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1.
Hematol J ; 2(4): 220-7, 2001.
Article in English | MEDLINE | ID: mdl-11920253

ABSTRACT

INTRODUCTION: In acquired immune deficiency syndrome patients, apoptosis of uninfected lymphocytes may contribute to development of immune deficiency. This process may involve recruitment of Fas by human immunodeficiency virus products. In line with this possibility, the viral envelope glycoprotein gp120 does not induce death of T cells from subjects with the autoimmune/lymphoproliferative syndrome displaying defective Fas function. This study evaluates the possibility that Fas function defects delay progression of HIV-induced immune deficiency. MATERIALS AND METHODS: The susceptibility to Fas-induced cell death was assessed on T cells from 18 'long-term non-progressor', four 'non-progressor', four 'progressor' asymptomatic HIV-1-infected, and nine AIDS patients using anti-Fas monoclonal antibodies. RESULTS: Fas-induced cell death was significantly lower in long-term non-progressors and non-progressors than in normal controls, progressors, and AIDS. The single-patient data showed that 9/18 long-term non-progressors and 3/4 non-progressors, but no progressors or AIDS were resistant to Fas. Analysis of the uninfected parents of two long-term non-progressors displaying decreased Fas-function showed that the mother of one of them and the father of the other displayed the same Fas function defect as their children. Fusion of T cells from Fas-resistant individuals with a Fas-sensitive cell line gave rise to Fas-resistant hybrid lines not carrying HIV, which suggests that the resistant phenotype is due to molecules exerting a dominant negative effect on a normal Fas system. CONCLUSION: These data suggest that Fas-resistance in long-term non-progressors may be due to inherited alterations of the Fas signaling pathway and may be a novel factor in delayed progression.


Subject(s)
Acquired Immunodeficiency Syndrome/diagnosis , HIV Long-Term Survivors , fas Receptor/physiology , Acquired Immunodeficiency Syndrome/genetics , Antibodies, Monoclonal , Apoptosis/genetics , Apoptosis/physiology , Case-Control Studies , Disease Progression , Family Health , Humans , Prognosis , Signal Transduction/genetics , Signal Transduction/physiology , T-Lymphocytes/metabolism , T-Lymphocytes/physiology , T-Lymphocytes/virology , fas Receptor/genetics , fas Receptor/immunology
2.
Eur J Immunol ; 30(12): 3463-7, 2000 12.
Article in English | MEDLINE | ID: mdl-11093165

ABSTRACT

The recently cloned CD28-like molecule ICOS displays striking similarities with H4, characterized some years ago in the mouse and recently in humans. Both molecules are selectively expressed by activated and germinal center T cells, display similar structure, and display co-stimulatory activities. H4 displays lateral association with the CD3/TCR and is expressed by mature thymocytes. In the mouse, H4 is also expressed at high levels by thymic NKT cells that are resistant to negative selection. The aim of this work was to evaluate whether H4 and ICOS are the same molecule using the C398.4A (binding human and mouse H4) and F44 (binding human ICOS) monoclonal antibody (mAb) in parallel experiments on human T cells. ICOS and H4 displayed the same expression pattern in a panel of T cell lines and the same expression kinetics in phytohemagglutinin-activated T cells. C398.4A completely blocked cell staining by F44, whereas F44 partially blocked C398.4A. H4 and ICOS immunoprecipitates displayed identical SDS-PAGE patterns and H4 immunoprecipitation completely removed ICOS from cell lysates. Finally, the C398.4A mAb specifically stained cells transfected with the human or mouse ICOS. These data prove that H4 and ICOS are the same molecule and that F44 and C398.4A bind partially different epitopes.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/analysis , Immunodominant Epitopes/analysis , Protozoan Proteins , Animals , Antibodies, Monoclonal/immunology , Antigens, Differentiation, T-Lymphocyte/physiology , Cell Line , Humans , Immunodominant Epitopes/physiology , Inducible T-Cell Co-Stimulator Protein , Mice , Precipitin Tests , T-Lymphocytes/chemistry
3.
FASEB J ; 13(15): 2265-76, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10593874

