ROLE OF NK CELLS IN IMMUNOTHERAPY AND VIROTHERAPY OF SOLID TUMORS.

Claudia Cantoni (a,b,c), Korneel Grauwet (d), Gabriella Pietra (a, e), Monica Parodi (a), Maria Cristina Mingari (a, b, e), Andrea De Maria (b, e, f, ), Herman Favoreel (d, ), Massimo Vitale (e, )(a) Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy(b) Center of Excellence forBiomedical Research (CEBR), University of Genova, Genova, Italy(c) Istituto Giannina Gaslini, Genova, Italy(d) Laboratory of Immunology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Belgium(e) IRCCS AOU San Martino-IST Genova, Genova, Italy(f) Department of Health Sciences (DISSAL), University of Genova, Genova, Italy() These authors share senior authorship

KEYWORDS: NK cells solid tumor tumor microenvironment - tumor escape immunotherapy virotherapy

Correspondence to:Massimo VITALE: massimo.vitale@hsanmartino.itClaudia CANTONI: claudia.cantoni@unige.it

ACKOWLEDGEMENTS:This work was supported by grants awarded by AIRC: IG project n. 15428 (M.V.); Ministero della Salute: Ricerca Finalizzata RF-2010-2316197 (A.D.M),RF-IG-20081200689 (M.C.M.); Programma nazionale di ricerca sullAIDS, Accordi di collaborazione scientifica 45G.11, 40H69 (A.D.M.); MIUR Progetto di Ateneo 2014(A.D.M); the Special Research Fund of Ghent University (grants 01J29110, 01J11611 and 01G01311) (H.F.); F.W.O.-Vlaanderen (grants 1.5.077.11N and G.017615N) (H.F.); the Hercules Foundation (grant AUGE-035) (H.F.), and an international mobility grant from F.W.O.-Vlaanderen (H.F.).

ABSTRACTAlthough NK cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors (TKi), Immunomodulatory Drugs (IMiDs), and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

INTRODUCTIONImmediately after their discovery, NK cells caught the attention of many researchers for their ability to kill a rather wide range of tumor cell types. From the outset, NK cells appeared as a ready to use cell population that could be employed against a variety of tumors without the need for selecting specific clones, as virtually all NK cells were endowed with a MHC-unrestricted wide specificity for tumor cells. The exploitation of these cells for effective immunotherapy seemed an achievable goal and promptly stimulated to investigate the mechanisms regulating their function. The brilliant intuition of Klas Karre (who postulated that NK cells were able to kill only when they missed the recognition of autologous MHC-I molecules on target cells) and the pioneering studies of different groups led quite rapidly to define the recognition mechanism of NK cells [1-4]. Human NK cells were demonstrated to recognize and kill tumor cells with down-regulated HLA-I molecule expression by using a complex group of HLA-I-specific inhibitory receptors (KIRs and NKG2A) and several major activating receptors (including NKG2D, DNAM-1 and the Natural Cytotoxicity Receptors - NCRs: NKp46, NKp30, NKp44) [5, 6]. The subsequent definition of the panel of activating receptor ligands (currently still incomplete) confirmed that, indeed, some of such ligands are commonly expressed by various tumor cell types (see Table 1).In spite of the substantial advance in our understanding of NK cell biology, the attempts to exploit these cells in immunotherapy have been frustrating for many years. The efficacy of the adoptive transfer of ex vivo activated NK cells and/or their in vivo activation by IL-2 infusion was challenged by the short lifespan of transferred mature NK cells, and by the clinical side effects induced by the cytokine [7-9]. In addition, IL-2 could also have paradoxical negative effects on the immunological response by inducing the expansion of CD25+ T regulatory cells (Tregs) or the Activation Induced Cell Death (AICD) on NK cells [10, 11]. Finally, at variance with in vitro established tumor cell lines, in several instances, primary tumor cells maintain the expression of HLA-I molecules and are thus protected from the attack of autologous NK cells. Important hints to overcome these problems were offered by Andrea Velardis group, who suggested that allogeneic NK cells could develop from donors stem cells following haplo-identical Hematopoietic Stem Cell Transplantation (HSCT) in patients with AML [12]. In the T-depleted haplo-HSCT, the HSC recipient can express HLA-I alleles that are not recognized by KIRs of donor NK cells (HLA/KIR mismatch). Such allogeneic NK cells expressing KIRs that fail HLA-I allele recognition exert an effective anti-leukemia activity in the patient. Thus, under this particular allogeneic setting, NK cells derived from donors progenitors overcome the limitations that initially hindered the design of effective NK-based immunotherapies. During the past 15 years, haplo-HSCT revealed itself as an extremely useful tool to cure high-risk leukemia patients lacking an HLA-matched donor. However, so far this approach has been mostly confined to hematological malignancies both in adults and in children. [13, 14].In solid tumors the picture may be more complex. The development of a tumor-orchestrated local microenvironment can heavily impact on the availability and function of NK cells within the tumor tissue [15-17]. A plethora of soluble factors and cellular networks, as well as peculiar physical and/or chemical conditions, can affect the ability of NK cells to recognize and kill tumor cells, or influence their ability to migrate from the blood to the tumor site. It should also be considered that NK cells present in the tumor microenvironment may directly originate from the local peripheral tissues. In this context, recent studies have shown that different peripheral tissues and secondary or tertiary lymphoid organs may contain members of an NK-related family of innate lymphoid cells (ILCs). These cells can use their receptors to fulfill peculiar functions, different from killing tumor cells [18-21]. This still expanding heterogeneous group of ILCs include: 1) conventional cytotoxic NK cells (recently proposed to be defined as cytotoxic ILC1), which constitute the large majority of peripheral blood (PB) NK cells (but are also present in tissues), release IFN- and TNF-, and functionally interact with different APC; 2) poorly cytolytic CD56brightCD16dim NK cells, which constitute the large majority of lymph nodal (LN) NK cells (but are also present in tissues and PB), release primarily IFN- and GM-CSF, and interact with DC, participating to the induction of Th1 polarization in LN; 3) NKp44+ NK22 cells (included in the ILC3 group), which are located in the MALT, release IL-22 and are involved in the mucosal homeostasis and defense; 4) decidual NK cells, which produce IFN-, IL-8, IP-10, SDF-1 and VEGF, and are thought to play an important role in the placental development and induction of maternal-fetal tolerance [22]. This complex repertoire of NK cell subsets with their different relative functions should also be considered to better exploit their functional capabilities in old and new therapeutic approaches, either to identify new possible beneficial synergies, or even to recognize still unnoticed NK-driven unwanted effects.Another well-known function of NK cells has also become important for tumor therapy, namely their ability to recognize and kill virus-infected cells [5-6]. This aspect of NK cell biology has come into play by the design of strategies to target tumor cells using oncolytic virotherapy [23]. In this context, NK cells may represent a double edged sword as the NK-mediated killing of virally infected tumor cells may either potentiate the anti-tumor effect of viruses or limit the infection spread within the tumor [24, 25] and therefore prevent success from oncovirotherapy.Thus, in contrast to the initial idea, the exploitation of NK cells for the cure of cancer is quite complicated and requires multiple approaches that should be adapted to the type of tumor and/or adopted therapeutic strategy.

