Activating natural cytotoxicity receptors of natural killer cells in cancer and infection

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Review

Activating natural cytotoxicity receptors of natural killer cells in cancer and infection Joachim Koch1, Alexander Steinle2, Carsten Watzl3, and Ofer Mandelboim4 1

Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Strasse 42–44, D-60596 Frankfurt am Main, Germany Institute for Molecular Medicine, Goethe-University Frankfurt am Main, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany 3 Leibniz Research Centre for Working Environment and Human Factors – IfADo, Ardeystrasse 67, D-44139 Dortmund, Germany 4 Lautenberg Center for General and Tumor Immunology, Hebrew University – Hadassah Medical School, P.B.O. 12272, Jerusalem 91120, Israel 2

Natural killer (NK) cells are central players in the vertebrate immune system that rapidly eliminate malignantly transformed or infected cells. The natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 are important mediators of NK cell cytotoxicity, which trigger an immune response on recognition of cognate cellular and viral ligands. Tumour and viral immune escape strategies targeting these receptor–ligand systems impair NK cell cytotoxicity and promote disease. Therefore, a molecular understanding of the function of the NCRs in immunosurveillance is instrumental to discovering novel access points to combat infections and cancer. NK cells in health and disease Human NK cells are important innate immune effector cells that provide rapid responses to tumour and infected cells [1]. Their importance is underscored by the fact that patients with NK cell deficiency suffer from severe recurrent systemic and life-threatening infections, in particular by herpes viruses such as human cytomegalovirus (HCMV) [2]. Strikingly, high activity of peripheral blood NK cells is associated with a 10% lower incidence of tumours for men and 4% for women [3], and their infiltration of certain tumour tissues is an indicator for better disease prognosis [4]. NK cells constitute 5–15% of the peripheral blood lymphocyte population and are also found in the liver, spleen, bone marrow, decidua, and lymph nodes in humans [5]. Unlike cytotoxic T lymphocytes (CTLs), NK cells kill without prior sensitisation via the polarised release of cytotoxic granules, which are loaded with perforin and granzymes [6,7]. Killing requires the formation of a complex immunological synapse between the target cell and the NK cell, which is highly organised in time and space [2,8,9]. NK cells are regulated by the integration of signals triggered by ligands binding to different activating and inhibitory germline-encoded surface receptors [1,4,7, 10–16]. The balance of activating and inhibitory receptor stimulation determines NK cell activation. The inhibitory Corresponding author: Koch, J. ([email protected]).

receptors on human NK cells comprise receptors that mostly recognise MHC class I molecules on the surface of target cells; however, some non-MHC class I ligands are also recognised [15]. Inhibitory receptors on human NK cells include the promiscuous immunoglobulin-like transcript (ILT)2 receptors, the killer immunoglobulin-like receptors (KIRs), which recognise different allelic groups of HLA-A, HLA-B, and mainly HLA-C molecules and the CD94–NKG2A receptor, which recognises HLA-E [17,18]. The activating receptors of human NK cells trigger cytolytic activity mainly against malignantly transformed cells and virus-infected cells, but also against some nonstressed self-cells such as dendritic cells (DCs) and pancreatic b-cells [4,12,15]. Upon activation, NK cells secrete distinct cytokines such as interferon (IFN)g and tumor necrosis factor (TNF)a [12]. NK cells express several activating receptors, including natural-killer group 2, member D (NKG2D), activating KIRs, NKp80, CD94/NKG2C, DNAX accessory molecule (DNAM)-1 and 2B4 [7]. NKG2D recognises several related ligands including MHC class I chain-related protein (MIC)A, MICB, and UL16-binding proteins (ULBPs) [19–22]. The interaction of NKG2D with these ligands and the signalling pathways downstream of NKG2D are well characterised [19–21] and are therefore not discussed here. NKp80, like NKG2D, is a homodimeric C-type-lectin-like receptor encoded in the natural killer gene complex (NKC). In contrast to NKG2D, NKp80 ligates an activation-induced C-type lectin (AICL) receptor, which is genetically linked to the NKp80 locus and preferentially expressed by myeloid cells, promoting a mutual crosstalk between NK and myeloid cells [23,24]. CD94/NKG2C is suggested to play a role in HCMV, HIV1, and hantavirus infections [25–27], whereas DNAM-1, recognising poliovirus receptor (PVR, CD155) and Nectin-2 (CD112) [28], is involved in HCMV infection [29] and the killing of tumour cells [30]. Another group of major activating receptors is represented by the immunoglobulin-like NCRs, NKp30 (also known as NCR3, NCTR3, and CD337), NKp44 (also known as NCR2, NCTR2, and CD336), and NKp46 (also known as NCR1, NCTR1, and CD335) (Figure 1) [12,17]. The