ABSTRACT

CD38 displays lateral association with the HIV-1 receptor CD4. This association is potentiated by the HIV-1 envelope glycoprotein gp120. The aim of this work was to evaluate the CD38 role in T cell susceptibility to HIV-1 infection. Using laboratory X4 HIV-1 strains and X4 and X4/R5 primary isolates, we found that CD38 expression was negatively correlated to cell susceptibility to infection, evaluated as percentage of infected cells, release of HIV p24 in the supernatants, and cytopathogenicity. This correlation was at first suggested by results obtained in a panel of human CD4(+) T cell lines expressing different CD38 levels (MT-4, MT-2, C8166, CEMx174, Supt-1, and H9) and then demonstrated using CD38 transfectants of MT-4 cells (the line with the lowest CD38 expression). To address whether CD38 affected viral binding, we used mouse T cells that are non-permissive for productive infection. Gene transfection in mouse SR.D10.CD4(-).F1 T cells produced four lines expressing human CD4 and/or CD38. Ability of CD4(+)CD38(+)cells to bind HIV-1 or purified recombinant gp120 was significantly lower than that of CD4(+)CD38(-) cells. These data suggest that CD38 expression inhibits lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent viral binding to target cells.-Savarino, A., Bottarel, F., Calosso, L., Feito, M. J., Bensi, T., Bragardo, M., Rojo, J. M., Pugliese, A., Abbate, I., Capobianchi, M. R., Dianzani, F., Malavasi, F., and Dianzani, U. Effects of the human CD38 glycoprotein on the early stages of theHIV-1 replication cycle.


Subject(s)
Antigens, CD , Antigens, Differentiation/physiology , HIV-1/physiology , NAD+ Nucleosidase/physiology , T-Lymphocytes/virology , Virus Replication , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antigens, Differentiation/genetics , CD4 Antigens/genetics , CD4 Antigens/metabolism , HIV-1/metabolism , Humans , Membrane Glycoproteins , Mice , NAD+ Nucleosidase/genetics , Phenotype , Transfection , Tumor Cells, Cultured
4.
AIDS Res Hum Retroviruses ; 15(14): 1255-63, 1999 Sep 20.
Article in English | MEDLINE | ID: mdl-10505674

ABSTRACT

CD4 cross-linking by HIV gp120 triggers CD4+ T cell death. Several authors have suggested that this effect is mediated by CD95, but this possibility is debated by other authors. In a previous work, we found by co-capping that gp120(451) and gp120MN, but not gp120(IIIB), induce lateral association of CD4 with CD95 on the T cell surface. In this work, we used fluorescence resonance energy transfer to confirm that CD4/CD95 lateral association is induced by gp120(451), but not gp120(IIIB). Moreover, we found that gp120 ability to induce the CD4/CD95 association correlates with ability to induce cell death, since gp120(451) and gp120MN induced higher levels of cell death than did gp120(IIIB) in PHA-derived CD4+ T cell lines. CD95 involvement in gp120-induced cell death was confirmed by showing that gp120(451) and gp120MN did not induce death in CD4+ T cells derived from patients with autoimmune/lymphoproliferative disease (ALD) and decreased CD95 function. Cell death induced by gp120MN was inhibited by a recombinant CD95/IgG.Fc molecule blocking the CD95/CD95L interaction. However, inhibition was late and only partial. These data suggest that the gp120-induced CD4/CD95 association exerts a dual effect: an early effect that is independent of CD95L and may be due to direct triggering of CD95 by gp120, and a late effect that may be due to sensitization of CD95 to triggering by CD95L. In line with the former effect, cell treatment with gp120MN activated caspase 3 in the presence of Fas/IgG.Fc, which shows that cell death induced by gp120MN independently of CD95L uses the same pathway as CD95.