NK CELLS AND THE TUMOR MICROENVIRONMENTThe presence of an NK cell infiltrate has been observed to variable extent in several types of solid tumors including melanomas, gastrointestinal stromal tumors (GIST), colorectal, renal, lung and breast cancers [26-34]. In some cases, GIST and renal carcinoma lung metastases displayed high levels of NK cell infiltration, which correlated to a better prognosis [29, 32]. The type and the size of the NK cell infiltrate in neoplastic tissues can depend on several factors, including the specific chemokines produced in the tumor microenvironment and the chemokine receptor pattern expressed by different NK cell subsets. In lung and breast cancers it has been shown that poorly cytotoxic CD56bright NK cells frequently represent the major NK cell subset recruited at the tumor site [31,33]. These tumors expressed high levels of mRNA coding for CXCL9 and CCL19, two chemokines are recognized by chemokine receptors selectively expressed by CD56bright cells (CXCR3 and CCR7 respectively). On the other hand, some studies also report a significant presence of CD56dim NK cells in lung tumors and in melanoma nodal metastases [30, 34]. In the latter case, cells from tumor tissue were demonstrated to produce IL-8, which, indeed, can preferentially attract CD56dim NK cells. Another important issue is the location of the NK cell infiltrate within the tumor tissue. In colorectal cancer studies it has been recently shown that, in spite of the presence of chemokines that are able to recruit both CD56bright and CD56dim NK cell subsets, NK cells are localized within the stroma, rather than being in direct contact with neoplastic cells [35]. A similar NK cell localization has been detected in melanoma and in GIST [32, 36, 37]. These findings suggest that additional poorly identified factors may influence NK cell recruitment to the tumor nest.Besides affecting NK cell migration into the neoplastic tissue, the tumor microenvironment can also influence the efficacy of NK cells within the tumor. Different mechanisms can contribute to tumor escape from an NK cell-mediated attack: 1) the induction of an immunosuppressive milieu; 2) the inhibition of NK cell effector functions; and 3) the selection of tumor cells with an altered expression of ligands for NK cell receptors [15, 17, 38]. Different immune cells have been shown to interact with NK cells and regulate their function. In a TH1/M1-polarized context, this interaction results in an improved anti-tumor immune response [39, 40]. Unfortunately, immune cells in the tumor microenvironment are frequently altered or polarized towards a type 2 response [41] and may inhibit/modulate the capacity of NK cells to interact with and kill tumor cells [15, 17]. Recent studies performed on hepatocellular carcinoma have shown that myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) from patients are able to modulate NK cell functions [42, 43]. One important mechanism accounting for the suppression of NK cells is the production of several factors, including TGF-, IDO, IL-4, and PGE2 [44-47], which can hinder NK cell activation or induce down-regulation of NK activating receptors responsible for recognition and killing of tumor cells. For example, TGF- inhibits the expression of NKp30 and NKG2D activating receptors [45], while IL-4 decreases the ability of NK cells to produce cytokines and to interact with DCs [46]. Both IDO and PGE2 can affect the expression of different activating receptors on NK cells, with a consequent decrease in NK cell cytotoxic activity against tumor cells [36, 47]. These soluble factors can be produced by immune and/or non-immune cells within the tumor microenvironment. For example, Tregs can dampen NK cell function by competing for IL-2 availability, but also by exposing membrane-bound TGF- [48, 49]. Among non-immune cells, fibroblasts associated to tumor tissues (TAF), obtained from melanoma metastases, have been shown to prevent the IL-2-induced up-regulation of NKp44, DNAM-1 and NKp30 receptors. This effect was mediated both by direct cell-to-cell contact and by PGE2 release [36]. A similar behavior has been observed in TAFs derived from colorectal cancer (HCC) and hepatocellular carcinoma [50, 51]. The relevance of NK cell-fibroblast interactions in vivo is further suggested by the finding that in melanoma metastases and GIST lesions NK cells can localize in proximity to fibroblasts surrounding tumor nests [32, 36, 37].A substantial suppressive role is also exerted by tumor cells through several mechanisms. We have recently demonstrated that melanoma-derived primary cell lines could inhibit the expression of several activating NK receptors, thus decreasing NK cell-mediated killing of tumor cells. This effect was mediated by IDO expression and/or PGE2 release [52]. Different tumor cell types can release MHC class I chain-related gene A (MICA), a ligand for NKG2D activating receptor, by shedding, thus inducing down-regulation or unresponsiveness of NKG2D [53]. Ovarian cancer cells can interfere with NK cell functions by inducing NKG2D down-regulation through the release of macrophage inhibitory factor (MIF), or by expressing the glycoprotein MUC16, which can inhibit synapse formation between NK cells and tumor cells [54, 55]. Tumor cells can also affect the ability of NK cells to infiltrate the tumor. It has been recently shown that neuroblastoma cells could modulate the chemokine receptor repertoire of NK cells through TGF- production [56]. Tumor cells from different cancers can release B7-H6, a ligand for NKp30 activating NK receptor, through ectodomain shedding mediated by cell surface proteases ADAM-10 and ADAM-17, thus reducing NK cell-mediated recognition [57]. Finally, hypoxia, a condition frequently associated to the tumor microenvironment, can influence the immune response against the tumor. As far as NK cells are concerned, we have recently demonstrated that the hypoxic status can deeply inhibit both the expression and the function of different activating receptors. NK cells, however, were not affected by hypoxia in their ability to perform Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) [58]. Comment by Korneel: Hypoxia can also alter ligand presentation of the tumor cells (Yamada N., Int J. Oncol, 2012)(is not discussed)More info on NK side:Hypoxia impairment of NK cells can be reduced by IL2 expression(Sarkar, S., Plos One, 2013)The anti-tumor activity of NK cells and their prolonged interaction with cancer cells can also exert a selective pressure that may favor the generation of tumor cells displaying an abnormal expression of ligands specific for activating or inhibitory NK cell receptors. Our group has shown that in vitro co-culture of melanoma cells with NK cells resulted in an IFN--dependent up-regulation of HLA-I expression on melanoma cells, which was responsible for the (acquired) resistance of these cells to NK-mediated killing [26]. Tumor cells may also escape from NK-mediated recognition by modulating cell surface expression of ligands for activating NK receptors, as in the case for NKG2D ligands [53]. Activating receptors expressed on NK cells may be important for tumor immunoediting. For example, it has recently been shown that tumors that developed in NKp46-deficient mice expressed higher levels of NKp46 ligands as compared to tumors that developed in wild type mice [59]. The up-regulation of ligands able to transmit inhibitory signals to NK cells represents another possible mechanism of tumor escape from NK cell anti-tumor activity. In this context, it has been recently shown that Proliferating Nuclear Cell Antigen (PCNA), frequently overexpressed in different tumor types, can engage the activating NKp44 receptor and induce a paradoxical inhibitory signal [60]. NK cell cytotoxic function can also be inhibited through the engagement of CD300a, an inhibitory receptor recognizing phosphatidylserine exposed on certain tumor cells [61].

ROLE OF NK CELLS IN THERAPIES TARGETING SOLID TUMORS

Although cancer immunotherapy strategies have mainly relied on the activity of tumor antigen specific T cells, in recent years the therapeutic use of NK cells to treat malignancies has rapidly moved forward [62, 63]. In this section, we will review the NK cell-based therapeutic approaches that are being currently explored in either clinical or pre-clinical contexts. These approaches include: a) in vivo stimulation of endogenous NK cells against tumors (by the use of cytokines, antibodies or immuno-modulatory drugs); b) adoptive transfer of in vitro expanded and activated NK cells (both in autologous and allogeneic settings); c) genetic modification of NK cells. The stimulation of different NK cell functions, including cytotoxicity against tumor cells and IFN- production can be induced by the use of several cytokines including IL-2, IL-15, IL-12, IL-18 and interferons . The administration of recombinant IL-2 resulted in remarkable cytokine-related toxicity (such as vascular leak syndrome and heart toxicity) and limited clinical benefits (possibly due to the induction of Tregs) [8, 64], These issues prompted the search for other cytokines able to effectively boost NK cells. IL-15, IL-12, and IL-18 are being evaluated both in preclinical [65] and clinical cancer models [66-68]. While these cytokines display limited anti-cancer activity when used as single agents, a recent report in mice showed that poorly functional NK cells, infiltrating MHC class I-deficient tumors, could be effectively boosted by the combined delivery of IL-12 and IL-18 [69]. This study warrants the evaluation of the possible use of this cytokine combination in humans. The efficacy of NK cells in targeting tumor cells may be also increased by intervening with the inhibitory interaction between KIRs and HLA-I molecules, that are possibly expressed on tumor cells. In this context, a human monoclonal antibody (mAb) (1-7F9/IPH2101) able to block KIRs that recognize HLA-C molecules has been recently developed [70]. The use of lirilumab (a recombinant version of 1-7F9) has been shown to increase NK cell-mediated killing of HLA-C positive tumor cells in an animal model [71].The therapeutic potential of PB NK cells can also be exploited for the treatment of both hematological and solid tumors thanks to their ability to mediate ADCC through crosslinking of CD16. The NK cell-mediated ADCC has been suggested to be relevant to the therapeutic effect of Rituximab or Cetuximab in mAb-based tumor-targeted therapies [72]. Along this line, several reports indicated that NK cell function could be harnessed by the administration of mAbs recognizing tumor antigens [73].Besides cytokines and antibodies, several immunomodulatory drugs are able to reinstate NK cell effector function either directly or indirectly. Among these drugs, Lenalidomide (a synthetic derivative of Thalidomide) has been extensively tested in patients. Interestingly, Lenalidomide not only directly acts on NK cells (by promoting both their natural cytotoxicity and ADCC function) but it can also sensitize tumor cells to NK-mediated killing (by up-regulating the expression on tumor cells of ligands recognized by activating NK receptors) [63]. Along this line, several chemotherapeutic drugs (such as etoposide and dacarbazine), histone deacetylases inhibitors (such as VPA), HSP-90 inhibitors and proteasome inhibitors are able to induce the expression of TRAIL and NKG2D-Ls on tumor cells [74]. Finally, also the TKi are being evaluated for their effects on the NK-mediated tumor cell killing [reviewed in 74]. The interest on the TKi was stimulated by the initial observation that Imatinib therapy of GIST patients could induce an increased NK cell function that correlated with a positive therapy response. The subsequent studies led to a rather complex picture showing that different members of the TKi family can modulate the NK cell function through combined, even contrasting, actions, ranging from the suppression of Tregs or the enhancement of the NK/DC cross-talk, to the reduction of NKG2D-L expression on tumor cells.Perhaps the most interesting approach to increase the efficacy of NK cells is the search for possible synergies between the above described strategies. For example, the synergistic effect of the KIR blockade by Lirilumab and the Rituximab-mediated ADCC has been recently tested in a KIR transgenic murine model [71]. A clinical trial using IPH2101 anti-KIR mAb combined with lenalidomide is in progress in multiple myeloma [75].The early studies employing ex vivo lymphokine-activated killer (LAK) cells showed no clear benefit of this form of therapy compared with IL-2 administration [8, 76]. In a recent study, Rosenberg and colleagues [77] reported that a lymphodepleting chemotherapy, consisting of fludarabine and low-dose cyclophosphamide, might promote homeostatic expansion of autologous adoptively transferred NK cells. Interestingly, these cells expressed CD16 and thus could mediate ADCC without cytokine activation [77].Autologous NK cells may be inhibited by recognition of self HLA-I molecules expressed by tumor cells. Therefore, the use of allogeneic NK cells in a KIR/HLA-I mismatch setting may be tentatively appealing, especially in view of the promising results obtained with HSCT in hematologic malignancies [12, 13]. The adoptive transfer of allogeneic NK cells is under investigation in solid tumors, as witnessed by recent clinical trials evaluating the safety and the efficacy of this approach [78-80]. However, the suppressive effects related to the local microenvironment as well as the use of mature NK cells (instead of HSCT) may complicate the issue.The poor ability of transferred NK cells to home to tumor sites represents another important negative factor that might explain, at least in part, the weak anti-tumor effect of adoptive NK cell therapy against solid malignancies. Recently, it has been shown that NK cells, when exposed to IL-15 and glucocorticoids, express high levels of CXCR3 thus increasing their potential to infiltrate CXCL10+ tumors [81].Finally, another intriguing new therapeutic strategy, not yet arrived in clinical testing, is the tumor targeting with genetically modified NK cells engineered to express chimeric antigen receptors specific for tumor antigens (CAR) [82] or cytokines.

