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The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.

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.

The relationship between the process of neural induction and the control of tyrosinase gene expression in the cells that derive from the neural crest of amphibians has been examined at the molecular level. [3H] Tyrosinase cDNA was utilized as a probe to measure the levels of tyrosinase RNA transcripts present in Rana pipiens embryos from the time of fertilization through Stage 25 of cleavage (operculum complete, 240 h) and to correlate these levels with those published for the rate of tyrosinase protein synthesis. R. pipiens [3H]tyrosinase cDNA was synthesized from a purified tyrosinase mRNA template using avian myeloblastosis virus reverse transcriptase and was enriched for full length copies by preparative polyacrylamide gel electrophoresis. This cDNA product was estimated to represent greater than 90% by length of tyrosinase mRNA and hybridized to tyrosinase mRNA to greater than 97% within two orders of magnitude of R0t values. The extent of hybridization of [3H]tyrosinase cDNA to total embryonic RNA throughout early development paralleled the rate of synthesis of tyrosinase protein. Tyrosinase RNA transcripts were first detected at Stage 12-13 (0.0032% of total RNA) and rose to maximal levels by Stage 19 (0.011%). This represents a 50-fold increase from preinduction levels. These results are consistent with a model which predicts that one of the early events in the development of neural crest derivatives is the transcriptionally dependent accumulation of functionally mature tyrosinase mRNA.

Anti-murine interleukin 2 (IL-2) receptor monoclonal antibodies (mAb) were made from rats immunized with murine cytotoxic lymphocytes. One mAb, designated M7/20, strongly inhibited the proliferation of both IL-2 dependent CTLL-2 cells and concanavalin A (Con A)-induced T-cell blasts. Inhibition was linearly dependent on the concentrations of both M7/20 and IL-2. Utilizing FACS analysis, M7/20 was shown to bind selectively to mitogen-activated T lymphocytes and, to a lesser degree, to activated B lymphocytes. 125I-Labeled M7/20 binding assays indicated that 48-hr Con A-induced T-cell blasts possessed 89,000 binding sites/cell with a Kd of 1.2 X 10(-9) M. Competitive binding analyses indicated that M7/20 and IL-2 occupy the same or overlapping cell surface sites. Preliminary biochemical characterization of M7/20 immunoprecipitates of detergent extracts from both surface-iodinated and internally D-[3H]glucosamine-labeled T lymphoblasts indicated that the murine IL-2 receptor is an N-glycosylated 58,000-Da glycoprotein. Together these results suggest that mAb M7/20 binds at or near the IL-2-binding epitope on the murine IL-2 receptor and, thus, upon manipulation may act as an IL-2 agonist.

The cellular response to many external stimuli is initiated by soluble mediators or factors which interact with specific membrane receptors. The consequences of these interactions are activational signals which are then transduced across the plasma membrane resulting in an altered cellular response. The mechanism by which signals generated at the cell membrane are transduced and translated into the activational program of the cell is currently an area of intense interest. Perhaps the most actively studied cell with respect to receptor-mediated control of growth and differentiation is the lymphocyte. Phenotypically and functionally, lymphocytes are divided into two types: T and B lymphocytes. Activation of each type involves a complex series of receptor-mediated signalling events. Ligand interaction with the clonally distributed antigen receptor has been shown to generate primary activation signals

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.

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.

A syngeneic monoclonal antiidiotype directed against the idiotype of an antireovirus type 3 hemagglutinin demonstrates several of the biological actions of the original viral hemagglutinin and binds to rat and murine cortical neurons grown in dissociated cell culture. Receptor-bearing neurons appear within 24 hours of plating in cultures from mouse or rat cortex taken on embryonic day 15; these neurons are demonstrable for the duration of the culture life span (4 to 8 weeks). When cortical cultures are incubated with antiidiotype before or during exposure to reovirus, the antiidiotype protects neurons from type 3 infection without inhibiting infection of nonneuronal cells with either type 3 or type 1. Thus an antibody directed against a viral receptor can prevent infection of receptor-bearing cells without directly neutralizing the virus.

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.

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.

The monoclonal anti-idiotypic antibody (mAb2) 87.92.6 directed against the 9B.G5 antibody specific for the virus neutralizing epitope on the mammalian reovirus type 3 hemagglutinin was previously demonstrated to express an internal image of the receptor binding epitope of the reovirus type 3. Furthermore, this mAb2 has autoimmune reactivity to the cell surface receptor of the reovirus. The nucleotide and deduced amino acid sequences of the 87.92.6 mAb2 heavy and light chains are described in this report. The sequence analysis reveals that the same heavy chain variable and joining (VH and JH) gene segments are used by the 87.92.6 anti-idiotypic mAb2 and by the dominant idiotypes of the BALB/c anti-GAT (cGAT) and anti-NP (NPa) responses. [GAT; random polymer that is 60% glutamic acid, 30% alanine, and 10% tyrosine. NP; (4-hydroxy-3-nitrophenyl)-acetyl.] Despite extensive homology at the level of the heavy chain variable regions, the NPa positive BALB/c anti-NP monoclonal antibody 17.2.25 binds neither 9B.G5 nor the cellular receptor for the hemagglutinin. Amino acid sequence comparison between the viral hemagglutinin and the 87.92.6 mAb2 light chain "internal image," reveals an area of significant homology indicating that antigen mimicry by antibodies may be achieved by sharing primary structure.