Subject(s)
Apoptosis , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/physiology , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Membrane Glycoproteins/metabolism , fas Receptor/metabolism , Apoptosis/immunology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Caspase 3 , Caspases/metabolism , Cells, Cultured , Fas Ligand Protein , Humans , Protein Binding
5.
Eur J Immunol ; 29(9): 2863-74, 1999 09.
Article in English | MEDLINE | ID: mdl-10508261

ABSTRACT

We have previously characterized mouse H4 (mH4), a surface glycoprotein recognized by the C398.4A monoclonal antibody. We now show that C398.4A also binds its human putative homolog (hpH4). Both hpH4 and mH4 (1) are selectively expressed by activated T cells and mature thymocytes, (2) are disulfide-linked dimers of two chains (29/37 kDa in humans, 25/29 kDa in mice), whose N-deglycosylation produces a single band at 20 - 21 kDa, and (3) display a low association with CD4 and the TCR. The expression pattern of hpH4 and its biochemical features showed that it is different from other known activation molecules, and this was confirmed when analysis of the tryptic digest of the hpH4 29-kDa band by peptide mass searching using matrix-assisted laser desorption ionization mass spectrometry did not reveal any significant homology with other molecules. In normal lymphoid tissue, hpH4 is expressed by T cells located at the periphery of lymph node germinal centers and paracortical areas. In T cell neoplasia, expression of hpH4 clusters with a subset of peripheral T cell lymphomas with a large-cell component, and with cases of angioimmunoblastic T cell lymphomas. Overall, these data provide evidence for a novel T cell activation molecule that could help in the phenotypic categorization of T cell malignancies.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/chemistry , Lymphoma, T-Cell/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/immunology , Cell Differentiation/immunology , Humans , Lymphocyte Activation/immunology , Lymphoid Tissue/immunology , Lymphoid Tissue/metabolism , Lymphoid Tissue/pathology , Lymphoma, T-Cell/immunology , Mice , Organ Specificity/immunology , Sequence Homology, Amino Acid , T-Lymphocyte Subsets/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Tumor Cells, Cultured
6.
Int Immunol ; 11(7): 1085-92, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10383941

ABSTRACT

CD44 is a family of mucin-like membrane proteins generated by alternative splicing of several exons, and participate in T cell adhesion and activation. CD44-mediated signaling involves activation of p56(lck) and leads to ZAP-70 phosphorylation. The aim of the present study was to identify the signaling pathways that follow CD44-triggered ZAP-70 phosphorylation and the molecular mechanisms underlying the CD44 interaction with p56(lck). We found that CD44 cross-linking by mAb in CD4(+) peripheral blood T cells promotes formation of a trimeric complex of Grb2, phospholipase (PLC)-gamma1 and a 36-38 kDa phosphoprotein, and the activation of PLC-gamma1. The amount of inositol triphosphate and the time kinetics of its generation were comparable to those following CD3 cross-linking. Co-capping, co-immunoprecipitation and fluorescence resonance energy transfer experiments showed that CD44 associates with CD4 and CD3 on the cell surface. This association suggests functional interplay between the CD4-TCR complex and CD44. In line with this possibility, we found that CD4 triggering by gp120, a natural ligand of CD4, potentiates CD44-mediated adhesion to hyaluronic acid. Moreover, Ca2+ mobilization induced by CD44 cross-linking by mAb was higher in a subclone of the HUT78 cell line expressing CD4 than in a non-expressing subclone.


