Robert C. Nowinski

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.

The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses

Abstract Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of C57BL/6 mice that were immunized with the AKR leukemia K36. Approximately 10% of the hybrid cells produced immunoglobulins that reacted in antibody binding assays with AKR MuLV. By the combined use of low-density passage and cloning, seven independent cell lines were isolated. These cells produced antiviral antibodies at a level of 3–15 μg/ml of culture fluid. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of tumors (hybridomas) that secreted extremely high levels of monoclonal antibodies (5–15 mg/ml) into the serum or ascites fluid. Five of the hybrid cell lines produced immunoglobulins of the IgM subclass, one produced IgG2a, and one produced IgG2b. In high-resolution two-dimensional polyacrylamide gels, these immunoglobulins showed the limited heterogeneity in heavy and light chains that would be expected for monoclonal products. Radioimmune precipitation assays demonstrated that the monoclonal antiviral antibodies reacted with the p15(E) protein of ecotropic MuLV; these antibodies did not react with the p15(E) protein of xenotropic MuLV. In contrast, rabbit antiserum prepared against purified p15(E) reacted equally well with ecotropic and xenotropic MuLV. The sera or ascites fluids from hybridoma-bearing mice had antibody titers 75- to 100-fold higher than the sera from conventionally immunized mice or rabbits. Serological analysis demonstrated that the monoclonal antibodies reacted with the cell surface of virus-producing leukemia cells, but not with normal thymocytes. Furthermore, monoclonal anti-pl5(E) antibodies of the IgG2a subclass mediated lysis of the virion of ecotropic MuLV in the presence of complement.

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells.

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.

Monoclonal antibodies against murine leukemia viruses: Identification of six antigenic determinants on the p15(E) and gp70 envelope proteins

Abstract Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.

Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies.

Monoclonal antibodies produced by hybrid cells in culture are chemically homogeneous reagents that react with constant avidity to single antigenic determinants (epitopes). As such, these antibodies are remarkably specific probes for small regions of complex antigenic proteins. We have used a panel of monoclonal antibodies in competition binding assays to investigate the arrangement of six epitopes on the envelope proteins of AKR leukemia virus. It was reasoned that if two epitopes were adjacent to each other on a single protein, the binding of antibody to one epitope would sterically hinder the binding of antibody to the other epitope. On the other hand, if the two epitopes were at distant sites on the protein, the binding of antibody to one epitope would not influence the binding of antibody to the other epitope. The results of these competition binding assays demonstrated the presence of two distinct antigen sites on both the gp70 and p15(E) envelope proteins. With the gp70 protein, one antigen site contained the gp70b and gp70c epitopes; the other antigen site on this protein contained the gp70a epitope. With p15(E), one of the antigen sites contained the p15(E)b and p15(E)c epitopes, while the other site contained the p15(E)a epitope. These findings demonstrate the utility of this type of serological analysis for the study of the tertiary structure of individual viral proteins.

Oncornaviruses produced by murine leukemia cells in culture.

Abstract Leukemia cells from mice of the AKR and C58 strains produced leukemia viruses (MuLV) in culture that failed to induce syncytia in the XC plaque assay (XC − ), were poorly infectious for mouse embryo fibroblasts, and yielded low levels of infectious progeny upon the establishment of infection. In host range analysis the majority of these viruses were N-ecotropic, although one leukemia cell line also produced xenotropic MuLV. Viral isolates from five different leukemia cell lines were oncogenic upon inoculation into newborn AKR mice. In contrast, viruses that occurred in high titer in the tissues and sera of normal AKR mice were not oncogenic in vivo and in culture they efficiently induced (with N-ecotropism) syncytia in the XC plaque assay (XC + ). These findings demonstrate that the transforming agent for spontaneous leukemogenesis differs in biological properties from the endogenous MuLV that is commonly observed in mice of high leukemic strains.

Selective neutralization of ecotropic murine leukemia virus by monoclonal antibodies: Localization of a site on the gp70 protein associated with ecotropism☆

Abstract A panel of nine monoclonal antibodies against the gp70 and p15(E) envelope proteins of MuLV were tested in parallel for their ability to bind to virus and neutralize infectivity. Although each of the antibodies bound to N-ecotropic MuLV, only antibody against the gp70 f epitope neutralized infectivity. These results were interpreted as evidence for a specific site on the gp70 protein of ecotropic MuLV, in proximity of the gp70 f epitope, which was responsible for the ecotropic-specific binding of virus to the surface of cells.

