Inhibitory checkpoints in human natural killer cells: IUPHAR Review 28.

Immune checkpoint inhibitors have revolutionized cancer therapy leading to exceptional success. However, there is still the need to improve their efficacy in non-responder patients. Natural killer (NK) cells represent the first line of defence against tumours, due to their ability to release immunomodulatory cytokines and kill target cells that have undergone malignant transformation. Harnessing NK cell response will open new possibilities to improve control of tumour growth. In this respect inhibitory checkpoints expressed on these innate lymphocytes represents a promising target for next-generation immunotherapy. In this review, we will summarize recent evidences on the expression of NK cells receptors in cancer, with a focus on the inhibitory checkpoint programmed cell death protein 1 (PD-1). We will also highlight the strength and limitations of the blockade of PD-1 inhibitory pathway and suggest new combination strategies that may help to unleash more efficiently NK cell anti-tumour response.

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

IL15 induces a potent antitumor activity in NK cells isolated from malignant pleural effusions and overcomes the inhibitory effect of pleural fluid.

Natural Killer (NK) cells are capable of recognizing and killing cancer cells and play an important role in tumor immunosurveillance. However, tumor-infiltrating NK cells are frequently impaired in their functional capability. A remarkable exception is represented by NK cells isolated from malignant pleural effusions (PE) that are not anergic and, upon IL2-induced activation, efficiently kill tumor cells. Although IL2 is used in various clinical trials, severe side effects may occur in treated patients. In this study, we investigated whether also other clinical-grade cytokines could induce strong cytotoxicity in NK cells isolated from pleural fluid of patients with primary or metastatic tumors of different origins. We show that PE-NK cells, cultured for short-time intervals with IL15, maintain the CD56bright phenotype, a high expression of the main activating receptors, produce cytokines and kill tumor cells in vitro similarly to those treated with IL2. Moreover, IL15-activated PE-NK cells could greatly reduce the growth of established tumors in mice. This in vivo antitumor effect correlated with the ability of IL15-activated PE-NK cells to traffic from periphery to the tumor site. Finally, we show that IL15 can counteract the inhibitory effect of the tumor pleural microenvironment. Our study suggests that IL15-activated NK cells isolated from pleural fluid (otherwise discarded after thoracentesis) may represent a suitable source of effector cells to be used in adoptive immunotherapy of cancer.

Zoledronic acid boosts γδ T-cell activity in children receiving αß+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αß+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αß T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 µM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.

The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance.

The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), acts as a sensor of oxidative or electrophilic stresses and plays a pivotal role in redox homeostasis. Oxidative or electrophilic agents cause a conformational change in the Nrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which, through its binding to the antioxidant/electrophilic response element (ARE/EpRE), regulates the expression of antioxidant and detoxifying genes such as heme oxygenase 1 (HO-1). Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumour progression, aggressiveness, resistance to therapy, and poor prognosis. This review focuses on the Nrf2/HO-1 stress response mechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies.

Pseudorabies Virus US3 Protein Kinase Protects Infected Cells from NK Cell-Mediated Lysis via Increased Binding of the Inhibitory NK Cell Receptor CD300a.

UNLABELLED: Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. IMPORTANCE: Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this novel NK cell evasion strategy may contribute to the design of improved herpesvirus vaccines and may also have significance for other PAK- and CD300a-modulating viruses and cancer cells.

Identification of diverse innate lymphoid cells in human decidua.

Innate lymphoid cells (ILCs) are developmentally related cells that play an important role in innate defenses and tissue remodeling. So far, only natural killer (NK) cells have been identified and functionally characterized in human decidua where they contribute to induction of immune suppression, neo-angiogenesis, and tissue building/remodeling. The presence of other ILC subsets in human decidua has not been yet characterized. Here we identify in human decidua, during early pregnancy, two subsets of decidual group 3 ILC (ILC3), including lymphoid tissue inducer (LTi)-like cells and natural cytotoxicity receptors (NCRs)(+)ILC3 and interferon-(IFN)γ-producing ILC1, different from NK cells. Decidual LTi-like cells produced interleukin -17 (IL-17) and tumor necrosis factor (TNF), while NCR(+)ILC3 released IL-22 and IL-8. Importantly, NCR(+)ILC3 and LTi-like cells established functional interactions with stromal cells. Decidual LTi-like cells differentiated into NCR(+)ILC3, whereas they marginally contributed to NK cell generation. Our data suggest that decidual ILC3 may play a role in innate defenses and in vessel and tissue building, thus contributing to maintenance of pregnancy.

KIR and KIR ligand polymorphism: a new area for clinical applications?

