. Semin Immunol (2014),

2.2. Direct recognition of BCG by human NK cells

A number of experimental evidences indicate that NK cellsare the main responsible of the anti-tumor responses induced

ARTICLE IN PRESSG ModelYSMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminars in Immunology xxx (2014) xxx–xxx 3

Fig. 1. TLR-mediated activation of human NK cells. NK cells express different functional TLRs: TLR2 recognizes bacterial lipoproteins and lipoteichoic acids is present, forexample, 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 NKcells 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 twocell 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. Stimulia , shars rectly

D ultip

btieNrofpiafac[

Chm[rr[

aitdTi

cting on TLR7 or TLR9 are able to simultaneously activate both NK cells and pDCsupport the activation of TLR-responsive NK cells. Thus, although NK cells can be diCs and pDCs) and the cytokine milieu play a crucial role in the activation of their m

y Mycobacterium bovis (bacille Calmette-Guérin, BCG) in thereatment of superficial bladder carcinoma [49–52], as well asn innate immune protection from tubercolosis [53,54]. How-ver, the molecular mechanisms of interaction between humanK cells and mycobacteria have yet to be fully clarified. Some

ecent studies would indicate that BCG, through the engagementf TLR2, can directly induce the acquisition of potent effectorunctions by NK cells [29,47,55,56]. These include the ability tootentiate the cytolytic activity against both tumor cells and

DCs, and to release proinflammatory cytokines (including IFN-�nd TNF-�), that, in turn, mediate the maturation of DCs, thusavoring the induction of adaptive Th1 immune responses. Remark-bly, different from other TLR-mediated signaling, the release ofytokines by NK cells does not strictly require the presence of IL-1229,56].

Notably, in response to BCG stimulation, both CD56bright andD56dim NK cell subsets release IFN-� [29]. In this context,owever, some authors suggest that the CD56bright subset is pri-arily involved in the release of IFN-�, in response to BCG

56]. Accordingly, a recent report would support the crucialole of IFN-� produced by CD56bright NK cells in mycobacte-ial infection and in BCG immunotherapy of bladder cancer57].

Recently, Esin et al. have shown that different componentsbundant in mycobacterium tuberculosis cell wall may directlynteract with the NKp44 NCR [58] and TLR2 [47]. However, only

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

he interaction via TLR2 promotes cell activation and IFN-� pro-uction by resting NK cells, whereas NKp44, expressed uponLR2-mediated NK cell activation, may promote later signal result-ng in prolonged NK cell activation [47].

ing these TLRs. In particular, IFN-�, released by pDCs upon TLR engagement, mayactivated by some TLR agonists, the cross-talk with other innate cells (in particularle effector functions as explained in the figure box.

All these findings suggest that a direct interaction with extracel-lular mycobacteria may represent an efficient stimulus to induceNK 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 toCpG-ODNs, i.e. the ligands of TLR9 receptor. In particular, it has beendemonstrated that KIR3DL2 works as a sensor for microbial prod-ucts and as a chaperon for TLR9 ligands [60]. KIR3DL2, expressed atthe NK cell surface, can bind CpG-ODNs and shuttle them to earlyendosomes where TLR9 translocates upon CpG-ODNs cell stimula-tion. Following CpG-ODNs binding, TLR9 leads to NK cell activationand cytokine secretion (Fig. 1).

Also other members of the KIR family (KIR3DL1, KIR3DS1,KIR2DL4) are able to bind CpG-ODNs, however, functional assaysconfirmed that IFN-� production in response to CpG-ODNs, ismostly confined to NK cells expressing KIR3DL2+. This suggeststhat the other CpG-ODN-binding KIRs do not mediate efficient CpG-ODNs shuttling to TLR9-rich intracellular compartments [61].

KIR3DL2 is known as an HLA-specific receptor that binds HLA-A*03 and A*11 allotypes. However, it is characterized by lowinhibitory capacity and its interaction with HLA ligands is highlydependent on the peptide(s) bound to HLA-A molecules [62].

an NK cell response to pathogens. Semin Immunol (2014),

Together with KIR2DL4 and KIR3DL3, KIR3DL2 is a frameworkgene which is present in all KIR haplotypes [63]. The capabilityof KIR3DL2 described above may provide novel important clueto understand the driving force leading to the conservation of

ING ModelY

4 s in Im

tfor

2

bcNtTr

toobptt

f[of

abdmTorsSgso

3

3

cvsi[

irHvdk

Nli

np

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

he KIR3DL2-encoding gene in all haplotypes, in spite of the lowrequency of HLA-A3 or -A11 alleles in humans [1]. Thus, the needf a prompt NK-mediated reaction to microbial products may rep-esent an important factor of selective genetic pressure [60].

.4. Correlation between TLRs and anti-HIV/HCMV responses

HCMV and HIV can activate the innate immune system throughoth TLR-dependent and -independent pathways [19,64]. The pre-ise role of TLR in early recognition and control of HIV and HCMV byK cells or other immune cells, has not been fully elucidated, even

hough different innate immune receptors, including TLR2, TLR3,LR7, TLR8 and TLR9, have been reported to play a role in innateesponses against these viruses.

In particular, HIV, similar to other ssRNA viruses, was describedo trigger TLR7/TLR8 expressed on pDC/DCs and induce the releasef proinflammatory cytokines capable of promoting the activationf other innate cells, including NK cells [65–67]. In addition, it haseen possible to demonstrate a direct binding of gp120 to TLR9 onDCs. This interaction could suppress pDC activation, preventinghe TLR9-mediated production of proinflammatory cytokines andhe pDC-induced stimulation of NK cytotoxicity [68].

The HIV-mediated DCs triggering results in production of dif-erent cytokines at different time intervals after acute infection69]. Remarkably, the early cytokine storm preceding the peakf viremia includes cytokines capable of increasing the NK cellunction.

It has been reported that gB and gH glycoproteins of HCMV,n enveloped dsDNA virus, can stimulate TLR2-expressing fibro-lasts [70]. Along this line, a recent study shows that NK cells canirectly recognize HCMV virions through TLR2. Upon TLR2 engage-ent, NK cells become activated and produce IFN-� [64]. In mice,

LR2 and TLR9 have been shown to participate in the recognitionf viral particles (including envelope glycoproteins and viral DNA,espectively) [71–74], while TLR3 and TLR7 could be involved inensing CMV-derived products during the infection cycle [72,74].imilarly, TLR9 is likely to be involved in sensing HCMV as sug-ested by epidemiological and genetic studies showing that specificingle nucleotide polymorphism in TLR9 gene is highly predictivef susceptibility to HCMV infection [75].

. NK cells in HIV and HCMV infections

.1. NK cell responses in HIV infection

HIV has developed multiple strategies to evade detection by NKells, strongly suggesting that these cells exert a pressure on theirus [4,19,76,77]. However, also the opposite may be true, sinceeveral functional defects of NK cell have been observed in HIV-nfected patients, particularly in active and advanced disease stages15].

