Tumor cells express surface molecules that either switch off or switch on NK cell-mediated cytotoxicity. HLA class I molecules on tumors negatively regulate NK cell function by engaging immunoreceptor tyrosine-based inhibition motifs (ITIM)-bearing receptors (24) that include the inhibitory killer Ig-like receptors (KIRs, CD158), highly polymorphic clonally distributed receptors able to distinguish among different HLA-A, -B, and -C allotypes (25), and CD94/NKG2A heterodimers, specific for non-classical HLA-E (26). KIR and CD94/NKG2A are differently expressed in CD56bright CD16− and CD56dull CD16+ NK cell subsets, which represent sequential stages of maturation (27, 28). While high numbers of CD56bright CD16−, KIR−, IFNγ producing cells are found in non-reactive lymph nodes and tumor sites, the majority of NK cells circulating in the blood are mature CD56dull CD16+, KIR+, cytolytic (perforinhigh) cells (29). CD56bright can progress to CD56dull cells, which upon activation increase the cytotoxic and IFN-γ producing capabilities (28, 30).

Tumor cells switch on NK cell function by expressing at the cell surface non-MHC class I, "danger" molecules that are recognized by an array of activating NK receptors. These include both the FcγRIII (CD16) that mediates the antibody-dependent cytotoxicity (ADCC) and receptors that function in the absence of antibodies. These latter molecules mainly consist of the NKp46, NKp30, and NKp44 receptors, collectively termed natural cytotoxicity receptors (NCR), which are mostly NK-restricted, and NKG2D and DNAM-1 that are also expressed by T cell subsets (31). In the last decade, several groups dedicated many efforts to unveil the nature of the surface tumor ligands specifically recognized by the activating receptors. NKp46 still remains a receptor with "orphan ligand," while NKp44 has been shown to recognize a novel isoform of the mixed-lineage leukemia (MLL5) protein (32) and NKp30 binds to B7-H6 molecules (33). The NKG2D and DNAM-1 (CD226) receptors recognize MICA/B and ULBPs or, PVR (CD155) and Nectin-2 (CD112), respectively (31). DNAM-1 shares the capability of recognizing PVR with tactile (CD96) (34) and TIGIT (35). All the ligands identified so far represent *de novo* expressed danger signals or molecules that are expressed in healthy cells and over-expressed upon tumor transformation. In most cases, the expression on tumors of multiple ligands leads to the engagement of different activating NK receptors that cooperate in triggering the anti-tumor cytotoxicity (31).

When both inhibitory and activating receptors are engaged by the specific ligands on potential targets, the function of ITIMbearing inhibitory receptors dominates over activation. Thus, in an autologous setting NK lymphocytes spare healthy cells that express high, "protective" levels of HLA class I molecules while they kill tumors, such as NB, in which HLA class I expression is downregulated (36, 37). Moreover, NK cells kill allogeneic targets that express non-self MHC class I alleles. This might occur when two individuals are "KIR/KIR-ligand mismatched," i.e., individual A is characterized by fully functional, educated NK cell subset(s) expressing KIR specific for HLA class I alleles that are missing in individual B. In the context of HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT), the presence of a KIR/KIR-ligand mismatch in the donor versus recipient direction allows donor's NK cell subsets to kill recipient's (HLA class I high) residual leukemic blasts thus resulting into a graft-versusleukemia effect (GvL), as well as mature DC and activated T lymphocytes, thus preventing graft-versus-host-disease (GvHD) and graft-rejection, respectively (38).

In NB, initial data were obtained by the use, as target cells, of *in vitro* established NB cell lines (39). NK cells displayed strong cytolytic activity against different NB cells and NCR were involved in the mechanisms leading to killing (39). The susceptibility of human NB cell lines to NK-mediated killing was validated in the context of a metastatic model set up in NOD/SCID mice. In this experimental setting, repeated infusions of IL-2 or IL-15 activated NK cells resulted in both an increased mean survival time of HTLA-230-bearing mice and reduced BM infiltration (40). Considering that long term cultured cell lines might be poorly representative of original tumors, more interesting data originated from *in vitro* studies that use neuroblasts, freshly purified from BM aspirate of stage 4 patients (36) (**Figures 1** and **2**). In this study, allogeneic activated NK cells killed neuroblasts isolated from patients although at a lesser extent as compared to the NB cell lines used as control. According to the absence or negligible expression of HLA class I molecules, the NK-mediated lysis of neuroblasts did not increase in the presence of anti-HLA class I mAb. Different molecular mechanisms responsible for the reduced HLA class I expression in NB cells have been elucidated. The immunohistochemical analysis of high-risk human NB showed different abnormalities in the antigen processing machinery, which include defects in the expression of immunoproteasomal subunits LMP2 and LMP7 and of transporters of antigen processing (TAP) (37). *In vitro* treatment of NB cells with IFN-γ induced up-regulation of HLA class I expression (37). Although decreasing their susceptibility to autologous NK cells, this up-regulation of HLA class I molecules could enhance T cell-mediated recognition. In this context, a restoration of killing mediated by antigen specific (MAGE3) cytolytic T cells was observed upon cotransfection of NB cell lines with IRF1 and NF-kB p65, HLA class I transcriptional activators that are also induced by INFγ and TNFα, respectively (41). It cannot be excluded that also *in vivo* neuroblasts could acquire/upregulate HLA class I expression. For example, this might occur in the context of transplantation or following the anti-GD2 antibody-mediated therapy that engages FcγR + immune cells such as NK cells, which are capable of releasing high amounts of IFN-γ upon activation (**Figure 1**). This phenomenon was observed in a murine NB model, where recurrent tumors developed after an NK-dependent anti-tumor response induced by a humanized IL-2 immunocytokine targeted to GD2. In these mice, NB cells showed markedly enhanced MHC class I expression as compared with tumors growing in controls (42). The possible *in vivo* increase of HLA class I expression in NB cells could explain the benefit of a KIR/KIR-L mismatch in the NK versus NB direction (43–45).

Bone marrow-purified neuroblasts from different patients, although expressing in all instances low levels of HLA class I, displayed a great heterogeneity in terms of susceptibility to lysis by NK cells. The good or poor susceptibility to NK-mediated killing correlated with the presence or the absence of PVR expression on neuroblasts (**Figures 1** and **2A**)(36). Interestingly, neuroblasts lacking PVR expression were from patients in relapse, whereas those expressing PVR were from children at the onset of the disease,suggesting a possible correlation between PVR expression and NB status. The predominant role of DNAM-1/PVR interaction in NK-mediated killing is striking considering that, in most cases, killing of human tumors depends on the cooperation between

various activating receptors (31). In this context, it is of note that none of the BM-purified neuroblast fractions expressed major adhesion molecules such as ICAM1 or LFA-3 (36). Thus, in NB the presence of PVR, originally described as an adhesion molecule, could become critical in rendering tumor cells susceptible to NKmediated killing. Although PVR and nectin-2 are closely related molecules of the Nectin family, the presence/absence of nectin-2 on NB cells did not influence the susceptibility to NK-mediated killing. Nectin-2, which displays binding affinity to DNAM-1 similar to PVR (46), also shows homophilic recognition. This, by hampering its recognition by DNAM-1, may force the receptor to preferentially bind PVR (46). In line with the increased killing of PVR+ neuroblasts, mAb mediated masking of DNAM-1 significantly reduced the NK-mediated lysis of neuroblasts showing a pivotal role of DNAM-1 in NB cell killing. While a minor contribution of NKp46 and NKp30 activating receptors could be appreciated (36), consistent with the low expression of the specific ligands, NKG2D did not play a significant role in killing of BM-purified neuroblasts (**Figure 1**). The expression of NKG2D ligands has been also investigated in human primary NB and cell lines (47). *MICA, MICB*, and *ULBPs* transcripts were found in most tumors and cell lines. However, MICA surface expression was absent in primary tumors and present only in some NB cell lines. Importantly, the soluble form of MICA (sMICA) was detected in sera of most patients and in the culture supernatant of some cell lines and was shown to downregulate NKG2D surface expression

in peripheral-blood T cells and decrease NK cell-mediated killing of MICA+ NB cells (47) (**Figure 2B**). MICB was detected exclusively in the cytosol of NB cells, either primary tumors or cell lines. ULBP-1 was rarely detected, ULBP-2 was expressed by approximately 50% of primary tumors and cell lines, and ULBP-3 was absent in primary tumors but was expressed in most NB cell lines (47).

A crucial role of the DNAM-1 receptor has also been reported in myeloid and lymphoblastic leukemia (48), ovarian tumors (49), and different murine tumor models (50, 51). In particular, it has been shown that in response to chemical carcinogens, DNAM-1-deficient mice develop more DNAM-1 ligand+ fibrosarcoma and papilloma cells than wt mice. Thus, the study of the molecular mechanisms responsible for the modulation of DNAM-1 ligands appears to be highly relevant. In this context, it has been shown that malignant RAJI lymphoma cells present hypermethylated PVR promoter-associated CpG islands. The epigenetic status was partially reversed by hypomethylating agents that led to upregulation of both transcription and surface expression of PVR (52). PVR up-regulation has been shown to be dependent on DNAdamage response (DDR)-dependent pathways that are activated by oxidative stress (53). Moreover, modulation of PVR surface expression might occur during infections. Different pathogenderived stimuli such as LPS, poly I:C, and flagellin upregulated the surface expression of PVR (and nectin-2) in human DC (54) and in murine antigen-presenting cells (55) via the MYD88 and TRIF pathways. It has also been shown that expression of the human

immunodeficiency virus type 1 (HIV-1)Vpr protein increases PVR levels in Jurkat T cells. This is due to activation of ATR kinase that triggers the DDR pathway and G2 arrest. Moreover, Vpr induces a PVR upregulation in HIV-infected CD4+ T lymphocytes that overcomes the PVR downregulation induced by the HIV Nef protein (56). A negative regulation of PVR surface expression has been observed during cytomegalovirus (CMV) infection resulting in ineffective NK cell-mediated killing of infected cells (57). The UL141 viral glycoprotein is responsible for PVR downregulation, since it retains the immature form of PVR in the endoplasmic reticulum (57). Recently, it has been shown that UL141 binds to PVR with high affinity (58). Considering the role of PVR in determining high susceptibility to the NK-mediated attack, up-regulation of PVR surface expression might represent an appealing strategy to strengthen the immune responses against tumors. However, the dark side of PVR function cannot be disregarded. Indeed, although data in NB are missing, PVR has been described as a positive regulator of cancer progression, since it is capable of enhancing tumor cell invasiveness and migration (59). Thus, further studies are required to understand whether the immunological favorable effects of PVR up-regulation might balance the induction of a more aggressive tumor phenotype.

A tumor-promoting role has been shown also for B7-H3, a surface molecule originally identified on NB thanks to the generation of a specific monoclonal antibody (M5B14) (60). Studies on different tumor histotype indicate that B7-H3 might drive tumor cell development by different molecular mechanisms. These include the capability of B7-H3 of reducing the effect of the chemotherapeutic-induced apoptosis in breast (61) and pancreatic carcinoma (62), as well as that of promoting migration and invasiveness (63, 64). The latter properties have been confirmed *in vivo* in an orthotopic model that showed that xenografted B7-H3 silenced glioma cells invaded significantly less into the surrounding brain tissue as compared to wt tumors. Moreover, B7-H3 expressed on glioblastoma cells negatively regulated NK cell-mediated cytotoxicity (64). These observations are consistent with previous data demonstrating that B7-H3 expression on BM-purified neuroblasts decreased NK cell-mediated killing (60) (**Figure 1**). Thus, different experimental approaches demonstrated that, in humans, B7-H3 might block the NK-mediated attack by interacting with a still unknown inhibitory NK receptor. A soluble form of B7-H3 (sB7-H3) that results from MMPs cleavage of the surface protein was detected in sera of tumor patients and appeared to correlate with tumor burden (65, 66). Moreover, B7- H3 is present in NB cell lines-derived exosomes (67). The exosomeassociated form could inhibit NK cell function by engaging the B7-H3-specific inhibitory receptor (**Figure 2B**). Thus, B7-H3 is an interesting NB-associated molecule that combines immuneevading and tumor progression properties. This correlates with the negative prognostic value associated to high expression of B7- H3 in several tumor types (64, 66, 68–72). In particular, in primary NB (73), high B7-H3 surface intensity and percentage of positive cells correlated with poor event-free survival. Interestingly, the differences in event-free survival were observed also in patients with localized disease (stage 1–3), suggesting the need of a more careful follow-up/aggressive treatment of B7-H3+ low-risk patients. It should be mentioned that, in the mouse system, B7-H3 has been described as a "friend" in tumor immunology (74). In particular, intratumoral injection of an expression plasmid encoding mouse B7-H3 led to complete regression of 50% of tumors, which was mediated by NK and CD8+ T cells (75). It is to note that the mouse *B7-H3* gene codes for a molecule characterized by two Ig-like domains (2Ig-B7-H3) in the order IgV and IgC, while the predominant isoform in human tissues and cell lines is a longer molecule (4Ig-B7-H3) characterized by four Ig-like domains (IgV–IgC–IgV– IgC), which results from duplication of the exons encoding the IgV and IgC domains (76). The mouse B7-H3 has been shown to bind the TREM-like transcript 2 (TREML2, TLT-2) triggering receptor, which is expressed by myeloid cells, CD8+ T cells, and activated CD4+ T cells (77, 78). In humans, TREML2 does not appear to be a receptor of B7-H3 (79) and the predominant inhibitory role of B7-H3 strongly suggests the existence of a (still undefined) receptor that down-regulates NK cell function. However, the existence in the human system of specific receptors with opposite functions cannot be excluded. Indeed, human B7-H3 belongs to the B7 family that includes members such as B7–1 (CD80), B7–2 (CD86), B7-H2 (ICOS-L), B7-H1 (PD-L1), B7-DC (PD-L2), B7- H4 (B7S1, B7x), and BT3 (80), some of which represent specific ligands of receptors with either activating or inhibitory function. The *scenario* should become even more complex when considering that the B7-H3 inhibitory and activating receptors might be co-expressed in immune cells rather than expressed in different cell types/subsets.