Subject(s)
CD4 Antigens/physiology , CD4-Positive T-Lymphocytes/immunology , Hyaluronan Receptors/physiology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Signal Transduction/physiology , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/metabolism , Enzyme Activation , Humans , Hyaluronan Receptors/metabolism , Isoenzymes/metabolism , Lymphocyte Activation/immunology , Phospholipase C gamma , Phosphorylation , Phytohemagglutinins/pharmacology , Precipitin Tests , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/physiology , Type C Phospholipases/metabolism , ZAP-70 Protein-Tyrosine Kinase
7.
Blood ; 91(7): 2334-40, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9516131

ABSTRACT

GAS6 is a ligand for the tyrosine kinase receptors Rse, Axl, and Mer, but its function is poorly understood. Previous studies reported that both GAS6 and Axl are expressed by vascular endothelial cells (EC), which play a key role in leukocyte extravasation into tissues during inflammation through adhesive interactions with these cells. The aim of this work was to evaluate the GAS6 effect on the adhesive function of EC. Treatment of EC with GAS6 significantly inhibited adhesion of polymorphonuclear cells (PMN) induced by phorbol 12-myristate 13-acetate (PMA), platelet-activating factor (PAF), thrombin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not that induced by FMLP and IL-8. GAS6 did not affect adhesion to resting EC. Titration experiments showed that high concentrations of GAS6 were needed to inhibit PMN adhesion and that inhibition was dose-dependent at the concentration range of 0.1 to 1 microg/mL. One possibility was that high concentrations were needed to overwhelm the effect of endogenous GAS6 produced by EC. In line with this possibility, treatment of resting EC with soluble Axl significantly potentiated PMN adhesion. Analysis of localization of GAS6 by confocal microscopy and cytofluorimetric analysis showed that it is concentrated along the plasma membrane in resting EC and treatment with PAF induces depletion and/or redistribution of the molecule. These data suggest that GAS6 functions as a physiologic antiinflammatory agent produced by resting EC and depleted when proinflammatory stimuli turn on the proadhesive machinery of EC.


Subject(s)
Endothelium, Vascular/cytology , Granulocytes/cytology , Intercellular Signaling Peptides and Proteins , Proteins/pharmacology , Cell Adhesion/drug effects , Cells, Cultured , Cytokines/pharmacology , Endothelium, Vascular/metabolism , Granulocytes/metabolism , Humans , Membrane Proteins/pharmacology , Peroxisomal Biogenesis Factor 2 , Tetradecanoylphorbol Acetate/pharmacology
8.
J Immunol ; 159(4): 1619-27, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9257821

ABSTRACT

We have previously shown that HIV-1 glycoprotein 120 (gp120) induces CD4 association with several molecules on the surface of CD4+ lymphocytes. Since one of these molecules was CD38, involved in lymphocyte/endothelium interaction, this article examines the possibility that gp120/CD4 binding alters CD4+ T cell interaction with vascular endothelium in vitro and in vivo. Cocapping experiments showed that gp120 induced CD4 association with CD38, CD29, CD49d, and CD11a in peripheral blood CD4+ T cells. Two in vitro binding assays were used to evaluate the effect of gp120. A static binding assay, performed at 37 degrees C, evaluated stable interactions mediated by integrins, and a dynamic binding assay, performed at 4 degrees C on a rocking shelf, evaluated weak interactions mediated by constitutively active molecules such as selectins and CD38. Gp120 increased dynamic binding and inhibited static binding to the endothelium of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs suggested that the gp120 effect on dynamic binding involved CD38, CD31, and CD49d, whereas the effect on static binding involved CD11a and CD49d. In vivo experiments showed that treatment of 2D4 cells, a CD4- CD8- mouse T cell clone transfected with the human CD4, with gp120 increased their homing into the spleen, intestine, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Alteration of lymphocyte homing may contribute to immune deficiency in HIV-1+ patients by decreasing the probability of an encounter between Ags and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.


Subject(s)
CD4-Positive T-Lymphocytes/physiology , Endothelium, Vascular/cytology , HIV Envelope Protein gp120/physiology , HIV-1/immunology , Animals , Cell Adhesion , Cell Adhesion Molecules/physiology , Humans , Mice
9.
Int Immunol ; 9(8): 1141-7, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9263011

ABSTRACT

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.