1471-4906/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2013.01.003 Trends in Immunology xx (2013) 1–10

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NKp30 + B7H6 (3PV6)

NKp46 (1P6F)

NKp44

NKp30

(1HKF)

(3NOI)

I I T T DAP12 A A M M

D D R I CD3ζ T A M

I T FcεRlγ A M

S S

S S

D D R I CD3ζ T A M I T A M I T A M

D D R I T A M

I T A P M

I T P A M I T P A M

Src

Syk ZAP-70

PL

I T A M I T A M

I T FcεRlγ A M

LAT / NTAL

K D D

S S

Vav -2/3

S S

Cγ

PI3K

Cytoskeletal reorganisaon

Ca2+ flux TRENDS in Immunology

Figure 1. Structural determinants for ligand binding of natural cytotoxicity receptors (NCRs). Schematic representation of the domain organisation of the human NCRs, NKp30, NKp44, and NKp46. All NCRs are type I membrane proteins comprised of an ectodomain with one (NKp30 and NKp44) or two (NKp46) immunoglobulin-like domains connected to a transmembrane-spanning a-helix via a short stalk domain. Notably, NKp44 contains a single immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, which however may not be functional (white rectangle at the cytosolic tail of NKp44). For signalling, the NCRs associate with adaptor proteins via an opposing charge contact within the corresponding transmembrane segments (contacting amino acids in single-letter code). The intracellular immunoreceptor tyrosine based activation motifs (ITAMs) of the adaptor molecules are shown as boxes. For NKp30, the X-ray crystal structures of the unbound immunoglobulin-like extracellular domain and of the complex with the ectodomain of its cellular ligand B7-H6 are shown. For NKp44 and NKp46, the X-ray crystal structures of the unbound immunoglobulin-like extracellular domains are shown. The PDB accession numbers are annotated. Sialic acid moieties within the stalk domain of NKp44 contribute directly to ligand binding (indicated by arrow). Moreover, a large groove within the immunoglobulin-like domain of NKp44 and the hinge region between the two immunoglobulinlike domains of NKp46 are proposed binding sites for yet unknown cellular ligand proteins (indicated by arrows). A schematic representation of NCR-induced signalling pathways is depicted as described in the text. NCR engagement triggers SRC-family kinase-mediated ITAM phosphorylation, resulting in the recruitment and activation of the kinases ZAP70 and SYK. These kinases phosphorylate transmembrane adaptors such as LAT and NTAL (non-T cell activation linker), resulting in the recruitment and activation of phospholipase C (PLC)g-1/2, VAV-2/3 and phosphoinositide 3-kinase (PI3K), ultimately leading to degranulation and secretion of cytokines via Ca2+ flux and cytoskeletal reorganisation. Structural illustrations were made with PyMol software. DAP12, DNAX-activation protein 12. Abbreviations: ZAP, zeta chain-associated protein kinase; SYK, spleen tyrosine kinase; LAT, linker for the activation of T cells.

importance of the nonhomologous NCRs for NK cell activation is underscored by the fact that low expression of NCRs results in the resistance of leukaemia cells to NK cell cytotoxicity in patients with acute myeloid leukaemia (AML) [31–33]. Moreover, blocking of NCRs results in significantly decreased killing of malignantly transformed and infected cells in vitro [34]. The efficiency of tumour cell killing by NK cells has been shown to correlate with the expression level of the NCRs, defining the NCRbright or the NCRdull phenotype [32,35,36]. Although the NCRs are primarily expressed by NK cells, the expression of NKp44 and 2

NKp46 has been described in NK22 (also known as ILC22) cells; a particular cell subset in gut-associated mucosal tissue, which is characterised by the constitutive production of interleukin (IL)-22 [37–39]. In this review, we focus on the interaction of NCRs with recently identified ligands, and the role of these receptors in stimulating NK cell responses against transformed, virus-infected and some nonstressed normal cells in humans. We discuss how transformed and virus-infected cells can evade NK cell attack and how further understanding of the function of NCRs may aid the development of