# **TUMOR ESCAPE MECHANISMS IN NEUROBLASTOMA AND POSSIBLE NOVEL NK CELL-BASED IMMUNOTHERAPEUTIC APPROACHES**

In principle, downregulation of HLA class I surface expression allows NB to evade T cell-mediated attack operated by the host immune system (36, 37), while rendering tumor cells susceptible to NK cell-mediated recognition and killing (31). However, both the fast tumor progression in high-risk NB patients and the *in vitro* data highlighted the existence of additional escape mechanisms that dampen the NK cell-mediated anti-tumor activity as well. As discussed above, the lack of ligands for activating receptors such as PVR (36) (**Figure 2A**), MICA/ULBPs (36, 37), or adhesion molecules (36, 81) represents a strategy allowing NB cells to strongly reduce their susceptibility to NK-mediated killing. Thus, one could set up immunotherapeutic interventions aimed at upregulating the expression of these ligands. However, the role of PVR in tumor progression (59), as well as its expression in normal cells, should not be underestimated. In particular, DNAM-1 co-operates with NKp30 or NKp46 in the NK-mediated killing of autologous immature DC and unpolarized (M0) or M2-polarized macrophages, which express low, "non-protective" amounts of HLA class I molecules. Conversely, mature DC and M1-polarized macrophages express high levels of HLA class I molecules and are protected by the NK cell-mediated attack (54, 82). The NK-mediated selection of DC and macrophages with optimal antigen-presentation properties has been named "immunoediting" and results from the fine-tuning of inhibitory and activating signals provided by the ligands physiologically expressed by DC and macrophages. The forced over-expression of ligands for activating receptors, such as PVR, might dangerously alter the immunoediting process. Moreover, it should be taken into account that PVR is expressed in normal endothelium, where it plays a role in leukocyte extravasation (83, 84). Also in this case, up-regulation of PVR expression might overcome the HLA class I-mediated inhibitory signals and result in loss of self-tolerance toward autologous endothelium. On the other hand, it has been shown in primary glioblastoma that endothelial cells of proliferating tumor vessels express PVR at higher surface densities as compared to normal vessels (22). Moreover, several studied indicated that most tumor cells including CSC constitutively express levels of PVR higher than the normal counterparts (21). Although further studies are needed to dissect the possible side effects, therapeutic approaches focused on PVR might represent a chance in the treatment of highly aggressive tumors such as NB. Up-regulation of PVR in NB patients might increase the susceptibility to NK-mediated lysis of PVR+ NB and restored that of PVR− NB. In this context, it is of note that the lack of PVR expression was detected in given BM metastatic neuroblasts (36) while no data are available on PVR expression in primary tumors. Thus, it cannot be excluded that PVR− NB cells might originate because of the selection pressure occurring during the dissemination process and that primary tumors could contain PVR+ targetable neuroblasts. If so, a larger cohort of patients might benefit from PVR up-regulation therapies. Importantly, PVR might represent a powerful therapeutic target on both neuroblasts and NB-associated vessels. PVR has been originally identified as the receptor for the poliovirus, a highly contagious

virus that only affects humans. As already mentioned in a study performed in a poliovirus-susceptible animal model, the oncolytic treatment by a novel attenuated poliovirus eradicated PVR+ NB cells without signs of paralysis (85).

B7-H3 might represent an additional attractive molecular target in NB. Beside its tumor-promoting properties, human B7-H3 represents a shield protecting HLA class I low/− neuroblasts from the NK cell-mediated attack (60,64) (**Figure 1**). B7-H3 is expressed in normal endothelium, but is strongly upregulated in tumorassociated vasculature (86). Encouraging results derived from the first in-human intrathecal injection of radioiodinated anti-B7-H3 Ab (following surgery, craniospinal irradiation, and chemotherapy) in 21 NB patients with recurrent CNS metastasis (87). Seventeen patients remained CNS disease free and had a median survival time (33 months) significantly better than patients treated with standard protocols (6.6 months) (88). Anti-B7-H3 antibodies, possibly delivered using lipid-based (liposome) formulations (89–91), might represent a suitable option to target tumoricidal compounds to NB and to increase immune responses (**Figure 3**). Indeed,acting on different cell types,it might simultaneously block invasiveness/migration of tumor cells, destroy tumor-associated vessels, and strength the activity of NK cells. Moreover, anti-B7- H3 antibodies might induce NK cell-mediated ADCC against NB and destroy the B7-H3/inhibitory receptor interactions. An alternative approach to trigger NK cell function might consist in the use of bi-specific antibodies reacting with B7-H3 (on tumor cells) and activating receptors (on NK cells). In addition B7-H3 might be considered as a molecule alternative to GD2 in CAR-based immunotherapy (**Figure 3**). CAR are chimeric antigen receptors, formed by tumor-specific Ab single chain Fv fragments (scFvs) genetically fused through a transmembrane domain to the CD3ζ chain, which are transfected into cytolytic lymphocytes (92, 93). The use of CD19-specific CARs resulted in a significant clinical benefit in two children with B-cell precursor acute lymphoblastic leukemia (94). The NK92 cell line engineered to express the GD2-specific CAR showed increased cytolytic activity against NB cell lines and primary NB cells (95). Moreover, T lymphocytes equipped with GD2-specific CAR are being used in preclinical and phase I studies with clinical benefit in NB patients (96, 97). Essential requirement in CAR-based immunotherapy is the selection of an adequate tumor surface antigen with a documented tumor-promoting role (92). B7-H3 satisfies this criterion, thus representing a suitable candidate in CAR-based immunotherapy. Moreover, while GD2-specific CAR target a molecule that is easily shed by the NB cell surface (98), B7-H3-specific CAR would target a stable type I transmembrane surface molecule expressed by NB cells and NB-associated vessels. Infusion of engineered and/or activated (by cytokine or DC) (99) NK cells might be helpful in adoptive immunotherapeutic approaches of high-risk NB patients receiving autologous (5) or haplo-HSCT. Indeed, a gap of several weeks exists between the infusion of CD34+ stem cells and the appearance in peripheral blood of fully "armed" mature NK cells (38). During the early post-transplant period, lack of NK-mediated immunosurveillance could favor re-growth of residual NB cells escaping the preparative regimen to the allograft, this causing tumor relapses. Haplo-HSCT, which represents a new standard therapy in the cure of adult and pediatric leukemic

patients, could also increase the event-free survival of NB patients (45). Recently, a novel transplant strategy based on negative depletion of both α/β T lymphocytes and of CD19+ B-cells has been proposed for pediatric hematological disorders. This approach leaves in the graft an array of immune cells with anti-tumor properties, including mature armed donor-derived alloreactive NK cells and γ/δ T cells (38). Also in this type of transplant that preserves donor mature NK cells in the graft, engineered NK cells might support the anti-tumor responses. In the perspective of *in vivo* NK-based adoptive immunotherapies as adjuvant in different transplant strategies, recent studies optimized the *in vitro* expansion and activation of NK cells that preserved the anti-NB activity (100–103).

B7-H3-specific CAR that transduce activating signals via CD3 chain.

In addition to the escape mechanisms described above (47, 60, 104), NB cells produce soluble factors such as MIF (105, 106) and TGF-β (107) capable of dampening NK cell activity (108, 109) (**Figure 2B**). Initial data showed that *in vitro* conditioning of NK cells with rTGF-β1 resulted in downregulation of NKp30 and NKG2D activating receptors and in significant reduction of NK-mediated killing (109). Different studies confirmed *in vivo* the role of TGF-β in regulating NKG2D expression and function in NK cells (110, 111). Although knockout mice lacking TGF-β1 or TGF-β2 showed distinct phenotypes suggesting that each isoform could also have specific, non-overlapping functions (112), *in vitro* high concentrations of both rTGF-β1 and rTGF-β2 (>10 ng/ml) showed similar capability of inhibiting the expression of the activating NK receptors (113). It has been shown that NB cell lines released low/medium amounts of TGF-β1 (<3 ng/ml), which were unable to significantly downregulate NKp30 and NKG2D expression while clearly modulated the chemokine receptor repertoire of NK cells (113). Interestingly, non-MYCN-amplified NB cells produced amounts of TGF-β1 (and several other soluble factors)

significantly higher than MYCN amplified cells (113). NB-derived TGF-β1 upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated that of CX3CR1 in the CD56dim subset. Notably, a similar altered chemokine receptor repertoire was observed in peripheral-blood NK cells of stage 4 NB patients resulting in the appearance of a CX3CR1low CD56dim NK cell population. In patients, also a tendency to CXCR3 upregulation on CD56bright NK cells was appreciated. No significant differences in CXCR4 expression were detected in NK cells from NB patients as compared to healthy donors. This might be due to a general difficulty in detecting *in vivo* CXCR4 expression, which in particular in the BM, might be fully occupied by the CXCL12 (SDF1) ligand (114). In NK cells from NB patients, also a significant CXCR1 down-modulation was observed, that, however, was TGF-β1-independent (113). Thus, *in vivo*, NB cells can release TGF-β1 and other, still unknown soluble factor(s) that profoundly affect the expression of a number of chemokine receptors that play pivotal roles in NK cell BM homing, egress, interaction with endothelium, and recruitment into peripheral tissues. High concentrations of TGF-β1 might locally exert a paracrine effect on tumor-associated NK cells that might decrease their anti-tumor activity by downregulating NKp30 and NKG2D expression. In this context, it has been shown that CD56bright, immature, poor cytolytic NK cells represent the major subset present in tumor tissues (115, 116). The above data suggest that TGF-β1 antagonists and/or proinflammatory cytokines capable of overcoming the modulatory effect of TGF-β might represent a reasonable adjuvant therapy in the cure of different tumors, including NB. In this context, mouse models showed that use of IL-2 targeted to GD2 is associated with increased infiltration of NK (and CD8+ T cells) in subcutaneous NB (117).

Novel immunotherapeutic approaches should also consider the complexity of tumor microenvironment that is populated by different immune cell types that could be endowed with immunomodulatory functions. These cells include fibroblasts (118), regulatory T cells (Treg), myeloid derived suppressor cells (MDSC) (119), and tumor-associated macrophages (TAM) (120). Different NB mouse models demonstrated the immunosuppressive and tumor-promoting role of Treg (120) and MDSC (121). To date, however, data on their presence within the human NB tumor and on their clinical relevance are still scarce. A recent study showed that metastatic NB have higher infiltration of TAM than locoregional tumors, and that metastatic tumors in patients ≥18 months have higher expression of inflammation-related genes than those in patients <18 months (122). TAM are characterized by an"M2-like"functional phenotype and exert tumor-promoting and immunoregulatory properties, including the release of TGFβ1 (120) (**Figure 2**). M2-polarized macrophages still present a functional plasticity and they can be recommitted toward an M1-like tumor suppressing, immunostimulatory phenotype. In particular, it has been shown that microbial products such as LPS or BCG reverted their functional phenotype toward M1, which released immunostimulatory cytokines (IL-12, IL-18) and induced strong activation of autologous NK cells. NK cells upregulated CD69 and CD25 (that associates with CD122 to form the high-affinity receptor for IL-2), expressed CCR7 (a chemokine receptor involved in homing of NK cells to secondary lymphoid

organs), increased the anti-tumor cytolytic activity, and released high amounts of IFN-γ (82). Interestingly, it has been shown that a subset (30–40%) of M2 express a membrane-bound form of IL-18 (mIL-18) that is released upon TLR stimulation (123, 124). This soluble form of IL-18 (sIL-18) by acting in close cellto-cell contact is crucial for both IFN-γ release and expression of CCR7 by NK cells. These data suggest that reconverting the immunosuppressive TAM phenotype using apathogenic TLR ligands might represent an additional immunotherapeutic approach to fully activate immature tumor-associated NK cells.

# **CONCLUSION**

Progress in the understanding of NB cell biology will allow a more accurate stratification of patients, thus reducing toxic side effects of aggressive therapy in low-risk patients. High-risk patients who currently have a dismal prognosis could benefit from multidisciplinary therapeutic protocols that include novel NK cell-based immunotherapeutic strategies. The latter will take advantage of our knowledge about the presence/absence of NB-associated ligands interacting with activating/inhibitory receptors expressed by NK cells. Moreover, it should also take into account the multiple immunomodulatory strategies set up by NB and various immune cell types to impair the recruitment and activation of NK cells in the tumor microenvironment. The encouraging results emerged from haploidentical hematopoietic cell transplantation in pediatric hematological malignancies, might strongly motivate a re-evaluation of transplant approaches in the therapy of high-risk NB patients.

# **ACKNOWLEDGMENTS**

This work was supported by Investigator Grants (10643 and 11650) and special project 5x1000 (9962) from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), by Ministero dell'Istruzione, dell'Università e della Ricerca (M.I.U.R), and by Ministero della Salute 5 per mille and Ricerca Corrente, and Progetto di Ricerca Finalizzata RF-IGG-2008-12006685. Francesca Bellora is recipient of a fellowship awarded by A.I.R.C. (special project 5x1000, 9962). We apologize to the colleagues whose work could not be cited because of space constraints.

# **REFERENCES**

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its ligands PVR (CD155) and nectin-2 (PRR-2/CD112). *Int Immunol* (2004) **16**:533–8. doi:10.1093/intimm/dxh059

from peripheral blood mononuclear cells through STAT-3 activation. *Clin Exp Immunol* (2013) **172**:104–12. doi:10.1111/cei.12034

**Conflict of Interest Statement:** Alessandro Moretta is a founder and shareholder of Innate Pharma (Marseille, France). The remaining authors declare no conflicts of interest.