A number of studies indicate that HIV uses the Nef gene to evadennate and adaptive immune responses. Indeed, Nef may down-egulate the expression of the dominant T-cell receptor ligandsLA-A and -B molecules at the surface of infected cells thus pre-enting their recognition by T cells. On the other hand, HLA-C, theominant ligand of inhibitory KIR2D is not affected preventing theirilling by NK cells [78,79].

In addition, Nef may prevent both the expression of someKG2D ligands (including MIC-A, ULBP-1, and -2) and the DNAM-1

igand PVR on the surface of infected cells, thus impairing, at least

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

n part, the NK-mediated cytotoxicity [80].It has also been suggested that particular KIR/HLA combi-

ations may impact the outcome of HIV-infection [81–83]. Inarticular, individuals expressing the activating KIR3DS1 allele, in

PRESSmunology xxx (2014) xxx–xxx

conjunction with its putative HLA class I ligand, were character-ized by a slower disease progression as compared to controls [81].In addition, KIR3DS1+ NK cells have been reported to exert theiranti-viral activity preferentially against HIV-infected target cellsexpressing its putative ligand [84]. Moreover, different data indi-cated that given amino acid changes in the peptide associated withHLA-class I molecules could abrogate binding of inhibitory KIRs totheir HLA class I ligands, thus allowing target cell lysis [14,85–89].On the other hand, certain viral peptides could favor binding of anactivating KIR to its HLA ligand, thus increasing NK-cell cytotoxicity[76].

3.2. Presence of dysfunctional CD56neg NK cell subset in HIVinfection

Several studies support the notion that NK cells, in most infectedpatients, are unable to control the progression of HIV infection.This is associated with the expansion of dysfunctional NK cell sub-populations that are virtually absent in uninfected/aviremic donors[19,42].

Peripheral blood (PB) NK cells include two main subsets basedon the expression of the CD56 molecule: the minor NK cell sub-population characterized by a CD56bright phenotype and the majorsubset expressing a CD56dim phenotype [90]. CD56dim cells arestrongly cytotoxic and can produce high levels of proinflammatoryand antiviral cytokines following stimulation via activating NKreceptors [27,91–93]. Both chronic and active phases of HIV infec-tion 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 per-

turbations, including down-regulation of the major activating NKreceptors (e.g. NCRs), associated with relevant functional abnor-malities [42,97]. These cells appear to exert a limited abilityto control opportunistic infections and tumors occuring at latestages of HIV infection. They also display a reduced secretion ofcytokines such as IFN-�, TNF-�, and GM-CSF. The expression ofinhibitory NK receptors (including KIRs) was either normal orincreased in NK cells of viremic patients. As a consequence, thesereceptors could mediate an even greater inhibition of cytolyticfunction.

Several studies reported in the course of HIV infection, abnor-malities in the interaction between NK cells and DCs, occurringin peripheral tissue at inflammation sites [3,48,98–100]. Thisled to an impaired activation of NK cell and to a deficientkilling of iDCs by NK cells. These defects prevent a correctDC-mediated priming of autologous naive CD4+ T cells thuscompromising downstream antigen-specific, adaptive immuneresponses [101].

In line with the observation that only high levels of viral repli-cation during chronic HIV infection correlate with the appearanceof functionally impaired CD56neg NK cells, suppression of viralload by antiretroviral therapy resulted in progressive restorationof CD56 expression in PB NK.

Recent studies have shown that a decreased expression of thecellular marker Siglec-7 occurred at the initial stages of HIV infec-tion preceding down-regulation of CD56 [102].

In addition to abnormalities in the levels of expression of NKcell receptors in chronic viremic HIV-infected individuals, anotherfinding was the decrease of NKG2A+ NK cells and a dramatic expan-sion of NKG2C+ NK cells leading to a low NKG2A/C+ NK cell ratio.In these patients changes in the expression of NKG2C were shown

an NK cell response to pathogens. Semin Immunol (2014),

to be linked to a concomitant infection/reactivation with HCMVrather than to the HIV infection alone. Indeed, the fraction of NKcells expressing NKG2C in HCMV seronegative donors was low orundetectable, regardless of their HIV status [18,103,104] (Fig. 2).

ARTICLE IN PRESSG ModelYSMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminars in Immunology xxx (2014) xxx–xxx 5

Fig. 2. Virus-induced shaping of human NK cell phenotype and function. (Left) HCMV infection promotes differentiation of functional NK cells, characterized by a maturephenotype (CD56dimCD16brightNKG2A−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 expressionof 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 chronicallyH both

b

3

ilvbasNHrivNNNIN[[ibtti[

tHHiUbUctNc

IV-infected subjects and HCMV-infected HSCT recipients, aberrant NK cells lackingy a mature phenotype, but display impaired effector function.

.3. NK cell responses in HCMV infection

HCMV affects most humans (50–100% depending on geograph-cal location and socioeconomic conditions) [105] and can persistifelong after primary infection [106]. In immunocompetent indi-iduals, HCMV infection is usually asymptomatic, but it mayecome cause of life threatening complications in primary orcquired immunodeficiencies and in immunosuppressed patientsuch as transplanted recipients. During the HCMV-host interplay,K cells and T cells, which are primarily involved in controllingCMV infection [107], undergo a persistent reconfiguration of their

eceptor repertoire. In particular, it has been reported that HCMVs capable of inducing an expansion of NK cells expressing the acti-ating receptor CD94/NKG2C. Remarkably, this expanded NKG2C+

K cell subset is characterized by a mature phenotype, mostlyKG2A− and KIR+[103,108]. In most cases HCMV-induced NKG2C+

K cells express self-reactive KIRs, i.e. KIR specific for self HLA class molecules [109]. Increased proportions of KIR+ NKG2A− NKG2C+

K cells have also been described in subjects affected by HIV18,104], Chikungunya virus [110], Hantavirus [111] or HBV/HCV112] and EBV [113] infections. However, it is conceivable thatnduction of such NK cell phenotype in these patients may actuallye related to HCMV co-infection and reactivation. It is also possiblehat HCMV infection may prime NK cells inducing the differentia-ion of mature KIR+NKG2C+ NK cells that could undergo expansionn response to a subsequent virus infection, such as Hantavirus111], EBV [113] or HBV [112].

