Human natural killer cells: news in the therapy of solid tumors and high-risk leukemias

Abstract

It is well established that natural killer (NK) cells play an important role in the immunity against cancer, while the involvement of other recently identified, NK-related innate lymphoid cells is still poorly defined. In the haploidentical hematopoietic stem cell transplantation for the therapy of high-risk leukemias, NK cells have been shown to exert a key role in killing leukemic blasts residual after conditioning. While the clinical results in the cure of leukemias are excellent, the exploitation of NK cells in the therapy of solid tumors is still limited and unsatisfactory. In solid tumors, NK cell function may be inhibited via different mechanisms, occurring primarily at the tumor site. The cellular interactions in the tumor microenvironment involve tumor cells, stromal cells and resident or recruited leukocytes and may favor tumor evasion from the host’s defenses. In this context, a number of cytokines, growth factors and enzymes synthesized by tumor cells, stromal cells, suppressive/regulatory myeloid and lymphoid cells may substantially impair the function of different tumor-reactive effector cells, including NK cells. The identification and characterization of such mechanisms may offer clues for the development of new immunotherapeutic strategies to restore effective anti-tumor responses. In order to harness NK cell-based immunotherapies, several approaches have been proposed, including reinforcement of NK cell cytotoxicity by means of specific cytokines, antibodies or drugs. These new tools may improve NK cell function and/or increase tumor susceptibility to NK-mediated killing. Hence, the integration of NK-based immunotherapies with conventional anti-tumor therapies may increase chances of successful cancer treatment.

Introduction

Natural killer (NK) cells are effector cells of the innate immunity that are capable of killing virus-infected or tumor cells, as well as to produce cytokines [1]. They were discovered four decades ago on the basis of their ability to kill certain tumor cell lines without previous stimulation. Although NK cells are present primarily in peripheral blood (PB), spleen and bone marrow (BM), they have been found also in lymph nodes, gut, liver and uterus [2]. NK cells can migrate to inflamed tissues and secondary lymphoid organs (SLOs) where they contribute to the first line of defense against pathogens, primarily viruses [1, 3]. They can rapidly release cytokines and chemokines upon activation, thus contributing to the induction and/or amplification of inflammatory responses [4, 5]. NK cell function is finely regulated by signals deriving from an array of activating and inhibitory receptors [6–9].

In humans, NK cells can be divided into two main subsets according to the levels of surface expression of the CD56 antigen (CD56^dim and CD56^bright NK cells) [10]. These subsets also differ in the expression of several surface molecules, in their functional capabilities and tissue localization. CD56^dim are CD16⁺ and may express killer cell Ig-like receptors (KIRs), display high cytolytic activity and rapidly release cytokines upon receptor-mediated triggering. Conversely, CD56^bright are CD16^low/neg KIR⁻ and do not exert cytolytic activity, but secrete large amounts of cytokines, primarily upon cytokine-induced stimulation. CD56^bright and CD56^dim cell subsets also differ in the expression of chemokine receptors and adhesion molecules that explain their different tissue distribution. CD56^bright NK cells represent 10 % of PB NK cells, while they are highly enriched in SLOs, where they can migrate thanks to the expression of CCR7, CCR5, CXCR3 and CD62L. CD56^dim NK cells represent the large majority of PB NK cells. They express CX₃CR1, CXCR2 and CXCR1 that allow their recruitment to inflamed tissue [10–12]. The tissue distribution of CD56^dim and CD56^bright subsets correlates with the chemokine expression pattern of different tissues [13, 14].

