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REVIEW ARTICLE Human NK cells: surface receptors, inhibitory checkpoints, and translational applications

Simona Sivori 1, Paola Vacca2, Genny Del Zotto 3, Enrico Munari4,5, Maria Cristina Mingari1,6 and Lorenzo Moretta 2

NK cells play important roles in innate defenses against and in the control of tumor growth and . The regulation/ induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral , or tumor transformation. The HLA-class I- specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class- Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 (PD-) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk .

Keywords: Human NK cells, NK receptors, Inhibitory checkpoints, Immunotherapy

Cellular & Molecular Immunology (2019) 16:430–441; https://doi.org/10.1038/s41423-019-0206-4

INTRODUCTION AND GENERAL OVERVIEW OF HUMAN NK molecules in both humans and in mice. Remarkably, while the CELLS first identified human receptors specific for human leukocyte Natural killer (NK) cells play major roles in first-line innate defenses antigen (HLA)-cl I molecules belong to the immunoglobulin (Ig) against viral , tumor growth, and metastatic spread. Both superfamily (and were subsequently named killer-cell immuno- the constitutive expression of efficient, ready-to-function lytic globulin-like receptors, KIRs), the murine receptors (Ly49) are machinery, and the rapid release of interferon-gamma (IFN-γ) and members of the lectin family. Thus, humans and mice developed tumor necrosis factor-alfa (TNF-α) following cell activation allow two molecularly different receptors fulfilling the same function, prompt intervention by NK cells, resulting in target cell killing and i.e., the recognition of MHC-cl I molecules and the delivery of the initiation of an inflammatory response. inhibitory signals to NK cells, resulting in NK cell inactivation.5 The NK cells were discovered in the mid-70s;1,2 however, the discovery of KIRs was made possible by the availability of two mechanism by which they discriminate between tumor or - important technologies, namely, technology infected cells and healthy cells remained a mystery for many years. and a high-efficiency lymphocyte cloning system. The latter The missing self-hypothesis, proposed in the late 80s by Karre and technology allowed the clonal expansion of virtually 100% of Ljunggren,3 represented a true milestone, inspiring subsequent human T lymphocytes.6 Thus, it has been possible to analyze the groundbreaking discoveries in the early 90s. This hypothesis was frequency of cells endowed with a given function (e.g., cytolytic based on the finding that murine NK cells could kill a activity) and to establish correlations between the surface cell line that had lost major histocompatibility complex-class I phenotype and function of any T-cell population.7 After suitable (MHC-cl I) molecules, while parental MHC-cl I+ cells were resistant adaptation of this cloning technique, NK cells were also revealed to lysis. Thus, NK cells appear to sense the absence of MHC-cl I on to be clonogenic and suitable for precise functional studies. NK cells (i.e., the missing self).4 Research aimed to identify the cell clones were used for mouse immunization and the production molecular mechanism(s) involved in this phenomenon, leading to of monoclonal antibodies (mAbs) that were selected for their the discovery of inhibitory receptors specific for MHC-cl I ability to either inhibit or induce NK cell cytolytic activity or

1Department of Experimental Medicine (DIMES) and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy; 2Department of Immunology, IRCCS Bambino Gesù Children’s Hospital, Rome, Italy; 3Department of Research and Diagnostics, Istituto G. Gaslini, Genoa, Italy; 4Department of Diagnostics and Public Health, University of Verona, Verona, Italy; 5Department of Pathology, Sacro Cuore Don Calabria, Negrar, VR, Italy and 6UOC Immunologia, IRCCS Ospedale Policlinico San Martino Genova, Genoa, Italy Correspondence: Lorenzo Moretta ([email protected]) Received: 14 January 2019 Accepted: 22 January 2019 Published online: 18 February 2019

© CSI and USTC 2019 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 431 production. Two selected mAbs, originally defined as sensing different HLA-E-bound CMV peptides and undergo p58.1 and p58.28–10, recognized two highly homologous mole- selective proliferation only in response to given HLA-E-peptide cules expressed by partially overlapping NK cell subsets. These combinations.31 Such specific expansion, together with a number mAbs represented prototypes of HLA-cl I-specific inhibitory of adaptive characteristics, is compatible with the memory-like receptors, and were found to recognize allotypic determinants function of these cells, which can allow for prompt control of CMV shared by the two main groups of HLA-C alleles.10–13 reactivation.32 Other HLA-cl I-specific receptors were identified (see below). Of While NK cells have been known for over 45 years, other cells relevance, the HLA-E-specific NKG2A,14 expressed earlier than KIR that are developmentally related to NK cells were identified only during NK cell maturation, was recently found to be of great 10 years ago and collectively termed innate lymphoid cells potential interest as a target of checkpoint inhibitors in tumor (ILCs).33,34 Similar to NK cells, ILCs derive from CD34+ hemato- immunotherapy15,16(see below). poietic precursors and share with NK cells a common ID2+ Remarkably, the pool of mature NK cells is equipped with at lymphoid precursor.35 Absent or infrequent in PB from healthy least one inhibitory for self HLA-cl I antigens (whether KIR individuals, ILCs reside in mucosal tissues, skin, and lymphoid or NKG2A). Only very few peripheral blood (PB) NK cells lack such organs. Unlike NK cells, the other ILCs (termed “helper ILCs”) are receptors and are anergic. The repertoire of inhibitory NK noncytolytic and secrete typical sets of that differ for receptors is shaped during NK cell maturation and is the result each subset, including IFN-γ (ILC1), IL-5, IL-13, and small amounts of a process of selection termed NK cell “licensing” or “educa- of IL-4 (ILC2), IL-22 (NCR+ ILC3), IL-17, and TNF-α (NCRneg ILC3 or tion”.17–19 Accordingly, only NK cells expressing receptors for self LTi-like cells).36 Interestingly, helper ILCs mimic corresponding HLA-cl I molecules acquire full functional potential, while the CD4+ T-cell subsets in terms of the type of cytokines produced. remaining are either deleted or anergic. Therefore, these cytokines can shape not only adaptive but also “Off” signals are necessary to prevent NK-mediated autoreac- innate immune responses, although with different timing. In tivity, implying the existence of “on” signals responsible for NK cell general, ILCs contribute to innate defenses against various activation. Indeed, in the absence of inhibitory interactions, NK and are involved in tissue repair and homeostasis cells kill target cells and produce cytokines. Again, the use of NK and in lymphoid organogenesis, particularly during fetal life. While cell clones and the selection of mAbs were fundamental for the they will not be discussed further in this review, which is devoted discovery of prototypes that activate NK receptors, namely, to NK cells, it is worth noting that ILCs may share some markers NKp46,20–22 NKp4423 and NKp30,24 collectively termed “natural and the NCR activating receptors with NK cells.37 25