The preferential proliferation of NKG2C+ NK cells in responseo HCMV-infected fibroblasts demonstrated that this expansion isCMV-specific [103]. It is possible that NKG2C+ NK cells recognizeLA-E molecules upregulated in certain HCMV-infected targets

ncluding fibroblasts. Remarkably, the signal peptide of the HCMVL40 protein stabilizes HLA-E expression on HCMV-infected fibro-lasts, while other HCMV-derived peptides (including US2, US3,S6, US10 and US11) dampen the surface expression of classi-

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

al HLA class I molecules [114]. Thus, it is conceivable that, uponhe interaction with HCMV-infected cells, the expansion of matureK cells expressing inhibitory KIRs specific for self HLA-class Iould be favored by the lack of inhibitory interactions with clas-

CD56 and Siglec-7 surface expression can develop. These NK cells are characterized

sical HLA class I molecules. The stabilized surface expression ofHLA-E molecules in HCMV-infected cells, while favoring the expan-sion of NKG2C+ NK cells, would also inhibit cells expressing theHLA-E-specific NKG2A receptor. However, we cannot exclude thatsurface molecules, other than HLA-E, expressed by HCMV-infectedtargets 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 beenshown to directly bind to the CD94/NKG2C heterodimer as well[115]. This molecular interaction could play a role in triggeringLIR-1− NKG2C+ NK cells [116]. Therefore, NKG2C+ NK cells maycontribute to the control of HCMV-infected cells upon recognitionof HLA-E [117] or of still undefined ligands expressed by infectedtargets [103]. In contrast with this interpretation, HCMV infectedDCs down-regulate both HLA-I and HLA-E, become susceptible tokilling by KIR+ NK cells and are poorly susceptible to NKG2C+ NKcells [118,119].

3.4. NK cell maturation is skewed by HCMV infection towardhighly differentiated stages in HSCT recipients: emergence of“memory-like” NK cells?

The imprinting on the NK cell phenotype induced by HCMVinfection is more evident when T-cell immunity is impairedin the infected host, as in HIV-infected patients [18,104], con-genitally immunodeficient individuals [120,121] and patientsundergoing 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 devel-opment of highly differentiated NK cells characterized by theNKG2A−KIR+NKG2C+CD57+CD16+Siglec-7− signature. These NKcells were highly cytolytic and produced cytokines. However,in some HCMV-reactivating patients a unusual and hypofunc-tional CD56negCD16+Siglec-7− NK cell subset could be detected[122]. This subset was reminiscent of that previously described

an NK cell response to pathogens. Semin Immunol (2014),

in viremic HIV-infected patients undergoing HCMV reactivation[15]. Importantly, the acquisition of CD57 (a marker of terminalNK cell differentiation) [124,125] by some NKG2C+KIR+ NK cells[122,123] further confirmed the primary role of HCMV infection

ING ModelY

6 s in Im

itditett[

pNNrrsctyNsseIetmcHrtcpuDms[bctciH(ctmtiNbipvadnKHttoc

S

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

n driving NK cell maturation and acquisition of effector func-ion. High proportions of NKG2C+CD57+ NK cells have also beenetected in healthy HCMV seropositive individuals [108,126] and

n recipients of solid organ transplantation (SOT) [127]. Notably,his NK cell subset displays a poor natural cytotoxicity, how-ver, these cells can efficiently kill HCMV-infected targets, inhe presence of anti-HCMV antibodies because they maintainhe ability to respond to signaling through CD16 cross-linking126].

The high proportions of HCMV-induced, NKG2C+KIR+ NK cellsersisted over one year after HSCT [122,123]. This expandedKG2C+ NK cell subset is reminiscent of a population of Ly49H+

K cells found in MCMV-infected mice that is responsible for theecovery from the disease through the induction of a memoryesponse [128]. Importantly, in mice, the Ly49H receptor has beenhown to bind the m157 protein, expressed by MCMV-infectedells [128]. On the other hand, in humans, as discussed above,he nature of the putative viral ligands recognized by NKG2C haset to be identified. However, it has been shown that NKG2C+

K cells, transplanted from a seropositive donor, undergo expan-ion both in recipients experiencing HCMV reactivation and ineropositive recipients with detectable viremia. Moreover, suchxpanded, NKG2C+ NK cells had an increased capacity of producingFN-�, as compared to those isolated from seronegative recipi-nts [129]. This suggests that “primed” NKG2C+ NK cells exposedo the same viral antigens in seropositive recipients may exert a

ore efficient anti-viral activity by producing higher amounts ofytokines. Although this is not a classic recall response againstCMV-infected targets, it represents an example of “memory-like”

esponse, possibly contributing to the control of HCMV reactiva-ion in HCMV+ recipients. Of note, recent studies indicated that NKells exposed to multiple cytokines can acquire a “memory-like”henotype and release higher amounts of IFN-� following restim-lation with cytokines or NK-susceptible target cells [130,131].espite these observations, NKG2C cannot be considered a univocalarker of “memory-like” NK cells. Indeed, although NKG2C expres-

ion appears to be a hallmark of HCMV-induced NK cell expansions103], recent reports suggest that also other NK receptors mighte involved in driving HCMV-induced NK cell differentiation andontribute to shape the NK cell receptor repertoire. In this con-ext, recent data have shown that HCMV infection can drive NKell maturation even in patients receiving UCBT from donors carry-ng a homozygous deletion of the NKG2C gene. In these patients,CMV infection induced a rapid expansion of mature NK cells

NKG2A−NKG2C−KIR+) expressing activating KIRs that could effi-iently trigger NK cells. In the absence of NKG2C, it is conceivablehat activating KIRs may play a role in the HCMV-driven NK cell

aturation [138], possibly contributing to the control of infec-ions. In this context, in a cohort of HCMV seropositive healthyndividuals not carrying the NKG2C deletion, the expansion ofKG2A−NKG2C− NK cells expressing activating KIRs [109], haseen recently described. Along this line, a number of other stud-

es suggested that the presence of activating KIRs correlates withrotection against viral infections. A reduced risk of HCMV reacti-ation has been reported in SOT recipients expressing two or morectivating KIRs [132,133], as well as in patients given HSCT fromonors expressing two or more activating KIRs [134]. However, theature of the putative viral ligands recognized by the activatingIRs has yet to be identified. Thus, both the mechanisms promotingCMV-driven maturation and the anti-HCMV activity mediated by

his subset remain to be clarified. Moreover, it cannot be excludedhat HCMV infection may determine a continuous replenishment

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

f new mature NK cells rather than the expansion of long-living NKells [17].

Remarkably, a sharp down-regulation of the surface receptoriglec-7 has been detected in NK cells maturing in UCBT recipients

PRESSmunology xxx (2014) xxx–xxx

reactivating HCMV, also in cases lacking NKG2C [122,138]. Thesefindings suggest that the loss of Siglec-7 may be a typical feature ofHCMV infection (Fig. 2).