NK cells belong to the innate lymphoid cell family

In the last two decades, other innate lymphocytes have been identified. Together with NK cells, they constitute the innate lymphoid cell (ILC) family [15]. Recently, ILC have been classified into two main groups: cytotoxic ILC (i.e., NK cells) and helper ILC [16]. Three different groups of helper ILC have been identified: ILC1, ILC2 and ILC3. They are characterized by diverse patterns of cytokine secretion that are reminiscent of those of CD4⁺ T helper cell subsets [16]. ILC1 include interferon-γ (IFN-γ)-producing cells that are mainly located in mucosal tissues and undergo expansion in the intestine of Crohn’s disease patients [17, 18]. ILC2 produce type 2 cytokines and promote innate anti-parasites responses. On the other hand, they can also contribute to airways hyper-reactivity and atopic dermatitis [19]. ILC3 include both fetal lymphoid tissue inducer (LTi) cells, involved in lymphoid organ development, and adult ILC3, that produce interleukin (IL)-22 and IL-17 and provide innate host defenses against extracellular pathogens [20]. Adult ILC3 are also responsible for tissue homeostasis and are mainly located in the intestinal lamina propria and tonsils [15]. More recent studies revealed their presence in skin and decidual tissue [21, 22]. Different from NK cells, the other ILC subsets are devoid of cytolytic activity and of the ability to exert a direct anti-tumor activity. Very limited information is so far available on ILC infiltration in tumors. Since ILC produce pro-inflammatory cytokines, it is conceivable that if indeed they are present at the tumor infiltrate, they may contribute to the inflammatory tumor microenvironment. In this context, in a murine model, IL-22 produced by T cells and ILC3 contributed to the development of colon cancer [23]. However, it would be interesting to investigate whether ILC3 might also favor anti-tumor responses. The presence of ectopic lymphoid structures (ELS), in which anti-tumor-specific adaptive lymphocytes can be activated, correlates with a better prognosis in certain tumors [24]. In this context, it has been proposed that ILC3 with LTi activity might drive the formation of ELS at the tumor site [25].

Influence of the tumor microenvironment on NK cell recruitment and function at the tumor site

NK cell infiltrates have been detected in various tumors. Remarkably, high NK cell infiltration has been reported to correlate with a better prognosis, at least in some tumors [26]. However, some reports have shown that NK cells present at the tumor site are enriched in the poorly cytotoxic CD56^bright subset [27, 28]. Tumor-infiltrating NK cells and even PB NK cells, derived from cancer patients, display a reduced expression of the main activating receptors, resulting in a reduced anti-tumor activity [29–32]. Surprisingly, tumor-infiltrating NK cells are not in direct contact with neoplastic cells, but they are rather located within the stroma [33, 34]. Thus, despite the strong NK cytolytic activity against tumor and leukemic cells in vitro, the NK cell efficacy against tumors in vivo may be hampered by an inefficient migration capacity and by the effect of the tumor microenvironment.

Although it is likely that NK cells may represent a potent tool to fight cancer, definite NK-based immunotherapeutic strategies against solid tumors have not been successful so far. One of the reasons may be related to mechanisms of immune subversion and/or evasion occurring at the tumor site. Indeed, various soluble factors produced by tumor cells themselves or other cells, present in the tumor microenvironment, can modulate NK cell function. Thus, transforming growth factor-β (TGF-β), indoleamine 2,3-dioxygenase (IDO), IL-4, prostaglandin E2 (PGE2) can interfere with NK cell activation or induce down-regulation of activating NK receptors, which are responsible of NK cell-mediated recognition and killing of tumor cells [34–37].

Different cell types present in the tumor microenvironment can be polarized toward a type 2 response and/or produce suppressive factors. For example, regulatory T cells (Tregs) isolated from patients with gastrointestinal sarcoma inhibit NK cells through membrane-bound TGF-β [38, 39]. In addition, Tregs could also interfere with NK cell activation by competing for IL-2 availability [40]. Also cells of myeloid origin may affect NK cell function, as reported in two studies analyzing myeloid-derived suppressor cells (MDSC) or tumor-associated macrophages (TAM) isolated from patients with hepatocellular carcinoma (HCC) [41, 42].

A remarkable suppressive activity may also be exerted by tumor-associated fibroblasts (TAF). Thus, TAF derived from melanoma, HCC and colorectal carcinomas were shown to exert an inhibitory effect on IL-2-induced NK cell activation [34, 43, 44]. Upon interaction with NK cells, TAF sharply increased their release of PGE2, which, in turn, inhibited the IL-2-induced up-regulation of NKp44, DNAX accessory molecule-1 (DNAM-1) and NKp30-activating NK receptors.