ONCOLYTIC VIROTHERAPY AND SOLID TUMORSOver the past two decades, virotherapy has received increasing attention with regard to its potential application towards (particularly hard to treat) cancers with very poor prognosis, such as glioblastoma. The general idea behind oncolytic virotherapy is the use of (genetically engineered) viruses that selectively replicate in and lyse tumor cells, while sparing healthy tissue. Remarkably, this approach may be particularly interesting also in the cure of solid tumors, since virus replication and spread may enable the oncolytic vector to penetrate the tumor, and thereby enhance immune and therepautic drug penetration in combination therapies (insert old reference 86 and 87; Woller, 2014; Chiocca, 2014)..Comment by Korneel: 86Woller N, Grlevik E, Ureche C-I, Schumacher A, Khnel F. Oncolytic viruses as anticancer vaccines. Front. Oncol. 4, 188 (2014).87Chiocca EA, Rabkin SD. Oncolytic viruses and their application to cancer immunotherapy. Cancer Immunol. Res. 2(4), 295300 (2014).Since the first approval of an oncolytic virus, adenovirus based oncolytic virus H101 (Oncorine), for cancer treatment in China in 2005, in particular for head and neck cancer in combination with cisplatin-based chemotherapy, the field is rapidly moving forward (Yu W., 2007, Cancer Drug targets Clinical trials with oncolytic adenovirus in china) (Xia ZJ, Ai Zheng, 2004)(Garber K, 2006 Journal of national cancer institute) (insert old reference 83, Pol J., 2014). This is illustrated by different oncolytic vectors currently in different stages of clinical trials, as shown in table 2. Oncolytic adenovirus CG0070, modified to express GMCSF under control of the human E2F-1 promotor, is being prepared for phase II/III clinical trials to treat invasive bladder cancer and has provided promising results in phase I testing. In phase I testing, a total of 81% of treated patients responded to the treatment and the maximum tolerated dose was not reached, demonstrating the slumbering potential of oncolytic virotherapy (Burke JM, 2012, J.Urol).Comment by Korneel: http://www.ncbi.nlm.nih.gov/pubmed/15601557Comment by Korneel: Pol J, Bloy N, Obrist F, et al. Trial Watch:: Oncolytic viruses for cancer therapy. Oncoimmunology. 3, e28694 (2014). A broad range of oncolytic viral vectors (based on HSV-1, reovirus, adenovirus, rhabdovirus, parvovirus, Newcastles disease virus, vesicular stomatitis virus, vaccinia virus and measles virus) are being assessed in recently launched phase I/II trials to treat a variety of solid cancers. The oncolytic viruses ParvOryx, NDV and Reolysin are naturally occurring viruses selected due to their oncolytic antitumor potency and ability to preferably target and replicate in tumor cells, whilst other oncolytic viruses, such as MV-NIS, are based on attenuated laboratory strain viruses reported to preferably replicate in and lyse tumor cells (MV-NIS: Dingli D., 2004, Blood )(ParvOryx: Geletneky, BMC Cancer, 2012) (NDV: Mansour, M, Journal of Virology, 2011) (Reolysin: Galanes E. Molecular therapy, 2012). Several oncolytic viruses that are being developed to treat solid tumors have been genetically modified to increase tumor selectivity. The oncolytic virus ColoAd1 was generated by passaging a mixture of different adenovirus serotypes on target tumor representatives of breast, colon, prostate and pancreatic cancer to select for tumor specificity and oncolytic capabilities (Kuhn, 2008, Plos One). The oncolytic HSV-1-based virus HSV1716 lacks the viral virulence factor ICP34.5 which attenuates the virus since it loses the ability to dephosphorylate eukaryotic translation initiation factor 2, and, thereby, fails to suppress host cell-mediated shut-off of protein synthesis in healthy cells (HSV17161: MacLean AR, J Gen Virol, 1991(Harland, 2002, Gene therapy)(Papanastassiou V, Gene Ther, 2002)(Leib DA, PNAS, 2000)(Chou J., 1995, PNAS). However, by yet unidentified factors, HSV1716 was found to be less attenuated in various tumors cells, thereby providing some tumor selectivity (Randazzo BP, Virology, 1996). Comment by Korneel: http://www.ncbi.nlm.nih.gov/pubmed/14604966Comment by Korneel: http://www.ncbi.nlm.nih.gov/pubmed/10801979Comment by Korneel: http://www.ncbi.nlm.nih.gov/pubmed/7479831Although removal of virulence factors can drastically increase safety, severe attenuation of the oncolytic virus can also interfere with its oncolytic efficacy. To circumvent this problem, oncolytic viruses have been constructed that are specifically attenuated in non-tumor cells. For example, the oncolytic virus RQNestin34.5, a vector based on an ICP34.5 deleted-HSV-1 strain contains the ICP34.5 gene under control of the tumor selective nestin-promotor. As a result, this vector shows specifically increased replication in tumor cells and enhanced antitumor specificity and potency in mice, compared to the ICP34.5-deleted HSV-1 backbone (Kambara, Cancer Res, 2005). This rQnNestin34.5 vector is currently being investigated in phase I clinical trials (Ning J, 2014, frontiers in microbiology).Several oncolytic viruses currently assessed in clinical trials express additional genes with the intent to manipulate the extracellular tumor environment (such as the extracellular matrix), to modulate the hosts immunity or to analyze the oncolytic virus spread in vivo by reporter genes. Interestingly, the VCN-01 virus, a human adenovirus currently assessed in clinical phase I trials, is particularly designed to address current solid tumor specific restrictions. The dense extracellular tumor matrix has been identified as a potential additional hurdle in virotherapy of solid tumors (Reviewed in: Wojton, 2010, Cytokine growth factor Rev.). This interlocked meshwork of matrix proteins forms a physical barrier that not only suppresses access of NK cells and other immune cells, but also hinders spread and penetration of the oncolytic virus, leading to suboptimal therapeutic effects. In line with this, virus spread in solid tumors was reported to be limited to discrete tumor areas upon ex vivo infection of solid tumors with Newcastles disease virus, and in clinical tissues from patients treated with G207, an HSV1-based oncolytic virus with completed phase I/II trials (Yaakov, 2012, Journal general virology) (Markert JM, 2009, Mol Ther). In this respect, the oncolytic adenovirus-based vector VCN-01 was designed to degrade hyaluronic acid, a major constituent of extracellular tumor matrix, via expression of the PH20 hyaluronidase. In mice with prestablished tumors, expression of PH20 hyaluronidase by an oncolytic virus following intratumoral and systemic administration has been reported to increase efficacy and improved spread within the tumor, compared to the empty virus backbone (Guedan S, Mol Ther, 2010). Preclinical trials with VCN-01 in mice and hamsters also provided promising results, supporting selectivity, safety and antitumor activity of VCN-01 (Rodriguez-Garcia, 2015, Clin Cancer).

ONCOLYTIC VIROTHERAPY AND NK CELLSThe interaction of oncolytic viruses with the host immune system is particularly delicate and may have a critical impact on the therapeutic efficacy. On the one hand, oncolytic vector-triggered immune responses may be essential elements of the anti-tumor effects of the therapy. On the other hand, premature clearance of the self-replicating oncolytic vector by the host immune response may severely limit therapeutic potential [25]. Hence, one of the most important challenges towards the generation of successful self-replicating oncolytic viral vectors is to fine-tune the interaction of the vector with the host immune system in such a way that beneficial vector-triggered antitumor immune responses are maximized while allowing sufficient time for the vector to replicate and spread before being eliminated by the immune response. Particularly important players in this delicate balance are NK cells, displaying both potent anti-tumor and anti-viral activity.

Natural Killer cell virus interaction. The interaction of viruses with NK cells finds its frame within the co-evolutionary adaptation of the immune system to invading pathogens. After the first characterization of their mechanism of action and the observation of the invasive, severe, disseminated clinical course of otherwise localized viral infections (e.g. herpesviruses) in patients with NK cell deficiencies [88, 89], NK cells have been recognized as a relevant first-line defense against viruses. This original view has been radically upgraded in recent years, leading to the addition of several NK cell functional features including memory-like activity through selective subset expansion upon viral infection (HCMV, MCMV, Chikungunya-, Hantavirus) and inherent regulation of NCR expression/function [90, 91].NK cells can sense infected cells either through direct interaction with pathogen-associated molecular patterns via Toll-like receptors, or through recognition of viral and/or (virus-induced) cellular ligands of major activating NK cell receptors. Such activating interactions, combined with the reduced engagement of inhibitory NK-receptors due to a frequently decreased surface HLA-I expression on infected cells, can induce NK cell cytotoxic activity and cytokine secretion.NK cells directly interact with and bind to viral glycoproteins through the NCRs (NKp46, NKp30, NKp44) as described, for example, for influenza, West Nile, and dengue viruses [92-95] (see Table 3). In addition, NCRs recognize still undefined virus-induced cellular ligands. Whether some of these ligands could be shared with tumor cells is poorly known, even if there are hints suggesting that a newly identified NKp44 ligand, 21spe-MLL5, could be both expressed by tumor cells and induced on T cells by HIV gp41 peptides. Along this line, also NKG2D and DNAM-1 receptors recognize ligands on virally infected cells that are common to tumor cells: MICA/B and ULBPs, and PVR and Nectin-2 respectively. In this context, it has been shown that several viruses activate DNA damage response, a common pathway involved in the regulation of NKG2D and DNAM-1 ligand expression in infected and tumor cells [53, 96, 97].Viruses have developed strategies to evade NK cell control on their replication either targeting activating or inhibitory receptor expression/recognition or tampering with the expression of cellular ligands for NK cell activating receptors in the cells they are replicating, or simultaneously inducing combinations of these mechanisms (see table 4). Accordingly, in HIV infection NK cells are activated [98], show decreased NCR expression [99], decreased production of IFN- and perforin, increased proportions of noncytolytic and non-secreting CD56-CD16+ NK cells [100], increased expression of HLA-specific inhibitory receptors (KIRs) [101], reduced expression [102], or increased shedding [103, 104], of ligands for activating receptors. Herpesviruses may interact with and degrade ligands for activating NK cell receptors [105-107] or may encode viral proteins mimicking HLA class I molecules that will block NK cell function through interaction with KIRs (or NKG2A). Along this line, HCV may block NK cell function through triggering of CD81 on NK cells, or may decrease their production of IFN- upon IL-12 signaling [108, 109], or may induce their production of immunosuppressive cytokines such as IL-10 [110].Genetic individual variability however plays a relevant role in molding the generalized NK/virus interaction into different individual responses to the same invading pathogen. Inherent/genetic carriage of given HLA class I-specific inhibitory receptors may improve NK cell activity against viruses. In HCV infection, for example, the combination of specific KIRs and HLA-C alleles associates to higher likelihood to clear acute infection and to respond to pegylated IFN-a/ribavirin treatment. In addition chronically infected HCV patients display at least two different NK cell transcriptional signatures which are associated with different responses to antiviral treatment [91], thus suggesting that inherent/genetic NK cell regulations affect the response to viruses. This is further proven by the observation of different NKp30 or NKG2D surface modulation in patients clearing acute HCV infection or clearing chronic infection upon antiviral treatment [111-114]. Similarly, in HIV-1 infection a non-progressive clinical course is associated with KIR3DS1 and HLA Bw4 carriage [115] and with an inherent/genetic regulation of NKp44, NKp30 and NKp46 expression upon NK cell activation [116].Overall, therefore, the manipulation of virus/NK cell interaction for the purpose of oncolytic virotherapy may profit from addressing and generally interfering with the so far highlighted mechanisms, but also may require additional insight in individual NK cell responses to viral triggers. Indeed individual genetic and inherent patient differences may affect the intended outcome thus benefiting from integrated individualized strategies.