1234567890();,: cytotoxicity receptors” (NCRs). mAbs directed to these surface NK cell traffic in the circulation and towards tissues and molecules were selected based on their ability to modulate NK lymphoid organs is regulated by and clone-mediated cytotoxicity against selected tumor targets. It receptors, directing the given NK cell subsets to specific sites.38 became evident that NK cell activation is not due to a master Thus, while the expression of CD62L, CXCR3, and CCR7 will direct receptor (such as TCR and BCR in T and B cells, respectively) but, in CD56bright NK cells to secondary lymphoid organs (in response to most instances, is the result of the combined action of several CCL19 and CCL21), the expression of Chemerin R, CXCR1, CXCR2, dim receptors and coreceptors (see below). Triggering of the activating and CX3CR1 directs CD56 NK cells to inflammatory peripheral NK receptors requires the expression of appropriate ligands on tissues in response to their corresponding chemokine ligands potential target cells. Such ligands are overexpressed or expressed (Chemerin, IL-8, and Fraktalkine). Since CD34+ cells capable of de novo in stressed healthy cells and, more importantly, in cells generating NK cells have been detected in different tissues, that have undergone tumor transformation or viral infection.26 including thymus,39 tonsils,40,41 decidua42, and liver,43 it is Remarkably, tumors and certain viruses may induce the down- conceivable that at least some tissue-resident NK cells may derive regulation of activating NK receptors and/or their ligands on from these precursors, migrate from BM, and acquire unique target cells, thus favoring escape from the NK-mediated control. properties as a consequence of their particular tissue This is evident in a relevant percentage of tumors, particularly microenvironment.44 neuroblastoma, in which not only HLA-cl I molecules but also The following paragraphs will analyze in more detail the various ligands for most activating NK receptors may be downregulated.27 NK receptors, recently identified inhibitory checkpoints, and their This mechanism of tumor escape underscores the potential implications in the immunotherapy of tumors. In addition, we will importance not only of T cells but also of NK cells in the immune outline the fundamental role of NK receptors in the cure of high- response against neuroblastoma. Taken together, the expression risk leukemias in allogeneic hemopoietic stem cell transplantation of inhibitory and activating receptors in NK cells and their ligands (HSCT). on target cells revealed that NK cell activation/inactivation is controlled by different checkpoints.28 This notion is important for translational applications that exploit NK cell function in therapy INHIBITORY AND ACTIVATING NK RECEPTORS AND THEIR for or solid tumors (see below). ROLES IN TUMOR CELL KILLING Human NK cells have been divided into two major subsets HLA-specific NK receptors identified according to the surface density of the CD56 antigen.29 As mentioned above, important regulation of NK cell function is CD56bright cells represent only ~10% of PB NK cells, but they provided by inhibitory receptors specific for HLA-cl I molecules. predominate in tissues. These cells express NKG2A, are poorly Indeed, HLA-cl I molecule recognition on healthy cells by these cytolytic but secrete cytokines, primarily IFN-γ and TNF-α, and inhibitory NK receptors prevents NK cell-mediated attack. efficiently proliferate in response to interleukin (IL)-2 or IL-15. Human NK cells express two different classes of HLA-class I- CD56dim cells largely predominate in PB (~90%), expressing specific inhibitory receptors: members of the KIR/CD158 family NKG2A or KIR or both, and rapidly display potent cytolytic activity and the CD94/NKG2A (CD94/CD159a) heterodimer.5,45 KIRs are and cytokine production upon activation. Based on the expression type I transmembrane receptors specific for polymorphic HLA-A, B of receptors/markers, CD56dim NK cells are further subdivided into and C molecules,8–10,12,13 whereas NKG2A is a type II transmem- different stages of differentiation, characterized by a progressive brane receptor that recognizes HLA-E, a non-classical HLA decrease in proliferative capacity paralleled by an increase in molecule characterized by limited polymorphism.14 To transduce cytolytic activity.30 The most mature NK cells are KIR+ NKG2Aneg inhibitory signals, both types of inhibitory receptors contain ITIM and express CD57 and (in individuals seropositive for cytomega- motifs in their cytoplasmic tail. In addition, activating forms of KIRs lovirus, CMV) NKG2C, an HLA-E-specific activating receptor. Recent have been identified.46 Different from inhibitory KIRs, activating studies revealed that NKG2C+ NK cells display adaptive features, KIRs lack ITIM motifs in their cytoplasmic tail and have a

