Transformation by Simian Virus 40 of Spleen Cells from a Hyperimmune Rabbit: Evidence for Synthesis of Immunoglobulin by the Transformed Cells

Abstract

Spleen cells derived from a rabbit hyperimmunized with a Type III pneumococcal vaccine were exposed to simian virus 40 in vitro. After 114 days, transformed cells growing on the surface of one culture dish were observed and a cell line was established. The transformed cells had a morphology characteristic of cells transformed by simian virus 40, contained the simian virus 40-specific T antigen, and yielded infectious simian virus 40 upon cultivation with indicator cells in the presence of Sendai fusion factor. Transformed cells incorporated labeled amino acid into protein with the antigenic properties of rabbit immunoglobulin G.We have previously attempted to obtain a virus-transformed cell line capable of indefinite growth in vitro and continuous production of specific antibody (1). Lymph-node cells from immunized rats were exposed to simian virus 40 (SV40); four transformed cell lines were obtained, but none was found to synthesize immunoglobulin or immunoglobulin fragments. Attempts to establish a relationship between the transformed cells and lymphoid cells were inconclusive, and the identity of the target cell for transformation remained uncertain. In order to increase the likelihood of successful transformation of antibody-producing cells, resort was made, in the present experiments, to cells from a rabbit hyperimmuniized with a pneumococcal vaccine. Rabbits injected intravenously with pneumococcal vaccines produce large amounts of antibody of restricted heterogeneity (2). Furthermore, marked plasmacell infiltration of the spleen has been demonstrated (3). This report describes the infection with SV40 of spleen cells from one such rabbit, and the subsequent establishment of a transformed cell line capable of producing rabbit immunoglobulin of the IgG class. In the accompanying manuscript, the presence of a protein of markedly restricted heterogeneity that specifically binds the immunizing antigen is demonstrated (4). MATERIALS AND METHODSImmunization. Rabbit L-27 was kindly provided by Drs 6). When the rabbit was killed, the serum contained 11 mg of antibody against S3 pneumococcal polysaccharide per ml. Isoelectric focusing of purified antibody showed three major and one minor species (4).Initiation and Maintenance of Stationary Suspension Cultures. These procedures were performed essentially as described for rat lymph-node cells (1). Briefly, rabbit L-27 was killed. The spleen was removed, minced finely, and pressed through wire mesh. Cells were suspended in medium RPMI 1640, supplemented with 20% unheated fetal-calf serum, 2 mM glutamine and antibiotics, infected with virus (see below), seeded in 60-mm diameter plastic dishes, and maintained in the above medium at 370 in a humidified atmosphere of 5% CO2 in air. Half of the medium of each plate was changed weekly, and 3 days after each medium change each plate was supplemented with '/lo volume of fresh medium. Bonemarrow cells were obtained by aspiration, dispersed by repeated pipetting, and treated as above.Virus Transformation of Suspension Cu...

Transformation by Simian Virus 40 of Spleen Cells from a Hyperimmune Rabbit: Demonstration of Production of Specific Antibody to the Immunizing Antigen

“…1 and 2) suggests that the product of a single clone is being examined. The percentage of labeled protein bound to the S3 immunoadsorbent was similar to that precipitated with anti-IgG (1), suggesting that all nor nearly all the IgG produced represents antibody to S3. An antibody-producing cell line capable of indefinite growth in vitro may prove capable of providing large amounts of homogeneous antibody and, perhaps, cell-surface receptors for detailed structural studies.…”

“…The description, conditions of growth, radioisotope labeling, harvesting of cells, and preparation of cell extracts and culture fluids from an SV40-transformed rabbit spleen cell (TRSC-1), SV40-transformed rabbit kidney cell (TRK-73), and normal rabbit kidney cell (MA-11 1) lines have been described (1).…”

“…Radiolabeled antigens were prepared by [125I]iodination of the protein-polysaccharide conjugates (6). Cell extracts were mixed with the labeled polysaccharide derivative and subjected to agarose gel electrophoresis followed by autoradiography (1).…”

“…In the accompanying paper, we showed that spleen cells of a rabbit hyperimmunized with Type III pneumococcal vaccine were transformed with simian virus 40 (SV40) and that the transformed cells synthesized IgG (1). In this paper, a fraction isolated from culture fluid and cell extract by a specific immunoadsorbent was shown to consist of a single major band by isoelectric focusing.…”

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

“…The low incidence of rabbit lymphoid tumors explains the paucity of rabbit T-and B-cell lines. In fact, the only previously reported rabbit B-lymphocyte line in culture was one developed by Collins et al (33). The E -myc transgenic rabbits should provide a source of B-cell lines and these cells will provide the opportunity to study B-cell differentiation and immunoglobulin gene rearrangements.…”