Subject(s)
Antigens, CD/metabolism , CD4-Positive T-Lymphocytes/drug effects , HIV Envelope Protein gp120/pharmacology , Antibodies, Monoclonal , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1 , HLA Antigens/metabolism , Humans , Lymphocyte Subsets
10.
Blood ; 89(8): 2871-9, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9108407

ABSTRACT

Fas (CD95) is a transmembrane molecule that induces programmed cell death (PCD) of lymphocytes. We examined its function in children with chronic thrombocytopenia, serum autoantibodies, and lymphadenopathy and/or splenomegaly. We found that T-cell lines from six of seven patients with this autoimmune/lymphoproliferative disease (ALD) were relatively resistant to PCD induced by monoclonal antibodies to Fas. By contrast, Fas function was normal in control patients with typical chronic idiopathic thrombocytopenic purpura (ITP) without lymphadenopathy. The defect was not due to decreased Fas expression, nor to over-production of soluble forms of Fas. Moreover, it specifically involved the Fas system because PCD was induced in the normal way by methylprednisolone. Complementary DNA sequencing of the Fas gene did not identify any causal mutation in patients with ALD. This distinguished them from patients with the human autoimmune lymphoproliferative syndrome (ALPS), who carry mutations of the Fas gene. Moreover, patients with ALD did not show the peripheral expansion of CD4/CD8 double-negative T cells that characterizes the ALPS phenotype. Fas signaling involves activation of a sphingomyelinase-catalyzing production of ceramide. We found that ceramide-induced PCD was defective in patients with ALD and not in patients with typical chronic ITP. These data suggest that the ALD patient defect involves the Fas signaling pathway downstream from the sphingomyelinase and that Fas gene mutations and double-negative T-cell expansion are not the only signs of a defective Fas system.


Subject(s)
Apoptosis/genetics , Autoimmune Diseases/immunology , Lymphoproliferative Disorders/immunology , T-Lymphocyte Subsets/immunology , Thrombocytopenia/immunology , fas Receptor/physiology , Adolescent , Adult , Apoptosis/drug effects , Autoimmune Diseases/genetics , Ceramides/pharmacology , Child, Preschool , Consanguinity , DNA Mutational Analysis , DNA, Complementary/genetics , Female , Humans , Infant , Lymphocyte Activation/drug effects , Lymphoproliferative Disorders/genetics , Male , Methylprednisolone/pharmacology , Polymorphism, Single-Stranded Conformational , Purpura, Thrombocytopenic, Idiopathic/immunology , Signal Transduction , Sphingomyelin Phosphodiesterase/metabolism , Thrombocytopenia/genetics , fas Receptor/genetics
11.
Eur J Immunol ; 26(11): 2781-9, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8921969

ABSTRACT

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single-positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide-linked dimer, precipitating as a broad band at about 50-65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N-glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross-blocking, co-capping, co-modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co-stimulatory molecules such as CD69, CD2, Ly-6, CD25, OX-40, Mac-1 and LFA-1. By in vitro kinase assay, H4 was found to co-precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co-modulation and co-capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell-specific co-stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/physiology , Lymphocyte Activation , Receptor-CD3 Complex, Antigen, T-Cell/physiology , T-Lymphocytes/metabolism , Animals , Antibodies, Monoclonal/immunology , Cricetinae , Cricetulus , Immunophenotyping , Mice , Precipitin Tests , Protein-Tyrosine Kinases/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology
12.
J Immunol ; 156(2): 727-34, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8543826

