Silvia Pesce

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization

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+ 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‐1bright) 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 CD56dim but not CD56bright NK cells and is confined to fully mature NK cells characterized by the NKG2A−KIR+CD57+ phenotype. Proportions of PD‐1bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. Conclusions: We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD‐1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody‐mediated disruption of PD‐1/programmed death ligand interaction.

Human NK Cells Induce Neutrophil Apoptosis via an NKp46- and Fas-Dependent Mechanism

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.

Features of Memory-Like and PD-1(+) Human NK Cell Subsets.

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.

TLR/NCR/KIR: Which One to Use and When?

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.

KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts

Natural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7(+) cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2-specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7(+) cells. Co-incubation of KIR2DS1(+) fresh NK cells or NK-cell clones with HLA-C2(+) CCR7(+) lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2(+) allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1(+) NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions.

NK/DC crosstalk in anti-viral response.

In recent years, it has been emphasized the role of the crosstalk between natural killer (NK) cells and monocyte-derived dendritic cells dendritic cells (moDCs) in the regulation of the early phases of innate immunity innate immunity and of the subsequent adaptive immune responses. NK cells and DCs coordinate their response communicating through direct cell-to-cell contact and soluble factors. NK cells appear to contribute to the quality control of immature DCs (iDCs) undergoing maturation. On the other hand, DCs may shape the magnitude of innate immune responses by modulating the NK-mediated cytolytic activity against tumors or infected cells. Recent studies suggest that the cooperation between NK cells and DCs is also critical in several anti-viral responses. In particular, NK cells are capable of effectively counteracting viral immune evasion immune evasion strategies by eliminating infected DCs, that display impaired antigen presenting functions, thus indirectly favoring the development of adaptive immune responses to viral antigens cross-presented by healthy DCs.

Role of alloreactive KIR2DS1+ NK cells in haploidentical hematopoietic stem cell transplantation

In allo‐HSCT, donor‐derived, ″alloreactive″ NK cells have been shown to play a crucial role in the treatment of acute leukemia, contributing to eradication of leukemic blasts (GvL effect) and to clearance of residual recipient DCs and T lymphocytes (thus, preventing GvHD and graft rejection, respectively). Such alloreactive NK cells do not express CD94/NKG2A but express inhibitory KIRs, specific for HLA class I allotypes, present in the donor but lacking in the recipient. This review is focused on the role of the activating KIR2DS1 receptor (specific for the C2‐epitope of HLA‐C) in haplo‐HSCT. Recent data indicate that KIR2DS1 expression in HSC donors may represent a remarkable advantage in alloreactive NK responses. This is a result of a substantial increase in the NK‐mediated capability to kill, not only recipientsˈ leukemic cells but also DCs and T cell blasts. The beneficial effects mediated by alloreactive KIR2DS1+ NK cells may occur after de novo expression of CCR7 upon interaction with allogeneic, KIR ligand‐mismatched CCR7+ cells. As a consequence, they can be redirected to LNs, where they can prevent priming of donor T cells and induction of GvHD. Finally, KIR2DS1 expression may also significantly amplify the size of the alloreactive NK cell subset by switching a subset of “not alloreactive” NK cells into potent alloreactive cells.

Bridging Innate NK Cell Functions with Adaptive Immunity

Killer Ig-like receptors (KIRs) are major human NK receptors displaying either inhibitory or activating functions which recognize allotypic determinants of HLA-class I molecules. Surprisingly, NK cell treatment with CpG-ODN (TLR9 ligands) results in selective down-modulation of KIR3DL2, its co-internalization with CpG-ODN and its translocation to TLR9-rich early endosomes. This novel KIR-associated function may offer clues to better understand the possible role of certain KIRs and also emphasizes the involvement of NK cells in the course of microbial infections. NK cells are involved not only in innate immune responses against viruses and tumors but also participate in the complex network of cell-to cell interaction that leads to the development of adaptive immune responses. In this context the interaction of NK cells with DC appears to play a crucial role in the acquisition of CCR7, a chemokine receptor that enables NK cells to migrate towards lymph nodes in response to CCL19 and/or CCL21. Analysis of NK cell clones revealed that KIR-mismatched but not KIR-matched NK cells acquire CCR7. These data have important implications in haploidentical haematopoietic stem cell transplantation (HSCT), in which KIR-mismatched NK cells may acquire the ability to migrate to secondary lymphoid compartments (SLCs), where they can kill recipients antigen presenting cells (APCs) and T cells thus preventing graft versus host (and host vs. graft) reactions.

TLR-Stimulated Neutrophils Instruct NK Cells To Trigger Dendritic Cell Maturation and Promote Adaptive T Cell Responses

Polymorphonuclear neutrophils (PMNs) are innate effector cells with pivotal roles in pathogen recognition, phagocytosis, and eradication. However, their role in the development of subsequent immune responses is incompletely understood. This study aimed to identify mechanisms of relevance to the cross talk between human neutrophils and NK cells and its potential role in promoting adaptive immunity. TLR-stimulated PMNs were found to release soluble mediators to attract and activate NK cells in vitro. PMN-conditioned NK cells displayed enhanced cytotoxicity and cytokine production, and responded vigorously to ensuing stimulation with exogenous and endogenous IL-12. The neutrophil-induced activation of NK cells was prevented by caspase-1 inhibitors and by natural antagonists to IL-1 and IL-18, suggesting a role for the NOD-like receptor family pyrin domain containing-3 inflammasome. In addition, PMN-conditioned NK cells triggered the maturation of monocyte-derived dendritic cells, which promoted T cell proliferation and IFN-γ production. These data imply that neutrophils attract NK cells to sites of infection to convert these cells into an active state, which drives adaptive immune responses via maturation of dendritic cells. Our results add to a growing body of evidence that suggests a sophisticated role for neutrophils in orchestrating the immune response to pathogens.

Uptake of CCR7 by KIR2DS4⁺ NK cells is induced upon recognition of certain HLA-C alleles.

The KIR2DS4 receptor is the oldest KIR2DS expressed by human NK lymphocytes. The specificity of recognition of this receptor for various HLA class I alleles has been demonstrated; however it remains poorly understood whether these interactions may result in the activation of some specific functions in NK cells. Here, we examined the functional outcome of the KIR2DS4/HLA class I interaction by the use of an alternative functional system based on the ability of KIR2DS4 to regulate the mechanism of trogocytosis by NK cells. We demonstrate that KIR2DS4 can induce the uptake of CCR7 by KIR2DS4+ NKG2A+ NK cell clones after interacting with CCR7+ target cells expressing HLA-Cw4 and HLA-Cw6 alleles. However this interaction is not always sufficient to override the inhibition generated by NKG2A expressed on the same NK cells. The recognition of HLA-Cw4 was confirmed by experiments of cytotoxicity against HLA-C-transfected cells. We also show that, different from resting NK cells, the acquisition of CCR7 in response to IL-18 cannot occur in IL2-activated NK cells because of a marked downregulation in their IL-18Rα expression. As a consequence trogocytosis represents the major mechanism by which KIR2DS4+ activated NK cells acquire the expression of this chemokine receptor.