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer (NK) activity, allowing NK cells to sense and respond to human leukocyte antigen (HLA) class I downregulation, an important hallmark for viral infections and tumor transformation. KIR and HLA genes are located on different chromosomes and KIR/HLA class I interaction represents an example of genetic epistasis in which the presence of receptor/ligand pairs is necessary for the induction of functional activity, while the presence of one in the absence of the other is not sufficient to influence NK cell function. Due to the high degree of HLA class I and KIR gene variability, KIR/KIR-ligand (KIR-L) interactions are extraordinarily diverse. KIR polymorphism arises from both haplotypic and allelic variations and was shaped by natural selection. KIR variability affects NK cell education influencing the KIR repertoire, KIR expression, the strength of KIR/KIR-L interactions and the capability to deliver signals. Moreover, it may influence NK cell function during infections, autoimmune diseases, pregnancy and allogeneic transplantation. This review summarizes the genetic and functional features of KIR/KIR-L interactions and gives an overview of their potential relevance in clinical studies.

Natural killer alloeffector responses in haploidentical hemopoietic stem cell transplantation to treat high-risk leukemias.

Natural killer (NK) cells, a major cell type of the innate immunity, express surface receptors that regulate potent effector functions such as cytolytic activity and release of cytokines playing a central role in inflammatory response and immunoregulation. In this contribution, we briefly outline the major steps from the discovery of human leukocyte antigen (HLA)-class I-specific inhibitory receptors in humans to recent successful clinical applications in the cure of high-risk leukemias both in adults and in pediatric patients. A central role is played by 'alloreactive' NK cells originated from donor's CD 34(+) cells in eradicating leukemic cells in the setting of T-cell-depleted haploidentical hemopoietic stem cell transplantation. Because alloreactive NK cells play a central role also in preventing graft rejection and graft-vs-host disease, they may represent an ideal tool to treat patients affected by acute high-risk leukemias.

Activating and inhibitory killer immunoglobulin-like receptors (KIR) in haploidentical haemopoietic stem cell transplantation to cure high-risk leukaemias.

A number of experimental studies have shown that natural killer (NK) cells can eliminate cancer cells and the mechanisms involved in this effect have been uncovered during the last two decades. Clinical data from haploidentical haematopoietic stem cell transplantation (haplo-HSCT) revealed that NK cells were responsible for remarkably favourable effects in both adult and paediatric high-risk leukaemias. NK receptors specific for major histocompatibility complex (MHC) class I molecules, including killer immunoglobulin (Ig)-like receptors (KIR) and CD94/NKG2A, play a major role in the anti-leukaemia effect (mediating either inhibitory or activating signals). Haplo- HSCT requires a heavy conditioning regimen for the patient and the use of large numbers of T cell-depleted HSC to be grafted. After transplantation, natural killer cells develop from HSC shortly after engraftment and may include 'alloreactive' NK cells that kill leukaemic cells and prevent graft-versus-host disease (GvHD). Alloreactive NK cells are characterized by the expression of KIR that are not engaged by any of the human leucocyte antigen (HLA) class I alleles expressed by the patient. Their generation is dependent upon the existence of a KIR/HLA class I mismatch between donor and recipient. Novel important information on the function and specificity of different KIR has been obtained recently by the analysis of donor-derived alloreactive NK cells in a cohort of paediatric patients given haplo-HSCT to cure acute, high-risk leukaemias.

Alloreactive natural killer cells in targeting high-risk leukaemias.

Natural killer (NK) cells express surface receptors that regulate potent effector functions, such as cytolytic activity and release of cytokines, which play a central role in inflammatory response and immunoregulation. In this paper, major advances are outlined from the original discovery of HLA-class I-specific inhibitory receptors in humans to recent, particularly successful, clinical applications in the cure of high-risk, otherwise fatal leukaemias. The central role of donor-derived "alloreactive" NK cells in eradicating leukaemic cells in the T-cell-depleted haploidentical haematopoietic stem cell transplantation setting became evident. Since alloreactive NK cells seem to play a key role also in preventing graft rejection and graft-vs-host disease, they may be an ideal tool to treat high-risk leukaemias in the haematopoietic stem cell transplantation setting.

Small round blue cell tumours: diagnostic and prognostic usefulness of the expression of B7-H3 surface molecule.

AIMS: To assess whether the expression of B7-H3 surface molecule could improve differential diagnosis of small cell round tumours. METHODS AND RESULTS: One hundred and one well-characterized paraffin-embedded small round cell tumours, stored in the pathology archive of the Gaslini Institute, were immunohistochemically analysed with the 5B14 monoclonal antibody, which recognizes the surface molecule B7-H3. All lymphoblastic lymphomas and the blastematous component of Wilms' tumours were completely negative and a few Ewing's sarcoma and Burkitt's lymphoma specimens showed focal positivity, whereas 74% of neuroblastomas, 67% of rhabdomyosarcomas and 100% of medulloblastomas were positive. The pattern of immunoreactivity of 5B14 mAb observed in rhabdomyosarcoma, neuroblastoma and medulloblastoma specimens was limited to the cytoplasmic membrane, and in neuroblastomas areas of rosette formation or of ganglion differentiation were preferentially stained. Interestingly, in neuroblastoma patients high expression of the antigen recognized by the 5B14 mAb was associated with a worse event-free survival. CONCLUSIONS: The 5B14 mAb represents an additional tool for the differential diagnosis of small round cell tumours and might be useful in identifying neuroblastoma patients at risk of relapse who may take advantage of more careful follow-up.