ABSTRACT

Human CD38, a pleiotropic molecule with ADP-ribosyl cyclase activity, regulates activation and growth of several cell types. Its in vivo function is incompletely determined, mainly due to the lack of evidence concerning the existence of a single or multiple ligands. We recently observed that CD38 rules a selectin-type adhesion between lymphoid cells and HUVECs. A panel of murine mAbs raised against HUVEC included one (Moon-1) constantly blocking the CD38-mediated adhesion of several cell lines to HUVEC. Tissue distribution studies and an extended immunohistochemical analysis on the majority of normal human tissues revealed that the Moon-1 molecule displays a unique pattern of expression, being present at high levels on resting and activated vascular endothelium, on the majority of monocytes, platelets, NK cells, and to a lesser extent on T, B, and myeloid cells. The Moon-1 structure of an apparent molecular mass of 120 kDa proved to be a ligand for human CD38, as inferred by the direct binding observed when using a chimeric mouse CD8 alpha-human CD38 (mCD8 alpha-hCD38) molecule as a probe in Western blot experiments. Furthermore, Ab-induced modulation experiments highlighted an association between the Moon-1 molecule and human CD38 on the surface of cell lines coexpressing the two structures, which also share a common regulation system of surface expression. Finally, direct ligation of Moon-1 on T cell lines caused a relevant increase in the cytoplasmic concentration of calcium ([Ca2+]i).


Subject(s)
Antigens, CD , Antigens, Differentiation/metabolism , Carrier Proteins/physiology , Endothelium, Vascular/metabolism , N-Glycosyl Hydrolases/metabolism , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antibodies, Monoclonal/immunology , Blood Cells/metabolism , Carrier Proteins/biosynthesis , Carrier Proteins/immunology , Carrier Proteins/isolation & purification , Cell Adhesion , Gene Expression Regulation , Humans , Leukemia-Lymphoma, Adult T-Cell , Lymph Nodes/blood supply , Membrane Glycoproteins , Mice , Molecular Weight , Organ Specificity , Recombinant Fusion Proteins/metabolism , Signal Transduction , Tumor Cells, Cultured , Umbilical Veins
13.
Eur J Immunol ; 25(5): 1306-11, 1995 May.
Article in English | MEDLINE | ID: mdl-7539755

ABSTRACT

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4+ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4+ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59 CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26 and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4+ interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that th e associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.


Subject(s)
Antigens, CD/metabolism , Antigens, Surface/metabolism , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , HIV Envelope Protein gp120/pharmacology , HIV-1/immunology , Immunologic Capping/drug effects , Leukocyte Common Antigens/metabolism , Lymphocyte Activation , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/immunology , Antibodies, Monoclonal/immunology , Antigens, CD/immunology , CD59 Antigens , Complement System Proteins/physiology , Culture Media, Serum-Free , Dipeptidyl Peptidase 4/physiology , HIV Envelope Protein gp120/metabolism , HLA Antigens/immunology , Humans , Leukocyte Common Antigens/immunology , Macromolecular Substances , Membrane Glycoproteins/metabolism , Protein Binding/drug effects , Receptors, Antigen, T-Cell, alpha-beta/immunology , Signal Transduction , T-Lymphocyte Subsets/metabolism
14.
J Immunol ; 153(3): 952-9, 1994 Aug 01.
Article in English | MEDLINE | ID: mdl-7913116

ABSTRACT

CD38 is a type II transmembrane glycoprotein, which is widely used as a marker for immature and activated lymphocytes, as well as plasma cells. Although its functional role and natural ligand are not known, CD38 has been shown to transduce activation signals to lymphocytes. Our work shows that CD38 is preferentially expressed by CD4+CD45RA+ cells, but not by CD4+CD45R0+ cells. CD4+CD45RA+ cells are reported to respond poorly to stimuli acting through the CD3/TCR in vitro and to display unique migration pathways in vivo. Cross-linking of CD38 by mAb did not overcome the hyporesponsiveness of CD4+ resting/naive cells to several activation stimuli; in contrast, CD38 engagement by mAb specifically inhibited their binding with human vein endothelial cells. These data suggest that CD38 may play a role in lymphocyte migration. The same inhibitory effect was detected on the (human x mouse) hybrid cell line CP410.A10, which expresses human CD38, but not on its CD38- subclone CP14. CD38 mAb did not inhibit the conventional binding assay between endothelium and several human CD38+ T and B cell lines. However, the inhibition was apparent when the binding assay was performed at 4 degrees C on a rocking shelf, conditions that minimized integrin function. These data suggest that CD38 mediates weak cell binding to endothelium, which is effective even in dynamic conditions. These features are reminiscent of those exerted by selectins, which are adhesion molecules that account for leukocyte rolling on vascular endothelial cells and play an important role in lymphocyte homing.