Tumor cells themselves can directly influence NK cell function by a variety of inhibitory mechanisms. Our group showed that different melanoma-derived primary tumor cell lines could inhibit the surface expression of NKp30, NKp44 and natural killer group 2, member D (NKG2D) on NK cells. This effect is mediated by IDO and/or PGE2 [36]. Additional suppressive mechanisms have been described in tumors of different histotypes. Different tumor cells can shed a soluble form of MHC class I chain-related gene A (MICA) that, in turn, induces down-regulation of NKG2D receptor, leading to the inhibition of NK cell function [45]. NKG2D down-regulation may also be induced by a mechanism involving the release of macrophage migration inhibitory factor (MIF) as shown for ovarian cancer [46]. Ovarian tumor cells can also express or release the mucin 16 (MUC16) glycoprotein, capable of interfering with the formation of the immunological synapses between NK and tumor cells [47]. Tumor cells may also affect the ability of NK cells to infiltrate the tumor itself. In this context, it has recently been shown that neuroblastoma cells could modulate the NK cell chemokine-receptor repertoire through TGF-β production [48]. Importantly, also physical status of the tumor microenvironment may affect NK cell function, thus preventing their ability to eliminate tumor cells. Hypoxia (a condition that often characterizes tumor tissues) can significantly alter both the expression and function of major activating NK receptors with the remarkable exception of CD16. Accordingly, the NK-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) was only marginally affected by hypoxia, giving hints for a more extensive exploitation of antibody-based immunotherapies [49]. Tumor cells may develop different strategies to evade the NK cell-mediated attack. For example, as a consequence of chronic interactions with NK cells, tumor cells can display an altered expression of ligands for either inhibitory or activating receptors. Along this line, our group has recently shown that following co-culture with NK cells, melanoma cells up-regulate the surface expression of both classical and non-classical HLA class I molecules, thus becoming resistant to NK cell-mediated lysis [50]. In this experimental setting, a major role in the up-regulation of HLA class I expression on tumor cells is played by NK cell-derived IFN-γ. Another mechanism by which tumor cells may evade NK cell-mediated recognition is the modulation of NKG2D ligands at their surface. In addition, similarly to the NKG2D ligand MICA, soluble forms of HLA-B-associated transcript 3/BCL2-associated athanogene 6 (BAT3/BAG6) and B7H6 (both ligands of NKp30) have been recently described [51, 52]. A reduction in NK cell cytotoxic activity by tumor cells can also be mediated by inhibitory signals induced by the engagement of NKp44 receptor with its ligand termed proliferating cell nuclear antigen (PCNA), a molecule overexpressed in different tumor types [53, 54]. The presence of phosphatidylserine (PS) on apoptotic tumor cells represents an additional mechanism to dampen NK cell cytotoxicity since PS is recognized by the IRp60 (CD300a) inhibitory NK receptor [55, 56].

Hematologic malignancies frequently relapse after therapy, and this disease recurrence represents the main cause of treatment failure. It is well established that leukemia cells may escape immunesurveillance and that a fraction of them, primarily the leukemic stem cells (LSC), may be particularly resistant to different chemotherapeutic drugs [57–60]. Leukemia elusion of immune system may act through different mechanisms. Different reports revealed that mature NK cells isolated from PB of acute myeloid leukemia (AML) patients display a low expression of major activating NK receptors, paralleled by an increased expression of inhibitory receptors, low production of tumor necrosis factor-α (TNF-α) and IFN-γ and an impaired cytolytic activity [32, 61–63]. Lymphocytic leukemia cells may release soluble BAG6 (NKp30 ligands) capable of competing with the exosome-bound BAG6 for the induction of NK cell activation via NKp30 [51, 52]. Soluble inhibitory factors such as IDO or TGF-β may also play a significant inhibitory role [64, 65]. In this context, sera derived from human AML patients have been shown to contain microvesicles bearing TGF-β at their surface, resulting in impairment of NK cell function. Of note, both phenotypic and functional alterations of NK cells were reverted in patients undergoing successful therapy and achieving complete remission [65].

Increasing evidences supports the notion that also the BM microenvironment may play a relevant role in supporting leukemia progression [66, 67]. Malignant cells may contribute to generate aberrant BM niche. Thus, in an acute lymphoblastic leukemia (ALL) mouse model, leukemia blasts could disrupt the BM microenvironment and rebuild aberrant niches by inducing the transition of niche cells from nestin⁺ to alpha-SMA⁺ mesenchymal stem cells (MSC). This transition favors the protection of LSC and the selection of therapy-refractory subclones. Importantly, these data were further confirmed by the analysis of BM biopsies from ALL patients [68].