Cellular & Molecular Immunology (2019) 16:430 – 441 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 432 transmembrane domain carrying a charged amino acid residue47 important to underline that the physical association between that mediates the association with the ITAM-bearing molecule NKp44 and KARAP/DAP12 is essential for NKp44 surface expres- KARAP/DAP12. The role of activating KIRs in the immune response sion67 and that the decreased surface density of NKp30 and is only partially known. Specificity for HLA-cl I molecules has been NKp46 on “adaptive” NK cells is associated with the lack of FcεRI-γ demonstrated for only a few of them.46,48–54 expression in these cells.68 Inhibitory KIRs are characterized by long cytoplasmic tails, Different molecules can interact with the extracellular domains whereas activating KIRs have short cytoplasmic tails (“L” or “S” in of NCRs. Some of them are virus-derived molecules that can the nomenclature, respectively). Regarding Ig domain content, activate NK cell function against infected cells (such as influenza each KIR displays two or three extracellular Ig domains (“KIR2D” or virus-derived HA recognized by NKp46 or NKp4469) or induce “KIR3D” in the nomenclature, respectively). Two types of KIR2D can inhibitory signals (such as the HCMV-encoded pp65 be determined according to their extracellular Ig domain content. recognized by NKp30.70) Other NCR ligands (such as BAT3/BAG6, KIR2Ds of the first type are composed of D1 and D2 domains and MLL5, and PCNA) are represented by intracellularly localized include the majority of KIRs (KIR2DL1/L2/L3 and KIR2DS1/S2/S3/ that may reach the cell surface in response to stress or S4/S5), whereas KIR2Ds of the second type are composed of D0 during tumor transformation. For example, BAT3/BAG6 is typically and D2 domains and include KIR2DL4/L5.55 Notably, not only HLA located in the nucleus;71 however, in cells exposed to heat shock, class I but also KIRs are characterized by high levels of it can move to the plasma membrane and be secreted in polymorphism, which may affect given KIR/HLA interactions. In exosomes by tumors and stressed cells.71,72 Then, BAT3/BAG6- addition, certain KIR/HLA combinations have been shown to expressing exosomes can stimulate cytokine release by NK cells correlate with protection or susceptibility to infectious, auto- upon interaction with NKp30. MLL5 is expressed in the nuclei of immune, and reproductive disorders. normal cells, but 21spe-MLL5, an MLL5 isoform that functions as Another HLA-specific inhibitory receptor is represented by LIR- an NKp44 ligand, is located in the cytoplasm and at the cell 1/ILT2/CD85j.56 LIR-1 is a type I transmembrane protein belonging surface.73 Finally, the nuclear protein PCNA can function also as an to the Ig-like receptor superfamily that can interact with classical NCR ligand. Indeed, in normal cells, PCNA is involved in the (HLA-A, HLA-B, HLA-C) and non-classical (HLA-G) HLA-cl I processes of DNA replication/repair and cell cycle control, but in molecules.57–59 It can also bind UL18, a cytomegalovirus- tumor cells, it can be shuttled to the tumor cell surface and encoded HLA-cl I homolog that is expressed on CMV-infected expressed on tumor-derived exosomes, functioning as an NKp44 cells.56 Notably, high LIR-1 expression correlates with the ligand.74 acquisition of NK cell memory in CMV+ donors.60 -H6, another NKp30 ligand,75 is not expressed on healthy Another HLA-specific activating receptor is represented by cells, but it is frequently present on the cell surface of many tumor NKG2C, a receptor that, similar to NKG2A, binds HLA-E but with types through a Myc-mediated mechanism.76 Interestingly, its lower affinity.61,62 Altogether with inhibitory KIRs, CD94/NKG2A expression may also be acquired by normal cells upon TLR- prevents the response against cells with normal expression of mediated stimulation and in the presence of pro-inflammatory HLA-I molecules, whereas CD94/NKG2C is involved in the response cytokines.77 to human HCMV. Notably, NKG2A is primarily expressed by PB Recently, NKp46 and NKp44 have also been shown to recognize immature NK cells, whereas NKG2C is expressed only at late stages extracellular ligands. Indeed, NKp46 can bind to a soluble plasma of NK cell maturation.63 The terms “adaptive” or “memory-like” are glycoprotein called complement factor P/properdin, revealing currently employed to designate the human differentiated cross-talk between two partners in innate immunity in the NKG2Cbright NK cell subset that is characterized by the CD56dim response to Neisseria meningitidis infections.78 Moreover, NKp44 CD57+ KIR+ NKG2Aneg phenotype and that is expanded in HCMV+ has also been shown to recognize an extracellular ligand called donors.32,64,65 Similar to activating KIRs, NKG2C is coupled to the Nidogen-1 (NID1, also known as Entactin).79 The NKp44/NID1 ITAM-bearing molecule KARAP/DAP12. interaction results in reduced NKp44-mediated cytokine release by NK cells and induces relevant changes in the NK cell proteomic Activating NK receptors and coreceptors involved in tumor cell profile, suggesting an effect on different biological processes. killing and their ligands Importantly, it has been shown that tumors can orchestrate Human NK cells express several receptors that can trigger their different mechanisms to impair NCR function. Thus, hypoxia or function upon interaction with specific ligands on the surface of various soluble factors produced by tumor/tumor-associated cells transformed, virus-infected, or stressed cells (Table 1). (such as indoleamine 2,3 dioxygenase [IDO], tumor growth factor- The NCRs25 are among the major activating NK receptors and beta [TGF-β], prostaglandin E2 [PGE2]), or inhibitory NCR ligands consist of three elements, called NKp46/NCR1/CD335,20–22 NKp44/ (such as the soluble form of BAT3 or B7-H6)72,80 can induce NCR2/CD33623, and NKp30/NCR3/CD337.24 These molecules were decreases in NCR expression and function.81 Indeed, NCRlow NK classically described as germline-encoded receptors and are cells can be detected in PB and particularly in the tumor site in important for inducing NK cell cytotoxic function against tumors patients affected by solid and hematologic tumors. Notably, and infected cells. reduced expression/function of NCRs can also be detected in NK NKp46 and NKp30 are expressed on nearly all resting human NK cells from HIV-infected patients.82 cells, upregulated on activated NK cells and downregulated on Another important activating NK receptor is NKG2D, a type II “adaptive” NK cells that are found in CMV+ individuals. Unlike transmembrane and C-type lectin-like receptor, which may be NKp46 and NKp30, NKp44 is constitutively expressed only on expressed on cytotoxic T cells. NKG2D ligands are represented by CD56bright NK cells, but it is acquired by essentially all NK cells after ULBPs and MICA/B,83 which are HLA-cl I structural homologs that activation by cytokines. are upregulated in infected, stressed, and tumor cells.84,85 Notably, NCRs are type I transmembrane molecules belonging to the shedding of NKG2D ligands by tumor cells may represent a immunoglobulin-like family. mechanism of tumor escape. These receptors were named in accordance with their molecular Other molecules, including 2B4,86 NTB-A,87 DNAM-1,88 CD59,89 weights according to sodium dodecyl sulfate polyacrylamide gel and NKp80,90 function primarily as coreceptors; indeed, they are electrophoresis (NKp46, NKp30, and NKp44). Their capable of amplifying the NK cell triggering induced by NCRs or transmembrane domains contain a positively charged amino acid NKG2D. In addition, NK cells may express toll-like receptors (TLRs) that allows their association with the transmembrane regions of that, after interaction with bacterial or viral products and in the the TCR-ζ and/or FcεRI-γ (for NKp30 and NKp46) or KARAP/ presence of pro-inflammatory cytokines, induce potent NK cell DAP1266 (for NKp44) adaptor proteins. In this regard, it is activation.91–94

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Table. 1. Human NK cell receptors and their ligands

