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Biomedical subjects

M C Mingari

Publications and source records attributed to M C Mingari.

At least 37 records · Page 2Linked to original sources
PubMed2000

Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders.

Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.

Amino Acid Sequence↗
PubMed1999

Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells.

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33. In vitro proliferation and differentiation of cord blood-derived CD34(+) cells (induced by stem cell factor and granulocyte-macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.

Antibodies, Monoclonal↗
PubMed1999

Inhibitory receptors sensing HLA-G1 molecules in pregnancy: decidua-associated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor.

Trophoblastic cells lack classical HLA class I and class II molecules but express HLA-G1. Although this may prevent allorecognition by maternal T cells, it renders trophoblastic cells potentially susceptible to lysis by natural killer (NK) cells. As shown here, only a fraction of peripheral-blood NK cells in pregnant women express the HLA-G1-specific CD94/NKG2A and/or LIR-1 receptors. However, all NK cells isolated from maternal decidua during the first trimester expressed either one or both of these receptors. Perhaps more importantly, a fraction of cells expressed p49, an HLA-G1-specific inhibitory receptor, undetectable in peripheral-blood NK cells. p49 was expressed on virtually all NK cells isolated from placenta at term. Functional analyses revealed that the HLA class I-negative 221 lymphoblastoid cell line transfected with HLA-G1 was only partially protected from lysis by peripheral-blood NK cells isolated from pregnant women, whereas it was fully protected from decidual NK cells. As indicated by the addition of specific antibodies to cytolytic tests, all the above receptors contributed to HLA-G1 recognition by decidual NK cells, although p49 would appear to play a predominant role.

Animals↗
PubMed1999

Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes.

Different HLA class I-specific killer inhibitory receptors (KIR) are expressed in vivo by a fraction of activated T cells, predominantly CD8+, in which they may inhibit TCR-mediated cell functions. In an attempt to identify mechanisms leading to KIR expression in T cells, we analyzed the effect of transforming growth factor-beta (TGF-beta) in T cells responding to bacterial superantigens in vitro. We show that TGF-beta induces the expression of CD94/NKG2A in cells responding to toxic shock syndrome toxin 1 or to other staphylococcal superantigens. Remarkably, maximal CD94 expression occurred at (low) TGF-beta concentrations which have no substantial effect on lymphocyte proliferation. Maximal CD94 expression occurred when TGF-beta was added shortly after the cells were placed in culture. No expression could be induced in CD94/NKG2A-negative T cell clones. Although both CD4+ and CD8+ expressed CD94, the simultaneous expression of NKG2A was mostly confined to CD8+ cells. Monoclonal antibody-mediated cross-linking of CD94/NKG2A led to an impairment of T cell triggering via CD3, as determined in a redirected killing assay using the Fcgamma receptor-positive P815 murine target cells.

Animals↗
PubMed1999

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis.

Expression of HLA and CD1b molecules was investigated in the THP-1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2-3 days, cell surface expression of HLA class II and CD1b was drastically down-modulated, whereas HLA class I expression was up-modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down-modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down-modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN-gamma, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP-1 cells to process and present mycobacterial antigens to HLA-DR-restricted antigen-specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen-presenting molecules.

Antigen Presentation↗
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

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

Cytokine-induced expression of killer inhibitory receptors in human T lymphocytes.

Killer inhibitory receptors (KIRs) represent a new family of HLA-class I-specific receptors. KIRs are involved in the function of Natural Killer cells and allow these cells to discriminate between normal cells and cells with impaired expression of HLA-class I molecules. KIRs are also expressed by a subset of cytolytic T lymphocytes in which they may exert an inhibitory effect on TCR-mediated function. Here we review recent data indicating that cytokines such as IL-15, may induce the de novo expression of CD94/NKG2A (a KIR which operationally detects the expression of various HLA-class I alleles). The expression of CD94/NKG2A has been documented not only in CD34+ precursors undergoing maturation towards NK cells, but also in mature T cells which respond in vitro to superantigens or allogeneic cells.

Animals↗
PubMed1997

Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: impairment of specific cytolytic functions.

Human T lymphocytes have been shown to express inhibitory natural killer cell receptors (NKR), which can down-regulate T cell antigen receptor-mediated T cell function, including cytolytic activity. In the present study, we demonstrate that CD3+NKR+ cells can be identified in HIV-infected patients. HIV-specific cytolytic activity was analyzed in five patients in whom autologous lymphoblastoid B cell lines could be derived as a source of autologous target cells. Phytohemagglutinin-activated T cell populations that had been cultured in interleukin 2 displayed HIV-specific cytotoxic T lymphocyte (CTL) activity against HIV env, gag, pol, and nef in 3 of 5 patients. Addition of anti-NKR mAb of IgM isotype could increase the specific CTL activity. Moreover, in one additional patient, HIV-specific CTL activity was undetectable; however, after addition of anti-NKR mAb such CTL activity appeared de novo. Similar results were obtained by analysis of CD3+NKR+ clones derived from two patients. These data provide direct evidence that CD3+NKR+ cells may include antigen (HIV)-specific CTLs and that mAb-mediated masking of inhibitory NKR may revert the down-regulation of CTL function.