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Review novel therapeutic strategies using NK cells to fight viral infections and cancer. NCRs The NCRs are type I membrane proteins and belong to the immunoglobulin superfamily [35,40–43]. All of the NCRs comprise an extracellular ligand-binding domain, which binds to cellular and exogenously derived ligands, a transmembrane domain, and a short cytosolic domain (Figure 1). The NCRs lack a functional intracellular signalling domain and therefore associate with appropriate adaptor proteins via a charged residue in their transmembrane domain [12]. Discovering cellular ligands of the NCRs represents a great challenge due to several reasons as outlined in Box 1. Although the picture is not yet complete, many ligands have been recently identified (Table 1). For example, all of the NCRs bind cellular heparin or heparan sulfate proteoglycans, which are upregulated on cancer cells [44,45], and these interactions lead to NK-mediated killing. Accumulating evidence suggests that different cellular and pathogenassociated ligands are also recognised by the NCRs [15]. Furthermore, the NCRs mediate NK cell interaction with bacteria, however, little is known about the corresponding ligands. The ectodomains of the NCRs consist of a membrane proximal stalk domain and a distal ligand-binding domain. Interestingly, the stalk domain can be important for binding to some NCR ligands [46–48]. Multiple interaction sites within the ectodomain could thus explain how the NCRs can interact with structurally diverse ligands. In the following sections, we focus on the interaction of NCRs with tumour (Figure 2) and viral ligands, as well as some self-ligands that have been identified. NKp30 To initiate signal transduction, NKp30 associates with immunoreceptor tyrosine based activation motif (ITAM)containing adaptor proteins, such as disulfide-linked Box 1. Obstacles in identifying cellular NCR ligands Many laboratories have tried to identify cellular ligands of the NCRs. However, attempts to co-purify NCRs and their corresponding cellular ligands have mostly failed or have led to low recovery rates, thus challenging subsequent mass-spectrometric identification. An exception was the identification of B7-H6 as a cellular ligand of NKp30 [51]. Currently, the reason for these difficulties is unknown. However, several constraints might come from misfit purification schemes and properties of the receptor–ligand proteins. In this respect, the monovalent receptor–ligand interaction might be of low affinity, which would lead to a loss of the cognate ligands during purification. In addition, the single-membrane-spanning or large multimembrane-spanning ligand proteins might not be solubilised by the currently applied purification schemes. Another problem might be that the cognate NCR ligands are proteins with unusual properties and/or that the respective binding interfaces are subject to secondary modifications. Moreover, the structures recognised by the NCRs might not be proteins but small molecules such as sugars or lipids or complex structures of proteins and small molecules analogous to peptide–MHC complexes. A stable NCR– ligand interaction might require synergistic action of other receptor complexes, which is not reflected by the use of bivalent NCR–IgG1– Fc fusion proteins to fish for ligands. Finally, the situation is complicated by the expected presence of several different ligands for a given NCR on a single cell.

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homodimers of CD3z and possibly heterodimers of CD3z with the g-chain of the high-affinity Fc receptor for IgE (FceRI) [17,35] (Figure 1). The gene encoding NKp30 is located in the highly polymorphic telomeric end of the class III region of the human MHC locus and is transcribed into several NKp30 mRNA splice variants, leading to three constitutively expressed isoforms of NKp30 (NKp30a, NKp30b, and NKp30c), which are characterised by intracellular domains of different length [49,50]. NK cells express all three isoforms at a time with NKp30a and NKp30b being stimulatory, whereas NKp30c is immunosuppressive. In immunosurveillance of malignantly transformed cells, NKp30 recognises the tumour antigens B7-H6 [11,51] and BCL-2-associated athanogene 6 (BAG6, also known as BAT3) [52,53], which leads to NK cell killing of the tumour cell. Interestingly, B7-H6 is thought to be a tumour-specific ligand, because its expression has not been detected, so far, on healthy cells in the steady state [51]. Among the NCRs, NKp30 is the only receptor whose structure has been resolved in an unbound [54] and in a ligand-bound [55] state (Figure 1). Binding of B7-H6 to the extracellular immunoglobulin-like domain of NKp30 induces a slight conformational reorganisation of the ligand-binding interface. Notably, the interaction of NKp30 with B7-H6 is similar to that of CD28 (distantly homologous to NKp30) with B7-1 or B7-2 (which are structural homologues of B7-H6 [11]). In contrast to B7-H6, which is found on the plasma membrane of certain tumour cells, the cellular localisation of BAG6 varies. Binding of NKp30 to BAG6, present on the plasma membrane of immature DCs triggers NK cellmediated killing of immature but not mature DCs [52,53]. This observation corresponds to the concept that by killing immature DCs (cells that are associated with the induction of tolerogenic responses) [56], activated NK cells might select a more immunogenic subset of DCs during a protective immune response [57]. The stalk domain of NKp30 increases the binding affinity of the receptor for its cellular ligands BAG6 and B7-H6 [58]. Additionally, recent data suggest that the glycosylation status of NKp30 at its three extracellular N-core glycosylation targeting sites alters its binding affinity for B7-H6, and might thus provide a novel mechanism by which to modulate the ligand-binding properties of NKp30 and related NK cell cytotoxicity [58]. NKp30 also binds several viral ligands (Table 1), including the tegument protein pp65 of HCMV [59]. As part of an HCMV immune escape mechanism, binding of pp65 to NKp30 results in the dissociation of NKp30 from the CD3z chain, thereby inhibiting NKp30-mediated NK cell activation [59]. Similarly, recognition of vaccinia virus haemagglutinin (HA) inhibits NKp30-mediated functions [60]. NKp44 NKp44 [40,43] is a distant member of the family of triggering receptors expressed on myeloid cells (TREM), which are activating receptors involved in the innate inflammatory response and in sepsis [61]. Similar to TREM1, NKp44 is coupled to a dimer of the ITAM-containing adaptor 3