Molecule Ligand CD56bright CD56dim

Non-HLA-specific receptors Coreceptors CD59 LFA-2 (CD2) All PB NK cells NTB-A (CD352) NTB-A (CD352) All PB NK cells NKp80 AICL All PB NK cells DNAM-1 (CD226) Nectin-2 (CD112), PVR (CD155) All PB NK cells 2B4 (CD244) CD48 All PB NK cells Inhibitory PD-1 (CD279) PD-L1 (CD274), PD-L2 (CD273) − Subsets Siglec-7 (CD328) Ganglioside DSGb5 Most of PB NK cells IRP60 (CD300a) α-Herpes virus, Pseudorabide virus, Phosphatidylserine, Phosphatidylethnolamine All PB NKs Tactile (CD96) PVR (CD155) All PB NK cells IL1R8 IL-37 PB NK cells TIGIT PVR (CD155) PB NK cells TIM-3 Gal-9, PtdSer, HMGB1, CEACAM1 Subset of NK cells Activating NKp30 (CD337) B7-H6, BAG6/BAT3 ++ + NKp44 (CD336) 21spe-MLL5 - Nidogen-1 Activated NK cells NKp46 (CD335) CFP (properdin), viral HA and HN, PfEMP1 ++ + NKG2D (CD314) MIC-A, MIC-B, ULPBs All PB NK cells

FcγRIII (CD16) IgG −/++/++ . . and. al. checkpoints, et inhibitory Sivori receptors, S surface cells: NK Human HLA-specific receptors Inhibitory NKG2A/KLRD1 (CD159a/CD94) HLA-E + Subsets KIR2DL1 (CD158a) HLA-C2 − Subsets KIR2DL2/3 (CD158b) HLA-C1, few HLA-Bb − Subsets (CD158d)a HLA-G − Subsets KIR2DL5 (CD158f) ??? − Subsets KIR3DL1 (CD158e1) HLA-A-Bw4, HLA-B-Bw4 − Subsets KIR3DL2 (CD158k) HLA-A*03 and *11 − Subsets ILT2/LIR-1 (CD85J) Different MHC-I alleles . Subsets LAG-3 (CD223) MHC-II Activated NK cells Activating KIR2DS1 (CD158h) HLA-C2 − Subsets KIR2DS2/3 (CD158j) ??? − Subsets KIR2DL4 (CD158d)a HLA-G − Subsets KIR2DS4 (CD158i) HLA-A*11 and some HLA-C − Subsets KIR2DS5 (CD158f) ??? _ Subsets KIR3DS1 (CD158e1) HLA-Bw4, HLA-F − Subsets NKG2C (CD159a) HLA-E − Subsets 433 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 434 γ dim Finally, the CD16, recognizing the Fc portion of IgG antibodies specific for unhealthy cells, can trigger antibody- − + + + + + − 95 bright CD56 Subsets Subsets dependent cell-mediated cytotoxicity (ADCC). CD16 expression is restricted to mature CD56dim KIR+ NK cells.

The molecular basis of NK cell function NK cell function is regulated by the activating and inhibitory receptors illustrated above. The fine balance of signaling that occurs through them determines if NK cells kill their target cells or remain inactive. As discussed, autoreactivity in human NK cells is controlled by KIRs and NKG2A inhibitory receptors specific for self HLA-cl I molecules. KIRs and NKG2A are clonally expressed or coexpressed in NK cell subsets, thus creating repertoires of NK

bright cells with different phenotypic and functional characteristics that are capable of responding to different types of virally infected + ++ − ++ − − − − + CD56 Subsets of NK cells Subsets of NK cells and tumor-transformed self cells. Under normal conditions, the interactions between these inhibitory receptors and their specific HLA-cl I ligands inactivate NK cells, thus preventing cytolytic activity against healthy cells. During cancer progression, the transformed cells decrease or even lose the expression of HLA-I on their surface. In addition, they express ligands for activating NK receptors: two events necessary for the induction of antitumor NK cell responses. Indeed, ligands for the activating NK receptors are generally absent or expressed in small amounts in normal cells, while they are expressed de novo or upregulated at the cell surface in stressed normal cells and, in particular, in virus-infected or tumor cells. Therefore, signaling through activating receptors can overcome the signaling mediated by inhibitory receptors on diseased cells.25,96

NK INHIBITORY CHECKPOINTS AND THEIR LIGANDS IN SOLID TUMORS Inhibitory checkpoints in NK cells In addition to the HLA-cl I-specific inhibitory receptors that regulate NK cell function and prevent NK-mediated damage to healthy tissues, additional inhibitory checkpoints, responsible for maintaining immune cell homeostasis, have been described in NK cells, including PD-1, TIGIT, CD96, TIM-3, etc. Under pathological conditions, some of these checkpoint regulators,

β which are absent on resting NK cells, can be induced de novo and affect antitumor NK cell function upon interaction with their

164 specific ligands that are frequently expressed on the tumor cell 97–99

and MIP1 surface and facilitate tumor immune escape.

α PD-1, a member of the immunoglobulin superfamily, is a major checkpoint regulating T-cell activation and ensuring peripheral tolerance.100,101 However, when binding to its ligands (PD-L1 and PD-L2), which may be expressed on tumor cells, PD-1 may compromise the antitumor effector function favoring tumor immune escape. Notably, in mice, the blockade of PD-1 resulted in restoration of T-cell responses.102 Although the expression of PD-1 has been described on T, B, and myeloid cells, recent studies have shown that, under pathological conditions, it is also expressed by NK cells.103,104 PD-1+NK cells that can inactivate NK cell function were found in patients with CMV infections and cancer, including Kaposi sarcoma, ovarian carcinoma, and Hodgkin lymphoma.63,104–107 A recent study by our group revealed the presence of a pool of PD-1 proteins and mRNA in the cytoplasm of healthy donor NK cells. The molecular mechanisms regulating the expression of PD-1 on human NK cells have not been defined so far. However, it is conceivable that signals delivered by cells and/or soluble factors in the tumor microenvironment may play a major role (Mariotti et al. in press). continued In mice, PD-1 expression may be induced by glucocorticoids,

ammation sites fi CCR7 (CD197) CCL19, CCL21 CXCR3 (CD183) CXCL9, CXCL10, CXCL11 CXCR1 (CD181) CXCL8 (IL-8) L- (CD62L) GLyCAM-1, MadCAM-1 CXCR2 (CD182) IL-8-RB CX3CR1 Fraktalkine ChemR23 Chemerin CCR5 (CD195) RANTES, MIP1 CXCR4 (CD184) CXCL2 S1P5 S1P c-Kit (CD117) SCF (KL)

fl underscoring the importance of speci c signaling for its 108 2° lymphoid tissues In Others KIR2DL2/L3 weakly recognize also HLA-C2 alleles and few HLA-B alleles that bear the HLA-C1 epitope (e.g., HLA-B*4601 and HLA-B*7301)

