Biomedical subjects

Massimo Vitale

Publications and source records attributed to Massimo Vitale.

15 recordsLinked to original sources

PubMed2006

The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function.

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell-mediated immune responses. In this study, we also demonstrate that natural killer (NK)-cell function can be influenced by IDO. Indeed, l-kynurenine, a Trp-derived catabolite resulting from IDO activity, was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell-mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D, while it does not affect other surface receptors such as NKp30 or CD16. As a consequence, l-kynurenine-treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead, they maintain the ability to kill targets, such as dendritic cells (DCs), that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine, which is effective at both the transcriptional and the protein level, can be reverted, since NK cells were found to recover their functional competence after washing.

Antigens, CD↗

PubMed2006

Effector and regulatory events during natural killer-dendritic cell interactions.

The different cell types of the innate immune system can interact with each other and influence the quality and strength of an immune response. The cross talk between natural killer (NK) cells and myeloid dendritic cells (DCs) leads to NK cell activation and DC maturation. Activated NK cells are capable of killing DCs that fail to undergo proper maturation ('DC editing'). Encounters between NK cells and DCs occur in both inflamed peripheral tissues and lymph nodes, where both cell types are recruited by chemokines released in the early phases of inflammatory responses. Different NK cell subsets (CD56(bright)CD16(-) versus CD56(+)CD16(+)) differ in their homing capabilities. In particular, CD56(bright)CD16(-) NK cells largely predominate the lymph nodes. In addition, these two subsets display major functional differences in their cytolytic activity, cytokine production, and ability to undergo proliferation. NK cell functions are also greatly influenced by the presence of polarizing cytokines such as interleukin (IL)-12 and IL-4. The cytokine microenvironment reflects the presence of different cell types that secrete such cytokines in response to microbial products acting on different Toll-like receptors (TLRs). Moreover, NK cells themselves can respond directly to microbial products by means of TLR3 and TLR9. Thus, it appears that the final outcome of a response to microbial infection may greatly vary as a result of the interactions occurring between different pathogen-derived products and different cell types of the innate immunity system. These interactions also determine the quality and strength of the subsequent adaptive responses. Remarkably, NK cells appear to play a key role in this complex network.

Animals↗

PubMed2005

Early liaisons between cells of the innate immune system in inflamed peripheral tissues.

The crosstalk between natural killer (NK) cells and myeloid dendritic cells (DCs) results in NK-cell activation and DC maturation. Activated NK cells acquire the ability to kill DCs that have failed to undergo complete maturation ('DC editing'). Recent studies have revealed that this crosstalk can be promoted by pathogen-derived products that activate different innate immune cell types directly and simultaneously through their Toll-like receptors (TLRs). These cells include NK cells and DCs, as well as plasmacytoid DCs (PDCs) and mast cells. This crosstalk can have a great impact on the quality and strength of the subsequent adaptive immune response. Thus, NK cells have an important role in the defense against pathogens, acting as regulatory cells as well as effector cells.

Cytokines↗

PubMed2005

Human natural killer cells: Molecular mechanisms controlling NK cell activation and tumor cell lysis.

Natural killer cells represent a highly specialized lymphoid population with a potent cytolytic activity against virus-infected or tumor cells. Their function is regulated by a series of inhibiting or activating signals. The mechanisms by which NK cells kill susceptible target cells was thought to be elucidated after the discovery of inhibitory receptors specific for MHC-class I molecules: NK cells would kill those target cells that lack MHC-class I molecules. However, the actual scenario revealed more complex with the discovery of activating receptors and their ligands. Thus, in certain pathological conditions, corticosteroid treatment or exposure to TGFbeta, NK cells may under-express activating receptors. In addition, target cells may lack ligands for activating receptors and thus fail to activate NK cells upon cell-to-cell contact. This clearly implies that activation of NK cells and of their potent effector mechanism are under the control of different checkpoints.

Animals↗

PubMed2005

NK-dependent DC maturation is mediated by TNFalpha and IFNgamma released upon engagement of the NKp30 triggering receptor.

