Simona Carlomagno

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A, but lacking inhibitory killer Ig-like receptors

The cognate NK–DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte‐derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig‐like receptors (KIR). Consistent with these data, the expression of HLA‐E (i.e. the cellular ligand of CD94/NKG2A) was down‐regulated in iDC. On the other hand, HLA‐B and HLA‐C down‐regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A+KIR– NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA‐E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA‐E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.

The small subset of CD56brightCD16- natural killer cells is selectively responsible for both cell proliferation and interferon-γ production upon interaction with dendritic cells

The encounter of NK cells with dendritic cells (DC) undergoing maturation may result in the induction of NK cell proliferation. Whether such proliferation involves most NK cells or just a subset has yet to be determined. In the present study we analyzed the nature of such proliferating NK cells by combining carboxyfluorescein succinimidyl ester staining and double‐fluorescence cytofluorimetric analysis. Freshly isolated peripheral blood NK cells cultured with LPS and immature DC underwent proliferation; however, proliferating cells were confined to a minor NK cell subset. This subset is characterized by the CD56brightCD16–NKG2A+KIR– surface phenotype (KIR, killer Ig‐like receptor). This was further confirmed by the fact that, after cell sorting, only the CD56bright NK cells were able to proliferate in response to the DC stimulus, whereas the CD56dull were not. We also provide evidence that the CD56bright subset is the main source of IFN‐γ‐producing NK cells, upon interaction with DC. The CD56brightCD16– NK cells express a panel of surface molecules including CD62L, CCR7 and CXCR3 that may allow their homing either to secondary lymphoid compartments or to inflamed tissues. This implies that, in vivo, the interactions between DC undergoing maturation and CD56bright NK cells may occur in different tissues and have different functional implications.

Natural killer cells expressing the KIR2DS1-activating receptor efficiently kill T-cell blasts and dendritic cells: implications in haploidentical HSCT

In allogeneic HSCT, NK-cell alloreactivity is determined by the presence in the donor of NK cells expressing inhibitory killer cell Ig-like receptors (KIRs) that recognize HLA class I allotypes present in the donor but lacking in the recipient. Dominant KIR ligands are the C1 and C2 epitopes of HLA-C. All HLA-C allotypes have either the C1 epitope, the ligand for KIR2DL2/L3, or the C2 epitope, the ligand for KIR2DL1/S1. Here, we show that, in alloreactive NK-cell responses, KIR2DS1 expression represents a remarkable advantage as it allows efficient killing of C2/C2 or C1/C2 myelomonocitic dendritic cells (DCs) and T-cell blasts. When DCs or T-cell blasts were derived from C2/C2, Bw4/Bw4 donors, the activating signals delivered by KIR2DS1 could override the inhibition generated by NKG2A or KIR2DL2/L3 expressed on the same NK-cell clone. Furthermore, substantial lysis of C2/C2, Bw4/Bw6 targets was mediated by KIR2DS1(+) NK cells coexpressing KIR3DL1. Importantly, in the case of C1/C2 targets, KIR2DS1(+) NK cells were inhibited by the coexpression of KIR2DL2/L3 but not of NKG2A. Thus, KIR2DS1 expression in HSC donors may substantially increase the size of the alloreactive NK-cell subset leading to an enhanced ability to limit GVHD and improve engrafment.

Comparison of different CpG oligodeoxynucleotide classes for their capability to stimulate human NK cells.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG ODN) mimic the immunostimulatory activity of microbial DNA via Toll‐like receptor (TLR)9. Previous studies indicated that human NK cells express functional TLR3 and TLR9, since their cytokine release and cytolytic function could be incremented by poly(I:C) or ODN A/B, respectively. We have now evaluated the capability of a novel class of CpG ODN, termed ODN C, to modulate the function of human NK cells in the presence of exogenous cytokines. We show that NK cells isolated from peripheral blood and cultured with ODN C, in the presence of either IL‐12 or IL‐8, express higher levels of CD69 as compared to those stimulated with either ODN A or ODN B. Moreover, NK cells cultured with ODN C displayed higher cytolytic activity against tumor cell lines. These effects were not confined to freshly isolated peripheral blood NK cells since polyclonal NK cell populations that had been cultured in the presence of exogenous IL‐2 for several weeks also displayed higher cytolytic activity and cytokine release after culture in the presence of ODN C. Remarkably, NK cells displaying poor responses to ODN A/B were efficiently stimulated by ODN C.

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.