Biomedical subjects

Alessandro Moretta

Publications and source records attributed to Alessandro Moretta.

At least 55 records · Page 3Linked to original sources

PubMed2004

DNAM-1 and PVR regulate monocyte migration through endothelial junctions.

DNAX accessory molecule 1 (DNAM-1; CD226) is a transmembrane glycoprotein involved in T cell and natural killer (NK) cell cytotoxicity. We demonstrated recently that DNAM-1 triggers NK cell-mediated killing of tumor cells upon engagement by its two ligands, poliovirus receptor (PVR; CD155) and Nectin-2 (CD112). In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells. Moreover, the specific binding of a soluble DNAM-1-Fc molecule was detected at endothelial junctions. This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti-Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells. Because DNAM-1 is highly expressed on leukocytes, we investigated the role of the DNAM-1-PVR interaction during the monocyte transendothelial migration process. In vitro, both anti-DNAM-1 and anti-PVR mAbs strongly blocked the transmigration of monocytes through the endothelium. Moreover, after anti-DNAM-1 or anti-PVR mAb treatment, monocytes were arrested at the apical surface of the endothelium over intercellular junctions, which strongly suggests that the DNAM-1-PVR interaction occurs during the diapedesis step. Altogether, our results demonstrate that DNAM-1 regulates monocyte extravasation via its interaction with PVR expressed at endothelial junctions on normal cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2004

Analysis of the natural killer mediated immune response in metastatic renal cell carcinoma patients.

Metastatic renal cell carcinomas (MRCC) are considered as immunogeneic tumors on the basis of the clinical responses observed in patients treated by IL-2. However, renal cell carcinoma patients are also characterized by alterations of the immune response that may compromise the immunotherapeutic approaches. In our study, we have studied the phenotype and the functional capacities of peripheral NK cells in a panel of neprectomized metastatic renal cell carcinoma patients. NK cells were harvested by negative immunoselection from fresh peripheral blood samples. In most of MRCC patients analysed (23/28), the expression of NCR (NKp46 and NKp30) was similar to that of donors. Lytic capacities by activated immunoselected NK cells from MRCC patients assessed against K562 and 3 renal tumor cell lines were in the range of that observed in NK cells from normal donors. HLA-I- renal tumor cells UOK23 were killed with a good efficiency, whereas HLA-I renal tumor cells were more resistant. Although LFA-1/ICAM-1 interaction potentiates RCC cell lysis, HLA-I/NKR interaction clearly decreased RCC cell susceptibility to NK cells. In addition, proliferation of NK cells from MRCC patients in response to cytokines was altered.

Aged↗

PubMed2004

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic.

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.

Cell Line, Transformed↗

PubMed2004

Structure of the saccharide-binding domain of the human natural killer cell inhibitory receptor p75/AIRM1.

The high-resolution crystal structure of the functional N-terminal domain from the extracellular region of the human natural killer cell inhibitory receptor p75/AIRM1 or Siglec-7 has been determined at 1.45 A resolution; it was obtained from a crystal belonging to a primitive monoclinic space group, with unit-cell parameters a = 32.65, b = 49.72, c = 39.79 A, alpha = gamma = 90, beta = 113 degrees. The structure reported here belongs to a different space group than the previously described Siglec-7 structure and was obtained using a bacterial expression system. The structure unveils the fine structural requirements adopted by a natural killer cell inhibitory receptor of the Siglec family in target-cell recognition and binding.

Binding Sites↗

PubMed2004

Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors.

NKG2D and natural cytotoxicity receptors (NCR) are essential recognition structures that mediate NK cell activation. NKG2D and NCR signaling is achieved through membrane association with signaling adaptors. The adaptors that associate with NCR--such as CD3 zeta, FcR gamma and KARAP/DAP12--bear intracytoplasmic immunoreceptor tyrosine-based activation motifs that activate Syk protein tyrosine kinases. Human NKG2D associates with the DAP10 transmembrane adaptor, which bears a YxxM motif and activates the phosphatidylinositol 3-kinase pathway. In the mouse, a short NKG2D-S isoform, generated by Nkg2d alternative splicing, can associate with either DAP10 or KARAP/DAP12. Here, we report that neither short human NKG2D alternative transcripts nor NKG2D association with KARAP/DAP12 was detected in activated human NK cells. Despite these results, NK cell triggering by both recombinant soluble NKG2D ligands (MICA and ULBP-1) and anti-NCR cross-linking antibodies induced similar CD25 expression, NK cell proliferation and cytokine production. In contrast, NKG2D triggering by anti-NKG2D antibodies did not lead to any detectable activation signals. These data thus show that target recognition via NKG2D or NCR triggers all aspects of NK activation, and pave the way for further dissection of the signaling pathways induced by NK cell recognition of ULBP-1 and MICA.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Homophilic interaction of NTBA, a member of the CD2 molecular family: induction of cytotoxicity and cytokine release in human NK cells.

NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA(+) but not to NTBA(-) T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-gamma and TNF-alpha. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

CD2 Antigens↗

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

Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients.

One of the hallmarks of HIV-1 infection is represented by the finding of massive T cell activation in peripheral blood lymphocytes of infected patients. An impairment of NK cell function during HIV-1 infection is also detected, and is associated with decreased expression of natural cytotoxicity receptors (NCR). In this study we tried to determine whether also NK cells are affected by relevant activation and whether this could be associated with decreased NK cell function. In 18 viremic HIV-1-infected patients, freshly drawn purified peripheral NK cells displayed significant levels of activation with an incomplete pattern (HLA-DR(+)CD69(+)CD25(-)NKp44(-)). Activated (HLA-DR(+)CD69(+)) peripheral NK cells expressed an NCR(dull) phenotype as determined by cytofluorometric analysis in all the patients, and did not derive from a homogeneous/oligoclonal expansion in vivo as analyzed by expression of HLA-specific inhibitory NK cell receptors. As determined by cytotoxicity assays, activated NK cells showed a decreased cytolytic function in HIV-1-infected patients. Thus, the decrease in NK cell function observed during HIV-1 infection is associated not only with decreased NCR expression, but also with significant and incomplete NK cell activation in vivo. These results suggest a consistent continuous involvement of the innate immune response in the failure to control viral replication.

Biomarkers↗

PubMed2004

Commentary: Regulated equilibrium between opposite signals: a general paradigm for T cell function?

The co-signaling receptors specific for the different members of the B7 molecular family are cell surface glycoproteins that are essential to modulate and tune the TCR-mediated activation of T lymphocytes. The common characteristic is that their function appears to be dependent on the engagement of TCR by antigenic peptides presented in the MHC context by antigen-presenting cells. Interestingly, co-signaling molecules can be distinguished into costimulators and co-inhibitors, the prototype being represented by CD28 and CTLA-4, respectively. In the case of costimulators, the co-signals integrate the signal originated from the TCR resulting in optimal T cell activation (two-signal model). In the case of co-inhibitors, the co-signals would moderate and/or switch off the Ag-dependent T cell activation, thus acting as negative regulators of immune responses. The growing number of novel co-signaling molecules has recently highlighted the need to integrate the two-signal model with the emerging data on the different co-inhibitory interactions. Thus, a model has been proposed based on the idea that the TCR signal alone cannot take a full decision on the nature of the functional outcome following an antigen-specific stimulation and that this final event is governed by the co-signaling molecules.

Immune System↗

PubMed2004

Killer immunoglobulin-like receptors.

Killer Ig-like receptors (KIRs) are surface inhibitory receptors specific for allelic forms of human leukocyte antigen (HLA) class I molecules, which are expressed by natural killer (NK) cells and a subset of T lymphocytes. Upon engagement with HLA class I molecules, KIRs block NK cell activation and function. Cells lacking HLA class I molecules are promptly killed by NK cells because of the predominant effect of several activating NK receptors. The NK-mediated killing of these cells might represent an important defence mechanism, antagonizing spreading of pathogens and tumours. Evidence has been accumulated that KIR-encoding genes have evolved and diversified rapidly in primates and in humans. Similar to HLA loci, KIR sequences are highly polymorphic and, moreover, KIR haplotypes greatly vary in the number of the type of genes they contain. KIR gene expression is regulated by mechanisms of DNA methylation. As recently shown, the HLA class I regulated control of NK cell function can be exploited in an allogeneic bone marrow transplantation setting to eradicate acute myeloid leukaemias.

Animals↗

PubMed2004

Shaping of adaptive immunity by innate interactions.

In inflamed tissues, the reciprocal interaction between Natural Killer (NK) cells and Dendritic Cells (DC) results in a potent activating cross talk that leads to DC maturation and NK cell activation with acquisition of NK-mediated cytotoxicity against immature DC (iDC). We focused our studies on NK-mediated killing of monocyte-derived iDC and we provided evidence that NK cells that express CD94/NKG2A but not killer Ig-like receptors (KIR) are able to kill autologous iDC. Indeed HLA-E (i.e. the cellular ligand of CD94/NKG2A) is sharply reduced in iDC, whereas it is partially recovered in mDC. The latter are lysed only by a small fraction of NK clones characterized by low levels of CD94/NKG2A expression. Another NK receptor, whose surface density is crucial for the ability to kill iDC, is represented by NKp30, a member of the NCR (Natural Cytotoxicity Receptor) family. We showed that transforming growth factor beta1 (TGFbeta1) treatment results in specific downregulation of NKp30 expression. This effect profoundly inhibits the NK-mediated killing of DC suggesting a possible mechanism by which TGFbeta1-producing DC may acquire resistance to the NK-mediated attack.

Animals↗

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↗

PubMed2004

CD158k/KIR3DL2 is a new phenotypic marker of Sezary cells: relevance for the diagnosis and follow-up of Sezary syndrome.