Natural killer (NK) cells were recently shown to play a relevant role in the process of dendritic cell (DC) maturation. This function is exerted either by direct DC stimulation or through killing those DCs that did not properly acquire a mature phenotype. While killing of immature DCs is dependent on the function of the NKp30 triggering receptor, the mechanism by which NK cells induce DC maturation is still unclear. In this study, we show that also the NK-mediated induction of DC maturation is dependent on NKp30. Upon NK/DC interaction, resulting in NKp30 engagement, NK cells produced tumor necrosis factor alpha (TNFalpha) (and interferon gamma [IFNgamma]) that, in turn, promoted DC maturation. Masking of NKp30 with specific monoclonal antibodies (mAbs) strongly reduced maturation of DCs cocultured with NK cells. In addition, supernatant from NK cells stimulated via NKp30 induced DC maturation, and this effect was neutralized by anti-TNFalpha antibodies (Abs). This NKp30 function is controlled by the HLA-specific inhibitory NK receptors. Accordingly, the ability to promote maturation was essentially confined to NK cells expressing the killer immunoglobulin-like receptor-negative (KIR-) NKG2A(dull) phenotype. Finally, the analysis of perforin-deficient NK cells allowed the dissection of the 2 NKp30-mediated NK-cell functions, since NKp30 could induce cytokine-dependent DC maturation in the absence of NK-mediated DC killing.

Cell Differentiation↗

PubMed2004

Isolation of a novel KIR2DL3-specific mAb: comparative analysis of the surface distribution and function of KIR2DL2, KIR2DL3 and KIR2DS2.

In recent years an increasing number of sequences coding for new KIRs have been described. However, the limited availability of mAbs with unique KIR specificities has hindered an exhaustive assessment of their actual function, HLA-specificity, expression at the cell surface and distribution in different cell populations. In this study we report the generation of a novel mAb (ECM41) specific for KIR2DL3 molecules. By the use of cell transfectants expressing one or other KIR we show that this reagent allows discrimination of KIR2DL3 from other GL183 mAb-reactive molecules such as KIR2DL2 and KIR2DS2. Moreover we show that this novel mAb can be used to assess the surface expression and distribution of KIR2DL3 in different polyclonal NK populations and in NK cell clones. Along this line, we were able to analyze the HLA class I specificity of NK clones expressing either KIR2DL3 or KIR2DL2, two inhibitory receptors that were so far serologically undistinguishable. Finally, the combined use of GL183 and ECM41 mAbs in redirected killing assays allowed us to investigate the functional outcome of the simultaneous engagement of KIR2DL3 and KIR2DS2 in NK cell clones co-expressing KIRs that display opposite (inhibitory vs activating) function.

Antibodies, Monoclonal↗

PubMed2004

CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells.

Toll-like receptors (TLRs) are pattern-recognition receptors responsible for triggering cells of innate immunity. In this study we investigated the expression and function of TLRs 3 and 9 in human natural killer (NK) cells. In the presence of IL-12, freshly isolated NK cells responded to double-stranded RNA or unmethylated CpG DNA and expressed CD69 and CD25 activation markers. Because both markers were expressed by virtually all NK cells, this would suggest that most of them can be triggered by TLRs. Remarkably, NK cell stimulation also resulted in the induction of their functional program as revealed by IFN-gamma and tumor necrosis factor-alpha release and by up-regulation of cytolytic activity against tumor cells. IL-8 could efficiently substitute IL-12 in supporting NK cell responses to TLR-mediated stimulation. Importantly, freshly isolated NK cells acquired the ability to lyse immature dendritic cells after stimulation with double-stranded RNA and IL-12. However, responses to these stimuli were not restricted to fresh NK cells, because significant responses were also detected in polyclonal NK cells cultured in the presence of exogenous IL-2 for several weeks. The analysis of NK cell clones revealed some degree of heterogeneity in the ability to respond to TLR stimulation also among NK clones derived from a single donor. These data suggest that stimuli acting on TLR not only activate immature dendritic cells to release IL-12 but also render NK cells capable of receiving triggering signals from pathogen-associated molecules, thus exerting a regulatory control on the early steps of innate immune responses against infectious agents.

Cells, Cultured↗

PubMed2004

The small subset of CD56brightCD16- natural killer cells is selectively responsible for both cell proliferation and interferon-gamma 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 CD56(bright)CD16(-)NKG2A(+)KIR(-) surface phenotype (KIR, killer Ig-like receptor). This was further confirmed by the fact that, after cell sorting, only the CD56(bright) NK cells were able to proliferate in response to the DC stimulus, whereas the CD56(dull) were not. We also provide evidence that the CD56(bright) subset is the main source of IFN-gamma-producing NK cells, upon interaction with DC. The CD56(bright)CD16(-) 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 CD56(bright) NK cells may occur in different tissues and have different functional implications.

CD56 Antigen↗

PubMed2004

Different checkpoints in human NK-cell activation.

After the discovery, in humans and mice, of inhibitory natural killer (NK) receptors specific for MHC class I molecules, the mechanism by which NK cells kill tumor or virus-infected cells was thought to be clarified: NK cells would kill those target cells that have lost, or underexpress, MHC class I molecules. However, a more complex scenario has recently emerged. For example, certain NK cells express insufficient amounts of triggering receptors, and target cells can lack ligands for such receptors. Thus, it appears that the activation of NK cells and their potentially harmful effector functions are under the control of different checkpoints.