NK cell-dependent oncolytic vectorsFor some oncolytic viral vectors, the anti-tumor effect has been reported to largely depend on NK cell activity. For example, the oncolytic potential of Orf virus in murine models of lung cancer and of Sindbis virus in SCID mice bearing human ovarian carcinoma tumors stronglylargely depends on NK cell activity [117-118]. Increased anti-tumor activity of NK cells may occur due to virus-induced modulation of the NK cell activating/inhibitory ligand profile on the surface of infected cancer cells. Indeed, infection of glioma cells with the oncolytic agent myxoma virus leads to down-regulation of MHC I surface expression via the viral M153R protein and consequent increased NK cell-mediated lysis of human glioma tumors in SCID mice and prolonged mouse survival [119]. Also, parvovirus infection of colon carcinoma cells leads to increased NCR-dependent tumor cell killing by NK cells (Bhat & Rommelaere, 2013). . Besides such direct effect on NK cell-mediated lysis of tumor cells, viral infection may also indirectly lead to increased anti-tumor activity of NK cells. In this context, the oncolytic rhabdovirus Maraba MG1 infects conventional dendritic cells, which leads to maturation of these cells and consequently increased NK cell-mediated antitumor responses [120].Comment by Herman Favoreel: Bhat, R., & Rommelaere, J. (2013). NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells. BMC Cancer 13:367.

Genetic engineering of oOncolytic virotherapy withvectors to trigger enhanced NK cell activation Seen the important contribution of NK cells to the anti-tumor effect of different oncolytic viruses, several genetically engineered oncolytic vectors have been developed that showwith the aim of triggering enhanced NK cell responses, often based on the strategies explained higher in the NK cell-based immunotherapy section. Some of these have led to promising results. For example, a strong anti-tumor response was observed in a xenograft mouse model of human pancreatic ductal adenocarcinoma using genetically engineered oncolytic parvoviruses that express IL-2 or MCP-2/CCL7, which led to efficient recruitment of NK cells and monocytic cells to the tumor nest [121]. Beneficial effects of combining virotherapy with biological treatments using IL-2 have also been reported for oncolytic VSV [122, 123]. For Sindbis virus, viral expression of IL-12 led to an NK cell-dependent increase in anti-cancer efficacy in SCID mice bearing human ovarian carcinoma tumors [118]. In line with this, genetically engineered oncolytic adenoviruses expressing IL-12 or co-expressing IL-12 and IL-18 resulted in increased infiltration and activity of NK and T cells in tumor tissues in murine models of prostate adenocarcinoma and of melanoma respectively, and consequently increased mouse survival [124, 125]. Another oncolytic adenovirus engineered with a CpG-enriched genome resulted in a significant and NK cell-dependent increase in anti-tumor efficacy in a xenograft model of lung cancer [126]. Also, the use of an oncolytic vaccinia virus strain expressing an agonist antibody against the costimulatory molecule CD137 (4-1BB) in a mouse model of breast carcinoma resulted in increased antitumor efficacy that depended on NK cells, T cells and neutrophils [127].

NK cells and oncolytic viruses in cancer vaccination Some groups have also harnessed the ability of oncolytic viruses to trigger the anti-tumor immune response, including the NK cell response, in the design of cancer vaccines. Immunization of mice with colon carcinoma cells that were previously infected in vitro with an oncolytic VSV virus protected the animals from subsequent tumor challenge, an effect that was further enhanced by expression of GM-CSF by the virus [128]. There are also indications that the reciprocal interaction between DC and NK cells may be beneficial in the context of cancer vaccination using viruses. DC loaded with reovirus-infected human melanoma cells (but not reovirus-infected tumor cells alone) induced chemotaxis of NK cells and IFN- production and anti-tumor cytotoxicity in NK cells, which possibly may contribute to tumor regression [129]. In addition, vaccination of mice with dendritic cells (DC) harboring a non-transmissible recombinant Sendai virus prevented lung metastasis of neuroblastoma (but not prostatic cancer). Depletion studies indicated that NK cells were crucial for the protective effect [130].

Inhibitory effects of NK cells on oncolytic virotherapyAlthough, as indicated above, (enhanced) NK cell activity may contribute to the therapeutic effects of particular oncolytic viruses, the antiviral activity of NK cells may also lead to unwanted premature clearance of the virus, reducing its therapeutic potential. Such negative effect of NK cell activity is particularly relevant when the oncolytic virotherapy involves the administration of relatively low doses of virus that needs to undergo multiple rounds of replication to sufficiently spread within the tumor and reach maximal therapeutic effectiveness. In such cases, ideally, the antiviral effect of NK cells should be minimal, especially during the early stages of treatment, while retaining the anti-tumor activity of NK cells [131]. One approach to reach this goal is to genetically engineer oncolytic vectors so that they display an optimized activating/inhibitory balance towards NK cells, which may involve the introduction of (viral) NK cell evasive genes in the oncolytic viral genome.In rats bearing multifocal hepatocellular carcinoma and treated with oncolytic VSV, NK cell depletion resulted in a logarithmic elevation of intratumoral VSV titers and an enhanced antitumor response, indicating a suppressive effect of NK cells on the therapeutic effect of the oncolytic vector in this setup [132]. Interestingly, genetic engineering of herpesvirus immune evasion genes in the VSV genome was able to counteract NK cell activity and enhance the oncolytic potential of VSV in this animal model. Indeed, introduction of the equine herpesvirus 1 chemokine-binding viral gG glycoprotein in VSV reduced the number of NK and NKT cells in the tumors, resulting in a 1-log enhancement of intratumoral virus titers, increased tumor necrosis and prolonged animal survival without toxicities [133]. In a follow-up study, NK cell activity was more specifically suppressed via introduction of the human cytomegalovirus UL141 gene in VSV. UL141 encodes a viral protein that down-regulates CD155 and CD112, ligands for the NK cell activating receptor DNAM-1 [133, 134]. Again, recombinant VSV expressing UL141 resulted in a one-log elevation of intratumoral virus replication, enhanced tumor necrosis and prolonged survival [135].An optimized balance of NK activation/inhibition signals may be particularly relevant for herpes simplex virus 1 (HSV1)-based oncolytic vectors, as these typically depend on several viral replication rounds and efficient spread within the tumor to be effective [136]. Indeed, Alvarez-Breckenridge et al. elegantly demonstrated that NK cells are responsible for premature clearance of an HSV1-based oncolytic vector in a mouse glioblastoma model [24]. They showed that NK cell activity is triggered by HSV1-induced up-regulation of NCR ligands, and that NK cell-mediated clearance of the viral vector represents an important limitation in glioblastoma virotherapy [24]. In line with these data, the positive effects of epigenetic modulators like valproic acid and histone deacetylase inhibitors on HSV1-based oncotherapy in glioblastoma mouse models wereas found to correlate with reduced recruitment of NK cells to tumor-bearing brains and suppressed NK cell cytotoxicity [136]. Hence, it will be interesting to determine the oncolytic potential of future recombinant HSV1 oncolytic vectors that encode NK cell suppressing proteins. A particularly interesting candidate may be the gD glycoprotein of the porcine alphaherpesvirus pseudorabies virus (PRV), which is closely related to HSV1. PRV gD has been reported recently to interfere with DNAM-1 dependent NK cell-mediated killing through downregulation of the DNAM-1 ligand CD112 both in porcine and human cells, including human glioblastoma cells [107].

Combination of viro/chemotherapy and NK cellsThe future of oncolytic virotherapy, like that of NK cell-based oncotherapy, will probably involve its integration in combination therapy with more conventional approaches like chemotherapy [84]. Hence, future research will need to carefully investigate whether some therapy combinations may reinforce or rather counteract one another. Both types of outcome have been reported and may depend at least in part on synergistic or counteracting effects on NK cell activity by the combination therapy. On the one hand, in a mouse model of aggressive human ovarian cancer, the combination of Sindbis virotherapy with topoisomerase inhibitor chemotherapy resulted in synergistic NK cell-dependent effective therapy, which was not observed with either treatment alone [137]. On the other hand, although combining oncolytic VSV with cyclophosphamide chemotherapy increased VSV titers in mesothelioma tumors in mice, it unexpectedly reduced therapeutic efficacy compared to cyclophosphamide alone. This antagonistic effect appeared to involve a VSV-induced TGF--dependent suppression of cyclophosphamide-triggered NK cell activity [138].