3.5. HCMV infection could favor an anti-leukemic effect in HSCTrecipients

Recent studies suggested a correlation between early HCMVreactivation and reduction of leukemia relapses after allogenicHSCT in adult patients [135,136]. Accordingly, HCMV infec-tion/reactivation would be beneficial rather than detrimental inHSCT recipients. The HCMV-induced rapid maturation of functionalNK cells could favor an NK cell-mediated anti-leukemic activ-ity in the case of a KIR-mismatched haploidentical HSCT. In thistransplantation setting, the rapid emergence of large proportionsof KIR+NKG2A− NK cells would coincide with the appearance ofalloreactive NK cells, capable of efficiently killing leukemic blasts[137]. However, the positive effect of HCMV infection [135] maynot necessarily be related to NK cells but rather depend on adirect cytotoxic virus-mediated lysis of HCMV-infected leukemicblasts. In addition, HCMV infection may induce the expressionof ligands recognized by activating NK receptors (e.g. NCR) anddown-regulate HLA-class I molecules on leukemic blasts [135] thatbecome highly susceptible to NK cell-mediated lysis even in caseof a KIR-matched HSCT. Indeed, in this case, mature and functionalKIR+ NK cells expanded upon HCMV infection could efficiently killleukemic blasts lacking HLA-I. In addition, NKG2C+ NK cells couldcontribute to kill leukemic blasts expressing HLA-E or still unknownNKG2C ligands. As discussed above, although this is an intriguingpossibility, the actual role of NKG2C+ NK cells in protecting fromleukemia relapses has still to be proven.

4. Conclusions

During the past two decades, the central role played by NKcells in immune responses against different pathogens has beenprogressively elucidated. In early studies, NK cells were shownto provide innate defenses against viral infections. More recently,it became clear that NK cells are also capable of recognizingand providing efficient responses against other microorganismsdifferent from viruses (e.g. BCG, fungi). In particular, recogni-tion of pathogens through TLRs has been shown to representa crucial event in NK-mediated anti-microbial responses. Theability of NK cells to respond directly or indirectly to differ-ent pathogens offered a clue to understand the importance ofmulti-directional interactions among innate cells, showing thatinnate responses could not only limit the spreading of pathogens,but also influence downstream adaptive immune responses. Aremarkable finding was the recent demonstration that NK celldevelopment and function can be deeply influenced by certainviral infections, including primarily HIV and HCMV infections. Theimprinting induced by HCMV infection on the NK cell receptorrepertoire, as revealed by the HSCT setting, has also suggestedthat NK cells may keep memory of past infections, thus shar-ing features with adaptive immune cells. Whether virus-inducedskewing of NK cell differentiation and generation of “memory-like” NK cells could be beneficial in pathologic conditions requiresfurther investigation. In addition, both HIV and HCMV infectionmay induce the emergence of aberrant/hypofunctional NK cell sub-sets, thus compromising the efficacy of NK-mediated anti-viralresponses.

an NK cell response to pathogens. Semin Immunol (2014),

In conclusion, harnessing the ability of NK cells to sense andrespond to pathogens and their developmental plasticity in thecourse of viral infections still represents an important field of inves-tigation to design new NK-cell based therapies.

ING ModelY

s in Im

C

(fi

A

p(2

R

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

onflict-of-interest disclosure

Moretta A. is a founder and shareholder of Innate-PharmaMarseille, France). The remaining authors declare no competingnancial interests.

cknowledgements

Supported by grants awarded by Associazione Italiana Ricercaer la Ricerca sul Cancro (AIRC) - Special Project 5 × 1000 n. 9962A.M.); PRIN 2010 (A.M.); Progetto di Ricerca Fondazione Carige013 (E.M.); Progetto Ricerca Ateneo 2012 (M.D.C.).

eferences

[1] Marcenaro E, Carlomagno S, Pesce S, Moretta A, Sivori S. Bridging innate NKcell functions with adaptive immunity. Advances in Experimental Medicineand Biology 2011;780:45–55.

[2] Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of naturalkiller cells. Nature Immunology 2008;9:503–10.

[3] Moretta A. Natural killer cells and dendritic cells: rendezvous in abused tis-sues. Nature Reviews Immunology 2002;2:957–64.

[4] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nature ReviewsImmunology 2001;1:41–9.

[5] Bar E, Whitney PG, Moor K, Reis ESC, Leibundgut-Landmann S. IL-17 regulatessystemic fungal immunity by controlling the functional competence of NKcells. Immunity 2014;40:117–27.

[6] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immuneresponses. Nature Immunology 2004;5:987–95.

[7] Medzhitov R. Recognition of microorganisms and activation of the immuneresponse. 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. Recep-

tors for HLA class-I molecules in human natural killer cells. Annual Review ofImmunology 1996;14:619–48.

[11] Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, et al. Activat-ing receptors and coreceptors involved in human natural killer cell-mediatedcytolysis. Annual Review of Immunology 2001;19:197–223.

[12] Long EO, Kim HS, Liu D, Peterson ME, Rajagopalan S. Controlling natural killercell responses: integration of signals for activation and inhibition. AnnualReview of Immunology 2013;31:227–58.

[13] Della Chiesa M, Sivori S, Castriconi R, Marcenaro E, Moretta A. Pathogen-induced 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. Peptideantagonism as a mechanism for NK cell activation. Proceedings of the NationalAcademy of Sciences of the United States of America 2010;107:10160–5.

[15] Brunetta E, Hudspeth KL, Mavilio D. Pathologic natural killer cell subset redis-tribution in HIV-1 infection: new insights in pathophysiology and clinicaloutcomes. Journal of Leukocyte Biology 2010;88:1119–30.

[16] Alter G, Teigen N, Davis BT, Addo MM, Suscovich TJ, Waring MT, et al. Sequen-tial deregulation of NK cell subset distribution and function starting in acuteHIV-1 infection. Blood 2005;106:3366–9.

[17] Della Chiesa M, Falco M, Muccio L, Bertaina A, Locatelli F, Moretta A. Impactof HCMV infection on NK cell development and function after HSCT. Frontiersin 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 inpatients with human cytomegalovirus co-infection. AIDS 2010;24:27–34.

[19] Carrington M, Alter G. Innate immune control of HIV. Cold Spring HarborPerspectives 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 immu-nity: 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)2and TLR4 in human peripheral blood granulocytes: a critical role for mono-cytes in leukocyte lipopolysaccharide responses. Journal of Immunology2002;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 andExperimental 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

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

Medicine 1996;90:271–7.[25] Jarrossay D, Napolitani G, Colonna M, Sallusto F, Lanzavecchia A. Special-

ization and complementarity in microbial molecule recognition by humanmyeloid and plasmacytoid dendritic cells. European Journal of Immunology2001;31:3388–93.

PRESSmunology xxx (2014) xxx–xxx 7

[26] Supajatura V, Ushio H, Nakao A, Akira S, Okumura K, Ra C, et al. Differen-tial responses of mast cell Toll-like receptors 2 and 4 in allergy and innateimmunity. Journal of Clinical Investigation 2002;109:1351–9.

[27] Parolini S, Santoro A, Marcenaro E, Luini W, Massardi L, Facchetti F, et al. Therole of chemerin in the colocalization of NK and dendritic cell subsets intoinflamed tissues. Blood 2007;109:3625–32.

[28] Becker I, Salaiza N, Aguirre M, Delgado J, Carrillo-Carrasco N, KobehLG, et al. Leishmania lipophosphoglycan (LPG) activates NK cells throughToll-like receptor-2. Molecular and Biochemical Parasitology 2003;130:65–74.

