Inhibition of cellular DNA synthesis by reovirus occurs through a receptor-linked signaling pathway that is mimicked by antiidiotypic, antireceptor antibody.

Mammalian reovirus type 3 binds to a 67-kD surface glycoprotein on the membrane of neuronal cells. This interaction initiates the infective reovirus cycle. The physiological function of this virus receptor is not known, however, initial studies illustrate a striking structural and antigenic homology to the beta adrenergic receptor family. The earliest known pathologic effect of reovirus type 3 is an inhibition of host cell DNA synthesis within 8-10 h after virus attachment. The studies reported here demonstrate that binding and aggregation of reovirus receptor molecules provides the signal for this inhibitory process. Inhibition of DNA synthesis in the neuroblastoma cell line B104.G4 was unaffected by using replication-defective virus or when lysosomal processing of normal virus was blocked. Inhibition was mimicked by an antiidiotypic, antireceptor mAb. Inhibition was not observed when monovalent mAb fragments were bound to receptors, but was reconstituted when these fragments were aggregated by the addition of anti-Ig. The signal for the inhibitory effect was generated within the first 60 min after mAb binding. These observations demonstrate that reovirus and antiidiotypic pathogenicity can result from the perturbation of membrane proteins that may perform normal physiological functions.

Syngeneic monoclonal antiidiotype can induce cellular immunity to reovirus.

A syngeneic monoclonal antiidiotypic antibody was generated in BALB/c mice after repeated immunization with a BALB/c monoclonal anti-reovirus hemagglutinin (HA) antibody. The resultant syngeneic monoclonal antiidiotypic antibody, in the absence of adjuvant, was found to be capable of priming both BALB/c (H-2d, Igh-1a) and C3H/Hej (H-2k, Igh-1j) mice for Lyt-1+- and Lyt-2+-dependent responses against the mammalian reovirus. By the use of intertypic reassortants and variant virus analysis, the specificity of the response was finely mapped to the neutralization domain of the viral hemagglutinin (HA). Using purified monoclonal antiidiotype, we were able to compare the potency of antiidiotype to virus in terms of induction of immunity. 8 X 10(8) protein molecules were able to prime for cellular responses to reovirus. These studies indicate that in the reovirus system, T cells and B cells share idiotypic configurations, and that antiidiotypic antibodies of the type described herein may be useful in the development of vaccines against certain viral infections.

Neonatal exposure to thymotropic gross murine leukemia virus induces virus-specific immunologic nonresponsiveness.

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses.

Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs cardiac allograft survival in mice.

Administration of the monoclonal antibody M7/20, which binds to the murine interleukin-2 (IL) receptor, significantly prolongs cardiac allograft survival in two H-2-incompatible strain combinations of inbred mice. The results support the important role of the IL-2 receptor in the mechanism of graft rejection, and suggest its suitability as a target for immunosuppressive therapy.

Bone marrow CD34(+) cells and megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV.

CD34(+) cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34(+) cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34(+) cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34(+) cells and CD34(+)KIT(+) cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1alpha, and MIP-1beta and that IFN-gamma stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34(+)KIT(+) cells, even by RT-PCR. Conditioned media from CD34(+) cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34(+) cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34(+) cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.

Differential regulation of glycosylated phosphatidylinositol subtypes by insulin.

Glycosylated phosphatidylinositol (gly-Pl) molecules have been implicated as precursors for insulin-sensitive second messengers (1-4) and lipid-anchored membrane proteins (5-9). The relationship between the diverse functions of these lipids and their predicted structural heterogeneity within gly-Pl subtypes was examined in human T lymphocytes. Four subtypes of gly-Pl molecules were identified in T lymphocytes after separation over high-performance thin-layer chromatography by sensitivity to Pl-specific phospholipase C and nitrous acid. Antibody probes of the glycan domain of gly-Pl were developed and used to assess the partial sensitivity of gly-Pl to insulin action. This analysis showed that the effects of insulin are linked to differential utilization of only two of the four gly-Pl subtypes in T lymphocytes. Polar fragments of this reaction were identified in extracellular supernatants from insulin-treated cells. The biological significance of insulin-dependent gly-Pl hydrolysis was demonstrated by insulin and inositol phosphoglycan regulation of glucose metabolism in intact lymphocytes. These results support the hypothesis that multifunctional roles of gly-Pl are served by discrete gly-Pl populations and that metabolites of gly-Pl subsets participate as signaling elements in insulin action.