KIR2DL4 has been shown to have both inhibitory and activating functions regulation. Several murine tumor models showed that PD-1/ b a Table 1 MoleculeHoming receptors Ligand PD-L1 interactions can strongly suppress NK cell-mediated

Cellular & Molecular Immunology (2019) 16:430 – 441 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 435 antitumor immunity and that PD-1+ NK cells present at the tumor blocking can unleash NK cells and promote efficient antitumor site display an exhausted phenotype.109 activity.138 In humans, in addition to PD-1, other inhibitory checkpoints are expressed not only by T lymphocytes but also by NK cells. They PD-L1 and other checkpoint ligands include the T-cell Ig and ITIM domain (TIGIT) and CD96 (tactile). As illustrated above, PD-1 negatively regulates immune responses; These receptors are members of a group of Ig superfamily under physiological conditions, these interactions lead to periph- receptors that also include the activating receptor DNAM-1. Their eral T-cell tolerance, while in cancer patients they may impair T- corresponding ligands, usually upregulated in tumor cells, are the cell responses against tumor cells. In this context, immunotherapy poliovirus receptor (CD155), nectin-2 (CD112), and nectin-like with checkpoint inhibitors that disrupt the PD-1/PD-L1 interaction molecules.110,111 TIGIT expression has been reported in different has proven to be highly effective in different tumor types, malignancies in peritumoral lymphocytes, often together with PD- representing a true revolution in cancer therapy.139 Since the 1 and TIM-3, and is associated with CD8+ T-cell and NK cell approval of nivolumab and pembrolizumab in 2014, the number suppression.112 For example, TIGIT was upregulated in tumor- of PD-1/PD-L1 inhibitors has grown rapidly. Currently, there are six associated NK cells of patients with colon-rectal cancer. TIGIT PD-1/PD-L1 inhibitors that have been approved by the Food and knockout mice or the use of mAbs that block TIGIT could induce/ Drug Administration (FDA): three against PD-1 (pembrolizumab, potentiate NK and T-cell antitumor activity, prevent NK cell nivolumab and cemiplimab) and three targeting PD-L1 (atezoli- exhaustion and lead to the control of tumor growth. A recent zumab, durvalumab and avelumab). In parallel with this rapidly study provided evidence that TIGIT targeting with specific mAbs expanding arsenal, the spectrum of advanced malignancies for may represent a new promising tool that can be used alone or in which each of these agents is indicated has broadened, including combination with other checkpoints.113 Thus, it has been shown (pembrolizumab and nivolumab), non-small cell lung that TIGIT blockade may induce antitumor immune activity in cancer (pembrolizumab, nivolumab, and atezolizumab), urothelial preclinical models, and its combination with PD-1/PD-L1 inhibitors cancer (pembrolizumab, nivolumab, atezolizumab, durvalumab, is being explored.114 avelumab), renal cell carcinoma (nivolumab), head and neck KLRG1, another inhibitory C-type lectin-like receptor containing cancer (pembrolizumab and nivolumab), Hodgkin lymphoma one ITIM, is expressed by activated NK cells, , T cells, mast (pembrolizumab and nivolumab), microsatellite instability or cells, basophils, and eosinophils.115 The interaction between mismatch repair deficient (dMMR) cancers including dMMR KLRG1 and its corresponding ligand E-cadherin has been shown colorectal cancer (pembrolizumab, nivolumab), gastric cancer to inhibit human ILC2 function in vitro; however, its actual effect (pembrolizumab), hepatocellular carcinoma (nivolumab), Merkel in vivo remains to be established.116–118 In non-small cell lung cell carcinoma (avelumab)140, and cutaneous squamous cell cancers (NSCLC) and colon-rectal carcinoma (CRC), KLRG1 expres- carcinoma (cemiplimab).141 Although PD-1/PD-L1 inhibitors repre- sion was detected in tumor-associated ILC2s.119,120 sent a revolution in the field of oncology, a significant proportion Other inhibitory receptors expressed by NK cells include LAG-3 of patients do not benefit from this class of agents; therefore, (CD223) and TIM-3. LAG-3 is a negative costimulatory receptor predicting tumor responses to PD-1/PD-L1 blockade represents a homologous to CD4 that binds MHC-cl I molecules on antigen major issue. So far, the most widely recognized parameter of presenting cells (APC) with very high affinity. LAG-3 expression is tumor response is PD-L1 expression in tumor cells and/or immune associated with an exhausted profile of tumor-infiltrating- cells, for which four assays have been approved to guide lymphocytes (TIL), and its blockade could restore antitumor treatment decisions for different agents (Dako 22C3, Dako 28-8, immune function.121–123 While its inhibitory activity in T-cell Ventana SP142 and Ventana SP263).142 However, despite the activation, proliferation and homeostasis has been defined, the clinical utility of PD-L1 expression as a biomarker of the response effect of LAG-3 on NK cells remains poorly explored.124 TIM-3, also to PD-1/PD-L1 inhibitors, its predictive value still appears to be known as Hepatitis A virus cellular receptor 2 (HAVCR2), is a type 1 unsatisfactory, to the point that other parameters are being glycoprotein expressed by subsets of T lymphocytes (Th17 and explored (including tumor mutational burden [TMB] and the regulatory T cells), dendritic cells and mature NK cells. The main presence and the numbers of TIL and genetic polymorphisms, ligand of TIM-3 is Galectin-9,125 but other ligands have been among others). In this context, all possible limitations inherent to identified, such as phosphatidylserine (PtdSer), high mobility PD-L1 expression should be taken into account, including intra- group protein 1 (HMGB1), and related and intertumor heterogeneity as well as technical issues cell adhesion molecule 1 (CEACAM1).126–128 Previous studies have represented by the use of different mAbs and different diagnostic shown that in TILs of lung,129 gastric,130 head and neck materials (cytology versus diagnostic biopsies versus surgical carcinomas131, and in melanoma,132 TIM-3 expression is upregu- specimens), in order to improve the predictive potential of PD-L1 lated. Moreover, the increased TIM-3 expression on NK cells in immunohistochemistry.98,99,143–145 In any case, a significant melanoma or lung adenocarcinoma was shown to be associated proportion of patients would still be not eligible or not responsive with impaired NK cell effector function.133,134 TIM-3 is often to PD-1/PD-L1 axis inhibitors. For this reason, as illustrated above, coexpressed with PD-1 and is associated with T-cell exhaustion ongoing research is aimed at finding additional checkpoints other during chronic infection and cancer. Blockade of TIM-3 could than PD-1/PD-L1 and CTLA-4 to be exploited for targeting by restore T-cell effector function in preclinical models and result in therapeutic antibodies either alone or in combination. increased NK cytotoxicity.132,135 Importantly, while the TCR-mediated antitumor activity of T Another important inhibitory receptor, IL1R8 (also known as lymphocytes correlates with TMB, this is not the case for NK cells. TIR8 or SIGIRR), is a member of the IL1 receptor family and a Therefore, therapeutic blockade of inhibitory checkpoint or their component of the human IL-37 receptor.136,137 It has been shown ligands may still be effective in tumors with NK cell infiltration. For to play a physiologic role in the regulation of responses to this reason, there is a growing interest in the less-explored NK cell pathogens by dampening excessive inflammatory responses and compartment for therapeutic purposes.146,147 As indicated, tissue damage. A recent study has shown that IL1R8 is highly promising inhibitory checkpoints are LAG-3, TIM-3, TIGIT, HLA-E, expressed in a subset of NK cells, in which it exerts major and HLA-G, among others. inhibitory control of cell activation and function. In a murine In this context, two HLA-cl I b molecules expressed on tumors model, NK cells lacking IL1R8 could prevent the growth of a may be particularly relevant. carcinogen-induced hepatocellular carcinoma. This study pro- HLA-E is overexpressed in many tumors.148 As illustrated below, vided evidence that IL1R8 functions as an important inhibitory a recent work demonstrated that monalizumab, a humanized anti- checkpoint in NK cell activation and function. Accordingly, its NKG2A antibody, enhanced NK cell activity against various tumor