NK cells at the interface between innate and adaptive immunity.

In recent years a novel concept has emerged indicating that the actual role of natural killer (NK) cells is not confined to the destruction of virus-infected cells or tumors. Indeed, different NK subsets exist that display major functional differences in their cytolytic activity, cytokine production and homing capabilities. In particular, CD56(high) CD16(-) NK cells that largely predominate in lymph nodes, have little cytolytic activity but release high levels of cytokines whereas CD56(low) CD16(+) NK cells that predominate in peripheral blood and inflamed tissues, display lower cytokine production, but potent cytotoxicity. The latter is characterized by granule polarization and exocytosis of various proteins including perforin and granzymes that mediate target cell killing. The recruitment of CD56(low) CD16(+) NK cells into inflamed peripheral tissues is orchestrated by various chemochines including the newly identified Chemerin. At these sites, NK cells, upon engagement of different triggering receptors become activated and upregulate their cytokine production and cytotoxicity after interaction with myeloid dendritic cells (DCs). Importantly, during this interaction NK cells also mediate the 'editing' of DCs undergoing maturation. This process appears to play a crucial role in shaping both innate and adaptive immune responses. Indeed, only DCs undergoing this NK-mediated quality control would become fully mature and capable of inducing priming of protective Th1 responses.

Purification and HPLC-MS analysis of a naturally processed HCMV-derived peptide isolated from the HEK-293T/HLA-E+/Ul40+ cell transfectants and presented at the cell surface in the context of HLA-E.

A new method for isolation and characterization of peptides presented in the context of the nonclassical human leukocytes antigen (HLA) class I molecule HLA-E was developed. A combination of different chromatographic steps coupled with electrospray mass spectrometry allowed us to detect the presence of small amounts of a naturally processed human Cytomegalovirus (HCMV)-derived peptide isolated from the HEK-293T/HLA-E+/UL40+ transfected cells of from HELA cell line. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). This approach provides a versatile and sensitive method for direct identification of MHC class I-binding peptides that might be derive from different pathogen or tumor-associated proteins.

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C-->T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

NK cell activating receptors and tumor recognition in humans.

Natural killer (NK) cells have been known for many years as the lymphocyte subset characterized by the highest cytolytic potential against virus-infected and tumor-transformed cells. A surprisingly high number of surface molecules have been recognized that regulate human NK cell function. These include MHC-specific inhibitory receptors, which impair NK cells' ability to attack normal self-tissues, and activating receptors and coreceptors that allow them to recognize and kill transformed cells. The recent identification of some of the cellular ligands specifically recognized by these receptors/coreceptors contributes to elucidation of the mystery of the role played by NK cells in immune responses.

Surface receptors and functional interactions of human natural killer cells: from bench to the clinic.

The past 10years have witnessed dramatic progress in our understanding of how natural killer (NK) cells function and their role in innate immunity. Thanks to an array of inhibitory receptors specific for different HLA class I molecules, human NK cells can sense the decrease or loss of even single alleles at the cell surface. This represents a typical condition of a potential danger, i.e. the presence of tumor or virally infected cells. NK cell triggering and lysis of these cells is mediated by several activating receptors and coreceptors that have recently been identified and cloned. While normal cells are usually resistant to NK-mediated attack, a remarkable exception is represented by dendritic cells (DCs). In their immature form they are susceptible to NK-mediated lysis because of the expression of low levels of surface HLA class I molecules. The process of DC maturation (mDCs) is characterized by the surface expression of high levels of HLA class I molecules. Accordingly, mDCs become resistant to NK cells. A recent major breakthrough highlighted the role played by donor NK cells in allogenic bone marrow transplantation to cure acute myeloid leukemias. 'Alloreactive' NK cells derived from donor hematopoietic precursors not only prevented leukemic relapses, but also prevented graft rejection and graft-versus-host disease.

Human natural killer cell receptors: insights into their molecular function and structure.

NK cells express receptors characterized by opposite functions that finely regulate their activities. Among inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are still orphan receptors. On the contrary, various activating receptors are involved in the triggering of NK-mediated natural cytotoxicity. In general, their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Thus, the process of NK cell triggering mediated by Natural Cytotoxicity Receptors can be mainly considered as a non MHC-restricted mechanism. Here, a brief description of the molecular nature of these receptors, as well as, of their 3D-structures and of the implications for ligand recognition, is given.