Biomedical subjects

Michela Falco

Publications and source records attributed to Michela Falco.

15 recordsLinked to original sources

PubMed2006

Analysis of natural killer cells isolated from human decidua: Evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function.

While during the first trimester of pregnancy natural killer (NK) cells represent the most abundant lymphocyte population in the decidua, their actual function at this site is still debated. In this study we analyzed NK cells isolated from decidual tissue for their surface phenotype and functional capability. We show that decidual NK (dNK) cells express normal surface levels of certain activating receptors, including NKp46, NKG2D, and 2B4, as well as of killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A inhibitory receptor. In addition, they are characterized by high levels of cytoplasmic granules despite their CD56(bright) CD16- surface phenotype. Moreover, we provide evidence that in dNK cells, activating NK receptors display normal triggering capability whereas 2B4 functions as an inhibitory receptor. Thus, cross-linking of 2B4 resulted in inhibition of both cytolytic activity and interferon-gamma (IFN-gamma) production. Clonal analysis revealed that, in the majority of dNK cell clones, the 2B4 inhibitory function is related to the deficient expression of signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) mRNA. Moreover, biochemical analysis revealed low levels of SAP in the dNK polyclonal population. This might suggest that dNK cells, although potentially capable of killing, are inhibited in their function when interacting with cells expressing CD48.

Antigens, CD↗

PubMed2006

Structural basis for a major histocompatibility complex class Ib-restricted T cell response.

In contrast to antigen-specific immunity orchestrated by major histocompatibility complex (MHC) class Ia molecules, the ancestrally related nonclassical MHC class Ib molecules generally mediate innate immune responses. Here we have demonstrated the structural basis by which the MHC class Ib molecule HLA-E mediates an adaptive MHC-restricted cytotoxic T lymphocyte response to human cytomegalovirus. Highly constrained by host genetics, the response showed notable fine specificity for position 8 of the viral peptide, which is the sole discriminator of self versus nonself. Despite the evolutionary divergence of MHC class Ia and class Ib molecules, the structure of the T cell receptor-MHC class Ib complex was very similar to that of conventional T cell receptor-MHC class Ia complexes. These results emphasize the evolutionary 'ambiguity' of HLA-E, which not only interacts with innate immune receptors but also has the functional capacity to mediate virus-specific cytotoxic T lymphocyte responses during adaptive immunity.

Animals↗

PubMed2005

Distinctive lack of CD48 expression in subsets of human dendritic cells tunes NK cell activation.

CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.

Antigens, CD↗

PubMed2005

Identification of effector-memory CMV-specific T lymphocytes that kill CMV-infected target cells in an HLA-E-restricted fashion.

HLA-E-restricted T cells represent a minor cytolytic T lymphocyte (CTL) population characterized by the surface expression of HLA class I-specific inhibitory receptors and by the capability of killing a large panel of allogeneic target cells (therefore named NK-CTL). Here we show that this subset of T cells is present in a sizeable fraction in the peripheral blood of human cytomegalovirus (HCMV)-seropositive healthy individuals. We provide evidence that NK-CTL recognize in an HLA-E-restricted fashion a naturally processed CMV-derived peptide in the transporter associated with antigen processing (TAP)-2(-/-) UL40+ RMA-S cell transfectants. Moreover, we show that they recognize and kill HCMV-infected target cells. NK-CTL are characterized by the CD8beta(dull) (CD45RA+)(CD28-)(CD27-)(CCR7-)(CD56+) surface phenotype, thus suggesting that they belong to the effector-memory cell compartment. Consistent with the effector-memory phenotype, they promptly produce IFN-gamma, but not IL-2, upon interaction with the specific HCMV UL40-derived peptide. Our data suggest that HLA-E-restricted CTL may represent an additional effector cell type involved in defenses against HCMV, a virus which escapes the control exerted by conventional CTL or NK cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112).

On the basis of recent clinical and experimental data, natural killer (NK) cells appear to play a crucial role in eradication of acute myeloid leukemias. In the present study, by exploiting our current knowledge on NK receptors and their ligands on target cells, we investigated the interactions between NK and leukemic cells. We show that the size of the NK cell subset expressing the killer immunoglobulin-like receptor (KIR) not engaged by the HLA-class I alleles of the patient parallels the degree of NK cytotoxicity against leukemic cells. A sharp down-regulation of HLA-class I molecules has been detected in various leukemias and it was more frequent in myeloid than in lymphoblastic leukemias. Analysis of the ligands for triggering NK receptors revealed the consistent expression of Poliovirus receptor (PVR) and Nectin-2 in myeloid leukemias. In contrast, major histocompatibility complex class I-related chain molecules A/B (MICA/B) and UL1b-binding protein (ULBPs) were either absent or weakly expressed. Accordingly, NK-mediated lysis of these leukemias was dependent on DNAM-1 but not NKG2D. The major role of NKp46 and NKp30 was also confirmed. The expression of PVR and/or Nectin-2 was less frequent in lymphoblastic leukemias. In most leukemias, both CD48 and NTBA were down-regulated. The correlation found between marker expression and susceptibility to lysis may reveal useful information for NK-based immunotherapy.