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Table 1. Human NCRs and their known ligands Receptor

Ligand

Source

Ligand localisation

NKp30

B7-H6 BAG6

Tumour cells Stressed cells, tumour cells, DCs

pp65 PfEMP1 Viral HA Unknown Heparin or heparan sulfate Sialylated and sulfated proteoglycans Viral HA and HN

HCMV Plasmodium falciparum Poxvirus, vaccinia virus Variety of tumour cells All animal cells All animal cells

Plasma membrane Nucleus, plasma membrane, exosomes Cytosol Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane

NKp44

PCNA Unknown

NKp46

Unknown Heparin or heparan sulfate Unknown Unknown Viral HA and HN Unknown Unknown Heparin or heparan sulfate

Influenza virus, Sendai virus, Newcastle disease virus Tumour cells Bacterial cell wall components of mycobacteria and others Variety of tumour cells All animal cells CD4+ T cells during HIV-1 infection Fusobacterium nucleatum Influenza virus, poxvirus, Sendai virus, Newcastle disease virus Pancreatic b-cells and stellate cells Variety of normal and tumour cells All animal cells

Effect on NK cell activation + +

Refs

– + – –/+ + +

[59] [107] [60] [35] [44,45] [63]

Plasma membrane

+

[46–48,64]

Cytosol Plasma membrane

– +

[62] [65]

Plasma Plasma Plasma Plasma Plasma

membrane membrane membrane membrane membrane

–/+ + + + +

[18] [44,45] [66,67] [108] [31,48,76]

Plasma membrane Plasma membrane Plasma membrane

+ –/+ +

[71,72] [74,75] [44,45]

[51] [52,53]

PfEMP-1, Plasmodium falciparum erythrocyte membrane protein 1.

DNAX-activation protein (DAP)12 for downstream signal transduction [43]. Along with NKp30, NKp44 is encoded by the class III region of the human MHC locus, raising the question of whether these NCRs have related functions. However, in contrast to NKp30, the expression of NKp44 on NK cells is detected only after activation [40]. The extracellular domain of NKp44 adopts a V-shaped conformation with a large positively charged groove on one face of the domain (Figure 1). This might provide a binding site for a yet unknown ligand on tumour cells [18]. Recently, proliferating cell nuclear antigen (PCNA), which is overexpressed by cancer cells, has been reported as an inhibitory ligand of NKp44 [62]. Similarly to BAG6, PCNA is an intracellular protein, but can be recruited to the plasma membrane of tumour cells [52,53]. The efficiency of tumour cell killing correlates with the expression of NKp44 and NKp46 [32,36], and it seems as if different cellular ligands are recognised by the various NCRs, therefore, it is expected that stimulating cellular ligands of NKp44 remain to be discovered. Sialylated and sulfated cellular proteoglycans [63], influenza virus HA and other viral HA–neuraminidase (HN) proteins [46–48,64], and as-yet-unknown elements of the bacterial cell wall [65], have all been described as ligands of NKp44 that stimulate an NK cell response (Table 1). Sialic acid moieties attached to the stalk domain of NKp44 contribute directly to the binding of influenza virus HA and other viral HN proteins [46–48]. HIV-1-induced NK cell killing of noninfected CD4+ T cells has been described [66,67], which occurred after the recognition of cellular NKp44 ligand, whose expression was induced following an aborted fusion process preceding 4