*Received: 20 December 2013; paper pending published: 14 January 2014; accepted: 30 January 2014; published online: 12 February 2014.*

*Citation: Bottino C, Dondero A, Bellora F, Moretta L, Locatelli F, Pistoia V, Moretta A and Castriconi R (2014) Natural killer cells and neuroblastoma: tumor recognition, escape mechanisms, and possible novel immunotherapeutic approaches. Front. Immunol. 5:56. doi: 10.3389/fimmu.2014.00056*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Bottino, Dondero, Bellora, Moretta, Locatelli, Pistoia, Moretta and Castriconi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Natural killer cells modulation in hematological malignancies

#### **Céline Baier 1,2, Aurore Fino1,2, Carole Sanchez 1 , Laure Farnault 1,3, Pascal Rihet 1,2, Brigitte Kahn-Perlès 1,2 and Régis T. Costello1,2,3\***

1 UMR1090 TAGC, INSERM, Marseille, France

2 UMR1090 TAGC, Aix-Marseille Université, Marseille, France

3 Service d'hématologie, APHM, Hôpital de la Conception, Marseille, France

#### **Edited by:**

Massimo Vitale, IRCCS A.O.U. S.Martino-IST, Italy

#### **Reviewed by:**

Laurel L. Lenz, National Jewish Health, USA Jacques Zimmer, Centre de Recherche Public de la Santé, Luxembourg Nicolas Dulphy, Universite Paris Diderot, France Renato Zambello, Padova University School of Medicine, Italy

#### **\*Correspondence:**

Régis T. Costello, Service d'hématologie, APHM, Hôpital de la Conception, 147 Bd Baille, 13005 Marseille 13005, France e-mail<EMAIL_ADDRESS>
Hematological malignancies (HM) treatment improved over the last years resulting in increased achievement of complete or partial remission, but unfortunately high relapse rates are still observed, due to remaining minimal residual disease.Therefore, sustainment of long-term remission is crucial, using either drug maintenance treatment or by boosting or prolonging an immune response. Immune system has a key role in tumor surveillance. Nonetheless, tumor-cells evade the specific T-lymphocyte mediated immune surveillance using many mechanisms but especially by the down-regulation of the expression of HLA class I antigens. In theory, these tumor-cells lacking normal expression of HLA class I molecules should be destroyed by natural killer (NK) cells, according to the missing-self hypothesis. NK cells, at the frontier of innate and adaptive immune system, have a central role in tumor-cells surveillance as demonstrated in the setting of allogenic stem cell transplantation. Nevertheless, tumors develop various mechanisms to escape from NK innate immune pressure. Abnormal NK cytolytic functions have been described in many HM. We present here various mechanisms involved in the escape of HM from NK-cell surveillance, i.e., NK-cells quantitative and qualitative abnormalities.

**Keywords: hematological malignancies, natural killer cells, natural cytotoxicity receptors, immune escape, immunotherapy**

# **INTRODUCTION**

The natural killer (NK) cells are central players in innate immunity particularly regarding the surveillance against malignant tumors (1, 2). NK role in tumor-cells clearance is proved by allogenic stem cells transplantation, since a better engraftment and a low relapse rate are observed when the graft NK inhibitory receptors mismatch with recipient HLA molecules (3) (**Figure 1**). The triggering event of NK-cells activation and killing of target cells results from a balance between activating and inhibitory signals sent by membrane receptors that either enhance or block the NK-mediated cytotoxicity (4). Inhibitory signals arise from interaction between HLA-specific inhibitory receptors, as the killer immunoglobulin-like receptors (KIR), NK group protein 2A (NKG2A), or Immunoglobulin-like transcript 2 (ILT-2) with HLA class I molecules, whereas the absence or abnormal expression of the later molecules induces NK-cells cytotoxicity (5). The down-regulation of HLA class I molecules is an immune escape mechanism frequently used by tumor cells (6) that, accordingly, should not be recognized by the T-lymphocyte receptor (TCR). The absence of normal HLA class I molecule on tumor cells should lead to NK-cells activation, more efficiently when co-stimulatory molecules and ligands for NK activating receptor are present at tumor cell surface. The natural cytotoxicity receptors (NCR) NKp46/NCR1, NKp30/NCR3, and NKp44/NCR2 (7), NKG2D, DNAM-1 and also co-receptors such as 2B4/CD244 and NTBA, play a central role in NK activation. Once activated, NK lymphocytes kill tumor cells via FcgRIIIA (CD16) which can trigger

antibody-dependent cellular cytotoxicity (ADCC) on encountering target cells opsonized with IgG, via the Fas/Fas-L pathway and via cytotoxic granules (perforin/granzyme) secretion (1, 8).

Defects in NK-cell cytotoxicity have been described in most hematological malignancies (HM) (9–12) (**Table 1**). Interestingly, tumor-cells develop various escape mechanisms to NKcell surveillance and contribute to the dysfunction of NK-cell cytotoxicity (4).

# **NK-CELLS DYSFUNCTIONS IN HEMATOLOGICAL MALIGNANCIES**

#### **QUANTITATIVE ABNORMALITIES**

The first mechanism explaining tumor escape is the quantitative abnormalities of NK cells. In myelodysplastic syndrome (MDS) patients, decreased NK-cell cytolytic functions correlate with a low number of circulating NK cells and a high level of sIL-2R (13, 14). In chronic myelogenous leukemia (CML), functional NK-cell deficiency can be reversed *in vitro* by IL-2 (15), but this effect is progressively lost while a progressive decrease in NK-cell number is observed (16). In Philadelphia (Ph1)-negative myeloproliferative syndrome (MPS), NK cytotoxic activity is decreased, mostly in idiopathic myelofibrosis (IMF) patients. The percentage of NK cells is decreased in IMF and increased in polycythemia vera (PV) (17). We have confirmed that the percentage and absolute number of NK cells are significantly increased in PV, but we failed to detect any abnormalities in the expression of activating NK-cell receptors or cytotoxic functions (personal data, C. Sanchez). An

increase in the total number of NK cells in the peripheral blood has also been described in chronic lymphocytic leukemia (CLL) but still associated with defective cytolytic functions (18).

# **ALTERED ACTIVATING RECEPTORS PROFILES**

In acute myeloid leukemia (AML) the down-regulation of activating receptors NKp30/NCR3 and NKp46/NCR1 correlates with defective NK-cell cytotoxicity and poor leukemia prognosis (9,19). In patients attaining complete remission (CR) after chemotherapy, NKp46/NCR1 expression returns to normal levels while patients who do not achieve CR or who relapse maintain abnormal NCR expression (9, 19). The defect in NCRs expression could be potentiated by the low expression of NCR and NKG2D ligands by leukemic cells (20–22). Down-regulation of the NK activating receptors/co-receptor DNAM-1, 2B4/CD244, and CD94/NKG2C have also been reported in AML (23, 24). Leukemic blasts that express DNAM-1 ligands induce DNAM-1 down-regulation at the NK-cell surface (25), thus impending NK-cell functions.

In acute lymphoblastic leukemia (ALL), expression of the NKG2D activating receptor ligands MICA/B was only observed in NK sensitive T-ALL cell line, while NK-resistant B-ALLs did not express detectable amounts of MICA/B (26). Deficient engagement of other activating receptors may also contribute to ALL resistance to NK lysis, since B-ALL cells lose or express low levels of several other NK activating ligands such as UL-16 binding proteins (ULBPs), PVR (polio virus receptor, CD155), Nectin-2 (CD112), or CD48 (27).

In MDS, a pre-leukemic stage, Epling-Brunette et al. (13) have shown that expression of NKp30/NCR3 and NKG2D was decreased, in contrast with the data of Kiladjian et al. (28); this discrepancy could be related to the heterogeneity of MDS patients.

In CML patients, Boissel et al. (29) reported high serum sMICA levels and weak NKG2D expression on NK cells, that correlate with low NK-cell cytotoxicity capacities. Imatinib mesylate, the first inhibitor of tyrosine kinase used in CML, increases NKG2D expression and decreases MICA protein production and release, thus contributing to normal NK cytotoxicity through the restoration of a functional NKG2D signaling (29).

Monoclonal gammopathy of undetermined significance (MGUS) is a common disorder of aging and a precursor lesion to multiple myeloma (MM). In MGUS, tumor-cells express high levels of MICA, whereas low levels of sMICA are detected in peripheral blood (30). This explains the capacities of NK cells to kill MGUS tumor cells by interaction between MICA and NKG2D. Conversely, MM patients present high plasma level of sMICA while tumor-cells express low level of MICA, thus impending NK stimulation via NKG2D (31). This reveals that the alterations in the NKG2D pathway signaling are associated with the progression from MGUS to MM (30, 32). In peripheral blood from patients with MM a normal expression of the NCRs and NKG2D is observed, while 2B4/CD244 and the low-affinity Ig- Fc receptor CD16 display significantly weaker expression in comparison with healthy donors (31). Nonetheless, when NK are studied at the site of tumor location, i.e., bone marrow, (33) a drastic downregulation of three major activating NK receptors (NKp30/NCR3, NKG2D, and 2B4/CD244) is observed in comparison with bone marrow from healthy donors (34). This suggests that some NK abnormalities may be underestimated if only peripheral blood is studied.

In CLL, NK cells have weak cytotoxic functions which can be restored by stimulation with recombinant IFN-γ or IL-2 (35). We failed to detect a difference in NCR expression between patients and age-matched healthy donors (36), but we found a correlation with abnormal activating molecule expression and poor prognosis factors. In CLL decreased NK functions could be explained in part by absent or low level of MICA or ULBP (18, 37) at CLL lymphocyte cell surface, thus impeding NKG2D engagement.

# **ABNORMAL KIRs PHENOTYPE AND INHIBITORY MOLECULES**

Killer immunoglobulin-like receptor-mismatch in allogenic stem cells transplantation improves the disease-free survival in AML (38). In fact, NK-cell cytotoxicity is down-regulated by the engagement of HLA-specific inhibitory receptors, including KIRs and CD94 and NKG2A/B heterodimers. The analysis of KIR phenotype in AML patients shows that the frequency of particular inhibitory KIRs in association with their putative HLA class I ligands is significantly increased compared to healthy donors (10, 39, 40). This supports the hypothesis that AML blasts escape from immune surveillance according to the dominance of inhibitory over activating KIR signals. In ALL, the resistance of B-cell precursors to cytotoxicity is explained by the interaction between HLA-G and KIR2DL4 (26). Demanet et al. (41) have observed in ALL and CLL a selective down-regulation of HLA-A and HLA-Bw6 associated with HLA-Bw4 preservation, which provided an escape mechanism from NK-cell immune surveillance. Maki et al. (18) reported in CLL cells an increased expression of HLA-G1, a class I molecule that engages NK-cell inhibitory molecules and which has for ligand p49/KIR2DL4/CD158d (expressed on NK cells and a fraction of T cells), ILT-2 [expressed on NK, T, B cells, dendritic cells (DC), and monocytes], and ILT-4 [expressed on antigen-presenting cells (APC)] (42).

# **TUMOR ENVIRONMENT AND ROLE OF CYTOKINES**

One of the strategies used by tumor cells having an effect on NK-cell function is the production of inhibitory molecules, which decrease NK-cells number and inhibit NK-cell activation. Increasing evidence supports the role of the tumor microenvironment in conferring drug resistance, a major cause of relapse and incurability of cancers. Tumor microenvironment includes tumor-cells contact and interaction, but also production of soluble factors, which provide signals for tumor growth and survival or inhibition of NK.

# **ROLE OF CYTOKINES AND CELLULAR LIGANDS**

Several cytokines decrease NK-cell activation and cytotoxicity, such as the Transforming Growth Factor beta (TGF-β). High circulating TGF-β level correlates with poor prognosis in acute leukemia (43) and is linked to reduced NK-cell activity with reduced expression of NKp30/NCR3 and NKG2D (44). TGF-β antagonizes IL-15, a cytokine that induces NK-cell proliferation and activation. Thus, TGF-β inhibits the expression of both NK-cell activation

receptor molecules and components of the cytotoxic apparatus (45, 46). Several studies have also underlined that low INF-γ producing capacity of NK cells was correlated with loss of NK-cell cytotoxicity (47, 48).

A physiologic concentration of platelet-derived growth factor (PDGF) significantly inhibits human NK-cell cytotoxicity. Patients suffering from IMF and essential thrombocythemia have significantly elevated circulating levels of plasma PDGF. Pretreatment of normal NK cells with concentrated PDGF-containing plateletpoor plasma from patients with these diseases significantly inhibits NK cytotoxicity. This inhibitory effect is reversed by neutralization of plasma PDGF with anti-PDGF (17, 49). All these data suggest that PDGF is probably a key factor of NK functional deficiency in MPS.

Interaction between tumor cells and NK impairs NK-cellmediated cytotoxicity and thus induces tolerance to tumor invasion. Up-regulation of the immunosuppressive cell surface glycoprotein CD200 and of soluble GITRL (glucocorticoid-induced TNFR related protein ligand) on AML cells specifically compromises NK-cell anti-tumor responses (50, 51) and is a poor prognosis factor. AML cells exert direct immunosuppressive effects on NK cells mediated by immunosuppressive ligands or soluble factors and induce regulatory T lymphocytes (Treg) that weaken NK-cell responses (52). NK cells can also interact with DC leading to activation of Treg and inhibition of NK cells (53). Recently, ligand of NKp44/NCR2 (NKp44L) was identified as an isoform of mixed-lineage leukemia-5 (MLL5) (54). This ligand is not detectable in the normal tissues but is present in hematopoietic, non-hematopoietic tumor and transformed cells. The expression of MLL5 is a good prognosis factor in AML (55). Thus we can speculate that the prognostic value of MLL5 expression is linked to its capacity to activate anti-leukemia NK cells.

### **REACTIVE OXYGEN SPECIES**

Non-malignant phagocytic cells down-modulate lymphocyte functions by producing and releasing NADPH oxidase-derived reactive oxygen species (ROS) (56). Monocytic and myelomonocytic (French-American-British classification M4/M5 subtypes) AML cells, but not cells from myeloblastic (FAB class M2) or immature (FAB class M1) AML, produce ROS via the NADPH oxidase component gp91phox, and trigger extensive apoptosis of NK cells *via* a poly-[ADPribose] polymerase-1 dependent pathway, together with a down-regulation of NKp46/NCR1. This suggests a novel mechanism of immune evasion in myelo-monocytic and monocytic AML (57).