The role of the immune response in MULV-induced lymphomagenesis.

Although the exact mechanisms of murine leukemia virus (MuLV)-induced lymphomagenesis have yet to be elucidated, it is clear that the immune reponse to viral proteins plays a critical role in this disease process. The parameters for lymphomagenesis are governed by an inverse relationship between viral persistence and immune responsiveness. MuLV have evolved ways to avoid immune detection either by altering their own genome or by altering the host environment. In addition, the intrathymic replication of MuLV during thymocyte maturation and immune selection plays an important role in T cell repertoire development and immune inhibition. These viruses have served as a highly effective experimental model in understanding the many pathways by which MuLV have overcome immune detection and thereby led to lymphomagenesis.

Neonatal HIV-1 thymic infection.

The thymus is the primary site of T cell ontogeny and selection during fetal and neonatal development. Previous studies have established that the thymus is also a site of HIV-1 infection, as early as the first trimester of pregnancy. Alteration of the thymocyte maturation process by HIV-1 could impact on the peripheral T cell population and interfere with immune responses. A neonatal thymic organ culture system was established to study HIV-1 infection within the thymus. We have shown that this primary tissue isolate can support a productive HIV-1 infection. Infection occurred without detectable thymocyte cytopathology. The ability to infect the developing thymocyte within an intact micro environment will enable us to further establish the kinetics of acute HIV-1 thymic infection and its consequences on lymphocyte maturation.

Manipulation of the immune response by foreign gene expression in the thymus.

Retroviral gene transfer vectors have been developed for optimal in vivo gene therapy. Ideally, these vectors should target gene expression specifically to selected tissues or organs. Our studies focus on the development of retroviral vectors for gene delivery to the thymus. The goal of these studies is to utilize thymic expression of exogenous genes to manipulate the immune repertoire. We have characterized the selective thymic tropism of a molecular clone of Gross murine leukemia virus, GD-17, to thymic medullary epithelial cells using immunohistochemical staining and confocal microscopy. Specific expression of viral antigens in the thymus lead to the induction of immunologic tolerance to GMuLV proteins. This tissue specific vector may thus be used to study the requirements of epithelial mediated tolerance induction, and provide a more efficient tool for gene therapy.

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.

Diminished tyrosine protein kinase activity in T cells unresponsive to TCR stimulation.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32-3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32-3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32-3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 cross-linking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 zeta-chain, recently observed in J32-3.2. These data are consistent with the idea that src-related kinases phosphorylate the zeta-chain, which in turn recruits ZAP-70 required to sustain the signal.

The limited infectability by R5 HIV of CD34(+) cells from thymus, cord, and peripheral blood and bone marrow is explained by their ability to produce beta-chemokines.

