PubMed Health⌕ Search

Biomedical subjects

A Moretta

Publications and source records attributed to A Moretta.

At least 19 recordsLinked to original sources
PubMed1999

NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase.

To elucidate the role of the two conserved cis-proline residues of aspartate aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195A in which the two prolines were replaced by alanine. The crystal structures of P195A and P138A/P195A have been determined at 2.3-2.1 A resolution. The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, whereas the trans conformation is adopted at the 137-138 peptide bond. Quite surprisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein. All the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913]. The kinetics of renaturation of P195A, where the alanine retains the wild-type cis conformation, is faster than wild type, whereas renaturation of P138A, which adopts the trans conformation, is slower. We conclude that cis-prolines seem to have been retained throughout the evolution of aspartate aminotransferase to possibly play a subtle role in directing the traffic of intermediates toward the unique structure of the native state, rather than to respond to the needs for a specific catalytic or functional role.

Alanine↗
PubMed1999

The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein displays a broad specificity for different HLA class I alleles: analysis of LIR-1 + NK cell clones.

Leukocyte Ig-like receptor (LIR)-1 is a member of the Ig superfamily which has been shown to bind the human cytomegalovirus MHC class I homologue UL-18 protein. In this study, we have analyzed the expression and function of LIR-1 in human NK cells. We show that LIR-1 is expressed by a subset of NK cells variable in size among different donors. When compared to the known HLA class I-specific NK receptors, the expression of LIR-1 was found to be partially overlapped with that of CD94-NKG2A or with that of killer inhibitory receptors (KIR) belonging to the Ig superfamily. The use of the soluble form of UL-18 molecule revealed, in double fluorescence analysis, a selective binding to LIR-1 + cells while no correlation was observed between expression of either KIR or CD94-NKG2A molecules and ability to bind UL18. We further determined whether LIR-1 could also function as receptor for HLA class I molecules. To this end, we assessed the capability of LIR-1 + NK cell clones of lysing HLA class I- target cells transfected with different class I alleles, including HLA-A, -B, -C and -G alleles. Data revealed that LIR-1 functions as a broad HLA class I-specific inhibitory receptor recognizing different alleles coded for by different HLA loci.

Alleles↗
PubMed1998

Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity.

NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

Amino Acid Sequence↗
PubMed1998

NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis.

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non-major histocompatibility complex (MHC)-restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)-mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcgammaR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3zeta or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.

Biomarkers↗
PubMed1998

HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells.

A fraction of human T lymphocytes, predominantly CD8+, express receptors for HLA class I molecules typical of natural killer cells (natural killer receptors or NKRs) that inhibit T cell receptor-mediated functions. Herein, we analyzed possible mechanism(s) leading to the expression of NKRs by T cells responding to superantigens or allogeneic cells in vitro. We show that, in the presence of interleukin 15 (IL-15), T cells (depleted of NKR+ cells) responding to toxic shock syndrome toxin 1 de novo express CD94, a molecule that is part of a heterodimeric NKR with a broad specificity for different HLA class I alleles. Maximal CD94 expression occurred when IL-15 was added shortly after the cells were placed into culture, and CD94 expression started 4-6 days after addition of IL-15. Although both CD4+ and CD8+ cells expressed CD94, the simultaneous expression of NKG2A (i.e., the other component of the CD94/NKG2A inhibitory NKR) was confined to CD8+ cells. Similar data were obtained in T cell populations activated in mixed lymphocyte cultures in the presence of IL-15. The expression of CD94/NKG2A led to an impairment of allo-specific cytolytic activity by mixed lymphocyte culture-derived T cell populations or clones. Remarkably, cytolysis could be restored by the addition of anti-CD94 mAb, i.e., by masking the inhibitory NKRs.

Antigens, CD↗
PubMed1998

Activity and phenotype of natural killer cells in peptide transporter (TAP)-deficient patients (type I bare lymphocyte syndrome).