CONCLUSIONS AND FUTURE PERSPECTIVESAttempts at exploiting NK cell adoptive use or NK cell manipulation for a successful immunotherapy and/or virotherapy of solid tumours (Figure 1) overall have been so far stymied by a combination of factors including too early clearance (and inefficient tumor penetration/intratumoral spread) of oncolytic viruses, poor NK cell penetration or localization in proximity of tumor cells and susceptibility of infiltrating NK cells to be manipulated and disarmed. The information and lessons learnt from these failures, together with new insights gathered in NK cell biology in different health conditions will provide an improved basis for future Immuno/oncovirotherapy. In particular, the in depth knowledge of the role of NK cells in the present and future therapeutic approaches is a prerequisite to identify crucial synergies (figure 1). In this context, strategies to increase NK cell:tumour interaction and direct killing, and tumour-targeting drugs or mAbs are being presently evaluated. Manipulation of viruses used for oncotherapy will be targeted at reducing too early virus clearance by NK cells thus allowing speread to the whole tumor thus allowing improved killing. All these strategies and new methods will need to carefully take into account the phenotypic and functional characteristics of NK cells in individual patients. Finally, evidences showing that a very high NK cell phenotype diversity exists in each individual will also need to be considered in future work. Indeed, standardized therapies using NK cell manipulation in any given group of patients may conflict with different interindividual characteristics involving NK cell function and phenotype in these patients and therefore may lead to treatment failure independent from an intrinsic usefulness of the immuno/virotherapy regimen. New techniques of NK cell or virus manipulation (e.g. single-cell transcriptional analysis, mass-cytomery, etc.) will provide useful tools to optimally individualize immunotherapeutic regimens exploiting NK cell:tumor interaction.

EXECUTIVE SUMMARY NK cells and the tumor microenvironment. Tumor microenvironment can regulate the availability and location of NK cells within the tumor tissue Tumor microenvironment can regulate the function of NK cells within the tumor tissue Tumor cells can edit their surface phenotype to escape NK cell attack Role of NK cells in therapies targeting solid tumors NK-based immunotherapy mAb-mediated tumor targeting Drug-mediated tumor targeting and IMiDs Role of NK cells in oncolytic virotherapy NK cells-virus interaction NK cell-dependent oncolytic vectors Oncolytic virotherapy with enhanced NK cell activation NK cells and oncolytic viruses in cancer vaccination Inhibitory effects of NK cells on oncolytic virotherapy Combination of viro/chemotherapy and NK cells

REFERENCES1. *Krre K. How to recognize a foreign submarine. Immunol Rev. 155, 5-9 (1997).*The story of how the Missing Self recognition hypothesis has been conceived as told by the author himself.

2. *Moretta A, Vitale M, Bottino C, et al. P58 molecules as putative receptors for majorhistocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J. Exp. Med. 178, 597-604 (1993).3. *Litwin V, Gumperz J, Parham P, Philips J, Lanier LL. NKB 1: a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J. Exp. Med. 178, 597-604 (1993).*The first two papers describing HLA-I specific NK receptors.

4. *Sivori S, Vitale M, Morelli L, et al. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J. Exp. Med. 186, 1129-1136 (1997).*The first paper describing an activating receptor specifically expressed by human NK cells.

5. **Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science 331, 44-49 (2011).**a comprehensive review describing the most recent insights on the NK cell functions and the panel of NK receptors involved in their control.

6. Moretta L, Bottino C, Pende D, Castriconi R, Mingari MC, Moretta A. Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 18, 151-158 (2006).7. Zhang Y, Wallace DL, de Lara CM, et al. In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection. Immunology 121, 258-265 (2007).8. Kammula US, White DE, Rosenberg SA. Trends in the safety of high dose bolus interleukin-2 administration in patients with metastatic cancer. Cancer 83, 797-805 (1998).9. Overwijk WW, Schluns KS. Functions of C cytokines in immune homeostasis: current and potential clinical applications. Clin Immunol. 132, 153-165 (2009).10. Rodella L, Zamai L, Rezzani R, et al. Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells. Br J Haematol. 115, 442-450 (2001).11. Ghiringhelli F, Mnard C, Martin F, and Zitvogel L. The role of regulatory T cells in the control of natural killer cells: relevance during tumor progression. Immunol. Rev. 214, 229-238 (2006).12. *Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 94(1):333-339 (1999).*The seminal paper showing the potential of alloreactive NK cells coming from HSCT.

13. Locatelli F, Pende D, Mingari MC, et al. Cellular and molecular basis of haploidentical hematopoietic stem cell transplantation in the successful treatment of high-risk leukemias: role of alloreactive NK cells. Front Immunol. 4, 15 (2013).14. Knorr DA, Bachanova V, Verneris MR, Miller JS. Clinical utility of natural killer cells in cancer therapy and transplantation. Semin Immunol. 26, 161-172 (2014)15. Stojanovic A, Cerwenka A. Natural killer cells and solid tumors. J Innate Immun 3, 355-364 (2011).16. Baginska J, Viry E, Paggetti J, et al. The critical role of the tumor microenvironment in shaping natural killer cell-mediated anti-tumor immunity. Front Immunol. 4, 490 (2013).17. Vitale M, Cantoni C, Pietra G, Mingari MC, Moretta L. Effect of tumor cells and tumor microenvironment on NK-cell function. Eur. J. Immunol. 44, 1582-1592 (2014).18. Shi FD, Ljunggren HG, La Cava A, and Van Kaer L. Organ-specific features of natural killer cells. Nat. Rev. Immunol. 11, 658-671 (2011).19. Montaldo E, Vacca P, Moretta L, Mingari MC. Development of human natural killer cells and other innate lymphoid cells. Semin Immunol. 26, 107-113 (2014).20. Eberl G, Di Santo JP, Vivier E. The brave new world of innate lymphoid cells. Nat Immunol. 16, 1-5 (2015).21. Sivori S, Olive D, Lopez-Botet M, Vitale M. NK receptors: tools for a polyvalent cell family. Front Immunol 5, 617 (2014).22. Vacca P, Moretta L, Moretta A, and Mingari MC. Origin, phenotype and function of human natural killer cells in pregnancy. Trends Immunol. 32, 517-523 (2011).23. Chiocca EA, Rabkin SD. Oncolytic viruses and their application to cancer immunotherapy. Cancer Immunol Res. 2, 295-300 (2014).24. *Alvarez-Breckenridge CA, Yu J, Price R, et al. NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors. Nat. Med. 18, 1827-1834 (2012).* Review paper covering the beneficial and unwanted effects of NK cells in oncolytic virotherapy25. **Alvarez-Breckenridge CA, Yu J, Kaur B, Caligiuri MA, Chiocca EA. Deciphering the Multifaceted Relationship between Oncolytic Viruses and Natural Killer Cells. Adv. Virol. 2012, 702839 (2012).** Landmark paper showing that NK cells may be a limiting factor in oncolytic virotherapy by premature clearance of the viral vector.

26. Balsamo M, Vermi W, Parodi M, et al. Melanoma cells become resistant to NK-cell-mediated killing when exposed to NK-cell numbers compatible with NK-cell infiltration in the tumor. Eur. J. Immunol. 42, 1833-1842 (2012). 27. Burke S, Lakshmikanth T, Colucci F, Carbone E. New views on natural killer cell-based immunotherapy for melanoma treatment. Trends Immunol. 31, 339-345 (2010). 28. Delahaye NF, Rusakiewicz S, Martins I, et al. Alternatively spliced NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors. Nat. Med. 17, 700-707 (2011). 29. Remark R, Alifano M, Cremer I, et al. Characteristics and clinical impacts of the immune environments in colorectal and renal cell carcinoma lung metastases: influence of tumor origin. Clin Cancer Res. 19, 4079-4091 (2013).30. Platonova S, Cherfils-Vicini J, Damotte D. et al. Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma. Cancer Res. 71, 5412-5422 (2011). 31. Carrega P, Bonaccorsi I, Di Carlo E, et al. CD56(bright)perforin(low) noncytotoxic NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph. J. Immunol. 192, 3805-3815 (2014)32. Rusakiewicz S, Semeraro M, Sarabi M, et al. Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors. Cancer Res. 73, 3499-3510 (2013).33. Mamessier E, Sylvain A, Thibult ML, et al. Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity. J. Clin. Invest. 121, 3609-3622 (2011). 34. Ali TH, Pisanti S, Ciaglia E, et al. Enrichment of CD56(dim)KIR+CD57+ highly cytotoxic NK cells in tumour-infiltrated lymph nodes of melanoma patients. Nat. Commun. 4,5, 539 (2014)35. Halama N, Braun M, Kahlert C, et al. Natural killer cells are scarce in colorectal carcinoma tissue despite high levels of chemokines and cytokines. Clin. Cancer Res. 17, 678-689 (2011). 36. Balsamo M, Scordamaglia F, Pietra G, et al. Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity. Proc. Natl. Acad. Sci. U S A. 106, 20847-20852 (2009).37. Messaoudene M, Fregni G, Fourmentraux-Neves E, et al. Mature cytotoxic CD56(bright)/CD16(+) natural killer cells can infiltrate lymph nodes adjacent to metastatic melanoma. Cancer Res. 74, 81-92 (2014).38. Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and adaptive immunity to cancer. Annu. Rev. Immunol. 29, 235-271 (2011).39. Moretta A, Marcenaro E, Sivori S, Della Chiesa M, Vitale M, Moretta L. Early liaisons between cells of the innate immune system in inflamed peripheral tissues. Trends Immunol. 26, 668-675 (2005).40. Bellora F, Castriconi R, Dondero A, et al. TLR activation of tumor-associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells. Eur J Immunol. 44, 1814-1822 (2014). 41. Whiteside T. The tumor microenvironment and its role in tumor growth. Oncogene. 27, 5904-5912 (2008). 42. Hoechst B, Voigtlaender T, Ormandy L, et al. Myeloid derived suppressor cells inhibit natural killer cells in patients with hepatocellular carcinoma via the NKp30 receptor. Hepatology. 50, 799-807 (2009). 43. Sprinzl MF, Reisinger F, Puschnik A, et al. Sorafenib perpetuates cellular anticancer effector functions by modulating the crosstalk between macrophages and natural killer cells. Hepatology. 57, 2358-2368 (2013). 44. Kalinski P. Regulation of immune responses by prostaglandin E2. J. Immunol. 188, 21-28 (2012).45. Castriconi R, Cantoni C, Della Chiesa M, et al. Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc. Natl. Acad. Sci. U S A. 100, 4120-4125 (2003).46. Marcenaro E, Della Chiesa M, Bellora F et al. IL-12 or IL-4 prime human NK cells to mediate functionally divergent interactions with dendritic cells or tumors. J. Immunol. 17, 3992-3998 (2005).47. Della Chiesa M, Carlomagno S, Frumento G, et al. The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. Blood. 108, 4118-4125 (2006).48. Ghiringhelli F, Menard C, Terme M, et al. CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner. J Exp Med. 202, 1075-1085 (2005).49. Sitrin J, Ring A, Garcia KC, Benoist C, Mathis, D. Regulatory T cells control NK cells in an insulitic lesion by depriving them of IL-2. J. Exp. Med. 210, 1153-1165 (2013).50. Li T, Yang Y, Hua X. Hepatocellular carcinoma-associated fibroblasts trigger NK cell dysfunction via PGE2 and IDO. Cancer Lett. 318, 154-161 (2012). 51. Li T, Yi S, Liu W, et al. Colorectal carcinoma-derived fibroblasts modulate natural killer cell phenotype and antitumor cytotoxicity. Med. Oncol. 30, 663 (2013). 52. Pietra G, Manzini C, Rivara S, et al. Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity. Cancer Res. 72, 1407-1415 (2012).53. Raulet DH, Gasser S, Gowen BG, Deng W, Jung H. Regulation of ligands for the activating NKG2D receptor. Annu. Rev. Immunol. 31, 413-441 (2013).54. Krockenberger M, Dombrowski Y, Weidler C, et al. Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by downregulating NKG2D. J. Immunol. 180, 7338-7348 (2008).55. Gubbels JA, Felder M, Horibata S, et al. MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells. Mol. Cancer. 9, 11 (2010). 56. Castriconi R, Dondero A, Bellora F, et al. Neuroblastoma-derived TGF-b1 modulates the chemokine receptor repertoire of human resting NK cells. J. Immunol. 190, 5321-5328 (2013). 57. Schlecker E, Fiegler N, Arnold A, et al. Metalloprotease-mediated tumor cell shedding of B7.H6, the ligand of the natural killer activating receptor NKp30. Cancer Res. 74, 3429-3440 (2014).58. Balsamo M, Manzini C, Pietra G, et al. Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC. Eur. J. Immunol. 43, 2756-2764 (2013). 59. Elboim M, Gazit R, Gur C, Ghadially H, Betser-Cohen G, Mandelboim, O. Tumor immunoediting by NKp46. J. Immunol. 184, 5637-5644 (2010).60. Rosental B, Brusilovsky M, Hadad U, et al. Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44. J. Immunol. 187, 5693-5702 (2011). 61. Lankry D, Rovis TL, Jonjic S, Mandelboim, O. The interaction between CD300a and phosphatidylserine inhibits tumor cell killing by NK cells. Eur. J. Immunol. 43, 2151-2161 (2013).62. Ames E, Murphy WJ. Advantages and clinical applications of natural killer cells in cancer immunotherapy. Cancer Immunol Immunother. 63, 21-28 (2014). 63. Terme M, Ullrich E, Delahaye NF, Chaput N, Zitvogel L. Natural killer cell-directed therapies: moving from unexpected results to successful strategies. Nat Immunol. 9, 486-494 (2008). 64. Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol. 17, 2105-2116 (1999). 65. *Ni J, Miller M, Stojanovic A, Garbi N, Cerwenka A. Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors. J Exp Med. 209, 2351-2365 (2012). *An important study in mice showing that IL-12, IL-15 and IL-18 cytokine combination boost NK cell anti-tumor activity.