[29] Marcenaro E, Ferranti B, Falco M, Moretta L, Moretta A. Human NK cellsdirectly recognize Mycobacterium bovis via TLR2 and acquire the ability tokill monocyte-derived DC. International Immunology 2008;20:1155–67.

[30] Sivori S, Falco M, Della Chiesa M, Carlomagno S, Vitale M, Moretta L, et al.CpG and double-stranded RNA trigger human NK cells by Toll-like receptors:induction of cytokine release and cytotoxicity against tumors and dendriticcells. Proceedings of the National Academy of Sciences of the United States ofAmerica 2004;101:10116–21.

[31] Schmidt KN, Leung B, Kwong M, Zarember KA, Satyal S, Navas TA, et al. APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. Journal of Immunology 2004;172:138–43.

[32] Chalifour A, Jeannin P, Gauchat JF, Blaecke A, Malissard M, N’Guyen T, et al.Direct bacterial protein PAMP recognition by human NK cells involves TLRsand triggers alpha-defensin production. Blood 2004;104:1778–83.

[33] Hart OM, Athie-Morales V, O’Connor GM, Gardiner CM. TLR7/8-mediated acti-vation of human NK cells results in accessory cell-dependent IFN-gammaproduction. Journal of Immunology 2005;175:1636–42.

[34] Alter G, Suscovich TJ, Teigen N, Meier A, Streeck H, Brander C, et al. Single-stranded RNA derived from HIV-1 serves as a potent activator of NK cells.Journal of Immunology 2007;178:7658–66.

[35] Agaugue S, Marcenaro E, Ferranti B, Moretta L, Moretta A. Human natural killercells exposed to IL-2, IL-12, IL-18, or IL-4 differently modulate priming of naiveT cells by monocyte-derived dendritic cells. Blood 2008;112:1776–83.

[36] Morandi B, Bougras G, Muller WA, Ferlazzo G, Munz C. NK cells of human sec-ondary lymphoid tissues enhance T cell polarization via IFN-gamma secretion.European Journal of Immunology 2006;36:2394–400.

[37] Marcenaro E, Della Chiesa M, Bellora F, Parolini S, Millo R, Moretta L, et al. IL-12or IL-4 prime human NK cells to mediate functionally divergent interactionswith dendritic cells or tumors. Journal of Immunology 2005;174:3992–8.

[38] Sivori S, Falco M, Carlomagno S, Romeo E, Moretta L, Moretta A. Heterogeneityof TLR3 mRNA transcripts and responsiveness to poly(I:C) in human NK cellsderived from different donors. International Immunology 2007;19:1341–8.

[39] Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, et al.p46, a novel natural killer cell-specific surface molecule that mediates cellactivation. Journal of Experimental Medicine 1997;186:1129–36.

[40] Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E,et al. NKp44, a novel triggering surface molecule specifically expressedby activated natural killer cells, is involved in non-major histocompatibil-ity complex-restricted tumor cell lysis. Journal of Experimental Medicine1998;187:2065–72.

[41] Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, et al. Identifi-cation and molecular characterization of NKp30, a novel triggering receptorinvolved in natural cytotoxicity mediated by human natural killer cells. Jour-nal of Experimental Medicine 1999;190:1505–16.

[42] Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA,et al. Natural killer cells in HIV-1 infection: dichotomous effects of viremiaon inhibitory and activating receptors and their functional correlates.Proceedings of the National Academy of Sciences of the United States ofAmerica 2003;100:15011–6.

[43] Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M, MozziconacciMJ, Reviron D, et al. Defective expression and function of natural killercell-triggering receptors in patients with acute myeloid leukemia. Blood2002;99:3661–7.

[44] Gerosa F, Gobbi A, Zorzi P, Burg S, Briere F, Carra G, et al. The reciprocalinteraction of NK cells with plasmacytoid or myeloid dendritic cells pro-foundly affects innate resistance functions. Journal of Immunology 2005;174:727–34.

[45] Sivori S, Carlomagno S, Moretta L, Moretta A. Comparison of different CpGoligodeoxynucleotide classes for their capability to stimulate human NK cells.European Journal of Immunology 2006;36:961–7.

[46] Della Chiesa M, Romagnani C, Thiel A, Moretta L, Moretta A. Multidirec-tional interactions are bridging human NK cells with plasmacytoid andmonocyte-derived dendritic cells during innate immune responses. Blood2006;108:3851–8.

[47] Esin S, Counoupas C, Aulicino A, Brancatisano FL, Maisetta G, Bottai D, et al.Interaction of Mycobacterium tuberculosis cell wall components with thehuman natural killer cell receptors NKp44 and Toll-like receptor 2. Scandina-vian Journal of Immunology 2013;77:460–9.

[48] Marcenaro E, Carlomagno S, Pesce S, Moretta A, Sivori S. NK/DC crosstalkin anti-viral response. Advances in Experimental Medicine and Biology2012;946:295–308.

an NK cell response to pathogens. Semin Immunol (2014),

[49] Alexandroff AB, Jackson AM, O’Donnell MA, James K. BCG immunotherapy ofbladder cancer: 20 years on. Lancet 1999;353:1689–94.

[50] Brandau S, Riemensberger J, Jacobsen M, Kemp D, Zhao WC, Zhao XQ, et al.NK cells are essential for effective BCG immunotherapy. International Journalof Cancer 2001;92:697–702.

ING ModelY

8 s in Im

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

[51] Brandau S, Bohle A. Activation of natural killer cells by bacillus Calmette-Guerin. European Urology 2001;39:518–24.

[52] Suttmann H, Jacobsen M, Reiss K, Jocham D, Bohle A, Brandau S. Mechanismsof bacillus Calmette-Guerin mediated natural killer cell activation. Journal ofUrology 2004;172:1490–5.

[53] Vankayalapati R, Garg A, Porgador A, Griffith DE, Klucar P, Safi H, et al. Roleof NK cell-activating receptors and their ligands in the lysis of mononuclearphagocytes infected with an intracellular bacterium. Journal of Immunology2005;175:4611–7.

[54] Guerra C, Johal K, Morris D, Moreno S, Alvarado O, Gray D, et al. Control ofMycobacterium tuberculosis growth by activated natural killer cells. Clinicaland Experimental Immunology 2012;168:142–52.

[55] Esin S, Batoni G, Pardini M, Favilli F, Bottai D, Maisetta G, et al. Functionalcharacterization of human natural killer cells responding to Mycobacteriumbovis bacille Calmette-Guerin. Immunology 2004;112:143–52.

[56] Batoni G, Esin S, Favilli F, Pardini M, Bottai D, Maisetta G, et al. HumanCD56bright and CD56dim natural killer cell subsets respond differentiallyto direct stimulation with Mycobacterium bovis bacillus Calmette-Guerin.Scandinavian Journal of Immunology 2005;62:498–506.

