Decreased function of Fas in patients displaying delayed progression of HIV-induced immune deficiency.

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.

The T cell activation molecule H4 and the CD28-like molecule ICOS are identical.

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.

Expression of the novel T cell activation molecule hpH4 in HIV-infected patients: correlation with disease status.

We have described hpH4, a surface glycoprotein selectively expressed by activated T cells and mature thymocytes and displaying weak lateral association with CD4. The hpH4 expression pattern and biochemical features, together with analysis of its tryptic digest by peptide mass searching using MALDI-MS, suggested that it is a novel molecule. The aim of this work was to evaluate the peripheral blood T cell expression of hpH4 in HIV-infected patients and the interplay between HIV gp120 and hpH4, since both molecules interact with CD4. hpH4 expression during HIV-1 infection was evaluated by assessing 55 patients at various disease stages and following up 3 patients with primary infection and 3 patients with AIDS. hpH4 expression displayed a peak in the early phase of primary infection, dropped to control levels in the asymptomatic phase, and was newly expressed, at low levels, as AIDS developed. The expression kinetics were different than those shown by HLA-DR, CD25, and CD38. The most striking findings were the transient hpH4 expression peak displayed in the earliest stage, which was unique for hpH4. Incubation of T cells from normal donors with HIV gp120 induced transient hpH4 expression in resting CD4+ T cells and potentiated the hpH4 lateral association with CD4 in activated T cells. Moreover, hpH4 triggering inhibited gp120-induced death of CD4+ cells. Therefore, H4 expression may be a response to avoid apoptosis induced by HIV products.

The cell death-inducing ability of glycoprotein 120 from different HIV strains correlates with their ability to induce CD4 lateral association with CD95 on CD4+ T cells.

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.

Characterization of a novel human surface molecule selectively expressed by mature thymocytes, activated T cells and subsets of T cell lymphomas.

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.

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.

GAS6 inhibits granulocyte adhesion to endothelial cells.

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.

Modulation of lymphocyte interaction with endothelium and homing by HIV-1 gp120.

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.

gp 120s derived from four syncytium-inducing HIV-1 strains induce different patterns of CD4 association with lymphocyte surface molecules.

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.

Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation.

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.

Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor.

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.

Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120.

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.

Interaction between endothelium and CD4+CD45RA+ lymphocytes. Role of the human CD38 molecule.

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.