66. Vacchelli E, Galluzzi L, Eggermont A, et al. Trial Watch: Immunostimulatory cytokines. Oncoimmunology. 1, 493-506 (2012). 67. Tarhini AA, Millward M, Mainwaring P, et al. A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer. 115, 859-868 (2009). 68. Conlon KC, Lugli E, Welles HC, et al. Redistribution, Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and Cytokine Production During First-in-Human Clinical Trial of Recombinant Human Interleukin-15 in Patients With Cancer. J Clin Oncol. 33, 74-82 (2015). 69. *Ardolino M, Azimi CS, Iannello A, et al. Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J Clin Invest. 124, 4781-4794 (2014). *For the first time the authors showed that IL-12 and IL-18 treatment restores responsiveness of anergic NK cells present within MHC class I deficient solid tumors.

70. Romagn F, Andr P, Spee P, et al. Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells. Blood. 114, 2667-2677 (2009). 71. *Kohrt HE, Thielens A, Marabelle A, et al. Anti-KIR antibody enhancement of anti-lymphoma activity of natural killer cells as monotherapy and in combination with anti-CD20 antibodies. Blood. 123, 678-686 (2014). * this paper provids a rationale for a treatment regimen that includes mab-mediated KIR blockade and therapeutic mAbs

72. Pahl JH, Rusian EN, Buddingh EP et al. Anti-EGFR antibody Cetuximab enhances the cytolytic activity of natural killer cells towards osteosarcoma. Clin. Cancer Res. 18, 432-441 (2012).73. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 12, 278-287 (2012). 74. Krieg S, Ullrich E. Novel immune modulators in hematology: impact on NK cells. Front. Immunol. 3, 388 (2013).75. Benson DM Jr, Bakan CE, Zhang S, et al. IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect. Blood. 118, 6387-6391 (2011). 76. Law TM, Motzer RJ, Mazumdar M, et al. Phase III randomized trial of interleukin-2with or without lymphokine-activated killer cells in the treatment of patients with advanced renal cell carcinoma. Cancer. 76, 824-832 (1995). 77. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin Cancer Res. 17, 6287-6297 (2011). 78. Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood. 105, 3051-3057 (2005). 79. Geller MA, Cooley S, Judson PL, et al. A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer. Cytotherapy. 13, 98-107 (2011). 80. Iliopoulou EG, Kountourakis P, Karamouzis MV, et al. A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother. 59, 1781-1789 (2010). 81. Wennerberg E, Kremer V, Childs R, Lundqvist A. CXCL10-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo. Cancer Immunol Immunother. 64, 225-235 (2015). 82. Oberoi P, Wels WS. Arming NK cells with enhanced antitumor activity: CARs and beyond. Oncoimmunology. (8), e25220 (2013). 83. Pol J, Bloy N, Obrist F, et al. Trial Watch:: Oncolytic viruses for cancer therapy. Oncoimmunology. 3, e28694 (2014). 84. Miest TS, Cattaneo R. New viruses for cancer therapy: meeting clinical needs. Nat Rev Microbiol. 12, 23-34 (2014).85. Yu W, Fang H. Clinical trials with oncolytic adenovirus in China. Curr. Cancer Drug Targets. 7(2), 141148 (2007).86. Woller N, Grlevik E, Ureche C-I, Schumacher A, Khnel F. Oncolytic viruses as anticancer vaccines. Front. Oncol. 4, 188 (2014).87. Chiocca EA, Rabkin SD. Oncolytic viruses and their application to cancer immunotherapy. Cancer Immunol. Res. 2(4), 295300 (2014).88. Lopez C, Kirkpatrick D, Read SE, et al. Correlation Between Low Natural Killing of Fibroblasts Infected with Herpes Simplex Virus Type 1 and Susceptibility to Herpesvirus Infections. J. Infect. Dis. 147, 1030-1035 (1983).89. Orange JS. Natural killer cell deficiency. J. Allergy and Clin. Immunol. 132, 515-525 (2013).90. Petitdemange C, Becquart P, Wauquier N et al. Unconventional Repertoire Profile Is Imprinted during Acute Chikungunya Infection for Natural Killer Cells Polarization toward Cytotoxicity. PLoS Pathog. 7, e1002268 (2011).91. Marras F, Bozzano F, Ascierto ML, De Maria A. Baseline and dynamic expression of activating NK cell receptors in the control of chronic viral infections: the paradigm of HIV-1 and HCV. Front. Immunol. 5 (2014).92. Mandelboim O, Lieberman N, Lev M, et al. Recognition of hemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature. 409, 1055-1060 (2001).93. Arnon TI, Lev M, Katz G, Chernobrov Y, Porgador A, Mandelboim O. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur. J. Immunol. 31, 2680-2689 (2001).94. Hershkovitz O, Rosental B, Rosenberg LA, et al. NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells. J. Immunol. 183, 2610-2621 (2009). 95. Chisholm SE, Reyburn HT. Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors. J. Virol. 80, 2225-2233 (2006).96. Ferrari de Andrade L, Smyth MJ, Martinet L. DNAM-1 control of natural killer cells functions through nectin and nectin-like proteins. Immunol. and Cell Biol. 92, 237-244 (2014).97. Cerboni C, Fionda C, Soriani A, et al. The DNA damage response: a common pathway in the regulation of NKG2D and DNAM-1 ligand expression in normal, infected and cancer cells. Front. Immunol. 4, 508 (2014)898. Fogli M, Costa P, Murdaca G, et al. Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients. Eur. J. Immunol. 34, 2313-2321 (2004).99. De Maria A, Fogli M, Costa P, et al. The impaired NK cell cytolytic function in viremic HIV-1 infection is associated with a reduced surface expression of natural cytotoxicity receptors (NKp46, NKp30 and NKp44). Eur. J. Immunol. 33, 2410-2418 (2003).100. Mavilio D, Lombardo G, Kinter A, et al. Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection. J. Exp. Med. 203, 2339-2350 (2006).101. De Maria A, Mavilio D, Costa P, Dignetti P, Fogli M, Mingari MC. Multiple HLA-class I-specific inhibitory NK receptor expression and IL-4/IL-5 production by CD8+ T-cell clones in HIV-1 infection. Immunol. Lett. 72, 179-182 (2000).102. Ward J, Bonaparte M, Sacks J, et al. HIV modulates the expression of ligands important in triggering natural killer cell cytotoxic responses on infected primary T-cell blasts. 110, 1207-1214 (2007).103. Matusali G, Tchidjou HK, Pontrelli G, et al. Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells. FASEB J. 27, 2440-2450 (2013).104. Cerboni C, Neri F, Casartelli N, et al. Human immunodeficiency virus 1 Nef protein downmodulates the ligands of the activating receptor NKG2D and inhibits natural killer cell-mediated cytotoxicity. J. Gen. Virol. 88, 242-250 (2007).105. Lisnic VJ, Krmpotic A, Jonjic S. Modulation of natural killer cell activity by viruses. Curr. Opin. Microbiol. 13, 530-539 (2010).106. Jost S, Altfeld M. Evasion from NK cell-mediated immune responses by HIV-1. Microbes Infect. 14, 904-915 (2012).107. Grauwet K, Cantoni C, Parodi M, et al. Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cell-mediated lysis of infected cells. Proc. Natl. Acad. Sci. U S A. 111, 16118-16123 (2014).108. Crotta S, Brazzoli M, Piccioli D, Valiante NM, Wack A. Hepatitis C virions subvert natural killer cell activation to generate a cytokine environment permissive for infection. J. Hepatology 52, 183-190 (2010).109. Crotta S, Stilla A, Wack A, et al. Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein. J. Exp.Med. 195:35-42 (2002).110. De Maria A, Fogli M, Mazza S, et al. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic patients. Eur. J. Immunol. 37, 445-455 (2007).111. Amadei B, Urbani S, Cazaly A, et al. Activation of Natural Killer Cells During Acute Infection With Hepatitis C Virus. Gastroenterology 138, 1536-1545 (2010).112. Golden-Mason L, Cox AL, Randall JA, Cheng L, Rosen HR. Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C virus infection in high-risk individuals and inhibits replication in vitro. Hepatology 52, 1581-1589 (2010).113. Werner JM, Heller T, Gordon AM, et al. Innate immune responses in hepatitis C virus-exposed healthcare workers who do not develop acute infection. Hepatology 58, 1621-1631 (2013).114. Bozzano F, Picciotto A, Costa P, et al. Activating NK cell receptor expression/function (NKp30,NKp46,DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment. Eur. J. Immunol. 41, 2905-2914 (2011).115. Martin MP, Gao X, Lee J-H, et al. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31, 429-434 (2002).116. Marras F, Nicco E, Bozzano F, et al. Natural killer cells in HIV controller patients express an activated effector phenotype and do not up-regulate NKp44 on IL-2 stimulation. Proc. Natl. Acad. Sci. U S A. 10, 11970-11975 (2013).117. Rintoul JL, Lemay CG, Tai L-H, et al. ORFV: a novel oncolytic and immune stimulating parapoxvirus therapeutic. Mol. Ther. J. Am. Soc. Gene Ther. 20(6), 11481157 (2012).118. Granot T, Venticinque L, Tseng J-C, Meruelo D. Activation of cytotoxic and regulatory functions of NK cells by Sindbis viral vectors. PloS One. 6(6), e20598 (2011).119. Ogbomo H, Zemp FJ, Lun X, et al. Myxoma virus infection promotes NK lysis of malignant gliomas in vitro and in vivo. PloS One. 8(6), e66825 (2013).120. Zhang J, Tai L-H, Ilkow CS, et al. Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease. Mol. Ther. J. Am. Soc. Gene Ther. 22(7), 13201332 (2014).121. Dempe S, Lavie M, Struyf S, et al. Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment. Cancer Immunol. Immunother. CII. 61(11), 21132123 (2012).122. Kottke T, Diaz RM, Kaluza K, et al. Use of biological therapy to enhance both virotherapy and adoptive T-cell therapy for cancer. Mol. Ther. J. Am. Soc. Gene Ther. 16(12), 19101918 (2008).123. Kottke T, Galivo F, Wongthida P, et al. Treg depletion-enhanced IL-2 treatment facilitates therapy of established tumors using systemically delivered oncolytic virus. Mol. Ther. J. Am. Soc. Gene Ther. 16(7), 12171226 (2008).124. Freytag SO, Barton KN, Zhang Y. Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer. Gene Ther. 20(12), 11311139 (2013).125. Choi I-K, Lee J-S, Zhang S-N, et al. Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12R2 or IL-18R. Gene Ther. 18(9), 898909 (2011).126. Cerullo V, Diaconu I, Romano V, et al. An oncolytic adenovirus enhanced for toll-like receptor 9 stimulation increases antitumor immune responses and tumor clearance. Mol. Ther. J. Am. Soc. Gene Ther. 20(11), 20762086 (2012).127. John LB, Howland LJ, Flynn JK, et al. Oncolytic virus and anti-4-1BB combination therapy elicits strong antitumor immunity against established cancer. Cancer Res. 72(7), 16511660 (2012).128. Lemay CG, Rintoul JL, Kus A, et al. Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine. Mol. Ther. J. Am. Soc. Gene Ther. 20(9), 17911799 (2012).129. Prestwich RJ, Errington F, Steele LP, et al. Reciprocal human dendritic cell-natural killer cell interactions induce antitumor activity following tumor cell infection by oncolytic reovirus. J. Immunol. Baltim. Md 1950. 183(7), 43124321 (2009).130. Komaru A, Ueda Y, Furuya A, et al. Sustained and NK/CD4+ T cell-dependent efficient prevention of lung metastasis induced by dendritic cells harboring recombinant Sendai virus. J. Immunol. Baltim. Md 1950. 183(7), 42114219 (2009).131. Kaufmann JK, Chiocca EA. Glioma virus therapies between bench and bedside. Neuro-Oncol. 16(3), 334351 (2014).132. Altomonte J, Wu L, Chen L, et al. Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo. Mol. Ther. J. Am. Soc. Gene Ther. 16(1), 146153 (2008).133. Tomasec P, Wang ECY, Davison AJ, et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat. Immunol. 6(2), 181188 (2005).134. Prodhomme V, Sugrue DM, Stanton RJ, et al. Human cytomegalovirus UL141 promotes efficient downregulation of the natural killer cell activating ligand CD112. J. Gen. Virol. 91(Pt 8), 20342039 (2010).135. Altomonte J, Wu L, Meseck M, et al. Enhanced oncolytic potency of vesicular stomatitis virus through vector-mediated inhibition of NK and NKT cells. Cancer Gene Ther. 16(3), 266278 (2009).136. Alvarez-Breckenridge CA, Yu J, Price R, et al. The histone deacetylase inhibitor valproic acid lessens NK cell action against oncolytic virus-infected glioblastoma cells by inhibition of STAT5/T-BET signaling and generation of gamma interferon. J. Virol. 86(8), 45664577 (2012).137. Granot T, Meruelo D. The role of natural killer cells in combinatorial anti-cancer therapy using Sindbis viral vectors and irinotecan. Cancer Gene Ther. 19(8), 588591 (2012).138. Willmon C, Diaz RM, Wongthida P, et al. Vesicular stomatitis virus-induced immune suppressor cells generate antagonism between intratumoral oncolytic virus and cyclophosphamide. Mol. Ther. J. Am. Soc. Gene Ther. 19(1), 140149 (2011).139. Koch J, Steinle A, Watzl C, Mandelboim O. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection. Trends Immunol. 34, 182-191 (2013).140. Hecht ML, Rosental B, Horlacher T, et al. Natural cytotoxicity receptors NKp30, NKp44 and NKp46 bind to different heparan sulfate/heparin sequences. J. Proteom Res. 8, 712-720 (2009).141. Pogge von Strandmann E, Simhadri VR, von Tresckow B, et al. A. Human leukocyte antigen-B-associated transcript 3 is released from tumor cells and engages the NKp30 receptor on natural killer cells. Immunity. 27, 965-974 (2007). 142. Reiners KS, Topolar D, Henke A, et al. Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity. Blood. 121, 3658-3665 (2013). 143. Brandt CS, Baratin M, Yi EC, et al. The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer receptor NKp30 in humans. J. Exp. Med. 206, 1495-1503 (2009).144. Schlecker E, Fiegler N, Arnold A, et al. Metalloprotease-mediated tumor cell shedding of B7.H6, the ligand of the natural killer activating receptor NKp30. Cancer Res. 74, 3429-3440 (2014).145. Baychelier F, Sennepin A, Ermonval M, Dorgham K, Debre P, Vieillard. Identification of a cellular ligand for NKp44. Blood. 122, 2935-2942 (2013).146. Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J. Exp. Med. 198, 557-567 (2003).147. Arnon TI, Achdout H, Lieberman N, et al. The mechanosms controlling the recognition of tumor- and virus-infected cells by NKp46. Blood. 103, 664-672 (2004).148. Jarahian M, Watzl C, Fournier P, et al. Activation of natural killer cells by Newcastle disease virus hemagglutinin-neuraminidase. J. Virol. 83, 8108-8121 (2009).149. Jarahian M, Fiedler M, Cohnen A, et al. Modulation of NKp30- and NKp46-mediated natural killer cell responses by poxviral hemagglutinin. PLoS Pathog. 7, e1002195 (2011).150. Chisholm SE, Howard K, Gomez MV, Reyburn HT. Expression of ICP0 is sufficient totrigger natural killer cell recognition of herpes simplex virus-infected cells by natural cytotoxicity receptors. J. Infect. Dis. 195, 1160-1168 (2007).151. Schepis D, DAmato M, Studah M, Bergstrom T,Karre K, Berg L. Herpes simplex virus infection downmodulates NKG2D ligand expression. Scand. J. Immunol. 69, 429-436 (2009).152. Costa P, Sivori S, Bozzano F et al IFN-alpha-mediated increase in cytolytic activity of maturing NK cell upon exposure to HSV-infected myelomonocytes. Eur. J. Immunol. 39, 147-158 (2009).153. Arnon TI, Achdout H, Levi O, et al. Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus. Nat. Immunol. 6, 515-523 (2005).154. Magri G, Muntasell A, Romo N, et al. NKp46 and DNAM-1 NK-cell receptors drive the response to human cytomegalovirus-infected myeloid dendritic cells overcoming viral immune evasion strategies. Blood. 117, 848-856 (2011).155. Madrid AS, Ganem D. Kaposis sarcoma-associated herpesvirus ORF54/dUTPase downregulates a ligand for the NK activating receptor NKp44. J. Virol. 86, 8693-8701 (2012)156. Dupuy S, Lambert M, Zucman D, et al. Human herpesvirus 8 (HVV8) sequentially shapes the NK cell repertoire during the course of asymptomatic infection and Kaposi sarcoma. PLoS Pathog. 8, e1002486 (2012).157. Holder KA, Stapleton SN, Gallant ME, Russell RS, Grant MD. Hepatitis C virus-infected cells downregualte NKp30 and inhibit ex vivo NK cell functions. J. Immunol. 191, 3308-3318 (2013).158. Bhat R, Rommelaere J. NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells. BMC Cancer. 13, 367- (2013).159. Vieillard V, Strominger K, Debr P. NK cytotxicity against CD4+ T cells during HIV-1 infection: a gp41 peptide induces the expression of an NKp44 ligand. Proc. Natl. Acad. Sci. U S A. 102, 10981-10986 (2005).160. Fausther-Bovendo H, Sol-Foulon N, Candotti D, et al. HIV escape from natural killer cytotoxicity: nef inhibits NKp44L expression on CD4+ T cells. AIDS. 23, 1077-1087 (2009).161. Richard J, Sindhu S, Pham TNQ, Belzile J-P, Cohen A. HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing. Blood. 115, 1354-1363 (2010).162. A. Muntasell, C. Vilches, A. Angulo, M. Lpez-Botet, Adaptive reconfiguration of the human NK-cell compartment in response to cytomegalovirus: A different perspective of the host-pathogen interaction. European Journal of Immunology 43, 1133 (05/01, 2013).163. Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M, et al. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp.Med. 208, 1321 (2011).