[57] Portevin D, Young D. Natural killer cell cytokine response to M. bovis BCGis associated with inhibited proliferation, increased apoptosis and ultimatedepletion of NKp44(+)CD56(bright) cells. PLoS ONE 2013;8:e68864.

[58] Esin S, Batoni G, Counoupas C, Stringaro A, Brancatisano FL, Colone M, et al.Direct binding of human NK cell natural cytotoxicity receptor NKp44 tothe surfaces of mycobacteria and other bacteria. Infection and Immunity2008;76:1719–27.

[59] Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, di Donato C, et al. Thenatural killer cell receptor specific for HLA-A allotypes: a novel member ofthe p58/p70 family of inhibitory receptors that is characterized by threeimmunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. Journal of Experimental Medicine 1996;184:505–18.

[60] Sivori S, Falco M, Carlomagno S, Romeo E, Soldani C, Bensussan A, et al.A novel KIR-associated function: evidence that CpG DNA uptake andshuttling to early endosomes is mediated by KIR3DL2. Blood 2010;116:1637–47.

[61] Sivori S, Falco M, Moretta L, Moretta A. Extending killer Ig-like receptor func-tion: from HLA class I recognition to sensors of microbial products. Trends inImmunology 2010;31:289–94.

[62] Hansasuta P, Dong T, Thananchai H, Weekes M, Willberg C, Aldemir H, et al.Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific. EuropeanJournal of Immunology 2004;34:1673–9.

[63] Wilson MJ, Torkar M, Haude A, Milne S, Jones T, Sheer D, et al. Plas-ticity in the organization and sequences of human KIR/ILT gene families.Proceedings of the National Academy of Sciences of the United States ofAmerica 2000;97:4778–83.

[64] Muntasell A, Costa-Garcia M, Vera A, Marina-Garcia N, Kirschning CJ,Lopez-Botet M. Priming of NK cell anti-viral effector mechanisms by directrecognition of human cytomegalovirus. Frontiers in Immunology 2013;4:40.

[65] Diebold SS, Kaisho T, Hemmi H, Akira S, Sousa CRE. Innate antiviral responsesby means of TLR7-mediated recognition of single-stranded RNA. Science2004;303:1529–31.

[66] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7and 8. Science 2004;303:1526–9.

[67] Beignon AS, McKenna K, Skoberne M, Manches O, DaSilva I, KavanaghDG, et al. Endocytosis of HIV-1 activates plasmacytoid dendritic cells viatoll-like receptor-viral RNA interactions. Journal of Clinical Investigation2005;115:3265–75.

[68] Martinelli E, Cicala C, Van Ryk D, Goode DJ, Macleod K, Arthos J, et al. HIV-1gp120 inhibits TLR9-mediated activation and IFN-alpha secretion in plasma-cytoid dendritic cells. Proceedings of the National Academy of Sciences of theUnited States of America 2007;104:3396–401.

[69] Stacey AR, Norris PJ, Qin L, Haygreen EA, Taylor E, Heitman J, et al. Induc-tion of a striking systemic cytokine cascade prior to peak viremia in acutehuman immunodeficiency virus type 1 infection, in contrast to more modestand delayed responses in acute hepatitis B and C virus infections. Journal ofVirology 2009;83:3719–33.

[70] Boehme KW, Guerrero M, Compton T. Human cytomegalovirus envelope gly-coproteins B and H are necessary for TLR2 activation in permissive cells.Journal of Immunology 2006;177:7094–102.

[71] Krug A, French AR, Barchet W, Fischer JA, Dzionek A, Pingel JT, et al.TLR9-dependent recognition of MCMV by IPC and DC generates coordi-nated cytokine responses that activate antiviral NK cell function. Immunity2004;21:107–19.

[72] Tabeta K, Georgel P, Janssen E, Du X, Hoebe K, Crozat K, et al. Toll-like receptors9 and 3 as essential components of innate immune defense against mousecytomegalovirus infection. Proceedings of the National Academy of Sciencesof the United States of America 2004;101:3516–21.

[73] Szomolanyi-Tsuda E, Liang X, Welsh RM, Kurt-Jones EA, Finberg RW. Role forTLR2 in NK cell-mediated control of murine cytomegalovirus in vivo. Journalof Virology 2006;80:4286–91.

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

[74] Zucchini N, Bessou G, Traub S, Robbins SH, Uematsu S, Akira S, et al. Cuttingedge: overlapping functions of TLR7 and TLR9 for innate defense against aherpesvirus infection. Journal of Immunology 2008;180:5799–803.

[75] Carvalho A, Cunha C, Carotti A, Aloisi T, Guarrera O, Di Ianni M,et al. Polymorphisms in Toll-like receptor genes and susceptibility to

PRESSmunology xxx (2014) xxx–xxx

infections in allogeneic stem cell transplantation. Experimental Hematology2009;37:1022–9.

[76] Alter G, Altfeld M. NK cells in HIV-1 infection: evidence for their role in thecontrol of HIV-1 infection. Journal of Internal Medicine 2009;265:29–42.

[77] Marcenaro E, Carlomagno S, Pesce S, Della Chiesa M, Parolini S, Moretta A,et al. NK cells and their receptors during viral infections. Immunotherapy2011;3:1075–86.

[78] Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL,et al. The selective downregulation of class I major histocompatibility com-plex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity1999;10:661–71.

[79] Le Gall S, Erdtmann L, Benichou S, Berlioz-Torrent C, Liu L, Benarous R, et al.Nef interacts with the mu subunit of clathrin adaptor complexes and revealsa cryptic sorting signal in MHC I molecules. Immunity 1998;8:483–95.

[80] Zingoni A, Ardolino M, Santoni A, Cerboni C. NKG2D and DNAM-1 activatingreceptors and their ligands in NK-T cell interactions: role in the NK cell-mediated negative regulation of T cell responses. Frontiers in Immunology2012;3:408.

[81] Martin MP, Gao X, Lee JH, Nelson GW, Detels R, Goedert JJ, et al. Epistatic inter-action between KIR3DS1 and HLA-B delays the progression to AIDS. NatureGenetics 2002;31:429–34.

[82] Martin MP, Qi Y, Gao X, Yamada E, Martin JN, Pereyra F, et al. Innate part-nership of HLA-B and KIR3DL1 subtypes against HIV-1. Nature Genetics2007;39:733–40.

[83] Jennes W, Verheyden S, Demanet C, Adje-Toure CA, Vuylsteke B, NkengasongJN, et al. Cutting edge: resistance to HIV-1 infection among African female sexworkers is associated with inhibitory KIR in the absence of their HLA ligands.Journal of Immunology 2006;177:6588–92.

[84] Alter G, Martin MP, Teigen N, Carr WH, Suscovich TJ, Schneidewind A,et al. Differential natural killer cell-mediated inhibition of HIV-1 replica-tion based on distinct KIR/HLA subtypes. Journal of Experimental Medicine2007;204:3027–36.

