Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45.

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.

Enhancement of the immune response to hepatitis B virus vaccine by antigen specific IgM.

In the present study, modulation of antibody response induced by Hepatitis B virus vaccine-IgM complex was investigated. Purified IgM-type anti HBv monoclonal antibody (1B11) was complexed to commercially available HBv vaccine (GenHevac B Pasteur, France) at varying concentrations of HBsAg (0.5, 1, 1.5 microg of HBsAg) and used to immunize BALB/c mice. An enhanced humoral immune response was obtained with the HBv vaccine-IgM complex at all the doses compared with those immunized by vaccine alone and increased antibody levels were observed with increased concentrations of HBsAg in vaccine formulation. Immunization with HBv vaccine-IgM complex mostly generated IgG-type antibodies in the sera of mice, and also gave rise to the development of hybrid cells which predominantly produced IgG-type monoclonal antibodies. Hence, results from this study indicate that 1B11 can be effectively used to obtain a better immune response to HBv vaccine.

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity.

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.

Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth.

We report here the use of viral fusogenic membrane glycoproteins (FMGs) as a new class of therapeutic genes for the control of tumor growth. FMGs kill cells by fusing them into large multinucleated syncytia, which die by sequestration of cell nuclei and subsequent nuclear fusion by a mechanism that is nonapoptotic, as assessed by multiple criteria. Direct and bystander killing of three different FMGs were at least one log more potent than that of herpes simplex virus thymidine kinase or cytosine deaminase suicide genes. Transduction of human tumor xenografts with plasmid DNA prevented tumor outgrowth in vivo, and cytotoxicity could be regulated through transcriptional targeting. Syncytial formation is accompanied by the induction of immunostimulatory heat shock proteins, and tumor-associated FMG expression in immunocompetent animals generated specific antitumor immunity.

Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion.

Studies of naturally occurring polymorphisms of the CCR5 gene have shown that deletion of the functional receptor or reduced expression of the gene can have beneficial effects in preventing HIV-1 infection or delaying disease. Because these polymorphisms are found in otherwise healthy people, strategies that aim to prevent or limit expression of CCR5 should be beneficial in the treatment of HIV-1 disease. To test this approach we have developed a CCR5-specific single-chain antibody that was expressed intracellularly and retained in the endoplasmic reticulum. This CCR5-intrabody efficiently blocked surface expression of human and rhesus CCR5 and thus prevented cellular interactions with CCR5-dependent HIV-1 and simian immunodeficiency virus envelope glycoprotein. Intrabody-expressing cells were shown to be highly refractory to challenge with R5 HIV-1 viruses or infected cells. These results suggest that gene therapy approaches that deliver this intracellular antibody could be of benefit to infected individuals. Because the antibody reacts with a conserved primate epitope on CCR5 this strategy can be tested in nonhuman lentivirus models of HIV-1 disease.

A novel factor produced by placental cells with activity against HIV-1.

The factors controlling the dynamics of HIV-1 transmission from mother to infant are not clearly known. Previous studies have suggested the existence of maternal and placental protective mechanisms that inhibit viral replication in utero. Preliminary studies from our laboratory revealed that supernatant from placental stromal cells protected HIV-1-infected PBMC from virus-induced apoptosis and suppressed virus production. We have attempted to characterize the antiviral activity of this placental factor (PF) and delineate the stages of HIV-1 replication affected. This activity was not due to the presence of any known cytokine reported to have anti-HIV effect. Direct exposure to PF had no suppressive effect on the infectivity of cell-free HIV-1, and envelope-mediated membrane fusion appeared to be unaffected. Western blot analysis of HIV-1 from infected PBMC treated with PF revealed that expression of all viral proteins was reduced proportionately, both intracellularly and in released virions. However, exposure of HIV-1-infected cells to PF resulted in production of virions with 10-100-fold-reduced infectivity. PF-treated virions contained two- to threefold reduced ratios of cyclophilin A:Gag protein as compared with untreated virus. Reduced cyclophilin A content resulting in decreased binding of cyclophilin A to Gag could account, in part, for the observed reduction in infectivity. Our results suggest that placental cells produce an antiviral factor that protects the fetus during gestation and may have therapeutic potential.

Transplantation of Fu/HC-incompatible zooids in Botryllus schlosseri results in chimerism.

The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vasculature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allorecognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids between Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that (1) instead of the expected rejections (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in conjunction with the normal blastogenic cycle; and (2) donor-recipient chimerism is established after complete morphological elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony.