Another central issue is whether leukemia cells may influence survival of normal hematopoietic stem cells (HSC) and their differentiation into immune cells with potential anti-leukemia activity. This is particularly relevant in patients achieving remission or undergoing HSC transplant (HSCT), since leukemia cells, residual after chemotherapy, may interfere with the homing and differentiation of donor-derived CD34⁺ cells. In addition, the conditioning regimen and/or the occurrence of graft-versus-host disease (GvHD) may induce an inflammatory response in the BM, which would further interfere with the normal hematopoiesis. Previous reports showed that the serum levels of inflammatory cytokines or chemokines in AML patients may represent a prognostic factor and may correlate with the clinical outcome [69, 70]. Recently, we have shown that IL-1β-releasing AML blasts could inhibit the recovery of CD34⁺-derived CD161⁺CD56⁺ cells, resulting in a reduced generation of ILC3 and NK cells. In the context of HSCT, it is possible that IL-1β released by residual AML blasts may alter the BM microenvironment and suppress the proliferation of NK cell precursors. This, in turn, could be detrimental, primarily in patients receiving haplo-HSCT in which NK cells play a fundamental role in clearing leukemia blasts surviving the conditioning regimen [71].

NK cell therapy of tumors

The efficacy of NK cells in the immunotherapy of solid tumors has been evaluated in various clinical settings. Pioneering trials were based on the adoptive transfer of autologous lymphokine-activated killer (LAK) cells in combination with IL-2 infusion in patients with metastatic cancers [72]. Although responses could be detected in approximately 20 % of patients, further studies failed to clearly document benefits of this treatment over the IL-2 monotherapy [73]. The clinical use of LAK cells for targeting a variety of solid malignancies and the outcomes of cellular therapies have been reviewed in detail [74, 75]. In a recent study, Rosenberg and colleagues re-evaluated the efficacy of adoptively transferred autologous NK cells that had been activated in vitro with IL-2. Patients included in this study were affected by metastatic renal carcinoma and melanomas [76]. Before adoptive cell transfer (ACT), these patients received a lympho-depleting chemotherapy consisting of cyclophosphamide and fludarabine to promote the expansion of the adoptively infused cells. The adoptively transferred NK cells persisted for long time intervals in patients PB. However, no significant clinical benefit could be documented. Interestingly, although NK cells that had been adoptively transferred were found to be “anergic” (i.e., unable to exert natural cytotoxicity against tumor cells), they retained CD16 expression and could mediate ADCC in vitro even in the absence of cytokine reactivation. These data are important because they suggest that the combination of adoptive NK cell transfer and administration of monoclonal antibodies specific for tumor cells may indeed represent a valuable therapeutic approach.

Since autologous NK cells may be functionally inhibited by the interaction between self-HLA class I molecules on tumor cells and inhibitory NK receptors, the adoptive transfer of allogeneic NK cells may potentially represent a better approach. However, evidences of the actual efficacy of allogeneic NK cell treatment in patients with solid malignancies are still missing [77–79]. The impaired capability of infused NK cells to traffic to tumor lesions may in part explain the poor anti-tumor effect of adoptive NK cell therapy in solid tumors. As revealed by a recent study, CXCL10 plays a major role in the chemoattraction of infused NK cells in melanoma-bearing mice [80]. Along the same line, other studies reported that NK cells expanded in vitro in the presence of IL-15 and glucocorticoids express high level of CXCR3, thus suggesting that they may better home to tumor sites [81].

The activity of endogenous NK cells can be enhanced in vivo by systemic or local administration of cytokines, such as IL-2, IL-12 IL-15, IL-18 and IL-21. We have recently shown that NK cells isolated from malignant pleural effusions (PE-NK cells) are not anergic and, upon IL-2 activation, they could efficiently kill autologous tumor cells [82]. In addition, they rapidly release cytokines (IFN-γ and TNF) and undergo degranulation upon exposure to tumor cells. These data suggest the possibility of treating primary or metastatic pleural tumors with local infusion of IL-2 and/or autologous IL-2-activated PE-NK cells [83, 84]. However, clinical trials using high doses of IL-2 for advanced melanoma and renal carcinoma showed limited clinical benefits. This reflects both the severe toxic effect of IL-2 (such as the vascular leak syndrome) and the stimulation of Tregs. Thus, alternative cytokines (such as IL-15, IL-12 and IL-18), capable of effectively boosting NK cells without stimulating suppressive loops, are currently being tested in preclinical cancer models [85]. A recent study in mice by Ardolino et al. [86] revealed that NK cells infiltrating MHC class I-deficient tumors become anergic, and this condition can be reverted by treatment with IL-12 and IL-18. While results of different studies suggest that IL-12 and IL-18 have low anticancer activity when used alone, clinical studies combining these two cytokines have not been performed so far [87, 88]. Early-phase clinical trials are currently employing IL-15 as an alternative to IL-2 [89].