Cellular & Molecular Immunology (2019) 16:430 – 441 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 436 cells and rescued CD8+ T-cell function alone or in combination different therapeutic approaches are being explored to antag- with PD-1/PD-L1 axis blockade.15 HLA-G is physiologically onize such soluble inhibitors, a particularly relevant approach is to expressed by trophoblast cells, which are mainly involved in identify other inhibitory checkpoints expressed by T or NK cells maternal-fetal tolerance.149 HLA-G can interact with that, when blocked, may unleash the antitumor effector activity of immunoglobulin-like transcript 2 (ILT2) expressed on NK, T and these cells and possibly favor their proliferation. B lymphocytes, monocytes/macrophages, and dendritic cells and Most promising progress towards this goal has recently been with ILT4, which is expressed only by myeloid cells, as well as reported by André et al.,15 who showed that NKG2A may KIR2DL4 expressed on NK cells. Under normal conditions, HLA-G is represent an important checkpoint in tumor therapy. Indeed, virtually absent in adult tissues; by contrast, most tumors express NKG2A targeting, alone or in combination with other checkpoint HLA-G at different levels, either on their cell membranes or inhibitors or with mAbs specific for tumor antigens, led to released as soluble isoforms. Different studies have shown that impressive results both in preclinical and preliminary clinical HLA-G acts by impairing the cytolytic function and proliferation of studies in highly aggressive human tumors (Fig. 1). peripheral and uterine NK cells and of cytotoxic T cells, the As discussed above, specific mAb-mediated blocking of NKG2A maturation and function of dendritic cells while inducing Tregs and/or KIR can restore NK cytotoxicity, which is inhibited by the and myeloid suppressive cells, thus inhibiting different actors interaction with HLA-cl I. Importantly, both inhibitory receptors, involved in antitumor responses.150 Therefore, HLA-G plays a key while constitutively expressed by mature NK cells, have also been role in the induction of , representing an detected in cytolytic T lymphocytes.152,153 The expression of important escape mechanism of tumor cells. However, the CD94/NKG2A can be induced by prolonged antigenic T-cell therapeutic role of HLA-G-based immunotherapy is still virtually stimulation154 or exposure to TGF-β155, a cytokine released in unexplored. We speculate that the use of T or NK cells expressing the tumor microenvironment. Such ex novo NKG2A expression chimeric antigen receptors (CAR-T or CAR-NK) targeting HLA-G leads to the impairment of T-cell function.154,155 Thus, blocking of could represent a promising approach for the adoptive immu- NKG2A was expected to unleash not only NK cells but also T notherapy of different tumors. Studies in this context are in lymphocytes with potential antitumor activity. This was indeed progressing in our labs. proven in elegant preclinical studies in suitable murine models. Subsequently, by studying human cancers, André et al.15 showed that HLA-E (the NKG2A ligand) is expressed in many highly EXPLOITING NK CELLS FOR TRANSLATIONAL APPLICATIONS aggressive tumors (including those of the pancreas, colon, lung, The majority of current tumor immunotherapies are based on the head and neck, stomach, and liver). Moreover, most tumor cells use of mAbs that target tumor antigens or unlock inhibitory examined were HLA-E+. Analysis of lymphoid infiltrates showed checkpoints expressed by T lymphocytes or the generation of that NKG2A was expressed in NK cells and in CD8+ T lymphocytes, T cells expressing CARs specific for tumor antigens. However, which could also coexpress PD1. Experiments in vitro confirmed recent contributions reassessed or confirmed the importance of that IFN-γ production and the cytolytic activity of NK and T cells NK cells in tumor therapy. Indeed, as illustrated under the against HLA-E+ tumors were recovered, at least in part, by headings below, NK cells can fulfill unique antitumor activities blocking anti-NKG2A mAbs, and, in the case of coexpression with such as ADCC, thanks to the expression of CD16 or the killing of PD-1, better functional restoration was documented in combina- HLA-cl I-deficient tumor cells that are undetectable by cytolytic T tion with therapeutic mAbs against PD1/PD-L1 (Fig. 1). This lymphocytes. In addition, masking the HLA-cl I-specific KIR or combined treatment could also restore T-cell proliferation, NKG2A inhibitory receptors may induce NK cell-mediated cytolytic suggesting the possible generation of tumor-specific T-cell activity against HLA-cl I+ target cells. Finally, the generation of memory. CAR-NK cells with extensive proliferative capacity is now available Another promising immunotherapy is based on the combined and may be implemented with CAR- therapy, exploiting the use of anti-NKG2A and tumor antigen-specific mAbs (e.g., an anti- homing properties and functional characteristics typical of NK EGFR mAb). The antitumor effects of these mAbs are, at least in cells. part, dependent on the action of mature NK cells recognizing Ig- opsonized tumor cells via their CD16 to mediate ADCC (Fig. 1). In Targeting NKG2A alone or in combination with other therapeutic these in vitro experiments, the combined use of anti-NKG2A and mAbs anti-EGFR enhanced NK cell-mediated cytotoxicity against As underlined above, blocking monoclonal antibodies specific for antibody-opsonized tumor cells. In this context, in preliminary inhibitory checkpoints have revealed unprecedented potential in clinical studies, the use of monalizumab (anti-NKG2A) combined tumor therapy, representing a true revolution in the prognosis of a with the anti-EGFR cetuximab in an ongoing phase II trial provided subset of otherwise highly aggressive cancers, including NSCL encouraging results in patients with squamous cell carcinoma of cancer, metastatic melanoma, kidney tumors and others.139 In the head and neck (SCCHN). Notably, this study also emphasized spite of this major progress, the majority of patients do not the importance of harnessing NK cell antitumor activity, while respond to treatment with mAbs targeting the CTLA-4 or PD-1- immunotherapeutic strategies so far have been focused on mediated inhibitory pathways. This partial failure may be due to potentiating T-cell responses. When unlocked from NKG2A one or more mechanisms of tumor escape. Thus, as mentioned blocking, NK cells may not only potentiate their natural above, in many instances, tumor progression is accompanied by a cytotoxicity but also kill tumor cells via ADCC in the presence of partial or total loss of classical HLA-cl I antigens, which are tumor-specific therapeutic mAbs. necessary for tumor antigen presentation to cytolytic T lympho- cytes. In addition, the tumor microenvironment may greatly Role of NK cells in haploidentical HSCT for the cure of high-risk contribute to tumor escape. Indeed, an important mechanism is leukemia the release of cytokines (e.g., TGF-β) and soluble inhibitory factors HSCT is the life-saving therapy for acute leukemias with either (e.g., kynurenine, PGE2), not only from tumor cells but also from adverse cytogenetic or molecular characteristics or poor response tumor-infiltrating cells (e.g., myeloid-derived suppressor cells, M2 to chemotherapy or relapse. However, only 2 out of 3 patients macrophages, fibroblasts/MSC). Additionally, the hypoxic environ- requiring HSCT can find a suitable HLA-compatible donor. A novel ment typical of many tumors may play a substantial negative transplantation approach has been attempted to rescue such role.151 Of note, all of these mechanisms impair both T- and NK patients for whom no other valuable therapeutic option are cell function by targeting receptor-associated polypeptides that available. Thus, haploidentical HSCT, developed in the late 90s, is are crucial for the transmission of activating signals. Although based on the infusion of “megadoses” of highly purified CD34+

