Role of the ITAM-Bearing Receptors Expressed by Natural Killer Cells in Cancer.

Natural Killer (NK) cells are innate lymphoid cells (ILCs) capable of recognizing and directly killing tumor cells. They also secrete cytokines and chemokines, which participate in the shaping of the adaptive response. NK cells identify tumor cells and are activated through a net positive signal from inhibitory and activating receptors. Several activating NK cell receptors are coupled to adaptor molecules containing an immunoreceptor tyrosine-based activation motif (ITAM). These receptors include CD16 and the natural cytotoxic receptors NKp46, NKp44, NKp30 in humans. The powerful antitumor NK cell response triggered by these activating receptors has made them attractive targets for exploitation in immunotherapy. In this review, we will discuss the different activating receptors associated with ITAM-bearing cell surface receptors expressed on NK cells, their modulations in the tumor context and the various therapeutic tools developed to boost NK cell responses in cancer patients.

Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response.

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

Phase I Trial of Prophylactic Donor-Derived IL-2-Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor.

Background: NK cell-based immunotherapy to prevent relapse after allogeneic transplantation is an appealing strategy because NK cells can provide strong antitumor effect without inducing graft-versus-host disease (GVHD). Thus, we designed a phase-I clinical trial evaluating the safety of a prophylactic donor-derived ex vivo IL-2 activated NK cell (IL-2 NK) infusion after allo-HSCT for patients with hematologic malignancies. Methods: Donor NK cells were purified and cultured ex vivo with IL-2 before infusion, at three dose levels. To identify the maximum tolerated dose was the main objective. In addition, we performed phenotypical and functional characterization of the NK cell therapy product, and longitudinal immune monitoring of NK cell phenotype in patients. Results: Compared to unstimulated NK cells, IL-2 NK cells expressed higher levels of activating receptors and exhibited increased degranulation and cytokine production in vitro. We treated 16 patients without observing any dose-limiting toxicity. At the last follow up, 11 out of 16 treated patients were alive in complete remission of hematologic malignancies without GVHD features and immunosuppressive treatment. Conclusions: Prophylactic donor-derived IL-2 NK cells after allo-HSCT is safe with low incidence of GVHD. Promising survivals and IL-2 NK cell activated phenotype may support a potential clinical efficacy of this strategy.

Helper-like Innate Lymphoid Cells in Humans and Mice.

The innate lymphoid cell (ILC) family consists of natural killer (NK) cells, helper-like lymphoid cells (ILC1s, ILC2s, and ILC3s), and lymphoid tissue inducer (LTi) cells. Helper-like ILCs are considered the innate counterpart of T-helper cells because of similarities in their cytokine output and expression of key transcription factors. ILCs provide and regulate innate immune functions before the development of adaptive immunity. They are involved in host defense against pathogens, inflammation, tissue repair, and metabolic homeostasis. However, they can also be involved in inflammatory disorders and carcinogenesis. In this review, we summarize the latest research on ILC development and plasticity in humans and mice, focusing on the pathogenic role of helper-like ILCs in inflammatory disorders, such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA).

ß2-adrenergic signals downregulate the innate immune response and reduce host resistance to viral infection.

In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the ß2-adrenergic receptor (ß2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a ß2-AR agonist were more susceptible to MCMV infection. By contrast, ß2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the ß2-AR signaling could be used to increase resistance to infectious diseases.

Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.

Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

Activating and inhibitory receptors expressed on innate lymphoid cells.

Innate lymphoid cells (ILCs) are innate immune cells located in lymphoid and non-lymphoid tissues. They are particularly abundant at mucosal and barrier surfaces. Three major ILC subsets are present in humans and mice: group 1 ILCs (comprising natural killer (NK) cells and ILC1s), ILC2s, and ILC3s. ILCs are involved in the maintenance of homeostasis and the regulation of immunity. This review focuses on the extensive array of activating and inhibitory receptors expressed by ILCs for communication with other cell types and their environment in health and disease.

Host resistance to endotoxic shock requires the neuroendocrine regulation of group 1 innate lymphoid cells.