Antibodies, Monoclonal↗
PubMed1997

Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor.

Immature postnatal thymocytes were shown to contain precursors which, under suitable culture conditions, give rise to phenotypically and functionally mature natural killer (NK) cells. Here, we analyzed the effect of different cytokines for their ability to induce the expression of HLA class I-specific inhibitory receptor(s) during the process of NK cell development from immature thymocytes. From thymocyte cell suspensions depleted of CD2+, CD3+, CD4+, CD8+, CD56+, and CD16+ cells, we further removed cells expressing HLA class I-specific inhibitory receptors including CD94/NKG2-A, p58.1, and p58.2 by immunomagnetic bead separation. The resulting cells did not contain any of the above NK receptors as determined by immunofluorescence and flow cytometric analysis, as well as by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using appropriate sets of primers. Although different cytokines have been used, including interleukin (IL)-7, stem cell factor (SCF), IL-2, and IL-15, only IL-2 or IL-15 induced cell proliferation when used alone. Moreover, maturation towards CD3- CD56+ cells displaying cytolytic activity against the HLA class I- targets K562 or 221 was detectable in cultures containing IL-15 used alone or in combination with IL-7 or SCF. On the other hand, these CD3- CD56+ cell populations did not lyse HLA class I+ target cells, including autologous PHA blasts. Analysis of the expression of the various HLA class I-specific inhibitory NK receptors revealed the presence of high proportions of CD94/ NKG2-A+ cells, while the NK receptors belonging to the Ig superfamily were undetectable both by immunofluorescence and by RT-PCR analysis. The expression of CD94/NKG2-A appeared to be responsible for the inability of cells to lyse HLA class I+ target cells. Thus, addition of anti-CD94 monoclonal antibodies of IgM isotype resulted in lysis of autologous target cells. The use of 221 cells transfected with different HLA class I alleles as target cells confirmed the broad class I specificity of CD94/NKG2-A receptor. Our experiments indicate that IL-15 provides an appropriate stimulus to the expression of CD94/NKG2-A, but not of other class I-specific NK receptors in the process of maturation of NK cells from thymocyte precursors.

Alleles↗
PubMed1997

Inhibitory receptors for HLA class I molecules on cytolytic T lymphocytes. Functional relevance and implications for anti-tumor immune responses.

In recent years, the molecular mechanism by which natural killer cells lyze, or fail to lyze, different target cells has been elucidated. Natural killer cells express receptors which recognize MHC class I molecules on target cells. This interaction leads to inhibition of cytolytic activity, thus preventing lysis of target cells. The receptors belong to two distinct molecular types: (1) the lg superfamily which includes receptors (p58.1, p58.2, p70, and p140) which recognize specific HLA allotypes; (2) CD94 molecules which display a broad specificity for HLA class I molecules. Recently, a subset of cytolytic T lymphocytes has been shown to express the various natural killer cell receptors. Such T cells are detectable in peripheral blood, spleen, tonsils, and lymph nodes, but not in the thymus and cord blood. In some instances, two or more natural killer receptors can be coexpressed at the single cell level. Surface marker analysis has revealed that natural killer cell receptor-positive T cells always express a memory phenotype. Moreover, they are characterized by a skewed T cell receptor V beta repertoire. Further analysis of the T cell receptor VDI sequences revealed that natural killer cell receptor-positive, CD3-positive cells isolated from a given individual are oligoclonal or monoclonal in nature. Crosslinking of natural killer receptors leads to inhibition of different T cell functions, including non-specific lysis of appropriate HLA class I-negative target cells, T cell receptor mediated cytotoxicity, and cytokine production. The inhibitory effect on T cell receptor-mediated function has important implications. Thus, the expression of natural killer cell receptors as a consequence of chronic antigen stimulation may result in functional impairment of specific cytolytic T lymphocytes. Preliminary data indicate that this phenomenon may occur in tumor or virally infected patients. Remarkably, various patients with large granular lymphocyte expansions characterized by a CD3/ natural killer receptor-positive phenotype had chronic viral infections. The fact that antigen-specific cytolytic T lymphocytes may simultaneously express T cell and natural killer cell receptors, both recognizing HLA class I molecules but mediating opposite signals, offers new perspectives in our appreciation of the regulation of T cell responses and offers new clues for understanding the immunopathological events involved in certain diseases.

Antigens, Neoplasm↗
PubMed1997

HLA-class I-specific inhibitory receptors in human cytolytic T lymphocytes: molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells.