There is a wide range of immune-modulatory drugs that are able to restore NK cell function either directly or indirectly. Recently, the immune-modulatory potential of lenalidomide (a synthetic derivate of thalidomide) has been extensively investigated in patients with hematologic malignancies or solid tumors. Lenalidomide has been shown to increase both natural cytotoxicity and ADCC in NK cells. In addition, it may induce the expression of ligands recognized by activating NK receptors on tumor cells [90]. A number of chemotherapeutic drugs (including doxorubicin, etoposide and dacarbazine), as well as heat-shock protein 90 (HSP-90) inhibitors, histone deacetylases inhibitors (such as sodium valproate, VPA) and proteasome inhibitors (such as bortezomib), may also enhance the expression of TNF-related apoptosis-inducing ligand (TRAIL) and NKG2D ligands in tumor cells that become more susceptible to NK cell-mediated cytotoxicity [91].

Other important new strategies to improve NK cell efficacy in immunotherapy are based on blocking inhibitory signals. Among these strategies, the use of anti-KIR monoclonal antibodies that disrupt the KIR/HLA class I interaction at the tumor cell surface, is a novel attractive therapeutic strategy not only for hematologic malignancies but also for solid tumors [92, 93]. An overall view of the different approaches for an NK-based immunotherapy is described in Fig. 1.

Fig. 1.

NK cell-based immunotherapy. NK cell function can be improved by different in vivo strategies (a–c). These include: a induction of CD16-mediated antibody-dependent cytotoxicity (ADCC) and IFN-γ production by the use of tumor antigen-specific mAb; b enhancement of NK cell cytolytic activity by blocking inhibitory signals with anti-KIR mAb, c administration of NK stimulatory cytokines. The middle panels d, e show examples of NK cell-based adoptive therapy. Autologous or allogeneic NK cells can be collected from peripheral blood (d) or can be derived from hematopoietic stem cells (HSC) (e). After in vitro activation and expansion, NK cells can be subsequently infused into the recipient. Finally, immune-modulatory and chemotherapeutic drugs may improve NK cell function through the up-regulation of activating NK cell receptors expression and/or the expression of NK ligands on the tumor cell surface (f)

Recently, T cells genetically modified to express chimeric antigen receptors (CAR) have been shown to exert a more specific and efficient recognition of tumor cells both in vitro and in vivo. Thus, the successful use of these CAR-T cells in clinical trials prompted the development of NK cells expressing CAR to improve their anti-neoplastic activity. However, their use is still limited mainly to preclinical model [94].

NK-based therapy of acute leukemias

Allogeneic HSCT from an HLA-compatible donor has been increasingly applied to the therapy of high-risk leukemias and also of non-malignant severe disorders (e.g., primary immune deficiencies, inherited BM failure syndromes and some few selected metabolic disorders). However, in over 1/3 of patients in need of an allograft, no HLA-matched donor can be found. For these patients, HLA-haploidentical HSCT offers a suitable alternative, particularly for pediatric patients in whom either the father or the mother can be used as HSC donor. Given the high degree of HLA incompatibility of this transplant, an extensive T cell depletion is mandatory in order to prevent life-threatening GvHD [95, 96]. T cell depletion must be associated with a high-intensity, immunosuppressive and myeloablative conditioning and to the infusion of very high numbers of CD34⁺ cells, to reach sustained engraftment of donor hematopoiesis. Through this comprehensive approach, haplo-HSCT resulted in high percentages of successful engraftment without severe (grades II to IV) GvHD [97, 98]. Pioneering studies in adult AML demonstrated that in T cell-depleted HSCT from an HLA-disparate relative the graft-versus-leukemia (GvL) is mainly mediated by NK cells undergoing in vivo maturation from donor HSC. However, an efficient GvL in haplo-HSCT occurred in those patients receiving a transplant from a donor who had NK alloreactive against patient cells [99]. NK alloreactivity was found to be associated with the presence of a KIR/KIR ligand mismatch in the donor versus recipient direction. Thus, in donor/patient pairs with KIR/KIR ligand mismatch, the patient survival rate was ~60 % as compared to <5 % in the absence of mismatch [99]. A similar positive effect of alloreactive NK cells was obtained also in children treated with haplo-HSCT to cure high-risk ALL [100, 101]. Notably, the NK-mediated GvL effect was distinguishable from GvHD, consistently caused by residual donor T cells. Thus, also in an allogeneic setting in vivo, NK cells eliminate leukemia blasts but spare normal allogeneic tissues with the remarkable exception of hematopoietic cells. A likely explanation for this selective effect is either the lack or low expression of ligands recognized by the activating NK receptors in normal, non-hematopoietic cells. This model explains also why alloreactive NK cells efficiently kill patient dendritic cells and residual T cells, thus preventing GvHD and graft rejection, respectively.