Upon infection, the immune system produces inflammatory mediators important for pathogen clearance. However, inflammation can also have deleterious effect on the host and is tightly regulated. Immune system-derived cytokines stimulate the hypothalamic-pituitary-adrenal (HPA) axis, triggering endogenous glucocorticoid production. Through interaction with ubiquitously expressed glucocorticoid receptors (GRs), this steroid hormone has pleiotropic effects on many cell types. Using a genetic mouse model in which the gene encoding the GR is selectively deleted in NKp46+ innate lymphoid cells (ILCs), we demonstrated a major role for the HPA pathway in host resistance to endotoxin-induced septic shock. GR expression in group 1 ILCs is required to limit their IFN-γ production, thereby allowing the development of IL-10-dependent tolerance to endotoxin. These findings suggest that neuroendocrine axes are crucial for tolerization of the innate immune system to microbial endotoxin exposure through direct corticosterone-mediated effects on NKp46-expressing innate cells, revealing a novel strategy of host protection from immunopathology.

Complement factor P is a ligand for the natural killer cell-activating receptor NKp46.

Innate lymphoid cells (ILCs) are involved in immune responses to microbes and various stressed cells, such as tumor cells. They include group 1 [such as natural killer (NK) cells and ILC1], group 2, and group 3 ILCs. Besides their capacity to respond to cytokines, ILCs detect their targets through a series of cell surface-activating receptors recognizing microbial and nonmicrobial ligands. The nature of some of these ligands remains unclear, limiting our understanding of ILC biology. We focused on NKp46, which is highly conserved in mammals and expressed by all mature NK cells and subsets of ILC1 and ILC3. We show here that NKp46 binds to a soluble plasma glycoprotein, the complement factor P (CFP; properdin), the only known positive regulator of the alternative complement pathway. Consistent with the selective predisposition of patients lacking CFP to lethal Neisseria meningitidis (Nm) infections, NKp46 and group 1 ILCs bearing this receptor were found to be required for mice to survive Nm infection. Moreover, the beneficial effects of CFP treatment for Nm infection were dependent on NKp46 and group 1 NKp46+ ILCs. Thus, group 1 NKp46+ ILCs interact with the complement pathway, via NKp46, revealing a cross-talk between two partners of innate immunity in the response to an invasive bacterial infection.

Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.

Manufacturing Natural Killer Cells as Medicinal Products.

Natural Killer (NK) cells are innate lymphoid cells (ILC) with cytotoxic and regulatory properties. Their functions are tightly regulated by an array of inhibitory and activating receptors, and their mechanisms of activation strongly differ from antigen recognition in the context of human leukocyte antigen presentation as needed for T-cell activation. NK cells thus offer unique opportunities for new and improved therapeutic manipulation, either in vivo or in vitro, in a variety of human diseases, including cancers. NK cell activity can possibly be modulated in vivo through direct or indirect actions exerted by small molecules or monoclonal antibodies. NK cells can also be adoptively transferred following more or less substantial modifications through cell and gene manufacturing, in order to empower them with new or improved functions and ensure their controlled persistence and activity in the recipient. In the present review, we will focus on the technological and regulatory challenges of NK cell manufacturing and discuss conditions in which these innovative cellular therapies can be brought to the clinic.

Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30.

Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, binding to the receptor-binding site of the ligand. Here, we investigated the inhibitory mechanism of an antibody (17B1.3) against human B7-H6, a stress-induced cellular ligand for the natural killer (NK) cell receptor NKp30. Binding of this antibody to B7-H6, a transmembrane protein expressed on tumor and other stressed cells, but not on normal cells, prevents NK cell activation via NKp30. We determined the crystal structure of antibody 17B1.3 in complex with the ectodomain of B7-H6 to 2.5Å resolution. Surprisingly, 17B1.3 binds to a site on B7-H6 that is completely distinct from the binding site for NKp30, such that 17B1.3 does not block the NKp30-B7-H6 interaction. We then asked whether 17B1.3 prevents signaling by binding to a putative site for B7-H6 dimerization. However, structure-based mutations designed to disrupt potential B7-H6 dimerization through this site did not diminish NKp30-mediated cell activation. We conclude that the bulky 17B1.3 antibody most likely acts by sterically interfering with close cell-cell contacts at the NK cell-target cell interface that are required for NK cell activation. A similar inhibitory mechanism may apply to other antibodies, including therapeutic antibodies that block signaling through cell surface receptors whose ligands are also cell surface proteins.

Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.

Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

Impact of ß2 integrin deficiency on mouse natural killer cell development and function.

Natural killer (NK) cells are innate immune cells that express members of the leukocyte ß2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that ß2 integrin-deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit(+) cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, ß2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

Pattern of DAP12 expression in leukocytes from both healthy and systemic lupus erythematosus patients.