[85] Correa I, Raulet DH. Binding of diverse peptides to MHC class I moleculesinhibits target cell lysis by activated natural killer cells. Immunity1995;2:61–71.

[86] Malnati MS, Peruzzi M, Parker KC, Biddison WE, Ciccone E, Moretta A, et al.Peptide specificity in the recognition of MHC class I by natural killer cellclones. Science 1995;267:1016–8.

[87] Peruzzi M, Parker KC, Long EO, Malnati MS. Peptide sequence requirementsfor the recognition of HLA-B*2705 by specific natural killer cells. Journal ofImmunology 1996;157:3350–6.

[88] Rajagopalan S, Long EO. The direct binding of a p58 killer cell inhibitoryreceptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibitspeptide selectivity. Journal of Experimental Medicine 1997;185:1523–8.

[89] Zappacosta F, Borrego F, Brooks AG, Parker KC, Coligan JE. Peptides isolatedfrom HLA-Cw*0304 confer different degrees of protection from natural killercell-mediated lysis. Proceedings of the National Academy of Sciences of theUnited States of America 1997;94:6313–8.

[90] Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cellsubsets. Trends in Immunology 2001;22:633–40.

[91] Fauriat C, Long EO, Ljunggren HG, Bryceson YT. Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood2010;115:2167–76.

[92] Marcenaro E, Pesce S, Sivori S, Carlomagno S, Moretta L, Moretta A.KIR2DS1-dependent acquisition of CCR7 and migratory properties by humanNK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts. Blood2013;121:3396–401.

[93] De Maria A, Bozzano F, Cantoni C, Moretta L. Revisiting human natural killercell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid pro-ducers of abundant IFN-gamma on activation. Proceedings of the NationalAcademy of Sciences of the United States of America 2011;108:728–32.

[94] Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, et al.Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunc-tional NK subset expanded in HIV-infected viremic individuals. Proceedingsof the National Academy of Sciences of the United States of America2005;102:2886–91.

[95] Gonzalez VD, Falconer K, Michaelsson J, Moll M, Reichard O, Alaeus A, et al.Expansion of CD56− NK cells in chronic HCV/HIV-1 co-infection: reversion byantiviral treatment with pegylated IFNalpha and ribavirin. Clinical Immunol-ogy 2008;128:46–56.

[96] Wilk E, Kalippke K, Buyny S, Schmidt RE, Jacobs R. New aspects of NK cell sub-set identification and inference of NK cells’ regulatory capacity by assessingfunctional and genomic profiles. Immunobiology 2008;213:271–83.

[97] De Maria A, Fogli M, Costa P, Murdaca G, Puppo F, Mavilio D, et al. The impairedNK cell cytolytic function in viremic HIV-1 infection is associated with areduced surface expression of natural cytotoxicity receptors (NKp46, NKp30and NKp44). European Journal of Immunology 2003;33:2410–8.

[98] Gerosa F, Baldani-Guerra B, Nisii C, Marchesini V, Carra G, Trinchieri G. Recip-rocal activating interaction between natural killer cells and dendritic cells.Journal of Experimental Medicine 2002;195:327–33.

[99] Mavilio D, Lombardo G, Kinter A, Fogli M, La Sala A, Ortolano S, et al. Char-

an NK cell response to pathogens. Semin Immunol (2014),

acterization of the defective interaction between a subset of natural killercells and dendritic cells in HIV-1 infection. Journal of Experimental Medicine2006;203:2339–50.

[100] Marcenaro E, Ferranti B, Moretta A. NK-DC interaction: on the usefulness ofauto-aggression. Autoimmunity Reviews 2005;4:520–5.

ING ModelY

s in Im

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

ARTICLESMIM-991; No. of Pages 9

M. Della Chiesa et al. / Seminar

101] Fogli M, Mavilio D, Brunetta E, Varchetta S, Ata K, Roby G, et al. Lysisof endogenously infected CD4+ T cell blasts by rIL-2 activated autologousnatural killer cells from HIV-infected viremic individuals. PLoS Pathogens2008;4:e1000101.

102] Brunetta E, Fogli M, Varchetta S, Bozzo L, Hudspeth KL, Marcenaro E, et al. Thedecreased expression of Siglec-7 represents an early marker of dysfunctionalnatural killer-cell subsets associated with high levels of HIV-1 viremia. Blood2009;114:3822–30.

103] Guma M, Budt M, Saez A, Brckalo T, Hengel H, Angulo A, et al. Expansionof CD94/NKG2C+ NK cells in response to human cytomegalovirus-infectedfibroblasts. Blood 2006;107:3624–31.

104] Guma M, Cabrera C, Erkizia I, Bofill M, Clotet B, Ruiz L, et al. Humancytomegalovirus infection is associated with increased proportions of NK cellsthat express the CD94/NKG2C receptor in aviremic HIV-1-positive patients.Journal of Infectious Diseases 2006;194:38–41.

105] Dowd JB, Aiello AE, Alley DE. Socioeconomic disparities in the seroprevalenceof cytomegalovirus infection in the US population: NHANES III. Epidemiologyand Infection 2009;137:58–65.

106] Ho M. Epidemiology of cytomegalovirus infections. Reviews of Infectious Dis-eases 1990;12(Suppl. 7):S701–10.

107] La Rosa C, Diamond DJ. The immune response to human CMV. Future Virology2012;7:279–93.

108] Guma M, Angulo A, Vilches C, Gomez-Lozano N, Malats N, Lopez-Botet M.Imprint of human cytomegalovirus infection on the NK cell receptor reper-toire. Blood 2004;104:3664–71.

109] Beziat V, Liu LL, Malmberg JA, Ivarsson MA, Sohlberg E, Bjorklund AT, et al.NK cell responses to cytomegalovirus infection lead to stable imprints in thehuman KIR repertoire and involve activating KIRs. Blood 2013;121:2678–88.

110] Petitdemange C, Becquart P, Wauquier N, Beziat V, Debre P, Leroy EM,et al. Unconventional repertoire profile is imprinted during acute chikun-gunya infection for natural killer cells polarization toward cytotoxicity. PLoSPathogens 2011;7:e1002268.

111] Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M, et al.Rapid expansion and long-term persistence of elevated NK cell numbersin humans infected with hantavirus. Journal of Experimental Medicine2011;208:13–21.

112] Beziat V, Dalgard O, Asselah T, Halfon P, Bedossa P, Boudifa A, et al. CMV drivesclonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronichepatitis patients. European Journal of Immunology 2012;42:447–57.

113] Saghafian-Hedengren S, Sohlberg E, Theorell J, Carvalho-Queiroz C, NagyN, Persson JO, et al. Epstein-Barr virus coinfection in children boostscytomegalovirus-induced differentiation of natural killer cells. Journal ofVirology 2013;87:13446–55.