Because alloreactive NK cells play a key role for the successful clinical outcome of leukemia patients undergoing T cell-depleted haplo-HSCT, we described that phenotypic identification and determination of the size of this cell subset in potential donors can be relevant for the selection of the best possible donor. In addition, analysis of NK cytotoxicity could provide direct information on the efficacy of NK cells to kill leukemia blasts [102, 103]. In our view, the phenotypic assessment of alloreactive NK cell subsets is still the most reliable criteria for donor selection. Indeed, by combining suitable anti-KIR monoclonal antibodies (mAbs) in a two-color cytofluorimetric assays, it is possible to identify and define the size of alloreactive NK cell populations [103, 104]. While the original studies were focalized on the analysis, in donor NK cells, of inhibitory KIR specific for HLA class I alleles absent in patient cells, further studies outlined the importance of identifying relevant activating KIR. In this context, the recent availability of mAbs allowing discrimination between inhibitory and activating KIR resulted in an even more accurate definition of the alloreactive NK cell subset. Indeed, the identification of activating KIRs resulted important for the clinical outcome when donors expressed KIR2DS1 and the patient expressed HLA-C2 alleles, representing the ligand of such activating KIR [103, 105, 106]. As mentioned above, NK alloreactivity occurs only in a fraction (around 50 %) of donor/patient pairs. In an attempt to compensate, at least in part, the lack of alloreactivity, additional selection criteria were implemented. These additional criteria revealed to be crucial for a better clinical outcome of those donor/patient pairs with no donor alloreactive NK cells. Thus, KIR gene repertoire analysis aimed to detect the KIR B/X genotype and the relative B content value, i.e., criteria that are based on the presence of activating KIR genes, resulted in a useful tool and correlated with improvement of disease-free survival in adult patients with AML [107, 108]. Similar beneficial effect was established for KIR B/X genotype and high B content score in pediatric patients with high-risk ALL [109]. These results suggest that, in general, the expression of activating KIR plays a favorable role in preventing disease recurrence.

Finally, a retrospective analysis on children and young adults with acute leukemia given T cell-depleted HSCT intriguingly suggested that the mother was a better donor than the father [110], and therefore, for donor selection, also this criterion is routinely applied by our group. This important observation is in line with other studies reporting less GvHD and higher overall survival in patients transplanted from mother than father [111, 112]. Moreover, it has been reported that transplantations from haploidentical donors to a recipient mismatched for maternal HLA antigens are associated with less GvHD than transplantations from haploidentical siblings mismatching for paternal antigens [112]. Since during pregnancy bidirectional cell traffic between mother and fetus occurs [112], a possible explanation of these clinical results is that, in some instances, maternal immune system might become tolerant to the paternal antigens expressed by the fetus and the developing fetal immune system might acquire tolerance to non-inherited maternal antigen (NIMA). Notably, reciprocal microchimerism in both mother and offspring can persist for a long time, contributing to NIMA specific tolerance in the fetus and tolerance to paternal HLA antigens in the mother [111, 112].