#### **MATERIAL TRANSFER DURING CELL–CELL CONTACT**

Tumor membrane-derived microvesicles (tMV) are important mediators of cell-to-cell communication. These circular membrane fragments are enriched in various bioactive molecules and directly stimulate cells as a kind of "signaling complex." An important mode of communication between carcinoma cells and immune cells involves tMV, also known as exosomes, ectosomes, or microparticles. These microvesicles carry lipids, proteins, mRNAs, and microRNAs and travel short or long distances to deliver undegraded and undiluted material to other cells (58). Microvesicles

present in AML patients' sera contain TGF-β that down-regulates the expression of NKG2D and thus interfere with NK-cell activation. Nonetheless, IL-15 protects NK cells from adverse effects of tMV and could thus contribute to maintain their anti-tumor response (59). Baj-Krzyworzeka et al. (60) have observed that tMV carry mRNA of tumor cells and transfer some of them to monocytes and modify their activity. This type of mRNA transfer could participate to NK inactivation and tumor escape to innate immunity.

# **IMMUNOTHERAPY APPROACHES**

Modulation of NK-cell function by down-regulation of receptors and/or ligand corresponds to an immune escape mechanism for tumors. Restoring the expression of activating receptors on NK cells, or corresponding ligands on cancer cells, is an effective approach to cancer immunotherapy in order to improve diseasefree survival after therapy. Currently, in most AML patients, the induction treatment leads to CR, defined as microscopic disappearance of leukemic disease along with the return of normal hematopoiesis. However, many patients in CR relapse with poor prospects of long-term survival.  In AML, a recipient of haploidentical allogenic transplant with a NK HLA-specific receptormismatch is associated with a favorable prognosis because this increases the anti-leukemic graft reactivity (61, 62). Rapid recovery of NK cells after hematopoietic stem cell transplantation has been associated with a reduction in the rates of relapse and acute graft-versus-host disease (GvHD) (63). The transfer of NK cell from haploidentical origins into AML recipients is a potent immunotherapy intervention that is, unfortunately, associated with a significant transplant-related morbidity and mortality that limit its use (64).

The use of cytokines is another therapeutic approach to enhance NK-cell cytotoxicity. IL-2, IL-12, Il-15 (65), and IL-18 have been used in culture to increase cell cytotoxic prior to the injection of NK cells in cancer patients (66). Another strategy consists in restoring normal NCR expression since these molecules are pivotal for the anti-leukemia response. A phase III study in 320 AML patients has demonstrated that immunotherapy with histamine dihydrochloride (HDC) and IL-2 decreases and delays relapses in AML (67). HDC suppresses or inhibits ROS formation in mononuclear and polymorphonuclear myeloid cells. This prevents from oxygen radical-induced NK apoptosis, restores NK-cell capacity to respond to IL-2, and improves NK proliferation and production of immuno-stimulatory cytokines (56, 68–71). Moreover, in presence of HDC, cytotoxic functions of NK cells remain intact due to the preserved expression of the activating receptors NKG2D and NKp46/NCR1.

DNA methylation also has a key role in the control of gene activity in cancer cells. Two agents are currently used in MDS treatment: 5-azacytidine (Vidaza) and 5-aza-20-deoxycytidine (Decitabine) (72). These two hypomethylating agents up-regulate NKG2D ligands MICA/B leading to enhanced NK-cell cytotoxicity (73, 74). Conversely we have observed the down-regulation of 2B4/CD244 in NK from AML patients treated with 5-azacytidine, that could

have an opposite effect, i.e., cytotoxicity down-regulation (Leclerq et al. personal data).

The immune-modulatory drugs (IMIDs) such as thalidomide and lenalidomide are used in MM and MDS treatment and have anti-angiogenic and anti-inflammatory properties. They also act as IMIDs by cytokine release and activating effector cells by enhancing ADCC and NK-cell cytotoxicity thanks to the up-regulation of NKp46/NCR1 (75–77).

Bortezomib is a proteasome inhibitor used in MM treatment. Physiologically, proteasome is involved in protein degradation. Its inhibition by the drug interferes with tumor growth and with innate immunity. Bortezomib is involved in the down-regulation of HLA I molecules and in the up-regulation of NKG2D, TRAIL, and DNAM ligands, thus leading to increased NK-cell cytotoxicity against plasma cell (78, 79).

Another attractive therapeutic approach consists in blocking the NK inhibitory receptors. A phase 1 trial has tested the IPH2101, a fully humanized IgG4 anti-KIR monoclonal antibody, in patients with relapsed/refractory AML (clinical trial registration number NCT01256073) and MM (clinical trial registration number NCT00999830). IPH2101 promotes immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. No evidence of autoimmunity was observed. These findings suggest that IPH2101 is safe and tolerable and that this approach warrants further development in MM and AML (80–82) as we are waiting for clinical results.

Several studies revealed ADCC as one major mode of action of antibody-based therapeutics and stimulated more interest in how to mobilize, expand, and activate NK cells in humans (83). In CLL, the ADCC pathway via the Fc receptor (FcgRIIIa) CD16 at surface of NK cells is pivotal in the clinical effect of mAbs such as rituximab or ofatumumab which mediate ADCC by NK cells (84). Second generation mAb are designed in order to maximize both direct apoptosis and ADCC. The role of ADCC is underlined by better clinical responses to rituximab when NK cells expressed the high-affinity form of the FcgRIII (85, 86).

Bi-specific NK-cell engagers (BiKE) simultaneously bind CD16α and c-Met (a receptor overexpressed in many tumors) and thus may increase NK-cell ADCC (87).

Another type of therapeutic strategy consists in taking advantage from anti-cancer drugs properties (**Table 1**). Chemotherapy drugs can be separated by their ability to inhibit (such as vinblastine, chlorambucil, docetaxel) or enhance (such as asparaginase, bleomycin, doxorubicin) NK-cell-mediated killing of target cells (88, 89). In cancer, epigenetic changes are also involved in dysregulating NK-cell ligand expression. Histone deacetylase inhibitors (HDACIs) such as trichostatin, are epigenetic anti-cancer agents that modulate innate immunity by the regulation of expression of NKG2D or DNAM-1 ligands. Indeed, HDACIs increase expression of ligands of these two activating receptors, MICA/B or PVR and Nectin-2 respectively, on acute leukemia cells (90, 91). This suggests that epigenetic drugs make tumor cells more sensitive to NK-cell-mediated lysis (92). However, it has also been demonstrated that HDACIs suppress NK-cell cytotoxic activity by down-regulation of NKp30/NCR3, NKp46/NCR1 (93). Thus, the same drug can have contradictory effects on NK cells.

# **CONCLUDING REMARKS**

The more precise and exhaustive analysis of NK dysfunction in HM has opened the way to novel therapeutic strategies involving either specifically developed drugs/antibodies or innovative use of "old" drugs such as IMIDs. Due to the complexity of the immune response and the putative opposed effects of a drug on the various partners of the immune network, data obtained by *in vitro* experiments or *in vivo* in animal models have to be evaluated in clinically and biologically carefully monitored clinical trials.

# **ACKNOWLEDGMENTS**

This work was supported by Grants from INSERM and Region Provence-Alpes-Côtes d'Azur "Bourse Doctorale Regionale 2011– 2014."

# **REFERENCES**

respond less to stimuli that recruit clonogenic natural killer cells, and exhibit decreased proliferation on a per cell basis. *Blood* (1996) **88**:2279–87.

leukemogenesis and therapeutic implications. *Curr Top Microbiol Immunol* (2010) **341**:149–72. doi:10.1007/82\_2010\_25

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 20 September 2013; accepted: 02 December 2013; published online: 19 December 2013.*

*Citation: Baier C, Fino A, Sanchez C, Farnault L, Rihet P, Kahn-Perlès B and Costello RT (2013) Natural killer cells modulation in hematological malignancies. Front. Immunol. 4:459. doi: 10.3389/fimmu.2013.00459*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Baier, Fino, Sanchez, Farnault, Rihet, Kahn-Perlès and Costello. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### **Salem Chouaib1\*, Gianfranco Pittari 2 , Arash Nanbakhsh1 , Hanadi El Ayoubi 2 , Sophie Amsellem3 , Jean-Henri Bourhis 4 and Jan Spanholtz 5**

1 INSERM U753, Institut de Cancérologie Gustave Roussy, Villejuif, France

2 Department of Medical Oncology, National Center for Cancer Care and Research, Hamad Medical Corporation, Doha, Qatar

3 Centre d'Investigation Clinique Biothérapies, Institut Gustave Roussy, Villejuif, France

4 Département d'Hématologie Clinique, Institut de Cancérologie Gustave Roussy, Villejuif, France

5 Glycostem Therapeutics, Hertogenbosch, Netherlands

#### **Edited by:**

Daniel Olive, INSERM UMR 891 Institut Paoli Calmettes, France

#### **Reviewed by:**

Jennifer Wu, Medical University of South Carolina, USA Yasmina Laouar, University of Michigan, USA

#### **\*Correspondence:**

Salem Chouaib, INSERM U753, Institut de Cancérologie Gustave Roussy, 39 rue Camille Desmoulins, Villejuif 94805, France e-mail: salem.chouaib@ gustaveroussy.fr

Blurring the boundary between innate and adaptive immune system, natural killer (NK) cells are widely recognized as potent anti-leukemia mediators. Alloreactive donor NK cells have been shown to improve the outcome of allogeneic stem-cell transplantation for leukemia. In addition, in vivo transfer of NK cells may soon reveal an important therapeutic tool for leukemia, if tolerance to NK-mediated anti-leukemia effects is overcome. This will require, at a minimum, the ex vivo generation of a clinically safe NK cell product containing adequate numbers of NK cells with robust anti-leukemia potential. Ideally, ex vivo generated NK cells should also have similar anti-leukemia potential in different patients, and be easy to obtain for convenient clinical scale-up. Moreover, optimal clinical protocols for NK therapy in leukemia and other cancers are still lacking. These and other issues are being currently addressed by multiple research groups. This review will first describe current laboratory NK cell expansion and differentiation techniques by separately addressing different NK cell sources. Subsequently, it will address the mechanisms known to be responsible for NK cell alloreactivity, as well as their clinical impact in the hematopoietic stem cells transplantation setting. Finally, it will briefly provide insight on past NK-based clinical trials.

**Keywords: NK cells, NK-based immunotherapy, acute myeloid leukemia, NK cell expansion, hematopoietic stem cell transplantation**

# **INTRODUCTION**

Natural killer (NK) cells play a key role in the immune response to infections and malignancies by direct cytolysis of infected or transformed cells and by secretion of potent immune mediators. NK cell killing depends on the overall balance of inhibitory and activating signaling mediated by an array of surface receptors recognizing cognate ligands on putative targets. Besides their lytic activity, NK cells are cytokine-producing cells. Several reports highlight the fact that NK cells are also regulatory cells engaged in reciprocal interactions with dendritic cells, macrophages, T cells, and endothelial cells (1). NK cells have a central role in tumor-cell surveillance (2) as demonstrated in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) (3–8).

Inhibitory NK receptors with specificityfor HLA class I antigens are well described. Among these, killer Ig-like receptors (KIR) bind several HLA class I ligand groups (9), while the CD94–NKG2A/B heterodimer recognizes HLA-E (10). Several activating receptors are also described. Activating NKG2D is a major mediator of spontaneous cytotoxic activity. Natural cytotoxicity receptors (NCRs), including three molecules (i.e., NKp46, NKp30, and NKp44) specific for mostly unknown ligands (11, 12), mediate cell lysis of many cancer cells. Escape mechanisms from NK cell surveillance may involve NK cell quantitative deficiency and NK cell functional impairment.

Recent studies suggest that NK cell-based immunotherapy may continue to be an effective approach for patients with leukemia and emerging strategies are currently under investigation, initially based on adoptive transfer of NK cells.

This review will first describe current laboratory NK cell expansion and differentiation techniques by separately addressing different NK cell sources. Subsequently, it will address the mechanisms known to be responsible for NK cell alloreactivity.

# **NK CELL DIFFERENTIATION AND EXPANSION**

#### **NK CELL EXPANSION AND NK CELL GENERATION METHODS**

Successful NK cell immunotherapies require an adequate number of NK cells. In addition to NK cell number, NK cell purity and function are key factors for a clinically efficacious NK cell product (13). Various methods of NK cell expansion have been explored (14). In general, NK cells can be produced from peripheral blood (PB), umbilical cord blood (UCB), bone marrow (BM), embryonic stem cells (hESC), or induced pluripotent stem cells (iPCS). While short-term NK cell culturing does not normally enhance the functional capabilities for *in vivo* transferred NK cells, long-term expansion methods may yield large numbers of functional NK cells, which may potentially benefit cancer patients (15). Several alternative protocols for NK cell *ex vivo* expansion for adoptive immunotherapy have been reported to date. However, only some strategies have been developed under good manufacturing practice (GMP) conditions. In addition, substantial variability in NK cell expansion efficiency, phenotype, and function has been observed among different protocols and among individual donors (16–20).

# **EXPANSION OF NK CELLS FOR CLINICAL PURPOSES ISOLATED FROM PERIPHERAL BLOOD HUMAN**

Several protocols for the expansion of PB NK cells are currently available, and others are under development. Various feeder cell-based systems have been used for NK cell expansion from peripheral blood mononuclear cells (PBMC), including thirdparty Epstein–Barr virus transformed lymphoblastoid B cell lines (EBV–BLCL), genetically modified K562 cells, or irradiated autologous cells (21–24). *Ex vivo* expansion of bulk peripheral NK cells using third-party EBV–BLCL feeders approximately yields a 180-fold NK cell expansion after 2 weeks of culture (22). Another expansion technique, yielding clinical valuable amounts of NK cells, is based upon K562 cell feeder double-transduced with IL-15 and 4-1BB (CD137) co-stimulatory ligand (K562–mb15–41BBL) (23). K562 cells transduced with IL-21 have also been used as feeder cells in NK co-culture systems (25). While K562–mb15– 41BBL have been shown to expand and functionally enhance PB NK cells, K562 genetically engineered with membrane-bound IL-21 allow an even higher proliferation and cytotoxicity of expanded NK cells, which also display longer telomeres and less senescence (25). To expand CliniMACS-purified PB NK cells, autologous irradiated feeder cells have also been used as feeder cells in culturing systems containing human serum, IL-2, IL-15, and anti-CD3 antibody (21).