A subset of cytolytic T lymphocytes has been shown to express receptors of the NK type (NKR) which can inhibit T cell cytotoxicity induced via the TCR-CD3 pathway. In this study, by the analysis of full length cDNA amplified from representative T cell clones, we show that NKR belonging either to the lg superfamily, including p58.1, p58.2, p70 and p140, or to the C-type lectin superfamily (CD94/NKG2A), display sequences which are identical to those of the corresponding NKR expressed by CD3-NK cells. Moreover, a fragment of cDNA encoding the NKG2A protein was consistently amplified from all CD94+ T cell clones analyzed. Since different NKR types can be expressed by T cells, we analyzed whether individual T cells could co-express more than one NKR. Analysis of either resting or activated (and cultured) T cell populations revealed that two or more NKR can be co-expressed by single T cells. Moreover, by the analysis of T cell clones, we show that co-expressed receptors are functional and can inhibit independently the TCR-induced cytolytic function. Finally, we investigated whether NKR+ T lymphocytes were also present in lymphoid tissues. No such cells were found in thymus or cord blood, thus further supporting the notion that they represent memory T cells. On the other hand, they were present in all the peripheral tissues analyzed including spleen, lymph nodes and tonsils.

Animals↗
PubMed1997

Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes.

Human NK cells express several specialized inhibitory receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Receptors specific for groups of HLA-C (p58), HLA-B (p70) and HLA-A (p140) alleles belong to the Ig superfamily with two or three Ig-like domains in their extracellular portion, and a long cytoplasmic tail containing ITIM motifs and associated with a non-polar transmembrane portion. In contrast, the CD94/ NKG2-A receptor complex is composed of type II proteins with a C-type lectin domain which displays a more broad specificity for different class I alleles. Recently, activatory forms of the HLA-C-specific receptors have been identified in some donors. They are virtually identical to the inhibitory forms in their extracellular portions, but display a short cytoplasmic tail lacking ITIM motifs associated with a Lys-containing transmembrane portion (p50). A subset of activated T-lymphocytes, primarily CD8+ and oligoclonal or monoclonal in nature, express NK-type class I-specific receptors. These receptors exert an inhibitory activity on T-cell receptor-mediated functions and may provide an important mechanism of down-regulation of T-cell responses.

HLA Antigens↗
PubMed1997

Expression of HLA class I-specific inhibitory receptors by chronically activated cytolytic T lymphocytes: useful or harmful to the host?

T cells, primarily chronically activated cytolytic T lymphocytes (CTL), can express human leukocyte antigen class I-specific inhibitory receptors previously identified in natural killer cells. Engagement of these receptors leads to inhibition of CTL function. Although the receptor expression may represent a safety device to prevent autoimmunity, it may result in the inability to control viral spread or tumour growth, i.e. two main functions of CTLs. The physiopathologic implications of this event are discussed.

Animals↗
PubMed1996

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

CD3 Complex↗
PubMed1996

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10.

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-gamma or IL-12. The resulting CD4+ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-gamma and tumor necrosis factor (TNF)-beta in response to stimulation with phorbol 12-myristate 13-acetate (PMA) + anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4+ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-gamma, TNF-beta or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-gamma. However, upon addition of exogenous IL-12, the resulting CD4+ thymocyte clones produced TNF-beta, IFN-gamma, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4+ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.

CD4-Positive T-Lymphocytes↗
PubMed1996

Expression of human NKRP1A by CD34+ immature thymocytes: NKRP1A-mediated regulation of proliferation and cytolytic activity.

In this study, we show that NKRP1A is expressed and functions on a subset of immature human thymocytes. We took advantage of the monoclonal antibody (mAb) 191B8 that was obtained by immunizing mice with cultured human thymocytes characterized by an immature surface phenotype [CD2- CD3- CD4- CD8- stem cell factor receptor (SCFR)+] and expressing cytoplasmic CD3 epsilon chain. The 191B8 antibody homogeneously reacted with the immunizing population but not with most unfractionated thymocytes. It stained a minor population of resting immature thymocytes co-expressing CD34, SCFR, or both. Following culture of the CD34+ or CD34- fractions of CD2- CD3- CD4- CD8- purified immature thymocytes with recombinant interleukin-2 (rIL-2), the 191B8-defined antigen was expressed on virtually all cells even when 191B8+ cells were removed from the starting population. On the other hand, no 191B8+ cells were detected in fresh or cultured thymocytes expressing a more mature phenotype. Biochemical analysis of 191B8 mAb-reactive molecules revealed, under non-reducing conditions, two bands displaying apparent molecular masses of 80 and 44 kDa and a single band of 44 kDa under reducing conditions. Digestion with proteases indicated that the 80-kDa form represented a homodimeric form of two 44-kDa molecules, while deglycosylation with N-glycanase suggested the existence of four N-glycosylation sites. Transfection of COS7 or NIH3T3 cells with hNKRP1A cDNA showed that the 191B8 mAb recognized NKRP1A as shown by both immunofluorescence analysis and immunoprecipitation experiments. Functional studies showed that the 191B8/NKRP1A molecule mediated strong inhibition of the cytolytic activity of cultured CD2- CD3- immature thymocytes against a panel of tumor target cells. More importantly, 191B8 mAb induced proliferation of CD2- CD3- fresh thymocytes which was not increased by rIL-2. Thus, we propose that NKRP1A molecules, which are expressed in highly immature thymocytes, may play a regulatory role in their growth and function.

Antigens, CD34↗