Another recent important progress in haplo-HSCT regards the method of HSC selection. In the conventional haplo-HSCT based on the use of highly purified, positively selected, CD34⁺ cells, the late appearance (6–8 weeks after transplantation) of KIR⁺ mature NK cells may be critical. Indeed, high residual tumor burden and/or rapidly proliferating blasts may result in early leukemia relapses [101]. In addition, life-threatening infections may frequently occur early after transplantation. In an attempt to reduce these serious risks, a new protocol has been developed in which mature, KIR⁺ donor NK cells are given to the patient at transplantation, together with HSC. This approach is based on the removal of α/β TCR⁺ T cells and of CD19⁺ B cells [113, 114]. While α/β TCR⁺ T cell depletion prevents the risk of GvHD, removal of B cells highly reduces the risk of Epstein–barr virus (EBV)-related B cell lymphomas, frequently occurring in immune-compromised individuals. Importantly, by using this protocol, other relevant mononuclear cell types are infused into the patient. Among these cells, γ/δ TCR⁺ T cells appear of particular interest because they may potentially kill leukemia cells. Indeed, similarly to NK cells, they express DNAM-1 and NKG2D activating receptors that bind to ligands expressed on tumor cells. In addition, via their Vγ9/Vδ2 TCR⁺, T cells recognize phosphoantigens that may be expressed at the leukemia cell surface. These data strongly suggest that γ/δ TCR⁺ T cells may play a substantial role in leukemia cell killing [115, 116]. In this type of graft manipulation, the criteria for donor selection also include the higher absolute numbers of NK cells and γ/δ TCR⁺ T cells detectable in different potential donors. Importantly, preliminary data would indicate that this novel transplantation protocol is particularly effective in pediatric patients, not only for high-risk ALL, but also for AML with a substantial improvement of the survival probability. In addition, this protocol has been successfully applied to a number of life-threatening non-malignant disorders [116, 117].

Another highly promising therapeutic approach for exploiting the anti-leukemia activity of NK cells is based on the use of masking mAbs specific for KIR. These humanized mAbs are now in phase II clinical trials in patients with multiple myeloma or AML. Since they induce a stable blocking of KIRs, they confer alloreactivity to any KIR⁺ cells [92]. It is possible and even likely that infusion of autologous NK cells and anti-KIR mAbs may result in a further improvement in the cure of hematologic malignancies, particularly in donor/patient pairs with no alloreactive NK cells and in elderly patients not eligible for HSCT.

In conclusion, the discovery of activating and HLA class I-specific inhibitory NK receptors and the major advances in understanding NK cell biology, together with the exciting clinical results obtained with NK-based tumor therapies, underline how cells of the innate immunity may play a crucial role in the cure of otherwise lethal malignancies.

Abbreviations

ACT

Adoptive cell transfer

ADCC

Antibody-dependent cell-mediated cytotoxicity

ALL

Acute lymphoblastic leukemia

AML

Acute myeloid leukemia

BAT3/BAG6

HLA-B-associated transcript 3/BCL2-associated athanogene 6

BM

Bone marrow

CAR

Chimeric antigen receptors

DNAM-1

DNAX accessory molecule-1

EBV

Epstein–barr virus

ELS

Ectopic lymphoid structures

GvHD

Graft-versus-host disease

GvL

Graft-versus-leukemia

HCC

Hepatocellular carcinoma

HSC

Hematopoietic stem cell

HSCT

Hematopoietic stem cell transplant

HSP-90

Heat-shock protein 90

IDO

Indoleamine 2,3-dioxygenase

IFN-γ

Interferon-γ

IL

Interleukin

ILC

Innate lymphoid cells

KIRs

Killer cell Ig-like receptors

LAK

Lymphokine-activated killer

LSC

Leukemic stem cells

LTi

Lymphoid tissue inducer

mAbs

Monoclonal antibodies

MDSC

Myeloid-derived suppressor cells

MICA

MHC class I chain-related gene A

MIF

Migration inhibitory factor

MSC

Mesenchymal stem cells

MUC16

Mucin 16

NIMA

Non-inherited maternal antigen

NK

Natural killer

NKG2D

Natural killer group 2, member D

PB

Peripheral blood

PCNA

Proliferating cell nuclear antigen

PE-NK cells

Pleural effusions

PGE2

Prostaglandin E2

PS

Phosphatidylserine

SLOs

Secondary lymphoid organs

TAF

Tumor-associated fibroblasts

TAM

Tumor-associated macrophages

TGF-β

Transforming growth factor-β

TNF-α

Tumor necrosis factor-α

TRAIL

TNF-related apoptosis-inducing ligand

Tregs

Regulatory T cells

VPA

Sodium valproate

Compliance with ethical standards

Conflict of interest

All the authors have no conflict of interest to disclose.