Subject(s)
Antigens, CD , Antigens, Differentiation/analysis , CD4-Positive T-Lymphocytes/immunology , Endothelium, Vascular/cytology , Leukocyte Common Antigens/analysis , T-Lymphocyte Subsets/immunology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , CD4-Positive T-Lymphocytes/cytology , Cell Adhesion , Flow Cytometry , Humans , In Vitro Techniques , Membrane Glycoproteins
15.
Blood ; 83(8): 2198-205, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-7909242

ABSTRACT

Peripheral blood (PB) T cells from 56 patients with B-cell chronic lymphocytic leukemia (B-CLL) were analyzed by two- and three-color immunofluorescence (IF) to determine the expansion of distinct T-cell subsets and their relationship with the clinical and biological features of the disease. We detected the expansion of an unusual T-cell subpopulation expressing lower CD4 or CD8 levels (CD4lo, CD8lo) than classic T cells (CD4hi, CD8hi). This subpopulation also expressed low levels of the CD3/TCR alpha/beta complex and was CD19-CD13-CD14-. A phenotypic analysis probing the activation level of CD4lo, CD8lo, CD4hi, and CD8hi cells showed that they comprised increased counts of HLA-DR+, CD11b+, CD45R0+, and CD45RA+ cells. Subset expansion ranged from 2.1- to 13.6-fold. Statistical analysis showed that the size of some of these subsets was correlated to intrinsic features of the tumor. First, CD4loHLA-DR+ cell counts were higher in patients with stage A than those with stages B and C disease. Second, CD8loHLA-DR+ cell counts were higher in patients in stable remission than in those at diagnosis. Third, CD4loHLA-DR+, CD4loCD45R0+, CD4loCD45RA+, and CD4hiCD11b+ cell counts were higher in patients whose tumor cells expressed high levels of surface immunoglobulin (sIg) than in those expressing low levels. The involvement of CD4lo and CD8lo cells in most of these correlations suggests that they may be tumor-reactive cells. Similar cells described in human and murine autoimmune disease have been shown to be autoreactive anergic cells, which may derive from nonclassic pathways of T-cell development.


Subject(s)
CD4 Antigens/analysis , CD8 Antigens/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , T-Lymphocyte Subsets/immunology , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , CD11 Antigens , HLA-DR Antigens/analysis , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukocyte Common Antigens/analysis , Middle Aged , Phenotype
16.
J Immunol ; 151(8): 3961-70, 1993 Oct 15.
Article in English | MEDLINE | ID: mdl-7691935