Many PB NK expansion strategies hold promise for NK-based immunotherapies. However, even using identical protocols, NK cell expansion yields and purity are typically inconsistent, and significant donor-to-donor variation is common. Moreover, complete absence of any residual viable tumor feeder in all final cell products is a critical requirement for large-scale NK cell therapy applications and their pharmaceutical translation.

The type of disposable cell culture systems for NK cell culturing also appears to influence the characteristics of the final cell product. Currently used disposable cell culture systems include flasks, bags, or WAVE® bioreactors. Compared to flasks, use of bioreactors allow a 10-fold higher NK cell expansion after 3 weeks of culture (26), at the expense of a reduced purity of the final product, which also contains T cells (CD3+/CD56−) as well as NKT cells (CD3+/CD56+). Presence of T cells limits the application of this cell product to the autologous setting in the absence of downstream T-cell depletion.

### **NK CELL GENERATION FROM UMBILICAL CORD BLOOD**

Umbilical cord blood is thought to be an excellent source for cell therapy applications. Initial work on positively selected cord blood NK cells, cultured on a feeder layer of mesenchymal stromal cells using a combination of IL-2, IL-15, Flt-3L, and IL-3, resulted in a mere 60-fold median expansion (27). In consideration of the low starting NK cell number in standard cord blood units, this approach is not feasible to generate NK cell numbers needed for a therapeutic NK cell product. Additionally, NK cell differentiation from CD34+ hematopoietic stem cells (HSC) has been addressed (28). Initially, research in this field focused on the *ex vivo* generation of NK cells from BM CD34+ cells (29–33), but later also involved CD34+ cells derived from UCB (34–39), a particularly rich source of HSC. These studies used different combinations of growth factor and cytokine mixtures, BM stroma cells, and culture media with or without animal or human sera. These culture systems generally contain components of animal origin; moreover, they fail to yield significant numbers of mature NK cells. For these reasons, it is unlikely that they will be used for clinical applications. Interestingly, the system reported by Silva et al. contained human serum without stroma cells and was therefore favored for clinical scaling-up (40). This NK cell expansion method was later abandoned, once stroma cells were recognized as a potential critical component for the enhancement of NK cell proliferation (40). In 2007, Kao et al. showed that serum-free expanded CD34+ cells may be differentiated into a NK cell product with an average purity of 40–60% after 5–7 weeks of culture, with a mean expansion rate of 300-fold. Such expansion, however, was obtained in the presence of fetal bovine serum (41). Later on, Vitale et al. studied the effects of methylprednisolone on CD34+ precursor cells and their ability to differentiate toward NK cells (38). They performed direct NK cell differentiation – i.e., no upfront CD34+expansion – using RPMI-1640 medium supplemented with human serum,fetal bovine serum, and a cytokine cocktail including IL-15 and IL-21. At day 25, they observed a 10-fold NK cell expansion, with an average purity of approximately 30%.

In 2010, a novel cell culture technology for the *ex vivo* expansion and NK differentiation of UCB-derived CD34+ cells was developed. This technique was based upon a clinical-grade serumfree culture medium and a mixture of heparin and cytokines, in order to mimic the extra-cellular matrix BM microenvironment in the absence of feeder cells (42). This method yielded up to 1010 CD34+ cell-derived NK cells, generated in static cell culture bags and automated bioreactors. Importantly, NK cell products were not found to be contaminated by T cells, and may therefore be safely used in the allogeneic setting (43). Additionally, NK cells expressed high levels of activating receptors (e.g., NKG2D and NCR), and are able to efficiently kill myeloid leukemia and melanoma cell lines, as well as primary leukemia blasts. Furthermore, mouse studies have shown that they expand *in vivo*, initially home to the lung, and later to the blood, lung, spleen, liver, and BM and prevent outgrowth of local tumors in the BM (44). A phase I trial in elderly acute myeloid leukemia (AML) patients using NK cell products based upon this expansion technique is currently in progress (CCMO nr. NL31699 and Dutch Trial Register nr. 2818) (43).

Definitely, producing NK cellsfrom CD34+hematopoietic precursors can be practically advantageous, since stem cells can be isolated and frozen, and can overcome several obstacles posed by purified PB NK cells (15).

## **NK CELL GENERATION FROM HUMAN EMBRYONIC STEM CELLS OR INDUCED PLURIPOTENT STEM CELLS**

In contrast to the CD34+ UCB-derived NK cells, the generation of NK cells from embryonic stem cells or induced pluripotent stem cells is to date largely experimental (45). As previously mentioned, human NK cells can be differentiated from CD34+ HSC under certain culture conditions (31, 35, 42). Currently, optimizing the generation of CD34+ HSC from hESC and iPSC remains a major challenge to rationally approach hESC- and iPSCs-based NK cell therapy (45–48). hESCs or iPSCs derived CD34+ cells are known to be enriched for hematopoietic progenitors defined by colony-forming cells, which could serve as a suitable source for cell therapy (47). Some years ago, a method for the efficient generation of functional NK cells from hESC using a two-step procedure was described. NK cell products contained cytotoxic cells, displayed a mature NK cell phenotype, including CD16 and KIR, CD94/NKG2A, NKp46, NKp44, NKG2D, TRAIL, and FasL (49).

Engineering CD34+ cells, hESCs, or iPSCs to express chimeric antigen receptors (CAR) specific for tumor-associated antigens is a potentially promising immunotherapeutic strategy (48).

## **NK CELL LINES FOR CLINICAL USE**

Several authors have reported on the use of NK cell lines for therapeutic purposes. Unlike primary NK cells, whose *in vivo* life span is limited, NK cell lines are immortal. However, most NK lines are weakly cytotoxic to cancer targets. Conversely, the NK-92 cell line (Neukoplast, Conkwest, San Diego) has shown to mediate liable cytotoxicity and good expansion kinetics when cultured in bags or bioreactors. NK-92 has been well characterized in *in vitro* and shown to display remarkable anti-tumor activity in severe combined immunodeficiency (SCID) mouse xenotransplant models (50). Furthermore, it has been extensively tested in the clinical setting (51).

# **ALTERATION OF NK-MEDIATED CYTOTOXIC RESPONSE AGAINST LEUKEMIA**

The escape of hematological malignancies from NK cell immunity can be explained by general mechanisms involving the quantitative deficiency of NK cells, their qualitative impairments caused by increased inhibition, decreased activation signaling, or by the negative influence of tumor microenvironment. It is widely recognized that leukemia cells may oppose a variety of immune escape strategies, including immune suppression and phenotypic mimicry, to elude NK-mediated killing.

The balance between activating and inhibitory signals received by NK cells will determine the NK cell-mediated elimination of leukemia cells. Decrease in ligands for activating receptors on leukemia cells could abolish NK cell dependent cytotoxicity. Furthermore, interaction of other ligands with cognate inhibitory receptors on NK cells surface could diminish NK cell killing activity, granule mobilization, and interferon production. Accumulating evidence indicates that chronic and acute leukemia cells could modulate NK cell activity by secreting soluble and exosomal ligands for NK cell receptors (52, 53). A key strategy used by leukemia cells to escape elimination by NK cells is related to apoptosis, and altered expression of molecules involved in different apoptosis signaling pathways may result in resistance to NK-mediated killing (54–56). In this setting, deregulation of the mitochondrial apoptotic machinery, functional blocks of caspase cascade, downregulation or inactivation of proapoptotic molecules, and upregulation of antiapoptotic molecules may all potentially favor leukemia chemoresistance or relapse. Finally, mechanism of NK cell resistance may also include drug resistance. For example, it has

been shown that doxorubicin resistant cells increase expression of HLA class I on their surface (57).

# **CAPTURING NK CELL ALLOREACTIVITY IN HEMATOPOIETIC STEM CELL TRANSPLANTATION**

In the last 15 years, growing knowledge of NK tolerance to self, cancer immunosurveillance, and functional licensing has been extensively applied to HCT, in an effort to identify donors protecting from leukemia relapse through NK-based alloreactivity. Most studies investigating NK effects on transplantation outcome are rationally based upon one of the following three models: the missing-self recognition paradigm, the missing ligand model, and the activating receptor-based NK cell alloreactivity. This section addresses the mechanisms known to be responsible for NK cell alloreactivity, as well as their clinical impact in the HSCT.

# **RECOGNITION OF HLA CLASS I ANTIGENS BY KILLER IMMUNOGLOBULIN-LIKE RECEPTORS**

The KIR gene cluster is mapped on chromosome 19q13.4, within the 1 Mb leukocyte receptor complex (LRC). It contains four ubiquitous "framework" genes (one centromeric, *KIR3DL3*; one telomeric, *KIR3DL2*; and two central, *KIR3DP1* and *KIR2DL4*) that flank centromeric and telomeric gene motifs characterized by extensive variation in both gene content (type and number) and polymorphism (58–60). Based on *KIR* gene content, multiple KIR haplotypes are identified, and categorized into two distinct groups, A and B. Group A haplotypes contain genes exclusively encoding inhibitory receptors and the activating *KIR2DS4*, while group B haplotypes contain genes encoding both inhibitory and activating receptors. Both KIR A and B haplotypes possess centromeric and telomeric gene content motifs. Products of functional KIR genes are type I transmembrane receptors with two (KIR2D) or three (KIR3D) highly homologous, extra-cellular immunoglobulin domains (61, 62, 63). Due to their clonal distribution in the NK repertoire, an individual NK cell may express one or more KIR (64, 65).

Inhibitory KIR may recruit the SH2-domain-containing tyrosine phosphatase 1 protein (SHP1) (66–69) through a single or double immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in their long cytoplasmic tail (denoted L, i.e., KIR2DL; KIR3DL). Of several known inhibitory KIR, 2DL1, 2DL2, 2DL3, and 3DL1 are particularly relevant for HLA class I recognition. KIR2DL1 is specific for HLA-C2 group antigens (sharing the Asn77/Lys80 residues in the HLA-Cw heavy chain); KIR2DL2 and KIR2DL3 are specific for HLA-C1 group antigens (sharing the Ser77/Asn80 in the HLA-Cw heavy chain) (70, 71); and KIR3DL1 is specific for HLA-Bw4 ligands, sharing a group of sequence motifs in residues 77–83 of the heavy chain of certain HLA-B and HLA-A alleles (72–74).

Killer Ig-like receptor mediated activating signaling has also been identified. Unlike inhibitory KIR, they possess truncated portions that transduce activating signals via tyrosine phosphorylation of DAP12 and other proteins (75–77). Couples of cognate activating and inhibitory KIR, sharing almost complete homology (95–99%) in their extra-cellular domains, are recognized (75). Thus, activating KIR2DS1, KIR2DS2, and KIR3DS1 are, respectively, cognate receptors for the HLA class I-specific inhibitory KIR2DL1, KIR2DL2, and KIR3DL1. Counter-intuitively, the identification of natural ligands for activating receptors remains largely elusive. Ligands for activating KIR2DS2 and KIR3DS1 receptors have not been identified. While it cannot be excluded that these receptors recognize HLA class I/peptide complexes, current evidence indicates that they may not affect NK function by generating activating signaling when HLA-C1 and HLA-Bw4 are self-ligands. Unique among activating KIR, 2DS1 recognizes HLA-C2 group antigens, similar to its inhibitory homolog 2DL1.

# **THE MISSING-SELF RECOGNITION PARADIGM AND NK CELL ALLOREACTIVITY**

Inhibitory KIR interactions with cognate HLA class I ligands play a critical role in NK cell education and tolerance to self. In normal individuals, NK cells commonly possess KIR repertoires including one or more KIR with ligand specificity for self-HLA class I ligands (65). It is generally believed that such NK cells are rendered functionally competent, or licensed, by continuous signaling generated by inhibitory KIR upon interaction with self-HLA class I antigens (78, 79). HLA class I is critical to maintain NK tolerance to self, and cells failing to express sufficient levels of HLA class I ligands are promptly cleared by NK-mediated cytotoxicity. This phenomenon, known as missing-self recognition, was first postulated in a report by Kärre et al. describing that lack of MHC class I (H2) antigen expression rendered mice lymphoma cells highly sensitive to NK-mediated rejection (80).

Natural killer cells from donor-derived hematopoietic progenitor cells quickly reconstitute in HCT recipients (81–83). In the HSCT setting, HLA class I ligands of donor origin are believed to drive functional licensing. Reconstituted NK cells expressing one KIR for HLA class I present in the donor display stronger *in vitro* responsiveness than NK cells expressing one KIR for HLA class I present in the recipient, but absent in the donor (84). Additionally, CD107a externalization and IFNg production of NK cells reconstituting in recipients of donor unrelated or UCB grafts is markedly increased if they express KIR for donor self (85). This donor HLAbased NK education model implies, that the size of licensed donor NK cell is shaped by the frequency of inhibitory KIR-positive NK cells combined with the presence of cognate HLA class I ligands in the donor. Thus, KIR2DL1+ NK cells would acquire functional competence if donor is *HLA-C2*; KIR2DL2–3+ NK cells if donor is *HLA-C1*; and KIR3DL1+ NK cells if donor is positive for *HLA-A* or -*B* alleles possessing the Bw4 motif.