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls.

Expression of the CD85j (leukocyte Ig-like receptor 1, Ig-like transcript 2) receptor for class I major histocompatibility complex molecules in idiopathic inflammatory myopathies.

OBJECTIVE: Expression of class I major histocompatibility complex (MHC) molecules on the surface of muscle cells is a biologic feature of idiopathic inflammatory myopathies (IIM). Class I MHC-transgenic mouse models support a causative role for class I MHC expression by muscle cells in the pathogenesis of IIM. The muscle lesions are characterized by leukocyte infiltration. We undertook this study to analyze the expression in muscle lesions of various class I MHC-specific receptors on leukocytes and natural killer (NK) cells. METHODS: We generated a panel of cell transfectants to control the immunofluorescence analysis of class I MHC receptor expression. We then analyzed the expression of CD158 (killer cell Ig-like receptors [KIRs]) and CD85j (leukocyte Ig-like receptor 1, Ig-like transcript 2) on muscle sections prepared from 14 patients with IIM (5 with dermatomyositis [DM], 5 with polymyositis [PM], and 4 with sporadic inclusion body myositis [IBM]). RESULTS: We could not detect the presence of NK cells in inflammatory lesions. However, the class I MHC receptor CD85j, but no KIRs, was expressed by inflammatory cells infiltrating muscle lesions in IIM. CONCLUSION: CD85j is expressed in PM and sporadic IBM at the sites of partial invasion and in DM in perivascular inflammation, paving the way for dissecting the role of CD85j in the pathogenesis of inflammatory myopathies.

A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo.

Natural killer (NK) cells have been originally defined by their "naturally occurring" effector function. However, only a fraction of human NK cells is reactive toward a panel of prototypical tumor cell targets in vitro, both for the production of interferon-gamma (IFN-gamma) and for their cytotoxic response. In patients with IL12RB1 mutations that lead to a complete IL-12Rbeta1 deficiency, the size of this naturally reactive NK cell subset is diminished, in particular for the IFN-gamma production. Similar data were obtained from a patient with a complete deficit in IL-12p40. In addition, the size of the subset of effector memory T cells expressing CD56 was severely decreased in IL-12Rbeta1- and IL-12p40-deficient patients. Human NK cells thus require in vivo priming with IL-12/23 to acquire their full spectrum of functional reactivity, while T cells are dependent upon IL-12/23 signals for the differentiation and/or the maintenance of CD56(+) effector memory T cells. The susceptibility of IL-12/23 axis-deficient patients to Mycobacterium and Salmonella infections in combination with the absence of mycobacteriosis or salmonellosis in the rare cases of human NK cell deficiencies point to a role for CD56(+) T cells in the control of these infections in humans.

Human NK cell education by inhibitory receptors for MHC class I.

Natural killer (NK) cells recognize the absence of self MHC class I as a way to discriminate normal cells from cells in distress. In humans, this "missing self" recognition is ensured by inhibitory receptors such as KIR, which dampen NK cell activation upon interaction with their MHC class I ligands. We show here that NK cells lacking inhibitory KIR for self MHC class I molecules are present in human peripheral blood. These cells harbor a mature NK cell phenotype but are hyporesponsive to various stimuli, including MHC class I-deficient target cells. This response is in contrast to NK cells that express a single inhibitory KIR specific for self MHC class I, which are functionally competent when exposed to the same stimuli. These results show the involvement of KIR-MHC class I interactions in the calibration of NK cell effector capacities, suggesting its role in the subsequent "missing self" recognition.

Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/DAP12-transgenic mice.

KARAP/DAP12 is a broadly distributed transmembrane signaling polypeptide with an immunoreceptor tyrosine-based activation motif, and is non-covalently associated with a variety of activating surface receptors. We report here the characterization of transgenic mice that overexpress KARAP/DAP12 polypeptides in both myeloid and lymphoid compartments. KARAP/DAP12-transgenic mice present, in a transgene dose-dependent manner, a complex phenotype characterized by two independent and spontaneous hematological abnormalities: (i) a severe lymphopenia and (ii) a massive inflammatory syndrome associated with neutrophilia and lung infiltration by multinucleated macrophages. These myeloid abnormalities observed in KARAP/DAP12-transgenic mice indicate that KARAP/DAP12-driven signals are critically involved in inflammation, and constitute an essential target to control the resolution of inflammatory disorders based on monocytes/macrophages and neutrophils.