FIGURE LEGEND. Figure 1. Role of NK cells in tumor therapy: potential synergies. NK cells can play a pivotal role in different therapeutic strategies. In NK cell-based immunotherapies NK cells are directly exploited to kill tumor cells. In tumor-targeted therapies NK cells can participate to the anti-tumor therapeutic effects through different mechanisms: mAb-induced ADCC or drug-increased NK cell cytotoxicity. In virotherapy, NK cells may exert either a positive or a negative effect on the efficacy of therapy. Depending on several factors such as virus type, virus dose and virus replication rate, NK cells can either participate in clearing the tumor by killing infected tumor cells, or limit virus spread within the tumor by prematurely killing infected tumor cells. The search for possible synergistic effects among diverse therapeutic approaches (yellow arrows) may represent a valuable strategy to the design of future effective therapies.

Table 1: Major activating NK receptors and their ligand(s) on tumor cells

Activating NK receptorLigand(s) on tumor cellsRef.

NKp46 (CD335)Unknown

HSPGa4, 6, 139

140

NKp30 (CD337)BAT3b/BAG6c

B7-H6

HSPGa139, 141, 142

143, 144

140

NKp44 (CD336)PCNAd*

MLL5e isoform

HSPGa60

145

140

NKG2DMICA/B,ULBP1-653

DNAM-1 (CD226)PVR (CD155)Nectin2 (CD112)96, 146

* The interaction with NKp44 results in a paradoxical inhibitory signal. aHSPG: heparan sulfate proteoglycans; bBAT3: HLA-B-associated Transcript 3; cBAG6: BCL-2-associated athanogene 6; dPCNA: proliferating cell nuclear antigen; eMLL5: Mixed-lineage leukemia-5.

Table 2: Recently launched oncolytic clinical virotherapy trials in cancer patients (based on www.clinicaltrials.gov).Comment by Herman Favoreel: Moet hier niet based on bijstaan of grotendeels aangepast/andere opbouw ?Comment by Korneel: Ik heb via clin gov alle trials opgevraagd en geselecteerd voor diegeen die aan het recruitten zijn => recently launched. Op de site staan wel een paar fouten die ik tegengekomen heb, seneca valley virus stond nog op not yet recruiting terwijl ik de clinical trial was tegengekomen, deze heb ik eruit gelaten. (toen ik nog eens checkte was de status al veranderd naar not recruiting). Weeral die chinezen die ons aan het saboteren zijn!Het geeft inderdaad niet een volledig overzicht van alle clinical trials, maar geeft wel de progressie weer. Virusviral vectorIndication(s)PhaseStatusRouteNotesRef

CG0070human adenovirus expressing GMCSFNon-muscle invasive bladder cancerII/IIIRecruitingi.ves.as a single agentNCT01438112

DNX-2401oncolytic adenovirusglioblastomaIRecruitingi.b.p.combined with temozolomideNCT01956734

glioblastoma or gliosarcomaIRecruitingi.t.combined with IFN-NCT02197169

VCN-01human adenovirus expressing PH20 hyaluronidasesolid tumorsIRecruitingi.v.combined with gemcitabineNCT02045602

pancreatic cancerIRecruitingi.t.combined with gemcitabineNCT02045589

ColoAd1chimeric Ad11p/Ad3 adenovirusEpithilial solid tumors, bladder and colorectal cancerI/IIRecruitingi.v.as a single agentNCT02028442

overian carcinomaI/IIRecruitingi.per.as a single agentNCT02028117

ICOVIR-5oncolytic adenovirusmalignant melanomaIrecruitinge.v.as a single agentNCT01864759

MG1 Maraba /MAGE-A3rhabdo virus expressing tumor-associated antigen MAGE-A3solid tumorsI/IIRecruitingi.v.Combined with AdMa3, adenovirus expressing MAGE-A3, as priming componentNCT02285816

HF10attentuated HSV-1 virusunresectable or metastatic melanomaIIRecruitingi.tcombined with IpilimumabNCT02272855

HSV1716 SeprehvirICP34.5 lacking HSV-1 mutantgliomaIRecruitingi.t.c.combined with dexamethasoneNCT02031965

non-CNS solid tumorsIRecruitingi.v. or i.t.as a single agentNCT00931931

malignant pleural mesotheliomaI/IIRecruitingi.pl.as a single agentNCT01721018

MV-NISmeasles virus expressing thyroidal sodium-iodide symporterMultiple myelomaIIRecruitingi.v.as a single agentNCT02192775

malignant mesotheliomaIRecruitingi.pl.as a single agentNCT01503177

peritoneal, fallopian ovarian carcinomaIIRecruitingi.per.as a single agentNCT02364713

plasma cell myeolmaI/IIRecruitingi.v.combined with cyclophosphamideNCT00450814

head and neck cancerIRecruitingi.t.as a single agentNCT01846091

GL-ONC1attenuated vaccinia virus with fluorescent reporter geneshead and neck cancerI/IINot yet recruitingi.v.combined with cisplatin and radiation therapyNCT01584284

advanced solid tumorsIRecruitingi.v.as a single agentNCT00794131

REOLYSINoncolytic reovirusmetastatic colorectal cancerIRecruitingi.v.combined with FOLFIRI and bevacizumabNCT01274624

non-small cell lung cancerIINot yet recruitingi.v.combined with paclitaxel and carboplatinNTC00861627

CVAcoxsackievirus A21malignant melanomaIIRecruitingi.t.as a single agentNCT01227551

VSV-IFNVSV virus expressing IFNprimary hepatocellular carcinomaIRecruitingi.t.as a single agentNCT01628640

ParvOryxparvovirus H-1glioblastomaI/IINot yet recruitingi.v. or i.t. and i.t.c.combined with surgeryNCT01301430

NDVnew castle disease virusglioblastoma, sarcoma and neuroblastomaI/IINot yet recruitingi.v.as a single agentNCT01174537

Abbreviations: i.t., intratumoral; i.v., intravenous; i.per.: intraperitioneal; i.pl.: intrapleural; i.t.c.: into tumor resection cavity; i.b.p.: in brain parychema; e.v., endvenous; i.ves.: intravesical

Table 3: Natural Cytotoxicity Receptors and their viral ligands on infected cells

VirusActivating NK receptorViral ligand(s) on infected cellsRef.

Influenza virus(IV)NKp46

NKp44HAa (activation)

HA (activation)92

93

Sendai virus(SV)NKp46

NKp44HNb (activation)

HN (activation)147

93

Newcastle disease virus(NDV)NKp46

NKp44HN (activation)

HN (activation)148

Vaccinia virus(VV)NKp30

NKp46 HA (inhibition)

HA (activation)149

Dengue virus(DV)NKp44E-protein (activation)94

West Nile virus(WNV)NKp44E-protein (activation)94

aHA: hemagglutinin; bHN: hemagglutinin-neuraminidase.

Table 4: Effect of viral infection on activating NK receptors and their ligands

VirusEffect on activating NK receptorsEffect on ligand(s) on infected cellsRef.

Herpes simplex virus 1 (HSV-1)

Up-regulation of NKp46, NKp30, NKp44 on maturing NK cellsUp-regulation of NCR-L(mediated by ICP0 protein)

Down-regulation of NKp30-L and NKp46-L

Down-regulation of NKG2D-L150

24

151

152

Herpes simplex virus 2 (HSV-2), Pseudorabies virus(PRV)Degradation of Nectin2 (mediated by gD)107

Cytomegalovirus(CMV)Modulation of NKp30 function(mediated by pp65/UL83)

Down-regulation of NKp46-L and NKp30-L

Modulation of NKG2D-L (mediated by UL16, UL141, UL142, miR-UL112)153

154

105

Kaposis sarcoma-associated herpesvirus (KHSV)

Down-regulation of NKp44-L (mediated by ORF54/dUTPase)155

Human herpesvirus 8(HHV8)Down-regulation of NKp46, NKp30, NKG2DUp-regulation of NKG2D-L156

Vaccinia virusUp-regulation of NCR-L95, 149

Hepatitis C virus(HCV)Up-regulation of NKp46 and NKp30

Down-regulation of NKp30

Transient induction of NKp30 and NKG2D (acute infection and exposed-uninfected pts)110

91,, 157

111, 112

ParvovirusUp-regulation of NCR-L158

HIV-1Down-regulation of NCR

Expansion of CD56-CD16+NCR- subset

Up-regulation of NKp44-L (mediated by gp41S3peptide)

Intracellular retention of NKp44-L (mediated by nef)

Down-regulation of NCR-L

Up-regulation of NKG2D-L (mediated by vpr)

Shedding of NKG2D-L

Down-regulation of NKG2D-L (mediated by nef)99

159

160

102

102, 161

103

104

100

Myxoma Virus(MYXV)Down-regulation of Nectin-2119

HCMVExpansion of NKG2C+ subset162

Chikungunya virusExpansion of NKG2C+ subset90

HantavirusExpansion of NKG2C+ subset163