The resistance of human bone marrow (BM) CD34(+) cells to human immunodeficiency virus (HIV) infection is at this point not fully understood. Recently we reported that the chemokines MIP-1alpha, MIP-1beta, and RANTES secreted by BM-derived CD34(+) cells may compete with the macrophagotropic HIV (R5 HIV) strain for the CCR5 coreceptor.In this study we extended our previous observations and examined various lympho-hematopoietic CD34(+) cells isolated from thymus (Th), cord blood (CB), mobilized peripheral blood (mPB), and BM for the expression of beta-chemokines binding to CCR5, i.e., MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, and MCP-4, and the alpha chemokine SDF-1 (binding to CXCR4) as these chemokines may compete with the R5 and X4 HIV strains, respectively, for entry into cells. We found that Th-, CB-, mPB-, and BM-derived CD34(+) cells express mRNA transcripts for all the beta-chemokines tested but not for SDF-1. Using sensitive ELISA assays we found that although MIP-1alpha and MIP-1beta proteins were secreted by all the lympho-hematopoietic CD34(+) cells tested, RANTES was detectable only in media conditioned by BM- and CB-derived CD34(+) cells and not Th-derived cells. However, media conditioned by BM-, mPB- and Th-derived CD34(+) cells protected the T lymphocytic cell line (PB-1) from infection by the R5 but not the X4 HIV strain. Hence this study demonstrates that beta-chemokines are secreted by lympho-hematopoietic CD34(+) cells originating from various sources and that these endogenously secreted chemokines may limit entry of the R5 HIV strain into the cells by competing for the CCR5 coreceptor.

Selective thymocyte depletion in neonatal HIV-1 thymic infection.

OBJECTIVE: To determine the impact of HIV-1 infection on thymocyte development, and the role of thymic infection on the pathogenesis of neonatal HIV-1 infection. DESIGN AND METHODS: The consequences of thymic infection by HIV-1 were examined by comparative histologic and molecular analyses of an asymptomatic, HIV-1-seropositive 3-day-old subject, versus age- and treatment-matched controls. The presence of replicating virus was established by in situ hybridization with specific molecular probes to HIV-1. The distribution of thymocyte subsets was determined by quantitative flow cytometry following staining with antibodies to CD4 and CD8 cell surface proteins. RESULTS: The results show clear evidence of severe thymic involution, HIV-1 infection of thymocytes, and selective depletion of thymocyte subpopulations. The consequences of HIV-1 infection were a marked depletion of CD3+CD4+ CD8hi and CD3+CD4+CD8- cells. The phenotype of the residual thymic lymphoid population was predominantly that of immature CD3-CD4-CD8- double negative and CD3+CD4+CD8lo cells. CONCLUSION: Changes in the distribution of thymocyte subsets suggests a role for thymic involvement in the pathogenesis of HIV-1 infection in neonates.

Viral pathogenesis and immunity within the thymus.

Replication of viruses within the thymic microenvironment may have a unique impact on viral persistence and pathology. The author's laboratory has studied thymic infection by both human and murine retroviruses. For human lentiviruses, such as HIV-1, the consequences of persistent thymic replication are frequently a severe disruption of the normal processes of thymopoiesis and potentially of progression to AIDS. Murine retroviruses, such as Gross murine leukemia virus, establish persistent infection with less cytopathic, but no less devastating effects. These include the alteration of immune recognition to retroviral antigens by the peripheral immune response, the thymic persistence of virus, and the establishment of viral-induced thymic leukemia. This article summarizes the analysis of both the common and distinctive means of pathology induced by these two retroviral families with particular attention on the influence and impact of the thymus as a unique site of virus replication.

Regulation of lymphocyte growth by antagonists of interleukin-2 or its cellular receptor.

The dependence of T cell proliferation on the production, binding and utilization of the lymphokine growth factor IL-2 has fostered the development and testing of new classes of drugs which act to either inhibit IL-2 production or the interaction of IL-2 with its cellular receptor. We have reviewed evidence which documents the potent immunosuppressive effects of inhibitors of IL-2 synthesis and secretion, such as Ciclosporin A, and of anti-IL-2 receptor MAb. The similarities of the potency and specificity of these agents and their effectiveness in a wide range of clinical settings encourage further studies on their mechanism of action. Perhaps the most dramatic similarity is the induction of long-term nonresponsiveness after drug removal to antigens present at the time of drug therapy. This observation has profound importance both on clinical manipulation with these agents and n the origins and maintenance of self-tolerance.

