Background: Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. Objective: We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. Methods: We performed multiparametric cytofluorimetric analysis of PD-1 1 NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. Results: We provide unequivocal evidence that PD-1 is highly expressed (PD-1 bright ) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56 dim but not CD56 bright NK cells and is confined to fully mature NK cells characterized by the NKG2A 2 KIR 1 CD57 1 phenotype. Proportions of PD-1 bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their

In this study the phenotype and function of tumor-associated NK cells from peritoneal fluids of a selected cohort of patients with seropapillary ovarian carcinoma were analyzed. In > 50% of these patients, the expression of the activating receptor NKp30 in tumor-associated NK cells was substantially reduced as compared to autologous peripheral blood (PB) NK cells. The impaired expression of this receptor was associated with the presence of one of its cellular ligands (B7-H6), which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the peritoneal fluid. NK cells from patients expressing this NKp30low phenotype displayed an impaired interferon-gamma (IFNγ) production and cytolytic function when tested against target cells expressing surface B7-H6. Our data also suggest that in these patients, the defective expression and function of NKp30 may be induced by the chronic engagement of this receptor by soluble B7-H6 or by tumor cells expressing this ligand. The impairment of NK cell functions described herein could represent a novel mechanism by which the tumor microenvironment may contribute to the escape from immune surveillance.

The identification of inhibitory NK cell receptors specific for HLA-I molecules (KIRs and NKG2A) provided the molecular basis for clarifying the mechanism by which NK cells kill transformed cells while sparing normal cells. The direct interactions between inhibitory NK cell receptors and their HLA-I ligands enable NK cells to distinguish healthy from transformed cells, which frequently show an altered expression of HLA-I molecules. Indeed, NK cells can kill cancer cells that have lost, or under express, HLA-I molecules, but not cells maintaining their expression. In this last case, it is possible to use anti-KIR or anti-NKG2A monoclonal antibodies to block the inhibitory signals generated by these receptors and to restore the anti-tumor NK cell activity. These treatments fall within the context of the new immunotherapeutic strategies known as “immune checkpoint blockade.” These antibodies are currently used in clinical trials in the treatment of both hematological and solid tumors. However, a more complex scenario has recently emerged. For example, NK cells can also express additional immune checkpoints, including PD-1, that was originally described on T lymphocytes, and whose ligands (PD-Ls) are usually overexpressed on tumor cells. Thus, it appears that the activation of NK cells and their potentially harmful effector functions are under the control of different immune checkpoints and their simultaneous expression could provide additional levels of suppression to anti-tumor NK cell responses. This review is focused on PD-1 immune checkpoint in NK cells, its potential role in immunosuppression, and the therapeutic strategies to recover NK cell cytotoxicity and anti-tumor effect.

Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell–cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.

IntroductionThe function of natural killer (NK) cells is regulated by a finely tuned balance of signals delivered by inhibitory and activating receptors. 1,2 The inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules can dampen NK-cell effector function upon appropriate ligation. These include killer immunoglobulin (Ig)-like receptors (KIRs) in humans, Ly49 in mouse, and CD94/NKG2A heterodimers in both species. [3][4][5][6][7][8] In humans, KIRs can harbor 2 (KIR2D) or 3 (KIR3D) extracellular C2-type Ig-like domains. KIRs recognize groups of MHC class I molecules that are determined by amino acid position in the C-terminal portion of the MHC class I ␣1 helix. 9 On the other hand, CD94/NKG2A recognizes the nonclassical MHC class I molecule human leukocyte antigen E (HLA-E) in humans 10 and Qa1 in mice. 11 The interactions between MHC class I molecules and their inhibitory receptors are thought to play a major role in the mechanisms of self-tolerance during NK-cell effector phases. Indeed, missing or low self-MHC expression results in NK-cell activation and induction of target cell lysis.Early stages of tissue damage, secondary to invasion of pathogen, including viruses, are associated with the recruitment and local activation of both dendritic cells (DCs) and NK cells. 12,13 The majority of peripheral blood NK cells are characterized by the CD56 dull CD16 ϩ KIR and/or NKG2A ϩ surface phenotype and express constitutively chemokine receptors such as CXCR1, CX3CR1, and ChemR23. On the other hand, the minor peripheral blood CD56 bright CD16 Ϫ KIR Ϫ NKG2A ϩ NK-cell subset expresses C-C chemokine receptor type 7 (CCR7). 14-17 Based on their chemokine receptor profile it is conceivable that CD56 dull CD16 ϩ cells are recruited mainly into inflamed peripheral tissues, whereas CD56 bright CD16 Ϫ cells are attracted to secondary lymphoid compartments (SLCs) such as lymph nodes, in response to C-C chemokine ligand (CCL)-19 and CCL21. 18 Accordingly, in inflamed peripheral tissues, NK cells are represented mostly by CD56 dull CD16 ϩ cells and express CXCR1 and ChemR23, 17 whereas within normal noninflamed lymph nodes, NK cells are homogeneously characterized by the CD56 bright CD16 dull/Ϫ KIR Ϫ NKG2A ϩ surface phenotype. 14,15,19,20 These NK cells are localized in the paracortical T-cell areas and are high interferon-gamma producers but display low cytolytic activity. Although these different pathways of NK-cell recruitment reflect the constitutive expression of different sets of chemokine receptors, another mode of NK-cell migration to lymph nodes has recently been described for the CD56 dull CD16 ϩ NK-cell subset. When exposed to exogenous interleukin-18 (IL-18) these cells de novo express surface CCR7 and respond to CCL19 and CCL21. 21 On the basis of these observations it has been proposed that CD56 dull CD16 ϩ NK cells, recruited into inflamed tissues (where proinflammatory cytokines/ chemokines favor their interaction with DCs), 12,[22][23][24] may exert an instructive activity on na...

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1+ NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1−, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56dim than in CD56bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.

By means of a complex receptor array, Natural killer (NK) cells can recognize variable patterns of ligands and regulate or amplify accordingly their effector functions. Such NK receptors include old, rather conserved, molecules, such as toll-like receptors (TLRs), which enable NK cells to respond both to viral and bacterial products, and newer and evolving molecules, such as killer Ig-like receptors and natural cytotoxicity receptors, which control NK cytotoxicity and are responsible for the elimination of virus-infected or tumor cells without damaging self-unaltered cells. In addition, to rapidly gain new functions NK cells also can acquire new receptors by trogocytosis. Thus, NK cells may have adapted their receptors to different functional needs making them able to play a key role in the modulation of critical events occurring in several compartments of human body (primarily in SLCs but also in decidua during pregnancy). In this review, we will discuss on how the various types of receptors can be used to address specific functions in different immunological contexts.

Human NK cells are distinguished into CD56brightCD16− cells and CD56dimCD16+ cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56bright NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56dim NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56dim cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A−KIR+LIR-1+CD57+CD56dim NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain “memory-like” NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The “memory-like” NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.