Cellular & Molecular Immunology (2019) 16:430 – 441 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 437

1

TUMOR CELL

Monalizumab HLA-E (an-NKG2A mAb) 3

NKG2A

Monalizumab (an-NKG2A mAb) NK CELL PD-1 HLA-E CD16 Durvalumab (an-PD-L1 mAb) Cetuximab (an-EGFR mAb) TUMOR PD-L1 EGFR CELL Monalizumab (an-NKG2A mAb) TUMOR HLA-E CELL

2

Fig. 1 Effects of blocking NKG2A alone or in combination with other therapeutic mAbs disrupting the PD-1/PD-L1 axis or directed to tumor- specific antigens. (1) Cytolytic NK cells expressing the NKG2A inhibitory receptor acquire the ability to kill HLA-E+ tumor cells upon mAb- mediated masking of NKG2A. (2) NKG2A+ NK cells that also express PD1 are rescued from their anergic state and kill HLA-E+ PD-L1+ tumor cells upon disruption of the two inhibitory pathways with mAbs specific for NKG2A and PD-L1 (or PD-1), respectively. (3) The therapeutic activity of mAbs directed to tumor-specific antigens (in this case EGFR) is mainly mediated via CD16 (FcγRIII) expressed by highly mature cytolytic NK cells. However, in the case of HLA-E+ tumors, the coexpression of NKG2A may represent a major brake on NK-mediated function. Accordingly, masking of NKG2A may restore NK cell function and allow killing of tumor cells targeted by specific mAbs haemopoietic precursors isolated from a parent or a sibling. Given immunocompromised patients). Thus, the infused mononuclear the high degree of HLA incompatibility, deep T-cell depletion is cells contained, in addition to CD34+ progenitors, mature KIR+ mandatory to avoid severe, life-threatening graft versus host (possibly alloreactive) NK cells and TCRγδ T cells,159 plus different disease (GvHD). In this transplantation setting, NK cells of the myeloid cells. Notably, not only NK cells but also TCRγδ T cells can donor may express KIRs that are mismatched with the HLA-cl I kill leukemia blasts.160 The prompt availability of effector cells alleles of the patient. The first wave of lymphoid cells in patient PB capable of attacking leukemia and exerting control of viral occurs at ~2 weeks after transplant and is composed almost reactivation greatly improved the clinical outcome. Indeed, the exclusively of NK cells. However, these NK cells are CD56bright and overall survival probability was ~70% for AML and over 70% for express NKG2A but not KIR. More mature KIR+NK cells are ALL. Altogether, these data clearly indicate that NK cells exert detectable only after additional 4-6 weeks. In the absence of strong anti-leukemia activity. The ability of CD34+ cells from T cells, NK cells have been shown to play a prominent role in the different sources to give rise to ILC3s, including NCRnegILC3s with anti-leukemia effect.156 While NK cells per se may kill leukemia LTi-like activity, which contribute to tissue repair and regeneration cells that express low levels of HLA-cl I molecules, the anti- of lymphoid tissues damaged by chemo/radiotherapy during leukemia effect has been primarily related to NK cell alloreactivity conditioning, should also be carefully evaluated.161 consequent to a KIR-HLA mismatch (in the donor vs recipient direction).156 Studies by our group revealed a direct correlation between the size of the alloreactive population and the degree of CONCLUDING REMARKS anti-leukemia cytotoxicity in vitro as well as the survival A large body of experimental evidence, particularly from probability of the patient.157,158 Thus, clinical data in over 80 preclinical studies of both in vitro and in vivo animal models, pediatric patients receiving haplo-HSCT indicated the treatment indicates that NK cells play relevant roles in antitumor defenses. was particularly good for high-risk ALL with an ~70% 5-year This is due to their direct cytolytic activity against tumor cells and survival probability in the presence of NK alloreactivity and ~40% their ability to produce cytokines that shape effective (Th1) in the absence of alloreactivity. The overall survival was ~60%. The downstream adaptive responses. However, different mechanisms outcome was less favorable for AML (~40% and ~20% with and occurring primarily in the tumor microenvironment may greatly without NK alloreactivity, respectively, and 30% overall survival). impair NK cell function by compromising their antitumor activity, Although all these patients would have died in the absence of and they may even play an unwanted role in tumor promotion. HSCT, the overall survival was not satisfactory, particularly for AML This may occur when the NK cells present at the tumor site are few patients. Thus, a novel transplantation setting has been applied in and/or their function is compromised. The result may be NK- an attempt to fill the gap between the time of HSCT and the mediated tumor editing (i.e., selection of the most resistant tumor generation of mature KIR+ alloreactive NK cells. Indeed, most of cells)162 or the induction of epithelial/mesenchymal transition the deaths occurred in the first weeks after HSCT due to leukemia (that favors tumor spread and metastasis).163 In this context, any relapse or infections. This approach is based on the selective therapeutic approach that may favor/reconstitute NK cell function depletion of T cells expressing TCRαβ, which are responsible for and expansion may be useful in tumor therapy. Thus, mAb- GvHD, and B cells (to avoid B cell malignancies in mediated masking of HLA-cl I inhibitory receptors may allow the