A cautionary note regarding Ia and H-2 typing of murine lymphoid tumors

1 Genetics Curriculum, Department of Bacteriology andlmmunology, University of North Carolina, Chapel Hill, North Carolina 27514 2 Basel lnstituteJor Immunology, 456 Grenzacherstrasse, Basel, Switzerland 3 Fred Hutchinson Cancer Research Center, Seattle, Washington 4 Department of Genetics, Washington University, St. Louis, Missouri 5 Department of Microbiology, University of Southern California Medical School, Los Angeles, California 90033

Biosynthesis and metabolism of viral proteins expressed on the surface of murine leukemia virus-infected cells

Viral proteins expressed on the surface of the Gross virus-induced leukemia E♂G2 were detected by immune precipitation assays from detergent lysates of cells that were labeled by the [125I]lactoperoxidase method. These proteins included gp70 and two glycosylated polyproteins that contained antigens coded by the gag gene. The 95,000-dalton polyprotein (gpP95gag) reacted with antisera against p30, p12, and p10, while the 85,000-dalton polyprotein (gpP85gag) reacted with antisera against p30 and p12, but not against p10. Neither polyprotein reacted with antiserum against reverse transcriptase. GpP95gag and gpP85gag contained glucosamine, but not fucose, while gp70 contained both sugars. Tryptic peptide maps of the iodinated polyproteins indicated that gpP95gag and gpP85gag were highly related. Viral polypeptides, having identical electrophoretic mobilities as the cell surface gpP95gag and gpP85gag, were also detected by immune precipitation of lysates from cells that were metabolically labeled with radioactive methionine or arginine. In kinetic studies, gpP95gag was labeled rapidly following a 10-min pulse; in contrast, gpP85gag was not detected until late in the chase period, suggesting that this glycosylated polyprotein was a processed product of gpP95gag. Mapping of arginine-labeled tryptic peptides of different gag polyproteins supported this conjecture. In fact, peptide maps with isolated viral proteins further demonstrated that gpP95gag and gpP85gag contained peptides of p30,p15/p12, and p10. The presence of p10 peptides in both polyproteins was an unanticipated finding since the gpP85gag did not react in immune precipitation assays with anti-p10 serum. By peptide mapping, both of the glycosylated polyproteins also appeared highly related to Pr75gag, the intracellular precursor to virion gag proteins. Our data suggest that gpP95gag is a glycosylated cell surface form of the primary gag precursor Pr75gag, and that processing of the gpP95gag results in the production of gpP85gag.

Genetic and viral factors influencing the development of spontaneous leukemia in akr mice.

Abstract Factors influencing the development of spontaneous leukemia were studied in a genetic cross that was prepared between leukemia-prone AKR and leukemia-resistant C57BL/6 mice. Two hundred mice of the AKR x (C57BL/6 x AKR)F1 backcross were examined for (a) the production of endogenous N-ecotropic murine leukemia virus (MuLV), (b) the production of anti-viral antibodies, (c) phenotype at the major histocompatibility complex (MHC), (d) phenotype at the Fv-1(Gpd-1) region, and (e) mortality due to leukemia or other natural causes. Although mice of this cross contained at least one genetic complement of AKR ecotropic MuL V and were expected to produce high levels of infectious virus, a variety of secondary factors influenced the level of MuLV that was detectable in extracellular fluids. The titer of ecotropic MuLV in individual mice was found to relate strongly and simultaneously to the Fv-1(Gpd-1) phenotype and to the titer of anti-viral antibodies in sera. Thus, the levels of infectious MuLV were significantly lower in mice of the Fv-1n/b genotype and in mice that produced high titers of anti-gp70 antibodies. The production of anti-gp70 antibodies, on the other hand, was regulated independently by the MHC phenotype and the sex of the mouse. High antibody titers were found to be associated with the MHCb/k haplotype and with the female sex. Survival data derived during a 24 month period were used to determine associations between virus expression and death caused by thymic leukemia, nonthymic leukemia, and other natural causes. Regression analysis indicated that genes which mapped within the Fv-1(Gpd-1) region were the most important determinants of the risk of thymic leukemia mortality. Although there was a mild additive effect on mortality associated with the high production of ecotropic MuLV, it appeared that additional factors controlled by genes in the Fv-1(Gpd-1) region were most responsible for the dramatic susceptibility of AKR mice to thymic leukemias. Although the presence of these factors were indicated in this study, their mode of action was not defined. In addition, it was also found that the production of anti-viral antibodies influenced the mortality caused by thymic leukemias. This association, however, could be largely explained by the inverse relationship that existed between the level of detectable ecotropic MuLV and the titer of anti-gp70 antibodies. Last, it was noted that mortality caused by nonthymic leukemias was not significantly influenced by phenotypic variation in either the Fv-1(Gpd-1) region or in the expression of ecotropic MuLV. This finding suggested that leukemias of thymic and nonthymic origin had different etiologies.