Syncytium-inhibiting monoclonal antibodies produced against human T-cell lymphotropic virus type 1-infected cells recognize class II major histocompatibility complex molecules and block by protein crowding.

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).

Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.

Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.

A new method to generate quadromas by electrofusion and FACS sorting.

Bispecific monoclonal antibodies (bsMAbs) are unique molecules incorporating two different paratopes in a single antibody molecule. BsMAbs can be useful in different areas of research as well in clinical applications. Traditionally, bsMAbs are produced by hybrid-hybridomas that are generated by the fusion of two pre-established hybridomas. The development of such hybrid-hybridomas can be difficult and time-consuming. Here, we introduce a new technique to generate such hybrids, electro-FACS-fusion. In this procedure, before the electrofusion, one of the hybridomas is labeled with fluorescein isothiocyanate (FITC) and the other with tetramethylrhodamine isothiocyanate (TRITC). The mixture of cells is then electrofused, and cells exhibiting dual fluorescence are selected by fluorescence activated cell sorting (FACS). The fused cells are directly plated in microplates for clonal growth. Using this technique, we produced three new hybrid-hybridomas secreting bsMAb that could be used for the next generation of immunoassays.

Mouse myoblasts can fuse and form a normal sarcomere in the absence of beta1 integrin expression.

Antibody perturbation experiments suggested that migration, terminal differentiation and fusion of myoblasts are dependent on beta1 integrin expression. In addition, several studies have postulated that beta1 integrins have a role in the formation of sarcomeres. In the present report we have analysed skeletal myogenesis in wild-type/beta1-null chimeric mice and beta1-null embryoid bodies. Trunk and limbs of beta1-null chimeric mice contained muscle tissue composed of normal and beta1-null myoblasts indicating that all myotomic sublineages can form, migrate to their peripheral targets and fuse in the absence of beta1 integrin expression. Pure populations of beta1-null myoblasts and satellite cells isolated from beta1-null chimeric embryos and chimeric newborn mice, respectively, were able to differentiate in vitro and to fuse into multinucleated myotubes. Quantitative and qualitative comparisons between normal and beta1-null myoblasts revealed no apparent difference in their capacity to terminally differentiate and fuse. Furthermore, beta1-null myotubes developed sarcomeres which were indistinguishable from wild-type controls. When normal and beta1-null ES cells were differentiated into embryoid bodies, they contained fully differentiated myotubes with normal sarcomeres and normal deposition of costameric components. However, formation of beta1-null myotubes was delayed and was less efficient in beta1-null embryoid bodies than in wild-type controls. High expression of alphav integrin subunit at the tips of normal as well as beta1-null myotubes indicated that the lack of beta1 integrins did not result in a significant redistribution of alphav-containing receptors.

CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells.

CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil differentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macrophage-inflammatory protein-1alpha and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1alpha, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are differentially regulated by IL-5 in this model.

Neutralizing mechanism of a monoclonal antibody against Japanese encephalitis virus glycoprotein E.

The neutralization of Japanese encephalitis virus (JEV) was studied using JEV-specific neutralizing (NT) monoclonal antibody (MAb) 503 that recognizes the envelope glycoprotein. Analysis using radiolabeled JEV and observations by confocal laser microscopy and electron microscopy indicated that the NT and protection activities of MAb 503 did not result from the prevention of the first step of JEV infection, binding of virus to the cell surface. Treatment with MAb 503 strongly inhibited JEV-induced cell fusion and internalization of JEV into the cells, and resulted in enhanced release of JEV-RNA from the cells. These observations suggested that the NT activity of MAb 503 is involved in the later steps of JEV infection.

Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity.

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.

Monoclonal antibodies define a cellular antigen involved in HTLV-I infection.

The exact mechanism by which the human T cell leukemia viruses (HTLV) infects target cells remains unclear; although some molecules have been identified to be important in viral infection and entry. To investigate these phenomena, we generated a panel of monoclonal antibodies (MAb) against a B cell line (BJAB-WH) which is highly permissive for infection with HTLV. These MAb have been used to further characterize the membrane molecules important for HTLV infection. Three of these MAb designated 4.2.3, 3.3.10, and 11.2.3 were capable of inhibiting syncytium formation induced in human B and T cell lines (i.e., BJAB-WH and SupT-1, respectively) by co-culture with HTLV-I infected MT-2 cells. All of these MAbs immunoprecipitated a 80-85 kDa antigen from the lysates of metabolically labeled BJAB-WH but not from BJAB-CC/84, a noninfectible target cell. The binding of these MAb with different HTLV target cells was analyzed and compared with binding of polyclonal monospecific antisera to the same cell lines. A 80-85 kDa membrane glycoprotein was isolated with an immunoaffinity chromatographic column constructed with MAbs 4.2.3 and 3.3.10. This cellular antigen was capable of inhibiting HTLV I/MT-2 induced fusion. This is the first direct demonstration that a 80-85 kDa cellular glycoprotein is directly involved in HTLV I/II infection and syncytium formation.

Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection.

Dendritic cells (DCs) are potent professional APCs capable of presenting Ag in the context of costimulatory signals necessary for T cell activation. Although tumor cells express target Ags, they are generally incapable of stimulating an immune response. We show that the short term physical interaction of DCs and tumor cells, with or without cell fusion, results in rapid, efficient, and stable DC-tumor cell association. Immunization of naive mice with unselected, irradiated DC-tumor cell conjugates induces tumor-specific CD8+ cytotoxic T cells and protection from lethal tumor challenge. Furthermore, the immunogenicity of this cellular vaccine is dependent on the physical interaction of DCs and tumor cells before injection. Immunization with DCs and tumor cells after physical interaction can result in the regression of established tumors and persistent antitumor immunity. These results suggest that immunization with DC-tumor cell vaccines may be a simple, rapid, and potent strategy for tumor immunotherapy.

Intranasal immunization of a DNA vaccine with IL-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens.

A DNA vaccine constructed with the CMV promoter conjugated to env gp160 and rev genes has been shown to induce an effective Th1-type immune response when inoculated via an intramuscular route. In the present study, we obtained high levels of both humoral and cell-mediated immune activity by intranasal administration of this DNA vaccine. The production of mucosal IgA Ab in feces and vaginal fluid was stimulated significantly by intranasal DNA administration. This route of administration resulted in a significant level of HIV-1-neutralizing Abs in feces and serum. Cytokine assays revealed that intranasal administration of this DNA vaccine induces a Th2-type immune response. Interestingly, cationic liposomes greatly enhanced these activities. Abs against HIV-1 were present for at least 10 mo. Coadministration of the DNA vaccine with IL-12- and granulocyte/macrophage-CSF-expressing plasmids induced high levels of HIV-specific CTLs and an increase in delayed type hypersensitivity when administered by the intranasal route. These results clearly demonstrate that intranasal administration of this DNA vaccine with liposomes, together with IL-12- and/or granulocyte/macrophage-CSF-expressing plasmids, induces a strong level of anti-HIV-1 immune response.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate.

The beta-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4(+) target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4(+) CCR-5(+) HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the "nonsyncytium-inducing," primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the "syncytium-inducing" HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the beta-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1beta (MIP-1beta) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these beta-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and beta-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

[Role of the conformation changement of CD4 in the HIV-cell fusion]. / Rôle du changement de conformation du CD4 lors de la fusion VIH/cellule.

It is well known that the gp120-gp41 complex undergoes a conformational change after CD4 binding. It is likely that CD4 undergoes a conformational change as well. Recently, a calculation of the normal modes of the two N-terminal domains of CD4 has shown that a hinge-bending motion of one of these domains with respect to the other may occur. In the present study, results obtained previously are verified with two other normal mode calculations, starting from crystallographic structures of different origin. A scheme describing the first steps of the process leading to cell infection by human immunodeficiency virus (HIV) is then proposed. It rests upon the idea that CD4 and gp120-gp41 conformational changes allow for bringing the cell and virus membranes closer to each other.

Development and characterization of monoclonal antibodies specific for amylin.

Highly selective monoclonal antibodies to the peptide hormone human amylin have been produced and characterized. These antibodies are produced by hybridomas resulting from the fusions of BALB/c-derived myelomas and splenocytes from either inbred or outbred mouse strains. Certain of these antibodies recognize epitopes at the amino-terminus or the amidated carboxy-terminus, as well as conformational epitopes within the central region of the 37 amino acid peptide. Several of these antibodies show less than 0.1% cross-reactivity with related peptide hormones such as calcitonin and calcitonin gene-related peptide (CGRP) and have apparent affinities in the low nanomolar range. Antibody pairs were selected for use in two-site assays for the direct measurement of endogenous amylin and the synthetic human amylin analogue, pramlintide (25, 28, 29 tripro-human amylin), which is presently under clinical investigation for improving glucose control in patients with both Type I and Type II diabetes treated with insulin.