viral infection [66,67]. The expression of the NKp44 ligand on CD4+ T cells is induced by binding of the conserved 3S motif (SWSNKS) of the HIV-1 gp41 envelope protein to the complement receptor gC1qR on CD4+ T cells [67,68]. Protective 3S motif-specific antibodies have been described in about 30% of HIV-1-infected patients and these antibodies suppress NKp44 ligand expression and inhibit NK cell killing of CD4+ T cells at early stages of infection [69]. 3S-specific CD4+ T cells, which are essential for the production of 3S-specific antibodies, express high levels of NKp44 ligand and are sensitive to NK cell killing, therefore, the protective immune response mediated by 3Sspecific antibodies disappears during the course of the disease. NKp46 NKp46 [41,42] is the most specific marker of NK cells reported so far. For signalling, NKp46 associates with CD3z and also with the g-chain of FceRI (Figure 1) [43]. Unlike NKp30 and NKp44, the ectodomain of NKp46 comprises two immunoglobulin-like domains that are oriented in a defined angle of 858 relative to each other via a connecting hinge region [70], which might be a ligand binding site [18]. NKp46 recognises unknown ligands expressed on pancreatic b-cells [71] and on stellate cells in the liver [72], which triggers an NK cell response, leading to the development of type I diabetes [71] and to protection from liver fibrosis [72]. The expression of the ligands for NKp46 occurs early during pancreas development, and they are found on all b-cells derived from diseased and healthy mice, humans and sand rats [71,73]. However, diabetes

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Tumour cell BAG6

PCNA

BAG6 exosomes

BAG6 soluble

+

PCNA

B7-H6 – –

NKp46

NKp30

CD3ζ

NKp44

CD3ζ

DAP12

NK cell TRENDS in Immunology

Figure 2. Tumour-derived natural cytotoxicity receptor (NCR) ligands. Schematic representation of the known tumour-associated NCR ligands. B7-H6 is a transmembrane protein specifically expressed by tumour cells, which can stimulate NKp30-mediated natural killer (NK) cell activities. The nuclear protein BCL-2-associated athanogene 6 (BAG6) can be released by tumour cells in an exosomal or soluble form, which can activate or inhibit NKp30-mediated functions, respectively. The cytosolic protein proliferating cell nuclear antigen (PCNA) can also be released by tumour cells and inhibit the function of NKp44.

does not develop in all individuals due to the fact that under healthy conditions, NK cells are not found in the pancreas. In addition, an NKp46 ligand is expressed on other normal and on tumour cells [74,75] but the identity of this ligand remains unknown. Glycosylation plays a minor role in the recognition by NKp46 of mouse and human tumour cells and stellate cells in vitro [31,72,76], whereas b-cells and HA are recognised in a glycosylation-dependent manner, which suggests that NKp46 interacts with several distinct cellular ligands. HA of influenza and Sendai viruses were discovered as ligands for both NKp46 and NKp44 [48]. The recognition of HA by NKp46 is sialic acid dependent, leading to the killing of the infected cells. However, it seems as if the protein backbone is also important, because other sialylated NK cell receptors do not interact with HA [48]. N- and O-glycosylation of NKp46 is essential for binding of viral HA from several virus families such as influenza virus, poxvirus, and Newcastle disease virus [31,76]. Among the NCRs, NKp46 is the only receptor that has an orthologue in mice, termed NCR1 [64,77], as well as an orthologue in other species. This specific evolutionary conservation suggests that NKp46 is the primary NCR involved in pathogen and tumour recognition. As no orthologues of NKp30 and NKp44 and their ligands are found in mice, only the in vivo function of NKp46 can be assessed. By replacing exons 5–7 of Ncr1 with a GFP reporter cassette, Ncr1 knockout mice have been generated (Ncr1gfp/gfp) [64]. These mice exhibit an increase in tumour metastasis and are susceptible to influence virus infections [71,72,78,79]. Using random mutagenesis, another mouse named Ncr1Noe´/Noe´ has recently been generated in which

the tryptophan residue at position 32 of Ncr1 is mutated to arginine [77]. In contrast to the phenotype of Ncr1gfp/gfp mice, Ncr1Noe´/Noe´ mice exhibit Ncr1 receptor-independent hyper-responsiveness of NK cell functions [77]. In the Ncr1Noe´/Noe´ mice, the NCR1 protein is mutated and not deleted as in the case of Ncr1gfp/gfp mice, therefore, substantial differences exist at the DNA, RNA, and protein level [46,64,77], which might explain these different phenotypes. In the Ncr1gfp/gfp mice, the intact NCR1 DNA, RNA, and protein are missing, whereas in the Ncr1Noe´/Noe´ mice, all are present in a mutated form. Thus, it might be possible that, in the Ncr1Noe´/Noe´ mice, the mutation results in a ‘gain of function’ activity of NCR1. Future studies will reveal the exact mechanism that underlies this phenomenon. However, regardless of these major differences it is clear from both mouse models that NKp46/NCR1 play a key role in the recognition and elimination of viruses, tumours, and even bacteria. NCR signalling and signal integration NCR signaling pathways involve many of the known ITAM-dependent signaling molecules (Figure 1) [16]. These include SRC family kinase, which phosphorylate the ITAMs of NCR adaptor molecules, resulting in recruitment and activation of zeta chain-associated protein kinase 70 (ZAP70) and spleen tyrosine kinase (SYK). These kinases phosphorylate transmembrane adaptor molecules such as linker for the activation of T cells (LAT) and NTAL (non-T cell activation linker) leading to the association, phosphorylation, and activation of several signaling molecules, including phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC)-g1) PLCg2, VAV2 and VAV3 5