Rana pipiens tyrosinase mRNA was isolated from Stage 22 (tailfin circulation) embryos by indirect immunoprecipitation of embryonic polysomes using highly specific rabbit anti-tyrosinase and goat-(anti-rabbit) immunoglobulins. Analysis on sucrose gradients indicated that anti-tyrosinase bound specifically to embryonic polysomes of the 300-350 S class coincident with the location of nascent tyrosinase enzyme activity and tyrosinase mRNA. These same anti-tyrosinase-bound polysomes were fully immunoprecipitated by the addition of goat-(anti-rabbit) IgG. Poly(A+) RNA was obtained from phenol-extracted antibody. polysome complexes by sequential passage over oligo(dT)-cellulose. The final purification of tyrosinase mRNA was achieved by preparative sucrose gradient fractionation. Tyrosinase mRNA sedimented as a single 13 S peak in 5-30% sucrose gradients and tracked on sodium dodecyl sulfate-polyacrylamide gels as a single band of 4.5 X 10(5) Da (1275 nucleotides). When assayed in a cell-free translation system, this mRNA directed the synthesis of a single 35,000-Da protein which co-migrated with native tyrosinase on sodium dodecyl sulfate-polyacrylamide gels and which was greater than 98% immunoprecipitable by anti-tyrosinase immunoglobulin. Final purification was 4103-fold over the starting polysomal RNA.

Exposure of newborn BALB/c mice to murine leukemia virus (MLV) TR1.3 induces fusion of brain capillary endothelial cells (BCEC), loss of cerebral vessel integrity, hemorrhagic stroke, and death. Although TR1.3 infects endothelial cells in multiple organs, syncytia are only observed in BCEC. To determine if viral and cellular factors are responsible for selective syncytia formation, capillary endothelial cells (CEC) from multiple organs were assayed in vitro for MLV infection and cell fusion. Following incubation with virus, all CEC were infected to an equal extent as determined by expression of MLV envelope and infectious virus production; however, MLV-induced syncytia were only observed in TR1.3-infected BCEC cultures. These in vitro results mirror the in vivo pattern of TR1.3 MLV infection and neuropathology, and definitively show that selective fusion and pathology of BCEC by MLV is determined by properties unique to BCEC as contrasted to other endothelial cell types.

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, hetero topic 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.

Anti-idiotypic antibodies have proven to have unique applications as probes in both functional and biochemical studies of cell surface receptors. Anti-idiotypic receptor antibodies have been prepared to antibodies which bind to purified ligand, as in the case of insulin, retinol-binding protein, the mammalian reovirus receptor, and the neutrophil chemotactic receptor, and to natural ligand analogs, such as the beta-adrenergic antagonist alprenolol. These systems have documented the usefulness of anti-idiotypic antibodies in the quantitation and modulation of specific membrane receptors on a variety of cell types. Anti-idiotypic antibodies have also been utilized for the isolation of specific membrane receptors, e.g., reovirus and B-1H globulin receptors. Some anti-idiotypic receptor antibodies, e.g., insulin and reovirus systems, have been shown to mimic the physiological properties of ligand upon binding to cellular receptors. These antibodies enable a new dimension of both receptor based cellular studies and therapeutic regimens. This review focuses on the past use of anti-idiotypic antibodies as probes of cell surface receptors, and on the methodologies required for the successful application of anti-idiotypic antibodies for use in further membrane receptor studies, and of the genes which encode and regulate these receptors. We also discuss the use of anti-idiotypic antibodies in the understanding of and therapeutic approach to receptor related diseases.

The susceptibility of cells to viral infection is often determined during the early stages of interaction.1 This is particularly evident in the mammalian viruses, where infection and virus disease are largely determined by the ability of viruses to bind to the cells of a particular species. Even within a given species, the expression of different genes in different cell types or within the same cell type at different stages of development can result in the formation of different sets of specific virus receptors. The identification of virus receptors and the study of their interactions with ligands are therefore of critical importance in understanding viral tropism and subsequent disease patterns.2,3

An ideal antirejection therapy should, of course, be effective in controlling rejection as well as selectively target only those T-cells that are committed to participate in rejection of the donor graft. Conventional immunosuppressive drugs exact unwanted side effects upon non-lymphoid tissues. The introduction of monoclonal antibodies as pharmacologic tools have been long awaited as therapeutic use of T-cell specific monoclonal antibodies can obviate many side effects on non-lymphoid tissues by providing new opportunities for a more targeted form of immunosuppressive therapy. Nonetheless, the pan-T cell antibodies, used with considerable success in transplantation, react with all T cells, while an ideal therapy would target only those lymphocytes committed to the unwanted immune reaction.