CD158k molecules belong to the family of killer cell immunoglobulin-like receptors (KIR) that are expressed on a minor population of circulating NK and CD8+ T lymphocytes. Here, we report a strong positive correlation between the percentage of CD158k+ blood lymphocytes analyzed by flow cytometry and the percentage of atypical circulating cells (Sezary cells) determined by cytomorphology in a large group of patients with Sezary syndrome. Moreover, we show that circulating CD4+CD158k+ lymphocytes correspond to the malignant clonal cell population. Our findings suggest that the CD158k marker could be a useful tool for the evaluation of the circulating tumoral burden and the follow-up of patients with Sezary syndrome.

Biomarkers, Tumor↗

PubMed2003

Unravelling natural killer cell function: triggering and inhibitory human NK receptors.

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.

Cytotoxicity, Immunologic↗

PubMed2003

Expression and crystallographic characterization of the extracellular domain of human natural killer cell triggering receptor NKp46.

Human natural killer (NK) cells are regulated in their cytolytic activity by a delicate interplay between activating and inhibitory signals related to distinct families of triggering and inhibitory receptor proteins. NKp46 is a major NK cell-specific triggering receptor involved in the recognition and lysis of human and murine tumour and virally infected cells. It consists of an extracellular portion, composed of two Ig-like domains, a transmembrane segment and a small cytoplasmic domain. To shed light on the molecular-recognition events involved in NK cytotoxicity triggering mechanisms, the NKp46 extracellular region was cloned, overexpressed, refolded and crystallized. X-ray diffraction data could be collected to a resolution limit of 1.93 A. Crystals of the NKp46 extracellular region belong to the hexagonal space group P6(1) (or P6(5)), with unit-cell parameters a = b = 85.48, c = 59.91 A, gamma = 120 degrees; the asymmetric unit contains one protein chain (197 amino acids).

Amino Acid Sequence↗

PubMed2003

Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates.

Natural killer (NK) cells play a central role in host defense against various pathogens. Functional defects of NK cells in HIV-1 infection as a direct effect of abnormal expression or function of inhibitory NK receptors (iNKRs), activating natural cytotoxicity receptors (NCRs), and NKG2D have not yet been described. This study demonstrates an expansion of the functionally defective CD56-/CD16+ population of NK cells in viremic versus aviremic patients. We also demonstrate that in HIV-infected viremic patients, expression of iNKRs was well conserved and that in most cases, there was a trend toward increased expression on NK cells as compared with healthy donors. It was also demonstrated that the major activating NK receptors, with the exception of NKG2D, were significantly down-regulated. In contrast, the expression of iNKRs and activating receptors in HIV-infected individuals whose viremia was suppressed to below detectable levels by highly active antiretroviral therapy for 2 years or longer was comparable to that of healthy donors. Functional tests confirmed that the abnormal expression of the activating receptors and of iNKRs was associated with a markedly impaired NK cytolytic function. This phenomenon is not attributed to a direct HIV-1 infection of NK cells; thus, this study may provide insight into the mechanisms of impaired host defenses in HIV-1 viremic patients.

Antibodies, Monoclonal↗

PubMed2003

Functional and molecular characterization of a KIR3DL2/p140 expressing tumor-specific cytotoxic T lymphocyte clone infiltrating a human lung carcinoma.

T lymphocytes infiltrating a human lung carcinoma stimulated in vitro with autologous tumor cell line showed a TCRVbeta13.6(+) T-cell expansion. This subset was isolated using TCRVbeta-specific antibody and several T-cell clones were generated. All these clones expressed a unique Vbeta13.6-Jbeta2.7 TCR with the same junctional region strongly suggesting that they derived from the same cell. They were CD8(+)/CD28(-) and expressed the MHC class I binding killer cell Ig-like receptor (KIR)3DL2/p140, but not KIR3DL1/p70, KIR2DL1/p58.1 and KIR2DL2/3/p58.2. Sequence analysis indicated that KIR3DL2/p140 cDNA was identical to the previously reported 3DL2*002 allele except for two nucleic acid substitutions. Functional studies showed that KIR3DL2/p140(+) CTL secrete a significant level of IFNgamma and mediate an HLA-A2-restricted cytotoxicity against the autologous and some allogeneic tumor cells but not towards the autologous EBV-B cells. Strikingly, both the lytic and the cytokine secretion activities induced upon specific cell interactions were unaffected by anti-KIR3DL2/p140 antibody. In addition, crosslinking KIR3DL2/p140 molecules on CTL did not result into the modification of cytotoxicity and cytokine production triggered by anti-CD3 antibody. These results strongly suggest that, as opposed to distinct KIR expressed by CTL, the in vitro KIR3DL2/p140 engagement does not result into inhibitory (nor activatory) effects on tumor-specific CTL.

Antigens, CD↗