Cellular & Molecular Immunology (2019) 16:430 – 441 Human NK cells: surface receptors, inhibitory checkpoints, and. . . S Sivori et al. 438 + direct attack of NK cells towards HLA-cl I tumor cells. Importantly, 12. Colonna, M. & Samaridis, J. Cloning of immunoglobulin-superfamily members our group demonstrated that such inhibitory receptors, particu- associated with HLA-C and HLA-B recognition by human natural killer cells. larly NKG2A, also can be expressed on CD8+ CTLs following Science 268, 405–408 (1995). antigen- or cytokine-induced proliferation or exposure to TGF- 13. Wagtmann, N. et al. Molecular clones of the p58 NK cell receptor reveal β 152,154,155 immunoglobulin-related molecules with diversity in both the extra- and intra- . These data offered clues to interpret recent studies by – Andrè et al.15 showing the efficacy of blocking NKG2A in tumor cellular domains. Immunity 2, 439 449 (1995). fi 14. Braud, V. M. et al. HLA-E binds to receptors CD94/NKG2A, B and therapy. On the other hand, the recent nding that not only T but C. Nature 391, 795–799 (1998). also NK cells may express PD-1 revealed a new perspective, 15. Andre, P. et al. Anti-NKG2A mAb is a checkpoint inhibitor that promotes anti- especially for the therapy of HLA-cl I-negative tumors (invisible to tumor immunity by unleashing Both T and NK cells. Cell 175, 1731–1743 e1713 T lymphocytes). (2018). It should be underscored that the major therapeutic success of 16. Mingari, M. C., Pietra, G. & Moretta, L. Immune checkpoint inhibitors: Anti- haploidentical HSCT in the cure of high-risk leukemia is mostly NKG2A antibodies on board. Trends Immunol. 40,83–85 (2019). related to NK cell activity. Great expectations for the therapy of 17. Anfossi, N. et al. Human NK cell education by inhibitory receptors for MHC class – both hematologic malignancies and solid tumors are based on the I. Immunity 25, 331 342 (2006). use of NK cells expressing CARs specific for tumor antigens. These 18. Kim, S. et al. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436, 709–713 (2005). CAR-NK cells may complement/substitute CAR-T lymphocytes in 19. Joncker, N. T., Fernandez, N. C., Treiner, E., Vivier, E. & Raulet, D. H. NK cell view of their potent cytolytic activity and their particular homing responsiveness is tuned commensurate with the number of inhibitory receptors capacity. for self-MHC class I: the rheostat model. J. Immunol. 182, 4572–4580 (2009). 20. Sivori, S. et al. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J. Exp. Med. 186, 1129–1136 (1997). ACKNOWLEDGEMENTS 21. Pessino, A. et al. Molecular cloning of NKp46: a novel member of the immu- This study is dedicated to Alessandro Moretta, who sadly passed away on February noglobulin superfamily involved in triggering of natural cytotoxicity. J. Exp. Med. 17, 2018. His seminal discoveries of inhibitory and activating NK receptors made it 188, 953–960 (1998). possible to understand how NK cells function. Many of the issues described in this 22. Sivori, S. et al. NKp46 is the major triggering receptor involved in the natural review are based on his pioneering studies. We greatly miss his scientific insight and cytotoxicity of fresh or cultured human NK cells. Correlation between surface even more his humanity and smile. Supported by grants awarded by Associazione density of NKp46 and natural cytotoxicity against autologous, allogeneic or – Italiana per la Ricerca sul Cancro (AIRC)-Special Program Metastatic disease: the key xenogeneic target cells. Eur. J. Immunol. 29, 1656 1666 (1999). unmet need in oncology 5 per mille 2018 Id. 21147 (S.S. and L.M.), AIRC IG2017 Id. 23. Vitale, M. et al. NKp44, a novel triggering surface molecule specifically expressed 20312 (S.S.), AIRC IG2017 Id.19920 (L.M.), RC-2018 OPBG (P.V. and L.M.); 5 × 1000 by activated natural killer cells, is involved in non-major histocompatibility Italian Ministry of Health 2015 (M.C.M); and Ministero della Salute RF-2013, GR-2013- complex-restricted tumor cell lysis. J. Exp. Med. 187, 2065–2072 (1998). 02356568 (P.V.). 24. Pende, D. et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. 190, 1505–1516 (1999). ADDITIONAL INFORMATION 25. Moretta, A. et al. Activating receptors and coreceptors involved in human nat- ural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19, 197–223 (2001). Competing interests: The authors declare no competing interests. 26. Pazina, T., Shemesh, A., Brusilovsky, M., Porgador, A. & Campbell, K. S. 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