In HLA-mismatched HSCT, *HLA-C* allele groups (*C1* or *C2*), and/or the Bw4 epitope may be present in the donor and absent in the recipient. In this situation, the repertoire of licensed donor NK cells may include NK clones mediating missing-self allorecognition against host tissues. For example, KIR2DL1+ /KIR2DL2– 3 −/KIR3DL1− clones from a *HLA-C2* positive donor may display allorecognition of missing self in a *HLA-C2* negative recipient. Recognition of missing self-HLA class I may improve the outcome of HSCT. T-cell-depleted haplotype-mismatched graftsfrom NK alloreactive donors mediate strong graft vs. leukemia (GvL) effects in AML recipients, allowing for lower risk of relapse and better survival (3, 4). While in haplotype-mismatched grafts, the effect of KIR-ligand incompatibility is well established, studies on mismatched unrelated HSCT reported conflicting results. In

this setting, Giebel et al. showed that donor NK cell alloreactivity improves recipient survival (86). A protective effect of KIR-ligand incompatibility on post-transplantation relapse was later confirmed in myeloid malignancies (87) and multiple myeloma (88). However, most studies failed to show a beneficial effect of donor NK cell alloreactivity on the outcome of mismatched unrelated HSCT (89–92). Similarly, studies exploring the clinical impact of NK alloreactivity mediated by KIR-ligand mismatch in UCB grafts have yielded variable results (93, 94). These inconsistent observations are possibly influenced by complex variables, such as donor KIR genotype (95), disease category, type of conditioning T-cell depletion, post-transplantation immune suppression for graft vs. host disease (GvHD) prophylaxis. For example, T cells may dominate alloreactive phenomena in mismatched unrelated HSCT and counteract the clinical benefit of NK alloreactivity (91). Accordingly, *in vivo* T-cell depletion with anti-thymocyte globulin (ATG) has been shown (86, 88, 96) to enhance the favorable impact of NK cell alloreactivity on HSCT outcome.

# **THE MISSING LIGAND MODEL AND NK CELL ALLOREACTIVITY**

Killer Ig-like receptors and HLA genes are mapped on different chromosomes, and segregate independently according to a Mendelian inheritance pattern. Therefore, certain individuals may have*KIR* genes but not the corresponding HLA/KIR-ligand groups (97). KIR receptors are clonally distributed on NK cell surface, allowing for the possibility, that subpopulations of NK cells exclusively express KIR with ligand specificity for non-self-HLA class I ligands. These NK cells are not classically licensed by self-HLA class I ligands during their development, and are believed to be hyporesponsive to stimulation in physiologic conditions. During posttransplant immune reconstitution, however, this non-licensed status may be transiently suspended during post-transplantation immune reconstitution, and effector functions could indeed be mediated by donor NK cells expressing KIR with ligand specificity for non-self-HLA class I. An important implication of the missing ligand model is that NK alloreactivity would be observed even in the absence of donor/recipient KIR-ligand mismatch, a necessary condition for missing self-mediated NK alloreactivity.

Several studies exploring the effect of the missing ligand model on HSCT outcome indicate, that donors possessing inhibitory KIR but not the corresponding HLA class I ligand do mediate beneficial NK effects in HLA-identical siblings or HLA-matched unrelated recipients (5,98–100). Hsu et al. originally explored a cohort of 178 subjects receiving a T-cell-depleted graft from a HLA-identical sibling. In patients withAML and myelodysplastic syndromes (MDS), lack of one or more HLA ligand for donor KIR resulted in lower relapse and better survival (5). Following the identification of beneficial NK effects in the HLA-matched setting, the function of classically non-licensed NK cells has been directly explored in the context of post-transplantation reconstitution. In T-cell-depleted grafts from HLA-identical siblings, NK cells expressing KIR for non-self-HLA display strong IFNγ production and cytotoxicity to target stimulation during the first trimester post-transplantation (101). These findings have not been confirmed in a cohort of recipients of T cell-replete grafts from HLA-identical siblings. Here, reconstituted NK cells expressing KIR for non-self-HLA ligands displayed tolerance to self. Moreover, lack of self-HLA ligands

for donor inhibitory KIR was found to have no effect on HSCT outcome (102). Presence or absence of T cells in the graft may differentially affect self-tolerance of non-licensed donor NK cells post-transplantation. Regardless, the interpretation of these conflicting results demands further studies on tolerance to self of donor NK cells reconstituting in the HLA-identical host.

# **ACTIVATING RECEPTOR-BASED NK CELL ALLOREACTIVITY**

Because ligands for most activating KIR are currently unknown, studies reporting associations between activating KIR and HSCT outcome are not generally supported by the identification of an underlying immunological background mechanistically explaining the observed NK-mediated alloreactivity. In a cohort of 65 graft recipients from HLA-identical siblings, donors with genotypes containing both *KIR2DS1* and *KIR2DS2* genes provided protection from relapse (103). Donor activating KIR was later found to control CMV reactivation post-transplantation. Recipients of T cell-replete grafts were found to have a remarkable reduction of the incidence of CMV reactivation, if donor possessed more than one activating *KIR* genes (104). Confirmation of the protective effect against CMV reactivation by donor activating *KIR* was concomitantly reported by another group (105).

In 2009, Cooley et al. investigated the effect of different donor KIR haplotypes in 448 AML recipients of unrelated T cell-replete HSCT. Recipients of KIR B/x grafts (i.e., homozygous or heterozygous for KIR B group haplotypes) displayed a higher 3-year-overall survival (6). In a cohort of 1086 AML recipients of unrelated grafts, the same group later compared the contribution to HSCT outcome of donor centromeric and telomeric group A and B KIR haplotypes. Donors homozygous for centromeric B gene content motifs (Cen B/B) most strongly associated with low risk of relapse and prolonged survival (7). Among activating KIR, activating KIR2DS2 is mapped on the centromeric region of several B group haplotypes, and may thus mediate the clinical benefit observed for Cen B/B donors through interaction with an unknown ligand expressed on leukemia cells (7).

Recent studies investigated the effect of telomeric activating *KIR3DS1* and *KIR2DS1* genes on transplantation outcome. Patients receiving unrelated grafts from *KIR3DS1* donors exhibited a lower risk for grade II–IV GvHD and mortality (8, 106). Activating KIR2DS1 is found in approximately 1/3 Caucasians, and commonly occurs in individuals positive for *HLA-C2* (*C1/C2*; *C2/C2*) (107–109). KIR2DS1 expression occurs in more than 20% NK cells (109), and 2DS1 single positive (KIR2DS1SP) NK cells (i.e., lacking inhibitory KIR expression), may also be identified. In *HLA-C2* individuals, KIR2DS1SP NK cells may potentially display auto-reactivity to normal self-tissues. Compared with *HLA-C1* donors, KIR2DS1SP NK cells from *HLA-C2* homozygous individuals are hyporesponsive to a HLA-C2 positive target cell (108). Similarly, mice studies described hyporesponsiveness of activating receptor-positive NK cells resulting from *in vivo* chronic interaction with a viral ligand (110, 111). Recently, KIR2DS1SP NK clones displaying *in vitro* anti-HLA-C2 cytotoxicity have been identified in all *HLA-C* genotypes (112). In *C2:C2* individuals, these clones are significantly reduced in frequency. In contrast, anti-HLA-C2 reactive KIR2DS1SP clones from *C1:C2* individuals are common, andfunctionally indistinguishable to those obtainedfrom *HLA-C2* negative (*C1:C1*) donors (112). These observations indicate that tolerance development is affected by HLA-C2 ligand expression density, and are consistent with the "functional NK plasticity" phenomenon described in mice studies (113, 114). The effect of donor *KIR2DS1* on HSCT outcome has been assessed in a cohort of 1277 AML recipients of unrelated HLA-matched or 9/10 mismatched HSCT. Patients receiving grafts from *KIR2DS1* donors displayed a decreased rate of leukemia relapse (8). In agreement with experimental findings (112), *KIR2DS1*-mediated protection was observed in *HLA-C1* (*C1:C1* and *C1:C2*) donors, but not in *HLA-C2* homozygous donors, presumably due to the strong tolerogenic effect on KIR2DS1-positive NK cells mediated by the *HLA-C2* homozygous genotype (8).

# **NK CELL THERAPY MODALITIES**

Early studies by Miller et al. in 2005 have open the way to demonstrate the safety of adoptive transferred human NK cells in patients with AML. Human haplo-identical NK cells could be expanded *in vivo* and transferred in patients with poor prognosis AML. Patients had to be prepared with an immunosuppressive high dose alkylating and purine analog conditioning regimen they can promote recirculation of infused allogeneic NK cells. Further haplo-identical allogeneic NK cells infusions were applied in patients with poor prognosis Hodgkin lymphoma and various solid tumors, i.e., melanoma (115) and renal cell carcinoma. In order to promote NK cell expansion, a lympho-depletion preparative regimen is required for the patient in all studies reported. To date, adoptive immunotherapy with unstimulated or IL-2 activated NK donor NK cells infusion is used for patients undergoing haplo-identical hematopoietic progenitor cells transplantation to prevent from relapse. No serious and immediate adverse effects have been observed following allogeneic NK cells infusion.

Clinical scale protocols to collect, enrich, and expand purified NK cells are nowadays feasible from cord blood, human donor apheresis, and dedicated cell lines. These cell therapy procedures remain costly and time consuming. They need an experimental lab with GMP facilities together with the skills and allowance to perform the cell manipulation.

The currently defined cell product has optimized T-cell depletion to avoid GvHD and allogeneic NK cell expansion; but we are still lacking a clinical standardized procedure for the use of enriched and expanded NK cells (116).

The most common limitation of the procedure remains the inability of collected, enriched, activated, and expanded allogeneic NK cells to expand properly *in vivo*. At first, a huge loss of NK cells during the isolation procedure can be seen following T-cell depletion procedure. Then CD56+/CD3− NK cells may be rejected by cytotoxic T cells or suppressed by myeloid-derived suppressor cells or Tregs.

To date, ongoing clinical scale protocols aim to both select CD56+/CD3− NK cells and increase drastically the total number of NK cells. Another limitation comes from the use of high dose alkylating chemotherapy-based protocols prior to infusion of NK cells that can increase the toxicity and efficacy of NK cell-based immunotherapy. Infusion of allogeneic NK cells can be performed following standard doses regimen together with immune modulation with steroids and IL-15.

In the field of BM transplantation, infusion of donor-derived stimulated and expanded CD56+/CD3− allogeneic NK cells may be used in repetitive injections to promote the GvL effect. Further studies have to be designed to enhance the NK cell therapy efficiency: improving the NK cell selection, processing, *ex vivo* expansion, better designing of lympho-depletion protocols for the patient and infusion rate of NK cells for a optimized control of residual leukemic cells.

# **CONCLUSION**

Natural killer cells continue to attract a lot of interest in transplantation. However defects in NK cell cytotoxicity have been observed in all hematological malignancies and the escape of hematological malignancies from NK cell immunity can be explained by general mechanisms. The latter are common to all immune-effector cells involving the quantitative deficiency of NK cells, their qualitative impairments caused by increased inhibition, or decreased activation signaling.

Natural killer cell selection from leukapheretic products is a largely inadequate approach to obtain large numbers of cancerreactive NK cells. Given the clonal and stochastic distribution of activating and inhibitory receptors on NK cell surface, pools of cancer-reactive NK cells generally represent a minority of the bulk CD3−, CD56+ population. Several methods for *ex vivo* NK cell expansion have been attempted to date. However, none of the described approaches have definitively proved able to effectively circumvent the issue of limited NK cell availability and efficiency. Although an important anti-tumor role for alloreactive NK cells has been shown in patients with AML either after stem-cell transplantation or adoptive transfer of haplo-identical NK cells, their clinical efficacy in human trials has been modest, presumably due to tumor escape by alteration of NK cell function and resistance to killing. In this respect, the ability to manipulate not only the balance of activating and inhibitory receptors on NK cells but also their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives in NK cell-based immunotherapy. Sensitization of tumor cells to activated cytotoxic lymphocytes by up-regulating either TNF death receptors or effector-activating ligands on tumor cells combined with immunotherapy has been pursued to overcome tumor-cell resistance and establishes an effective anti-tumor response. Another therapeutic approach to enhance NK cell cytotoxicity is the use of cytokines, such as IL-2, IL-12, and IL-18. Chemokine manipulation is also of interest as molecules may both attract NK to the tumor-cell microenvironment and stimulate their cytotoxic properties. Another attractive approach to enhance NK cytotoxicity is to use monoclonal antibodies (mAbs). IPH-2101, a fully human IgG4 anti-KIR mAb (developed by Innate Pharma) is currently being tested in phase I and II clinical trials in patients with AML and multiple myeloma (117). Its blockade of inhibition could allow NK cell activation when activating ligands are present on target cells. It should also be emphasized that bispecific mAbs directed against both the target cells and NK cells are also currently under investigation. Anti-CD20 mAbs that have enhanced affinity for CD16 have been also developed, and they are more effective at NK activation than rituximab (118).

One limitation of the use of NK cells is due to the great capacity of leukemic cells to escape to NK recognition and killing. This resistance remains indeed a drawback in immunotherapy of hematological malignancies. In this regard, adoptive transfer of polyclonal or clonal NK cells with mismatch NK inhibitory receptors and HLA class I ligands would produce GvL (GvHD) in the absence of GvHD. The simultaneous differentiation of the effective killer effectors, the boosting of NK function, their expansion, and the sensitization of leukemic targets will offer potential opportunities in the treatment of hematological malignancies. Therefore, how to expand UCB stem cells for optimal downstream generation of terminally differentiated NK cells potentially alloreactive to leukemic cells is at present very challenging.

# **ACKNOWLEDGMENTS**

This work was supported by INCA and Qatar Foundation grants.