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Review [80–84]. Through these signaling proteins, Ca2+ flux and cytoskeletal reorganisation are induced, ultimately resulting in cellular cytotoxicity and the secretion of cytokines such as IFNg and TNFa [35,40,41]. NCRs are thought to function as monomeric receptors associating with a single dimeric adaptor protein [12]. Evidence exists for selective crosstalk between different NCRs, where triggering of one NCR can stimulate ITAM phosphorylation in the adaptor protein of a different NCR [85]. This suggests that the NCRs may be organised in larger receptor clusters to improve optimal ligand recognition and to stimulate cytotoxicity and cytokine secretion. Such clusters could exist constitutively on the surface of NK cells, or they might be initiated during the formation of the immunological synapse. However, there is currently no direct evidence available for the existence of such NCR clusters. ITAM-mediated signaling in NK cells can have different outcomes. CD16-mediated signaling through the adaptor molecule FceRIg can induce the activation of resting NK cells, whereas NKp30 and NKp46, which also signal through FceRIg, need the co-engagement of other activating receptors such as of 2B4 (also known as CD244) to activate resting NK cells [86]. The reason for this important difference is unknown, but in contrast to NKp30 and NKp46, CD16 associates with FceRIg without possessing a basic amino acid in its transmembrane domain [87], which could affect the structure of the signalling complex. Other activating NK receptors such as NKG2D, NKp80, and 2B4, trigger non-ITAM-based signalling pathways [88–90] and co-engagement of these receptors will generate additional signals that may either complement or enhance ITAMmediated NCR signalling by the NCRs [86]. The requirement for different signal qualities to govern appropriately NK cell reactivity also may explain in part the variety of activating NK cell receptors. Several ligands have been shown to inhibit NKp30 and NKp44 function [59,60,62]. It has been suggested that inhibition of NKp44 function by PCNA involves the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of NKp44 [62], which might signal in analogy to the ITIMs in the inhibitory NK cell receptors [12]. However, this ITIM has been shown to be nonfunctional for NKp44-mediated NK cell activation because it, although phosphorylated, does not recruit phosphatases such as Src homology region 2 domain-containing phosphatase (SHP)-1, SHP-2, and Src homology region 2 domain-containing inositol phosphatase (SHIP), and does not affect NKp44-mediated NK cell activation when mutated [91]. This suggests that activating and inhibitory ligands engage NKp44 differentially, resulting in the stimulation of different signalling events. In the case of NKp30 there are no data supporting an induction of inhibitory signalling by inhibitory ligands. Instead, inhibitory soluble ligands may simply block the receptor or prevent activating signals by dissociating the receptor from its signalling partner [59,60]. In summary, this demonstrates that although NCR signalling may use many of the pathways described for ITAM-dependent signals, they can also induce different signals dependent on the ligand with which they interact. 6