In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I-deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I- K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)- NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP- NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP- NK lymphocytes showed them to display a normal diverse repertoire of HLA class I-specific NK receptors. These receptors were expressed at normal levels, apart from the CD94-NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP- individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP- patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases.

Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Amino Acid Sequence↗
PubMed1998

The activating form of CD94 receptor complex: CD94 covalently associates with the Kp39 protein that represents the product of the NKG2-C gene.

Inhibitory receptor complexes formed by CD94 and NKG2-A (Kp43) molecules have been implicated in HLA class I recognition by human natural killer (NK) cells. Additional forms of CD94 receptors have recently been described in NK cells characterized by the lack of NKG2-A expression. These CD94 receptors were shown to display activating functions. Immunoprecipitation with anti-CD94 monoclonal antibodies (mAb) led to the identification, in these cells, of a 39-kDa (Kp39) molecule that was originally believed to represent an activating isoform of the CD94 molecules. In the present study we show that the Kp39 molecule is covalently associated with CD94 and displays a protein backbone (26 kDa) similar to that of NKG2-A (Kp43) glycoproteins. Peptide mapping analysis indicates that Kp39 and NKG2-A glycoproteins belong to the same molecular family. A novel NKG2-specific mAb (termed P25) has been generated that specifically reacts with both NKG2-A and NKG2-C molecules, but fails to recognize NKG2-E molecules. Analysis of polyclonal and clonal NK cells shows that P25 mAb reacts with all NKG2-A+ cells and with a fraction of CD94+ cells lacking the expression of NKG2-A. These data indicate that NKG2-C molecules are indeed expressed only in a subset of cells lacking the expression of NKG2-A. The CD94-associated Kp39 molecule can be detected only in NKG2-A- P25+ cells, i.e. cells expressing NKG2-C molecules. Indeed, reverse transcription-polymerase chain reaction analysis performed on a large panel of NK clones indicates that NKG2-A- P25+ NK clones express the NKG2-C transcript. Notably, the cytolytic activity of these clones can be triggered by the P25 mAb in redirected killing analysis. Finally, biochemical analysis of COS7 cells cotransfected with CD94 and NKG2-C demonstrates the identity between Kp39 and NKG2-C molecules. Altogether, our data demonstrate that NKG2-C molecules associate with CD94 to form an activating receptor complex in a subset of human NK cells.

Amino Acid Sequence↗
PubMed1998

The susceptibility to natural killer cell-mediated lysis of HLA class I-positive melanomas reflects the expression of insufficient amounts of different HLA class I alleles.

NK cells selectively lyse tumor cells which do not express one or more MHC class I alleles. The ability to discriminate between self normal or tumor cells is due to the expression of MHC class I-specific killer inhibitory receptors (KIR). In the present study we analyzed melanoma cell lines which were highly susceptible to NK cell-mediated lysis in spite of the expression of a complete set of HLA class I alleles. Quantitative analysis of the HLA class I expression using allele-specific monoclonal antibodies (mAb) revealed a down-regulation of all HLA class I molecules. Treatment of melanoma cells with IFN-gamma resulted in up-regulation of all HLA class I alleles that was paralleled by the acquisition of resistance to lysis. That resistance to lysis reflected the up-regulation of HLA class I molecules was revealed by the finding that mAb-mediated masking of either KIR or their HLA class I ligands completely restored the melanoma cell lysis. These results were obtained by the use of selected NK cell clones derived either from allogeneic or autologous donors. In addition, similar results were obtained using in vitro expanded autologous NK cell populations. Our data indicate that NK cells can lyse not only melanoma cells which have lost the expression of one or more HLA class I alleles but also cells expressing a decreased amount of class I molecules.

Alleles↗
PubMed1998

Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses.

Killer-cell inhibitory Receptors (KIRs) are a new family of major histocompatibility complex (MHC) class I-specific receptors. KIRs allow natural killer cells to identify and lyse self cells that do not express sufficient amounts of MHC class I molecules. Here, Maria Cristina Mingari and colleagues view the expression of KIRs by cytolytic T lymphocytes and their regulation by certain cytokines as a double-edged sword.