Impaired immune responsiveness is an essential component in persistent central nervous system infection with gross murine leukemia virus.

Exposure of newborn mice to Gross murine leukemia virus (GMuLV) results in persistent viral infection of the central nervous system (CNS) white matter. Animals exposed to virus as neonates showed a marked depression in GMuLV-specific B lymphocyte function as evidenced by significant decreases in adult and neonatal anti-GMuLV antibody levels. Immunohistochemical analyses showed that the sites of GMuLV infection in the CNS were also devoid of major histocompatibility complex (MHC) class I and II protein expression, although transplantation of GMuLV-infected brain tissue to the kidney capsules of immunocompetent mice induced a potent mononuclear cell graft infiltrate. These results indicate that persistent GMuLV infection of the CNS is linked to both impairment of anti-GMuLV peripheral immune responses and deficient antigen-presenting cell function within the CNS.

The effect of anti-interleukin-2 receptor monoclonal antibody on allograft rejection.

During immune response to an allograft, activated T cells express a number of cell surface activation antigens, among them the membrane receptor for the lymphokine interleukin 2 (IL-2). As the IL-2 receptor is not present on resting T cells, it offers an attractive target for potentially specific immunosuppressive therapy. The rat monoclonal antibody M7/20, which binds to the murine IL-2 receptor, was studied for its effect on allograft survival in two H-2-incompatible strain combinations in inbred mice. Treatment with M7/20 for 10 days markedly prolonged survival of vascularized, heterotopic heart allografts in both strain combinations, with indefinite graft survival in 50% of recipients. The same treatment significantly prolonged skin allograft survival in one of the two combinations. The results support the important role of the IL-2 receptor in the mechanism of graft rejection and confirm its suitability as a target for immunosuppressive therapy in transplantation.

Multifunctional roles of glycosyl-phosphatidylinositol lipids.

The results presented here indicate that GPI lipids are a structurally and functionally diverse molecular family. Despite new detailed information on the structures of GPI-anchored proteins, there is relatively scant information on the structure of free-GPI. Thus, little is known of the relationships between GPI structures and the mechanism of their biological effects. For example, there is no distinction at the structural level between hormone-sensitive free-GPI and those that serve as precursors for protein-GPI. Nor is there precise biochemical data on the mechanism and importance of free-GPI in hormone signaling, or the signaling roles that GPI anchors play in protein function. The T-cell activation cascade is an ideal system for studying both forms of GPI and their derivatives. The study of GPI molecules in T lymphocytes offers the exciting possibility of addressing questions on the structure, function, genesis, and regulation of both free- and protein-GPI molecules in a single cell type. The detection of multiple protein-GPI and free-GPI forms, and of hormone-sensitive GPI, provides the first approach to these issues. For the moment, the potential for biochemical signaling by intact GPI or its metabolites is enormous. If significant progress is to be made, the structures of hormone sensitive free-GPI must be elucidated. Only then can we precisely define the roles of these molecules in the regulation of cell metabolism and proliferation.

The role of diacylglycerol kinase activation and phosphatidate accumulation in interleukin-2-dependent lymphocyte proliferation.

The biological effects of interleukin-2 (IL-2) were examined in T lymphocytes. IL-2 binding induced the rapid activation of diacylglycerol kinase in both cytosolic and membrane subfractions. This enzyme utilized diacylglycerol from multiple endogenous and exogenous sources for the synthesis of phosphatidic acid. Phosphatidic acid was, in turn, shown to stimulate the accumulation of c-myc mRNA and augment cellular proliferation when added to IL-2-dependent cell lines. These results link previous observations of IL-2 and glycosylphosphatidylinositol-dependent diacylglycerol production to phosphatidic acid accumulation, and suggest that diacylglycerol kinase activation is part of an intricate IL-2 signaling cascade that utilizes phosphatidic acid as an effector molecule.