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Therefore, future studies will have to analyse carefully which signals are induced by which NCR ligand in order to understand fully the functionality of the NCRs. Tumour and viral NK cell immune escape Tumour mechanisms for evasion from NK cells Consistent with the observation that NK cells are an important component of antitumour immune responses, several tumour escape strategies to evade the NKG2Dinduced NK cell-mediated attack of these cells have been identified (reviewed in [10,13,20,92]). By contrast, only a few tumour immune escape mechanisms are known to target the NCR system (Figure 3). This could be due to the fact that most of the known NCR ligands are pathogen derived (Table 1) and knowledge of the molecular nature of tumour-associated NCR ligands is scarce. As mentioned above, reduced expression of NCRs is associated with several different forms of cancer such as melanoma, cervical cancer, human breast cancer, and AML [33,93–95]. Tumour-induced downregulation of NCR expression might therefore represent one immune escape mechanism [96]. Moreover, it has been demonstrated that, in the absence of NKp46, the expression of the unknown NKp46 ligand is increased on methylcholanthrene (MCA)induced tumours and that this leads to increased IFNg secretion [96]. Thus, MCA-induced tumours escape from NKp46-dependent immunosurveillance by a process termed immunoediting, thereby interfering with IFNg secretion, which can lead to substantial activation of both the innate and the adaptive immunity. Predominant expression of the immunosuppressive NKp30c isoform has been found in patients with gastrointestinal sarcoma [49]. NKp30c expression is associated with reduced survival of patients due to preferential production of the immunosuppressive cytokine IL-10 and reduced NKp30-mediated NK cell activation [49]. This demonstrates the importance of functional NKp30mediated NK cell stimulation for controlling gastrointestinal tumours. The cellular NCR ligands BAG6 and PCNA are interesting mediators of a tumour immune escape strategy. BAG6 is released with exosomes of various tumour cells [52,53], therefore, we hypothesise an NKp30-dependent tumour immune escape mechanism similar to that of exosomal ULBP3, which mediates downmodulation of NKG2D [97]. Furthermore, the inhibitory ligand PCNA may promote survival of cancer cells by inhibition of NKp44-induced NK cell-mediated attack [62]. The NKp30 ligand B7-H6 seems tumour specific in its expression [11,51]. It will be interesting to learn what drives B7-H6 expression because some tumours might interfere with this process to escape NKp30-mediated immune responses. B7-H6 possesses signalling motifs within its cytoplasmic tail. Engagement by NKp30 might thus induce unknown signals in tumour cells. Moreover, soluble B7-H6 variants might be generated by alternative splicing or ligand shedding and mediate tumour immune escape. The NCRs thus seem to play a critical role in antitumour immunity. This has been convincingly demonstrated in Ncr1gfp/gfp mice, which display a markedly enhanced

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BAG6 exosomes

Target cell BAG6

? ? pp65 B7-H6

NKp30

Influenza HA pp65 PCNA

CD

3ζ

Soluble influenza HA released with virions

CD3ζ

NKp46 CD3ζ lysosomal degradaon

PCNA

DA P12

?

PCNA

NKp44

NK cell TRENDS in Immunology

Figure 3. Immune evasion mechanisms targeting the natural cytotoxicity receptors (NCRs). Schematic representation of the effector proteins of tumour or virus-infected cells (target cells) and their interaction with signalling complexes of NKp30, NKp44, and NKp46. Exosomal and soluble BCL-2-associated athanogene 6 (BAG6) is released from tumour cells and blocks NKp30. Proliferating cell nuclear antigen (PCNA) is released from tumour cells and, after uptake into the natural killer (NK) cell, inhibits NKp44 signalling via the inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence in the cytoplasmic tail of NKp44. B7-H6 on the plasma membrane of tumour cells binds to NKp30, which in turn might not only activate NK cell cytotoxicity but also initiate tumour immune escape mechanisms due to downstream signalling of B7-H6 in the tumour cell. pp65 of human cytomegalovirus (HCMV) is released from HCMV-infected cells and results in dissociation of NKp30 from the CD3z chain, which in turn inhibits immunoreceptor tyrosine based activation motif (ITAM)-dependent signalling and NK cell activation. Along with new virions, influenza-virus-infected cells release haemagglutinin (HA), which is taken up by NK cells and induces lysosomal degradation of CD3z chains to inhibit NK cell activation via NKp30 and NKp46. DAP12, DNAXactivation protein 12.

development of various tumours and tumour metastases in the absence of NCR1 [64,78,79]. The identification of the entire spectrum of NCR tumour ligands is therefore crucial for a better understanding of NCR-mediated antitumour immunity and for the development of possible new anticancer treatments. Viral mechanisms for evasion from NK cells Many NCR ligands are of viral origin, supporting a role for NCR-mediated NK cell activation in antiviral immune responses. Indeed, several viral immune-evasion mechanisms affect the NCR system (Figure 3). As an example, upon HIV-1 infection, the expression of NCRs is reduced, leading to impaired clearance and thus accumulation of immature DCs, reduced cytokine secretion, and subsequent failure to activate NK cells efficiently [98–101]. Several viral NCR ligands inhibit NCR function (Table 1) and interestingly, the inhibitory mechanism of some of these ligands is similar. The HCMV tegument

protein pp65, one of the dominant antigens for HCMVspecific CTL responses, has been found to block NK cell cytotoxicity through interactions with NKp30 [59]. It is proposed that binding of pp65 results in dissociation of NKp30 from the CD3z chain, which is required for ITAM-dependent signalling [59]. A similar mechanism has recently been described with regard to the poxvirus HA, which unlike the influenza virus HA, interacts with NKp30 [60]. Influenza viruses use an immune-evasion mechanism that is based on the dual nature of HA. On the one hand, HA is an activating ligand of NKp46 and NKp44 when expressed by infected cells [48], but on the other hand, influenza-virus-infected cells release HA along with new virions, and this free HA is taken up by NK cells. Once inside NK cells, HA induces the lysosomal degradation of CD3z chains, thereby preventing the transduction of activating signals from NKp46 and NKp30 [102]. As a novel immune evasion strategy, viral miRNAs have been described to regulate the expression of NKG2D 7