Animals↗

PubMed2004

Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells.

Human Natural Killer cells express a large array of surface receptors that are involved in the regulation of the different NK cell functions. Some of these receptors, by sensing the expression of HLA class I on potential target cells, deliver negative signals that inhibit the NK-mediated killing. In the absence of such inhibitory interactions, other receptors are allowed to transduce activating signals that result in NK-mediated attack of tumor- or virus-transformed cells. The function of these main activating receptors is supported and enhanced by the simultaneous engagement of different co-receptors. Finally, the ability of NK cells to interact with normal, non-transformed other cells such as dendritic cells (DC) appears to crucially contribute to the regulation of both innate and adaptive immune responses.

Histocompatibility Antigens Class I↗

PubMed2003

Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of approximately 70 kD. The other mAb reacted with two distinct molecules of approximately 65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 delta/alpha (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1-dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants).

Amino Acid Sequence↗

PubMed2003

Expression and function of KIR and natural cytotoxicity receptors in NK-type lymphoproliferative diseases of granular lymphocytes.

Using monoclonal antibodies (mAbs) specific for different natural killer (NK) receptors, we studied the lymphocyte population from 18 patients with NK-type lymphoproliferative disease of granular lymphocytes (LDGL). The analysis of both resting and cultured NK cell populations demonstrated that these patients are frequently characterized by NK cells displaying a homogeneous staining with given anti-killer Ig-like receptor (anti-KIR) mAb (11 of 18 patients). In most patients NK cells were characterized by the CD94/NKG2A+ phenotype, whereas only a minor fraction of the cases expressed CD94/NKG2C. In 7 of these patients we could also assess the function of the various NK receptors. Remarkably those KIR molecules that, in each patient, homogeneously marked the NK cell expansion were found to display an activating function as determined by cross-linking with specific anti-KIR mAb. The KIR genotype analysis performed in 13 of 18 cases revealed that in NK-type LDGL certain activating KIRs, as well as certain infrequent KIR genotypes, were detected with higher frequencies as compared to previously analyzed healthy donors. Moreover, most KIR genotypes included multiple genes coding for activating KIRs. The analysis of non-HLA-specific triggering receptors indicated that the natural cytotoxicity receptors (NKp46, NKp30) were expressed at significantly low levels in freshly drawn NK cells from most patients analyzed. However, in most instances the expression of NKp46 and NKp30 could be up-regulated on culture in interleukin 2. Our data indicate that in NK-LDGL the expanded subset is frequently characterized by the expression of a given activating KIR, suggesting a direct role for these molecules in the pathogenetic mechanisms of this disorder.

Antibodies, Monoclonal↗

PubMed2003

Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells.

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor beta1 (TGFbeta1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFbeta1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFbeta1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFbeta1-producing dendritic cells may acquire resistance to the NK-mediated attack.

Antigens, CD↗

PubMed2003

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.

Antigens, CD↗

PubMed2002

Analysis of natural killer cells in TAP2-deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells.

Natural killer (NK) cells are characterized by the ability to kill cells that lack HLA class I molecules while sparing autologous normal (HLA class I(+)) cells. However, patients with transporter-associated antigen processing (TAP) deficiency, though displaying strong reductions of HLA class I surface expression, in most instances do not experience NK-mediated autoimmune phenomena. A possible mechanism by which TAP(-/-) NK cells avoid autoreactivity against autologous HLA class I-deficient cells could be based on either quantitative or qualitative defects of surface receptors involved in NK cell triggering. In this study we show that NK cells derived from 2 patients with TAP2(-/-) express normal levels of all known triggering receptors. As revealed by the analysis of polyclonal and clonal NK cells, these receptors display normal functional capabilities and allow the killing of a panel of NK-susceptible targets, including autologous B-LCLs. On the other hand, TAP2(-/-) NK cells were unable to kill either allogeneic (HLA class I(+)) or autologous (HLA class I(-) ) phytohemagglutinin (PHA) blasts even in the presence of anti-HLA class I monoclonal antibody. These data suggest that TAP2(-/-) NK cells express still unknown inhibitory receptor(s) capable of down-regulating the NK cell cytotoxicity on binding to surface ligand(s) expressed by T cell blasts. Functional analyses, both at the polyclonal and at the clonal level, are consistent with the concept that the putative inhibitory receptor is expressed by virtually all TAP2(-/-) NK cells, whereas it is present only in rare NK cells from healthy persons. Another possibility would be that TAP2(-/-) NK cells are missing a still unidentified triggering receptor involved in NK cell-mediated killing of PHA blasts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