ABSTRACT

CD73 is a molecule expressed by a subset of CD8+ human T lymphocytes and is involved in T cell activation. CD73 expression and function were analyzed in peripheral blood CD45RAhiCD45ROlo (naive) and CD45RAloCD45ROhi (memory) CD8+ cells. We found that CD73 was expressed by a majority of naive cells (74 +/- 12%), whereas fewer memory cells were CD73+ (29 +/- 10%). Moreover, CD73 was selectively expressed by the CD11b- subset of naive CD8+ cells, which were almost all CD73+. The same result was found on CD8+ cord blood lymphocytes, which prevalently display the naive phenotype. Naive CD8+ CD11b- cells were almost unresponsive to CD3 engagement, but this apparent anergy was completely overcome when CD3 and CD73 were simultaneously cross-linked by plastic-immobilized CD73 and CD3 mAb, showing that CD73 delivers an accessory signal that allows their activation via the CD3/TCR. This costimulatory signal was tenfold more potent than that induced by CD28 ligation. A phosphotyrosine analysis by Western blotting showed that cross-linking of CD73 induced the phosphorylation of two proteins with a molecular mass of approximately 28 and 100 kDa respectively, whereas ligation of CD3 induced phosphorylation of many substrates. When CD3 and CD73 were simultaneously triggered these substrates were hypophosphorylated. Because CD73 is linked to the cell surface by a GPI anchor, the transduction of this signal is probably mediated by a lateral interaction with transmembrane molecules. This hypothesis was assessed by cocapping, which showed that CD73 associates strongly with CD45RC, moderately with CD8, and weakly with CD3. These data suggest that CD73 signaling is coupled to both tyrosine kinase and phosphatase activities.


Subject(s)
5'-Nucleotidase/physiology , CD8 Antigens/analysis , Leukocyte Common Antigens/analysis , Lymphocyte Activation , T-Lymphocytes/immunology , CD3 Complex/physiology , Cells, Cultured , Humans , Phosphotyrosine , T-Lymphocyte Subsets/immunology , Tyrosine/analogs & derivatives , Tyrosine/physiology
17.
Eur J Immunol ; 22(2): 365-71, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1531636

ABSTRACT

Association of CD45 with surface molecules was investigated in human T lymphocytes by co-capping. CD45 appeared to be associated with the CD3/T cell receptor complex and with CD4 or CD8 molecules in memory, but not in naive T cells, as previously reported in the mouse. Associations of CD45 isoforms with accessory molecules were then identified with seven anti-CD45R monoclonal antibodies (mAb). An isoform-specific association pattern was observed: CD2 co-capped with CD45 molecules recognized by UCHL1 mAb (CD45R0). LFA-1 with molecules bound by 2H4 mAb (CD45RA), and both CD4 and CD8 with molecules reacting with MCA.347 mAb (whose isoform specificity was not known). Further information on the CD45 isoform(s) associated to CD4 and CD8 was sought by assessing the isoform specificity of MCA.347. Cross-competition experiments showed that it reacts with an epitope clearly different from those recognized by 2H4 and UCHL1, and only partially overlapping the PD7/26 epitope (CD45RB). Moreover, the competition between MCA.347 and PD7/26 was maximal in naive T cells and minimal both in memory T cells and in a subset expressing CD11b, a marker of granular lymphocytes. Immunoprecipitation experiments showed that MCA.347 binds to CD45 molecules with a molecular mass of 220, 205 and 190 kDa, the 190-kDa molecules not being recognized by 2H4, PD7/26 or UCHL1. These data indicate that MCA.347 recognizes amino acid sequences different from those coded by the exon A or B of the gene, and not expressed by CD45R0, suggesting that it binds to sequences coded by the exon C. In conclusion, this work shows that in human T cells different CD45 isoforms are associated to different surface molecules: LFA-1 is associated to CD45RA, CD2 to CD45R0 and CD4 and CD8 presumably to CD45RC. This peculiar behavior of CD45 suggests that it may play a crucial role in lymphocyte activation, probably by modulating the signals delivered to the cell by different receptor systems.


Subject(s)
Antigens, CD/metabolism , Histocompatibility Antigens/metabolism , Immunologic Memory , T-Lymphocytes/metabolism , Antibodies, Monoclonal/immunology , Antigens, CD/classification , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/metabolism , CD3 Complex , CD4 Antigens/metabolism , CD8 Antigens/metabolism , Histocompatibility Antigens/classification , Histocompatibility Antigens/immunology , Humans , Immunologic Capping , Leukocyte Common Antigens , Macromolecular Substances , Receptors, Antigen, T-Cell/metabolism , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/ultrastructure
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