Gene Expression Regulation↗
PubMed1998

Revaccination against hepatitis B virus of non-responding and low-responding infants immunised at birth. A parallel evaluation of rubella and tetanus vaccine.

The aim of the present study was to identify the true extent of the non-responsiveness in infants born from HBsAg-negative mothers, vaccinated against Hepatitis B Virus (HBV) at birth. Sixty-four non- and low-responding infants, selected from an initial cohort of 2009, were given two additional doses of recombinant HBV vaccine between the tenth and the twelfth month of age. A parallel evaluation was conducted on the response to anti-rubella and anti-tetanus vaccine. Only two infants remained non-responders, whereas 68% of the non-responders and 94% of the low responders after the primary vaccination schedule developed antibody titres over 100 mIU ml-1. No significant relationship between the specific antibody level against HBV and against rubella or tetanus 1 month after vaccination was observed.

Hepatitis B↗
PubMed1998

Does the emergence and persistence of donor-derived leukaemia-reactive cytotoxic T lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study.

The frequency of CTL precursors (CTLp) directed towards recipient-derived pre-transplant leukaemic blasts (LB) was measured in the peripheral blood of nine children with acute leukaemia and given BMT from either an HLA-identical sibling or a matched unrelated donor (MUD). Patients were evaluated at various time points between 1 month and more than 2 years after transplantation. A high frequency of donor-derived LB-reactive CTLp was detectable 2-6 months after BMT in all children and persisted for at least up to 18 months in the eight patients in haematological remission, while it rapidly declined in the only patient who relapsed. Generation of LB-reactive T cell clones obtained from some of these patients demonstrates that various T lymphocyte subsets, either HLA class I-restricted/TCR-dependent or HLA-unrestricted, contribute to this phenomenon. The in vitro GVL effect here described seems to be at least partially separated from GVHR, since no correlation was observed between the emergence of LB-reactive CTLp and the development and/or severity of GVHD. Development of LB-reactive CTL in the patients was independent of the frequency of these cells in the donor. These data suggest that donor-derived CTL activity specifically directed towards leukaemic blasts may develop in patients given allogeneic BMT and contribute to the maintenance of a state of haematological remission.

Bone Marrow Transplantation↗
PubMed1997

p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

Amidohydrolases↗
PubMed1997

Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor.

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) for major histocompatibility complex class I molecules. Engagement of these surface receptors inhibits NK cell cytotoxic programs. KIR can also be expressed on T cell subsets, and their engagement similarly results in inhibition of effector functions initiated by the CD3/T cell receptor complex. KIR genes belong to two distinct families: the immunoglobulin superfamily (IgSF KIRs) and dimeric C2 lectins (lectin-like KIRs). Whereas both IgSF (p58: CD158, p70, and p140) and lectin-like KIRs (CD94/NKG2A heterodimers) have been found in human, only lectin-like KIRs (all members of the Ly-49 family) have been described in the mouse. We have generated transgenic mice expressing an IgSF KIR, CD158b (p58.2), which recognizes HLA-Cw3. Our data show that CD158b is necessary and sufficient to confer specificity to NK cells, as well as to modulate T cell activation programs in vitro. In addition, we did not detect any adaptation of CD158b cell surface expression to that of HLA class I ligands in the CD158b x HLA-Cw3 double transgenic mice, in contrast to observations with Ly-49 in the mouse. Therefore, distinct strategies of selection/calibration appear to be used by IgSF and lectin-like KIRs. Finally, the transgenic expression of CD158b KIR prevents the in vivo rejection of H-2 mismatch bone marrow grafts, which express the cognate major histocompatibility class I HLA-Cw3 allele, demonstrating for the first time the in vivo implication of human IgSF KIRs in the negative regulation of NK cell function.

Animals↗