We have attempted to selectively target immunosuppressive therapy to those clones able to mount cardiac graft rejection. As recently activated, but not resting or memory T cells bear receptors for interleukin-2, we have treated rodent (mouse and rat) cardiac graft recipients with anti-interleukin-2 receptor monoclonal antibodies. Such treatment has been remarkably successful. Despite short courses of treatment, a significant number of recipients are permanently engrafted.

A cell-surface receptor for the mammalian reovirus type 3 hemagglutinin was isolated by using antiidiotypic anti-receptor antibodies. The receptor is a glycoprotein with a molecular mass of 67,000 daltons and a pI of 5.9. Evidence that the isolated structure represents the reovirus receptor was obtained by electrophoretic immunoblot studies, which demonstrated that the 67,000-dalton glycoprotein is the only cell-surface structure recognized by both reovirus type 3 and the anti-receptor immunoglobulin. Comparison of the reovirus receptor on murine thymoma (R1.1) and rat neuroblastoma (B104) cells indicated that similar structures on the cell surface are recognized by the reovirus type 3 and the anti-receptor antibodies as previously suggested from cellular and binding studies. This receptor was found on mouse, rat, monkey, and human cells. Furthermore, diverse tissue types, including lymphoid and neuronal cells, express the receptor structure. The receptor structure is discussed in terms of its role in mediating viral tropism and as an essential cell-surface protein.

Summary A combination of metabolic labeling and chemical or enzymatic modification was employed to isolate and biochemically characterize a set of glycosyl-phosphatidylinositol (gly-PI) molecules synthesized by T lymphocytes. Gly-PI displayed unique patterns of synthesis following mitogen activation relative to the phosphoinositides and major structural lipids. The increase with time in gly-PI was paralleled by the appearance of insulin receptors. Gly-PI molecules were sensitive to hydrolysis by a PI-specific phospholipase C and were rapidly (15 sec) degraded in response to insulin binding. The product of this hydrolysis is believed to be a novel inositol phosphate-glycan (IP-gly) that was shown to inhibit the activity of a cAMP-dependent protein kinase. These results demonstrate that T cells contain a structurally related set of gly-PI molecules, at least one of which is sensitive to insulin and may function as a second messenger of hormone action.

This paper describes the production of a rabbit polyclonal antibody against an insulin-sensitive glycophospholipid from rat liver membranes. The immunogen was a highly purified glycophospholipid-tetanus toxoid conjugate. Immunoglobulin purified from immune serum reacted with a glycophospholipid-ovalbumin conjugate, indicating specificity for the glycophospholipid hapten and not the protein carrier. By radioimmunoassay the antibody recognized the purified glycophospholipid antigen but not other phospholipids including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. The antigenic site appears to be the carbohydrate portion of the glycophospholipid. The antibody also reacted with glycophospholipid purified from two rat hepatoma cell lines. Analysis of partially purified liver glycophospholipid by thin-layer chromatography revealed over 20 orcinol- or fluorescamine-positive bands, but immunostaining identified only 1 band. The latter had an Rf identical to those of the original glycophospholipid isolated from rat liver and metabolically labeled material isolated from hepatoma cells. The antibody should prove useful in determining the role of the glycophospholipid and its metabolites in insulin action.

The study of ligand-receptor interactions has been greatly aided by the application of anti-idiotypic antibody probes which mimic the specificity of ligand binding. Anti-idiotypic antibodies which represent internal images of viral attachment proteins have been utilized in our laboratories as probes of the biochemistry, cell biology, and immunology of the mammalian reovirus and its cellular receptor. The basis of this approach rests on the network theory of immune regulation originally proposed by Niels Jerne (1). Current applications of this technique have been recently reviewed by us (2,3). In brief, anti-idiotypic anti-receptor antibodies have been produced which recognize receptors for cellular growth factors (4,5), physiological regulators (4,6), and virus binding proteins (7,8). In these examples, anti-receptor antibodies were defined by immunoprecipitation and purification of receptor molecules or by their capacity to modify receptor function or ligand signal delivery. Anti-idiotypic antibodies have also been successfully employed as immunological mimics of antigen-ligand. For example, anti-idiotypic antibodies were used to stimulate T and B cell-mediated immunity to a variety of parasite, bacterial, and viral pathogens (9–12).