114] Tortorella D, Gewurz BE, Furman MH, Schust DJ, Ploegh HL. Viral subversionof the immune system. Annual Review of Immunology 2000;18:861–926.

115] Kaiser BK, Pizarro JC, Kerns J, Strong RK. Structural basis for NKG2A/CD94recognition of HLA-E. Proceedings of the National Academy of Sciences of theUnited States of America 2008;105:6696–701.

116] Prod’homme V, Griffin C, Aicheler RJ, Wang EC, McSharry BP, Rickards CR,et al. The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+but activates LIR-1- NK cells. Journal of Immunology 2007;178:4473–81.

117] Heatley SL, Pietra G, Lin J, Widjaja JM, Harpur CM, Lester S, et al. Polymorphismin human cytomegalovirus UL40 impacts on recognition of human leuko-cyte antigen-E (HLA-E) by natural killer cells. Journal of Biological Chemistry2013;288:8679–90.

118] Magri G, Muntasell A, Romo N, Saez-Borderias A, Pende D, Geraghty DE,et al. NKp46 and DNAM-1 NK-cell receptors drive the response to humancytomegalovirus-infected myeloid dendritic cells overcoming viral immuneevasion strategies. Blood 2011;117:848–56.

119] Djaoud Z, David G, Bressollette C, Willem C, Rettman P, Gagne K, et al.Amplified NKG2C+ NK cells in cytomegalovirus (CMV) infection pref-erentially express killer cell Ig-like receptor 2DL: functional impact in

Please cite this article in press as: Della Chiesa M, et al. Humhttp://dx.doi.org/10.1016/j.smim.2014.02.001

controlling CMV-infected dendritic cells. Journal of Immunology 2013;191:2708–16.

120] Kuijpers TW, Baars PA, Dantin C, van den Burg M, van Lier RA, Roosnek E.Human NK cells can control CMV infection in the absence of T cells. Blood2008;112:914–5.

PRESSmunology xxx (2014) xxx–xxx 9

[121] Farnault L, Chambost H, Michel G, Thuret I, de Saint Basile G, Fischer A, et al.Persistence of natural killer cells with expansion of a hypofunctional CD56-CD16+KIR+NKG2C+ subset in a patient with atypical Janus kinase 3-deficientsevere combined immunodeficiency. Journal of Allergy and Clinical Immunol-ogy 2013;131:1230–3, 1230–3, 1233 e1231–2.

[122] Della Chiesa M, Falco M, Podesta M, Locatelli F, Moretta L, Frassoni F, et al.Phenotypic and functional heterogeneity of human NK cells developing afterumbilical cord blood transplantation: a role for human cytomegalovirus?Blood 2012;119:399–410.

[123] Foley B, Cooley S, Verneris MR, Pitt M, Curtsinger J, Luo X, et al.Cytomegalovirus reactivation after allogeneic transplantation promotes alasting increase in educated NKG2C+ natural killer cells with potent function.Blood 2011;119:2665–74.

[124] Lopez-Verges S, Milush JM, Pandey S, York VA, Arakawa-Hoyt J, Pircher H,et al. CD57 defines a functionally distinct population of mature NK cells inthe human CD56dimCD16+ NK-cell subset. Blood 2010;116:3865–74.

[125] Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA,et al. Expression patterns of NKG2A, KIR, and CD57 define a process ofCD56dim NK-cell differentiation uncoupled from NK-cell education. Blood2010;116:3853–64.

[126] Wu Z, Sinzger C, Frascaroli G, Reichel J, Bayer C, Wang L, et al.Human cytomegalovirus-induced NKG2C(hi) CD57(hi) natural killer cellsare effectors dependent on humoral antiviral immunity. Journal of Virology2013;87:7717–25.

[127] Lopez-Verges S, Milush JM, Schwartz BS, Pando MJ, Jarjoura J, York VA, et al.Expansion of a unique CD57+NKG2Chi natural killer cell subset during acutehuman cytomegalovirus infection. Proceedings of the National Academy ofSciences of the United States of America 2011;108:14725–32.

[128] Sun JC, Lopez-Verges S, Kim CC, DeRisi JL, Lanier LL. NK cells and immune“memory”. Journal of Immunology 2011;186:1891–7.

[129] Foley B, Cooley S, Verneris MR, Curtsinger J, Luo X, Waller EK, et al. Humancytomegalovirus (CMV)-induced memory-like NKG2C(+) NK cells are trans-plantable and expand in vivo in response to recipient CMV antigen. Journalof Immunology 2012;189:5082–8.

[130] Romee R, Schneider SE, Leong JW, Chase JM, Keppel CR, Sullivan RP,et al. Cytokine activation induces human memory-like NK cells. Blood2012;120:4751–60.

[131] Cooper MA, Colonna M, Yokoyama WM. Hidden talents of natural killers: NKcells in innate and adaptive immunity. EMBO Reports 2009;10:1103–10.

[132] Stern M, Elsasser H, Honger G, Steiger J, Schaub S, Hess C. The num-ber of activating KIR genes inversely correlates with the rate of CMVinfection/reactivation in kidney transplant recipients. American Journal ofTransplantation 2008;8:1312–7.

[133] Stern M, Hadaya K, Honger G, Martin PY, Steiger J, Hess C, et al. Telomericrather than centromeric activating KIR genes protect from cytomegalovirusinfection after kidney transplantation. American Journal of Transplantation2011;11:1302–7.

[134] Cook M, Briggs D, Craddock C, Mahendra P, Milligan D, Fegan C, et al.,Donor KIR. genotype has a major influence on the rate of cytomegalovirusreactivation following T-cell replete stem cell transplantation. Blood2006;107:1230–2.

[135] Elmaagacli AH, Steckel NK, Koldehoff M, Hegerfeldt Y, Trenschel R,Ditschkowski M, et al. Early human cytomegalovirus replication after trans-plant is associated with a decreased relapse-risk: evidence for a putativevirus-versus-leukemia effect AML patients. Blood 2011;118:1402–12.

[136] Manjappa S, Bhamidipati PK, Stokerl-Goldstein KE, Dipersio JF, Uy GL, West-ervelt P, et al. Protective effect of cytomegalovirus reactivation on relapseafter allogeneic hematopoietic cell transplantation in acute myeloid leukemiapatients is influenced by conditioning regimen. Biology of Blood and MarrowTransplantation 2014;20:46–52.

[137] Ruggeri L, Aversa F, Martelli MF, Velardi A. Allogeneic hematopoietic trans-plantation and natural killer cell recognition of missing self. Immunological

an NK cell response to pathogens. Semin Immunol (2014),

Reviews 2006;214:202–18.[138] Della Chiesa M, Falco M, Bertaina A, Muccio L, Alicata C, Frassoni F, et al.

Human cytomegalovirus infection promotes rapid maturation of NK cellsexpressing activating killer Ig-like receptor in patients transplanted withNKG2C−/− umbilical cord blood. Journal of Immunology 2014;192:1471–9.