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Review ligands promoting immune evasion [103]. Whether similar viral strategies exist to target NCR ligands is currently unknown; mostly due to the paucity of knowledge about the cellular NCR ligands. Future directions Much progress has been made in recent years on the characterisation of receptor–ligand interactions involved in NK cell activation, especially with regard to NKG2D and its ligands. Much less is known about the NCRs; probably because most ligands identified so far are pathogen derived and the identification of cellular ligands of the NCRs remains challenging (Box 1). The NCRs play an important role in NK cell biology. A special emphasis is placed on the NCRs not only because they act as important mediators of cytotoxicity towards malignantly transformed cells, virusinfected cells, bacteria, and even some healthy cells, but also because their expression is restricted primarily to NK cells. There are many outstanding questions to explore. For instance, what factors drove the evolution of the NCR receptor system that recognises structures that are fairly specific to a certain pathogen and do not constitute a molecular pattern common to many different pathogens? What is the benefit of carrying a germline-encoded receptor specific for only a few viruses? It is also not clear why many viruses that are recognised by the NCRs are not lethal to the host, although several of these interactions inhibit, rather than activate, the receptors. Another important question to ask is why do we even need the NCRs, considering that we have additional NK cell activating receptors such as NKG2D? Although mice have a homologue to NKp46, they lack a homologue of NKp44 and contain only a pseudogene of human NKp30 (except in the evolutionary ancestor Mus caroli) [64,77,104]. Perhaps the longer lifespan of humans compared with that of mice has forced NK cell evolution to generate receptors specific for the detection of tumours. The NCRs may have initially evolved to recognise mainly pathogen-derived ligands (as the mouse homologue of NKp46 does). They could then have been adapted to recognise tumour-specific structures such as B7-H6 in humans. However, recognition of tumours is always challenging, because the structures recognised may also be present on healthy cells. It has been demonstrated that NKp46 is involved in type 1 diabetes development. Why are NCR1-mediated autoimmune diseases not more common if the NCR ligands are also present on healthy cells? We suggest that under normal conditions, NCR-dependent autoimmune diseases do not develop because NK cells are excluded from the tissues that express NCR ligands. Alternatively, because two signals are needed to trigger properly the activity of NK cells [86], it is possible that NK cells are efficiently activated by the NCRs only when additional receptors are activated. Similarly, NKp44 function is controlled by other signals as the expression of NKp44 is induced only following NK cell activation. Concluding remarks NK cells hold promise for adoptive cancer immunotherapy, however, their use is challenged by immune escape strategies and the requirement of targeted delivery 8

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of the effector NK cells [105]. As a proof of concept, engineered clinically applicable human NK-92 cells carrying a chimeric antigen receptor (CD20-specific antibody fragment fused to the CD3z chain) have been shown to overcome resistance of lymphoma and leukaemia cells to NK cell-mediated killing [106], indicating that a similar strategy might be attainable using NCRs. The detailed molecular characterisation of activating and presumably inhibitory NCR functions will open up novel targeting strategies to harness NK cell functions in cancer such as manipulating the balance of activation and inhibition [105]. Acknowledgements The laboratory of J.K. is supported by institutional funds of the GeorgSpeyer-Haus and by grants from LOEWE Center for Cell and Gene Therapy Frankfurt funded by: Hessisches Ministerium fu¨r Wissenschaft und Kunst (HMWK) funding reference number: III L 4- 518/17.004 (2010) and the Wilhelm-Sander Stiftung (2010.104.1). The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health (BMG) and the Ministry of Higher Education, Research and the Arts of the State of Hessen (HMWK). Research of the laboratory of A.S. is funded by the Deutsche Forschungsgemeinschaft (DFG). The laboratory of C.W. is supported by grants from the DFG and the Bundesministerium fu¨r Bildung und Forschung (BMBF). The laboratory of O.M. is supported by the ISF, the ICRF and by the Israeli-ICORE. O.M. is a Crown Professor of Molecular Immunology.

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