# **REFERENCES**

of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by "group 2" or "group 1" NK clones. *J Exp Med* (1995) **182**:605–9. doi:10.1084/jem.182.2.605

incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. *Blood* (2005) **105**:2594–600. doi:10.1182/blood-2004-04-1441

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 09 January 2014; accepted: 23 February 2014; published online: 17 March 2014.*

*Citation: Chouaib S, Pittari G, Nanbakhsh A, El Ayoubi H, Amsellem S, Bourhis J-H and Spanholtz J (2014) Improving the outcome of leukemia by natural killer cell-based immunotherapeutic strategies. Front. Immunol. 5:95. doi: 10.3389/fimmu.2014.00095 This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Chouaib, Pittari, Nanbakhsh, El Ayoubi, Amsellem, Bourhis and Spanholtz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Cancer-induced alterations of NK-mediated target recognition: current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity

#### **Anne-Sophie Chretien1 , Aude Le Roy 2 , Norbert Vey 1,3,Thomas Prebet 3 , Didier Blaise1,4, Cyril Fauriat 1 and Daniel Olive1,2\***

1 Centre de Cancérologie de Marseille, INSERM, U1068, Institut Paoli-Calmettes, Aix-Marseille Université, UM 105, CNRS, UMR7258, Marseille, France

2 Centre de Cancérologie de Marseille, Plateforme d'Immunomonitoring en Cancérologie, INSERM, U1068, Institut Paoli-Calmettes, Aix-Marseille Université, UM 105, CNRS, UMR7258, Marseille, France

3 Département d'Hématologie, Institut Paoli-Calmettes, Marseille, France

4 Unité de Transplantation et de Thérapie Cellulaire, Institut Paoli-Calmettes, Marseille, France

#### **Edited by:**

Simona Sivori, University of Genoa, Italy

#### **Reviewed by:**

Alexander Steinle, Goethe University Frankfurt am Main, Germany Claudia Cantoni, University of Genoa, Italy

#### **\*Correspondence:**

Daniel Olive, 232 Boulevard de Sainte-Marguerite, Marseille, France e-mail<EMAIL_ADDRESS>
Despite evidence of cancer immune-surveillance, which plays a key role in tumor rejection, cancer cells can escape immune recognition through different mechanisms.Thus, evasion to Natural killer (NK) cell-mediated anti-tumor activity is commonly described and is mediated by various mechanisms, mainly cancer cell-induced down-regulation of NK-activating receptors (NCRs, NKG2D, DNAM-1, and CD16) as well as up-regulation of inhibitory receptors (killer-cell immunoglobulin-like receptors, KIRs, NKG2A). Alterations of NK cells lead to an impaired recognition of tumor cells as well as a decreased ability to interact with immune cells. Alternatively, cancer cells downregulate expression of ligands for NK cellactivating receptors and up-regulate expression of the ligands for inhibitory receptors. A better knowledge of the extent and the mechanisms of these defects will allow developing pharmacological strategies to restore NK cell ability to recognize and lyse tumor cells. Combining conventional chemotherapy and immune modulation is a promising approach likely to improve clinical outcome in diverse neoplastic malignancies. Here, we overview experimental approaches as well as strategies already available in the clinics that restore NK cell functionality.Yet successful cancer therapies based on the manipulation of NK cell already have shown efficacy in the context of hematologic malignancies. Additionally, the ability of cytotoxic agents to increase susceptibility of tumors to NK cell lysis has been studied and may require improvement to maximize this effect. More recently, new strategies were developed to specifically restore NK cell phenotype or to stimulate NK cell functions. Overall, pharmacological immune modulation trends to be integrated in therapeutic strategies and should improve anti-tumor effects of conventional cancer therapy.

**Keywords: cancer, immune escape, NK cell, NCR, NKG2D, KIR, immunotherapy**

# **INTRODUCTION**

Natural killer (NK) cells are key components of the innate immunity and substantially contribute to anti-tumor immune responses (1–3). The role of NK cells in immune surveillance is linked to many aspects of the NK cell biology. First, NK cells directly recognize and lyse cancer cells. Besides this direct effect, NK cells are also able to initiate anti-tumor immune responses via the secretion of various cytokines such as IFN-γ and TNF-α (1, 4).

Triggering of effector functions of NK cells is the result of a balance between activating and inhibitory signals provided by a large set of activating or inhibitory receptors. The most commonly described activating receptors involved in anti-tumor immunity are NKG2D, DNAM-1, and the natural cytotoxic receptors (NCR), NKp30, NKp44, and NKp46. Hence, NCR are NK-activating receptors of primary importance in immune surveillance and response in the context of cancer (5–7). NKp30, NKp46 are expressed by all NK cells, whereas NKp44 is only expressed by activated NK cells (8–11). The acquisition of NCR during NK cell maturation correlates with the acquisition of cytolytic activity against tumor target cells (12). NKG2D is an activating receptor also expressed by, but not restricted to, all NK cells. Ligands for NKG2D include proteins related to nonclassical HLA-I such as MICA, MICB, or the structurally related ULBP1–6 (13, 14). Inhibitory receptors belong to the killercell immunoglobulin-like receptors (KIRs) or to the C-type lectin CD94/NKG2A heterodimer (15). These receptors recognize HLA-I and the non-classical HLA-E and inhibit NK cell activation.

The fundamental role of NK cells in oncology has been widely demonstrated in both hematologic and solid neoplasms. The relevance of this concept is illustrated by many examples in clinical practice, such as the success of hematopoietic stem cell transplantation in hematologic malignancies (16–19), poor NK cell functions associated with increased incidence of cancer (20), the importance of NK cells for the response to chemotherapy and radiotherapy (21, 22), or the use of parameters related to NK cell functions as prognostic biomarkers (23–25). Thus, NK cells can be used as prognostic biomarkers, as well as therapeutic targets or therapeutic agents.

However, although NK cells can kill target cells spontaneously without prior stimulation, a delicate balance between inhibitory and activating signals tightly regulates their activation (1, 26). In the context of cancer, this balance is often deregulated through various mechanisms (27). First of all, cancer cells are able to induce a down-regulation of activating receptors (notably NCR and NKG2D,) as well as an up-regulation of the NK cell inhibitory receptors (23, 24, 28, 29). Then, tumor cells usually poorly express ligands for activating receptors, and/or overexpress ligands for inhibitory receptors (30–32). Finally, the release of various factors such as cytokines or reactive oxygen species (ROS) within the tumor microenvironment impairs the crosstalk between NK cells and dendritic cells (DCs), enhancing the phenomenon of tumor escape (33–35).

Many efforts have been developed in the past few years to restore NK cell functionality in cancer patients. In this review, we focus on NK cells as a cornerstone to restore or improve anti-tumor immunity. We overview different pharmacological strategies aiming at counteracting the effect of tumor cells on NK cell functionality (**Figure 1**). Taking into account the crucial importance of NK cells for maintenance of a prolonged response to treatment, therapeutic strategies improving or restoring NK cell functions in combination with standard treatment regimens are expected to broadly impact patients' clinical outcome.

# **INDUCING NATURAL CYTOTOXIC RECEPTORS EXPRESSION**

Natural cytotoxic receptors expression is classically downregulated during cancer progression, regardless of the type of cancer (23, 24, 28, 29). The mechanisms involved in NCR down-regulation still need to be further defined. Restoring NCR expression may render NK cells more efficient against tumor cells. So far, clinical strategies aiming at restoring NCR expression remain to be proposed. However, taking into account the strong prognostic value of NCR expression, therapeutic strategies aiming at inducing their expression is expected to improve clinical outcome. Therefore, targeting events interfering with the expression of these receptors is certainly a relevant therapeutic option (23, 25).  The release of TGF-β1 is done either by the tumor cell or by regulatory T cells (Tregs). Other tumor-released soluble factors are involved in NCR down-regulation, such as Activin-A, indoleamine dioxygenase (IDO), or prostaglandin E2 (PGE2) (34, 39, 40). Similarly to other activating receptors defect, the down modulation of NCR is somehow dependent on the pressure exerted by tumor cells, which reflects a pathway for tumor evasion. Hence, in acute myeloid leukemia (AML) patients, the low NCR expression acquired during leukemia development is restored in patients achieving complete remission (23). Some recently published data

suggest that NCR down-regulation is consecutive to NK activation in the tumor, leading to an exhaustion of the NK cells and a subsequent down-regulation of the NCRs (41).

# **CYTOKINES**

Amongst the efficient ways to improve NCR expression on NK cells, the use of cytokines, mainly IL-2, IL-15, and IL-21, may be promising. NK cell differentiation is cytokine-dependent (29). High baseline levels of circulating IL-2 constitute an independent prognostic factor for head and neck cancer patients (42).

# **IL-2**

IL-2 is FDA-approved for cancer indications, which is not the case for IL-15 and IL-21. Most clinical trials using cytokines alone or in combination with chemotherapy or radiotherapy are set with IL-2. Conclusions of clinical trials report modest anti-tumor activity when used in monotherapy. Among its diverse immunostimulatory potentials, IL-2 is able to induce expression of NKG2D and NKp46 on NK cells (43, 44). However, following IL-2 stimulation, the NK cytolytic functions do not seem to reach normal cytolytic activity when compared to healthy volunteers (44). Moreover, IL-2 fails to induce NK cell proliferation compared to healthy volunteers, and increases the rate of apoptotic NK cells (44). Some authors evidenced the critical role of IL-2 for the development and peripheral expansion of regulatory T cells (45), which is not the case for IL-15 and IL-21. Noteworthy, the use of IL-2, especially at high doses, might be limited to *ex vivo* expansion of NK cells for problems of *in vivo* toxicity (46).

# **IL-15**

IL-15 plays a major role in the proliferation, differentiation, survival, and functions of T and NK cells (29, 47). Exposure of NK cells to low doses of IL-15 significantly improved NKp30, NKp46, NKG2D, and NKG2C surface expression. Accordingly, this increase of receptor expression was correlated with an increase of natural cytotoxicity against autologous AML blasts (29, 48). In addition, in hematologic malignancies, low levels of circulating IL-15 after bone marrow transplantation were predictive of risk of relapse (49). In line, NK cell recovery in stem cell transplantation is strongly correlated with plasmatic concentrations of IL-15 (48).

IL-15 serum concentration increases dramatically following administration of cytotoxic agents (29, 49). For some authors, this elevation of serum IL-15 could be related to the depletion of lymphoid populations that normally consume circulating IL-15 or to inflammation induced by chemotherapy (48). *In vivo*, injections of the IL-15/IL-15Rα heterodimer result in significant expansion of γδ, CD8+ T, and NK cells (47). Recently, this cytokine has become available for use in early phase clinical trials as an alternative to IL-2 (29, 47). IL-15 is currently assessed as a therapy for various solid tumors including refractory metastatic melanoma, metastatic renal cell cancer. IL-15 is also assessed as an adjuvant of chemotherapy and vaccines strategies or prior to stem cell therapy and NK cells infusion.

# **IL-21**

IL-21 shares significant structural homology with IL-2 and IL-15 (50). In phase I trials, this cytokine shows a favorable safety

profile and signs of clinical activity (51). Although some reports demonstrated a deleterious effect of IL-21 by reducing activating receptor expression (NKG2D, NKp44), its main effect is to enhance NK cell functions. Hence, IL-21 is capable of inducing NK cell maturation and NKp46 and NKp30 expression (12, 52, 53). *Ex vivo*, IL-21 stimulates the production of IFN-γ and cytotoxic properties of NK cells (53). Several clinical trials reported the

effect of IL-21 therapy on immune system after administration in patients with metastatic melanoma and renal cell carcinoma (51). Although NK and T-cell numbers were temporarily decreased during administration of IL-21, the cells had higher expression of CXCR3, HMMR, IFN-γ, perforin, and granzymes at the mRNA level. Evidence of NK cell activation was further confirmed by enhanced ability of NK cells from patients to lyse K562 target cells (51). These results were confirmed in a phase II trial for metastatic melanoma (54).

## **IMMUNOMODULATORY DRUGS**

Immunomodulatory drugs (IMiDs) present another therapeutic option to increase activating receptors expression. Two molecules are currently developed in oncology: lenalidomide, FDA-approved in hematologic malignancies, and pomalidomide. These drugs present anti-angiogenic and anti-proliferative activity, and their effect on the immune system, particularly on NK cells, is probably part of their mechanism of action. For instance, immunomonitoring of patients treated with immunomodulatory drugs, IMiDs have been associated with an increased expression of NKp44 and NKp46, in multiple myeloma (MM), myelodysplastic syndrome but also in solid tumors (55, 56). Interestingly, this effect of lenalidomide may not be a direct effect on NK cells because this effect was not observed *in vitro* on purified NK cells (57). In this study, IMiDs-treated NK cells displayed a lower NKp46 expression, although this had no functional consequences on cytolytic functions of NK cells.

# **HISTAMINE**

Blocking phenomenon responsible for NCR down-regulation is another potential strategy to induce indirect NCR expression. Thus, ROS, PGE2, and IDO, which are present in the tumor microenvironment, appear to be relevant targets (33–35). Romero et al. demonstrated that histamine was able to prevent NKp46 and NKG2D down-regulation mediated by mononuclear and polymorphonuclear phagocytes ROS production (35). Moreover, histamine maintains the cytolytic activity of NK cells toward leukemic cells despite the presence of phagocytes. A phase III clinical trial assessed the efficacy of post-consolidation immunotherapy with IL-2 and histamine dihydrochloride for patients with AML in complete remission. This treatment was shown to significantly improve leukemia-free survival, with mild to moderate side effects (33).

# **INDUCING NKG2D EXPRESSION**

NKG2D down-regulation on circulating NK cells in cancer patients compared to healthy volunteers was described in various cancer types, including breast cancer, glioma, melanoma, and lung cancer (58–62).

### **CYTOKINES**

Few pharmacological agents are able to directly increase the expression of NK-activating receptors. Until now, the only described possibility to directly induce NKG2D expression on NK cells is the use of immunostimulatory cytokines. *Ex vivo*, IL-15 was shown to be able to induce a dramatic increase of NKG2D expression (63, 64). Although the use of IL-15 is still restricted to phase I and II clinical trials, conventional chemotherapies are able to induce a huge increase of the circulating IL-15 (29).

#### **TGF-**β **PATHWAY**

A second strategy allowing NKG2D restoration in the cancer context is indirect up-regulation by blocking the agents responsible for NKG2D down-regulation. For instance, stroma-derived factors in the tumor microenvironment, in particular TGF-β, display an immunosuppressive activity on most anti-tumor immune effectors, and an indirect immunosuppressive effect via the inhibition of MICA transcription (38, 65). Besides immune suppression, stroma-derived factors also present direct effects on the tumor cell since TGF-β promotes tumorigenesis and epithelial–mesenchymal transition (66). *In vitro*, TGF-β inhibits the expression of NKp30 and NKG2D (37) and blood concentration of TGF-β1 was shown to inversely correlate with NKG2D expression at the surface of NK cells of cancer patients and has been linked with impaired NK cytotoxicity (58, 60). TGF-β antagonizes the IL-15-induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of NK cell activation receptor molecules (67). Moreover,*ex vivo* addition of neutralizing anti-TGF-β monoclonal antibodies completely restores surface NKG2D expression at the surface of NK cells and partially restores NKp30 expression (60, 67). In addition, blocking TGF-β completely restores IFN-γ production by tumor-associated NK cells (67).