Cell Communication↗

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

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

HLA-E-restricted recognition of human cytomegalovirus by a subset of cytolytic T lymphocytes.

Natural killer (NK)-cytotoxic T lymphocytes (CTL) are a subset of CD8(+) cytolytic T lymphocytes that express human leukocyte antigen (HLA) class I-specific inhibitory receptors. They are detectable as monoclonal expansions in the blood of cytomegalovirus (CMV)-seropositive individuals displaying particular HLA-Cw allotypes. Similar to NK cells, they are capable of killing various allogeneic tumor cell lines, a function referred to as "NK-like activity." The mechanism underlying this unusual functional property has recently been clarified. Via their T-cell receptor, NK-CTL recognize the nonclassical HLA class I molecule HLA-E, which is characterized by a limited polymorphism and by the ability to bind peptides derived from the leader sequence of various HLA class I alleles as well as from few viral proteins. The analysis of the T-cell receptor avidity revealed that NK-CTL recognize with high avidity a CMV UL40-derived peptide. The HLA-E-restricted recognition of CMV by NK-CTL may represent an important immunologic strategy in defenses against this virus. Indeed, unlike conventional CTL, NK-CTL mediated lysis is apparently not affected by the downregulation of major histocompatibility complex class I that occurs during CMV infection. Because the CMV UL40-derived peptide is identical to the one generated from the leader sequence of various HLA-Cw alleles, NK-CTL are also able to display an "HLA-E-dependent alloreactivity" against allogeneic target cells expressing appropriate HLA-Cw alleles. This broad ability to recognize and kill allogeneic cells may pose serious problems in transplantation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes.

HLA-E-restricted T cell receptor alphabeta+ CD8+ cytolytic T lymphocytes (CTLs) exist as monoclonal expansions in the peripheral blood of some individuals. Here, we show that they recognize, with high avidity, peptides derived from the UL40 protein of different human cytomegalovirus (CMV) strains. Recognition results in the induction of cytotoxicity, IFN-gamma production and cell proliferation. Autologous cells pulsed with CMV-derived peptides become susceptible to lysis by HLA-E-restricted CTLs and induce their proliferation. The high avidity for CMV-derived peptides may explain how these cells are generated in vivo and suggest their possible role in the host defenses against CMV, a virus that evolved various mechanisms to down-regulate classical HLA class I molecules, thus escaping detection by conventional CTLs.

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

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity.

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.

Animals↗

PubMed2002

Identification of HLA-E-specific alloreactive T lymphocytes: a cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells.

Previous studies showed that a subset of CD8(+) cytolytic T lymphocytes expressing HLA class I-specific natural killer inhibitory receptors (iNKR) is characterized by the ability to recognize HLA-E and to mediate T cell receptor-dependent killing of different NK cell-susceptible tumor target cells. In this study, we show that this CD8(+) T cell subset can also undergo extensive proliferation in mixed lymphocyte cultures in response to allogeneic cells. Analysis of their cytolytic activity revealed a broad specificity against a panel of allogeneic phytohemagglutinin-induced blasts derived from HLA-unmatched donors. On the other hand, autologous and certain allogeneic phytohemagglutinin blasts were resistant to lysis. We used as target cells the transporter associated with antigen processing (TAP)-2(-/-) murine RMA-S cell line cotransfected with beta2-microglobulin and HLA-E*01033. On loading these cells with different HLA-E-binding peptides derived either from HLA class I leader sequences or viral proteins, we could show that HLA-E-specific cytolytic T lymphocytes recognized many, but not all, peptides analyzed. These data suggest that these cells may recognize, on allogeneic cells, HLA-E with peptides that are not present in the host of origin. In addition to their ability to proliferate in response to allogeneic stimulation and to lyse, most allogeneic cells may have important implications in transplantation and in antitumor immune responses.

Alleles↗

PubMed2002

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation.

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34(+) Lin(-) cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor.

Antigens, CD↗

PubMedDate not supplied

Human natural killer cell receptors: insights into their molecular function and structure.

NK cells express receptors characterized by opposite functions that finely regulate their activities. Among inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are still orphan receptors. On the contrary, various activating receptors are involved in the triggering of NK-mediated natural cytotoxicity. In general, their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Thus, the process of NK cell triggering mediated by Natural Cytotoxicity Receptors can be mainly considered as a non MHC-restricted mechanism. Here, a brief description of the molecular nature of these receptors, as well as, of their 3D-structures and of the implications for ligand recognition, is given.

Antigens, CD↗