Some approaches aiming at decreasing circulating TGF-β in patients are currently under investigation (68). These early stage clinical trials currently assess several approaches, mainly the use of anti-TGF-β monoclonal antibodies and antisense oligonucleotides. For example, fresolimumab (GC-1008), a fully humanized pan-neutralizing antibody directed against all the three isoforms of TGF-β, has been assessed in renal cell carcinoma and in metastatic melanoma (68, 69). In this phase I/II trial, fresolimumab was safe and well-tolerated with no dose-limiting toxicities and displayed encouraging results.

The impact of TGF-β blockade on immune parameters was recently assessed in patients with malignant pleural mesothelioma treated with fresolimumab (70). Fresolimumab had no effect in the expression of NK, CD4+, or CD8+ T-cell-activating and inhibitory markers, other than a decrease in the expression of 2B4 and DNAM-1 on NK cells, although TGF-β serum concentrations were markedly decreased. The authors conclude that acute changes in serum TGF-β concentration are not associated with the set of biomarker changes that were predicted based on animal models. No effect was detected on the expression of NKG2D nor NKp30, and the effect on DNAM-1 expression, although significant, was minor (70).

Another possibility to decrease TGF-β in the tumor milieu is the use of antisense oligonucleotides. Some of these compounds are currently in clinical evaluation. Belagenpumatucel-L, a therapeutic vaccine comprised of four TGF-β2 antisense gene-modified allogeneic NSCLC cell lines was assessed in grade III/IV NSCLC patients. In a phase II study, positive clinical outcomes were correlated with immune response to the vaccine and induction of immune enhancement of tumor antigen, but the effect on NK cells was not assessed (65). This compound is still currently investigated in non-small cell lung carcinoma in phases II and III trials.

Alternatively, SD-208, a TGF-β receptor I kinase inhibitors, restores the lytic activity of polyclonal NK cells against glioma cells in the presence of recombinant TGF-β or of TGF-β-containing glioma cell supernatant (71). This molecule is able to restore NKG2D expression on NK cells, whose expression was altered *in vitro* by cancer cell lines supernatants or direct inhibition with recombinant TGF-β (72).

To conclude, NKG2D expression has never been shown to present a prognostic value unlike NKG2D ligands expression, thus suggesting that the best strategy to target the NKG2D/NKG2D ligand system might be to induce ligands expression rather than the receptor itself.

# **INDUCING LIGANDS EXPRESSION FOR NK-ACTIVATING RECEPTORS**

The main ligands for NKG2D are the MHC class I chainrelated molecules MICA and MICB and the ULBP1–4. These ligands have been extensively studied in various malignancies. Ligands of DNAM-1 are CD112 (Nectin-2) and CD155 (Poliovirus receptor, PVR). Ligands of NCRs have been elusive for many years and although pathogen-related ligands have been suggested (hemagglutinins, heparate sulfates), only ligands for NKp30 have been identified. B7-H6, an Ig molecule from the family of B7 molecules has been identified as NKp30 ligand (73). B7-H6 is expressed by several cell lines and by primary tumors (74). Mechanisms of induction of B7-H6 expression have been described in non-transformed cells with TLR agonists as well as the pro-inflammatory cytokines TNFα and IL-1β (75). In primary tumors, recent experimental data suggest that B7-H6 expression is regulated by HDACs, in particular HDAC3 (74). In addition, BAG6/BAT3, a nuclear protein localized at the plasma membrane or on exosomes of tumor cells, has also been assigned as an NKp30 ligand (76). The importance of ligands expression for tumor cell recognition by NK cells is a key factor for anti-tumor immune response, as illustrated by the strong prognostic value of MICA/MICB, RAET1G, and ULBP2 expression in colorectal cancer and breast cancer (30–32). Tumor cells poorly express ligands for NK-activating receptors, and tumor ligands expression is inversely correlated with clinical stage (77).

# **HISTONE DEACETYLASE INHIBITORS**

Histone deacetylase inhibitors were successfully introduced as anti-cancer agents for their ability to block gene transcription and promote cell differentiation. These molecules induce cell cycle arrest and induce apoptosis of tumor cells, with minimal effects on normal tissue (78). Unexpectedly, their effect on anti-tumor immunity is part of their mechanism of action.

The main impact of these molecules on immunity is mediated through up-regulation of tumor antigens, in particular NKG2D ligands (79). HDACi-mediated immune modulation is also linked to the ability of these molecules to enhance immune recognition and lysis of the tumor cells by T cells and NK cells (79). To date, two molecules, romidepsin and vorinostat, have received approval from the FDA for the treatment of cutaneous T-cell lymphoma. *In vitro*, romidepsin, vorinostat, and sodium valproate were shown to increase MICA/B and ULBPs expression on various cancer cell lines and primary tumor cells, and render the target cells more sensitive to NK cell lysis (80–84). Depending on the authors, this mechanism was found to be GSK3- or ERK-dependent (81, 83).

Induction of MICA and MICB expression was associated with a shedding of the soluble forms of these NKG2D ligands, sMICA and sMICB (82). This raises the question of the potential counterbalancing of the clinical benefits in this particular case, since increase of the serum concentrations of sMICA and sMICB are responsible for NKG2D endocytosis and degradation, and represents a mode of T-cell silencing and immune escape (62, 82). Thus, Poggi et al. monitored NKG2D ligands shedding following treatment of AML patients treated with valproic acid. In this study, MICA, ULBP2, and ULBP3 expression on blasts was significantly increased after treatment with valproic acid. No ligand shedding was detected despite a strong up-regulation of the ligands on leukemic cells. Consequently, leukemic cells from patients treated with valproic acid, become able to trigger lytic granule exocytosis by autologous CD8+ T and NK cells (85).

However, some studies evidenced that HDACi down-regulate ligands for other NK cells-activating receptors, such as B7-H6, a ligand for NKp30, and impair tumor cell recognition by NK cells. These results were obtained with first and second generation HDACi (vorinostat, trichostatin A, valproic acid, and apicidin) on various cancer cell lines (74). Moreover, treatment of human NK cells with trichostatin A, valproic acid, or sodium butyrate affects the functional response of human NK cells, evidenced by a strong inhibition of IFN-γ secretion and a decreased ability to lyse target cells (86). Furthermore, the authors evidenced a downregulation of activating receptors NKG2D and NCRs on resting and cytokine-stimulated NK cells.

Another study assessed the effect of vorinostat and valproic acid on NK cells. At therapeutic concentration, these drugs induced the down-regulation of NKp30 and NKp46, and inhibited IL-2 activation of NK cells, thus suppressing their cytolytic activity toward leukemic cell lines. This effect seems to be mediated by the inhibition of NFκB. In addition, the authors showed that vorinostat was toxic to NK cells in the range of therapeutic concentrations (87).

### **DEMETHYLATING AGENTS**

The hypomethylating drugs decitabine and azacytidine are epigenetic drugs that are currently used in treatment of hematological malignancies (88). Besides their direct effect on the tumor cell, these drugs probably act through their impact on innate immunity. *In vitro*, both drugs induce ULBP1 and MICB on cell lines and primary tumor cells when incubated with either decitabine or 5-azacytidine (89, 90). This effect was related to promoter DNA methylation and DNA damage and correlates with enhanced NK cytotoxicity (90, 91).

However, DNA methylation is an important regulator of KIR expression by NK cells, potentially impacting on NK cell functions (92, 93). Hence, 5-azacytidine induces an increase in the percentage of KIR+ NK cells upon treatment with clinically relevant concentrations of 5-azacytidine, which correlated with an impaired granzyme B and perforin release, IFN-γ production, and decreased cytotoxicity (91, 94). However, this effect seems to be restricted to 5-azacytidine, since decitabine increases NK cell cytotoxicity and enhances IFN-γ production, in a dose-dependent manner (91). These results were confirmed in recent studies in different settings. Recently, Cerdeira et al. tested the effect of 5-azacytidine in hypoxic conditions with addition of TGF-β. Although the authors confirmed the impact of this drug on KIR expression, however, the cytotoxicity of NK cells cultured in these specific conditions was not affected (92).

For some authors, the results obtained *in vitro* in such settings are debatable. Indeed, since 5-azacytidine and decitabine are nucleoside analogs, these molecules require DNA replication to be incorporated into the DNA strand. *In vitro* studies using resting NK cells are therefore more likely to reflect the direct mRNA effect of such drugs than the effect of hypomethylation (88). Thus, Kopp et al. studied the effect of decitabine on proliferating NK cells. The authors show that decitabine negatively affects NK cell viability and proliferation in a dose-dependent manner. Simultaneous increase in KIR and NKp44 expression and decrease in NKG2D expression was evidenced. However, the impact on NK functionality in terms of toxicity was biphasic, with decreased toxicity at low doses and increased toxicity at high doses. Since the target cells used in these experiments lack class I HLA, this effect is independent of KIR up-regulation. Whether this increased cytotoxicity is maintained in the presence of HLA-positive targets remains to be determinate (88).

To conclude, further investigation is required to determine whether epigenetic drugs adversely affect NK cell survival, proliferation, or functions when administrated to patients.

### **DNA-DAMAGING AGENTS**

Some conventional chemotherapeutic agents can induce immunogenic cell death, e.g., tumor cell apoptosis and stress signals that lead to the surface expression of ligands for NKG2D and DNAM-1 (95, 96). This DNA damage pathway can be activated by several mechanisms, during the course of chemotherapy with DNAdamaging agents such as doxorubicin, mitoxantrone, cisplatin, and oxaliplatin (8, 95–99). This particular mode of cell death displays damage-associated molecular patterns, e.g., exposure of calreticulin endoplasmic reticulum proteins at the surface of the pre-apoptotic cell, as well as secretion of ATP (100).

The oncogenic stress induced by these DNA-damaging agents stimulates various aspects of anti-cancer immunity, including activation of NK cells via ULBP1, MICA/B, and PVR expression at the surface of the cancer cell in an ATM (ataxia telangiectasia, mutated), ATR (ATM- and Rad3-related) protein kinases, and/or P53-dependent manner (8, 96–99). Other agents are able to induce stress conditions, leading to the expression of ligands for NKG2D and DNAM-1, such as IMiDs and proteasome inhibitors (22). These results await clinical confirmation with immunomonitoring studies of patients undergoing DNA-damaging agent therapy.

# **TARGETING SOLUBLE LIGANDS FOR ACTIVATING RECEPTORS**

The expression of NKR ligands at the surface of cancer cells appears to be a good prognostic factor. However, the shedding of soluble ligands in the circulation strongly impairs NK cell functions and has been linked with tumorigenesis and tumor progression (101) and high serum concentration of ULBP2 presents a strong prognostic value in breast cancer, colorectal cancer, and melanoma (30–32). Noteworthy, the discovery of B7-H6 and BAG6, ligands for NKp30, included the detection of soluble forms, which may compete for cell–cell interaction with membrane-bound ligands, although only soluble/exosome-bound BAG6 has been detected in a cancer situation (75, 102).

The prototypical example of ligand shedding is the release of soluble MICA/MICB (sMICA/sMICB), typically by A disintegrin and metalloproteases (ADAMs) (103, 104). These proteases are overexpressed in malignant tissues compared to normal tissues (105, 106). As a consequence, serum concentrations of soluble ligands for NKG2D are elevated in various malignant conditions (103). The ligation of these soluble ligands induces internalization of NKG2D and its subsequent degradation, leading to an overall down-regulation of the receptor at the surface of NK cells. In various cancers, high levels of circulating ligands for NK-activating receptors correlated with a poor prognosis. Direct pharmacologic inhibition of these metalloproteases is still in preclinical evaluation.

# **SORAFENIB**

Sorafenib is a multi-target tyrosine kinase inhibitor targeting RAS/RAF/MAPK as well as VEGFR and PDGFR signaling pathways, implicated in cell proliferation and angiogenesis. Sorafenib is indicated in renal cell carcinoma, hepatocellular carcinoma, thyroid cancer and melanoma. *In vitro*, this molecule presents interesting off-target effects on ADAM9 expression as evidenced by a recent study on the human hepatocellular carcinoma cell line HepG2. In this study, sorafenib was able to strongly decrease ADAM9 expression at the proteic and transcriptional level, which correlated with a decrease of sMICA concentration in the culture supernatant and enhanced sensitivity to NK cell lysis. In addition, ADAM9 inhibition increases the expression of membranebound MICA on the tumor cell, enhancing the NK sensitivity of hepatocellular carcinoma cells (105).

Controversial data were published about effects of sorafenib on NK cells. NK cell function is inhibited by sorafenib as a consequence of impaired phosphorylation of PI3K and ERK, which directly control NK cell reactivity (107). Immunomonitoring of patients with renal cell carcinoma and melanoma treated with sorafenib failed to evidence modification of pERK1/2 expression in peripheral-blood NK cells after short-term or long-term administration (108). In addition, sorafenib may also positively (Th1) or negatively (DCs) impact other aspects of anti-tumor immunity (61, 109, 110). Whether this action is positive or negative remains to be determinated, as well as the overall "immune benefit" of such antagonistic effects on anti-tumor immunity, besides their direct pro-apoptotic effect on the tumor cell.

# **TARGETING INHIBITORY RECEPTORS**

Although activating NK receptors are crucial, triggering of NK cell effector functions is prevented by the expression of the inhibitory receptors KIR and NKG2A. Although in some examples of solid cancer, KIR and NKG2A expression is altered, generally expression is maintained and tumor cells may maintain sufficient amounts of HLA molecules to ensure inhibition of NK cells and evade killing. Moreover, some tumors display decreased expression of TCRdependent HLA molecules while maintaining a normal expression of KIR-dependent HLA molecules (111). High HLA-E expression has been observed in several solid cancers (112, 113) and leukemias (114). Consequently, as 20–70% of NK cells express NKG2A, HLA-E expression by tumor cells impairs the anti-tumor activity of a predominant proportion of NK cells.

# **ANTI-KIR MONOCLONAL ANTIBODIES**