Abstract
Mutagen treatment of mouse mastocytoma P815 produces highly inununogenic “tum−” variants. Most of these variants express potent transplantation antigens which are not present on the original P815 tumor cells. These tum− antigens, which appear to be specific for each variant, elicit a strong cytolytic T lymphocyte (CTL) response, but do not seem to induce a specific antibody response. As a first step in the isolation of the gene of a tum− antigen, we attempted DNA-mediated gene transfer. As a DNA recipient cell we used P1.HTR, a highly transfectable P815 cell line, whose selection has been previously described. For the detection of antigen-expressing cells in transfected populations we developed a procedure that relies on the ability of these cells to stimulate the proliferation of the relevant CTL. Using DNA from tum− variant P91 mixed with a plasmid carrying an antibiotic resistance gene, we obtained several independent transfectants expressing a tum− antigen, at a frequency of approximately 1 in 13 000 antibiotic-resistant transfectants. These transfectants express only one of the two tum− antigens that were identified on P91, suggesting that these tum− antigens correspond to different genes. We expect that the detection procedure described here will be-suitable for the identification of transfectants for any gene that determines the expression of an antigen recognized by CTL.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Albino, A. P., Graff, L. H., Kantor, R. R., McLean, W., Silagi, S., and Old, L. J.: DNA-mediated transfer of human melanoma cell surface glycoprotein gp130: Identification of transfectants by erythrocyte rosetting. Mol. Cell. Biol. 5: 692–697, 1985
Bernard, H.-U., Krämmer, G., and Röwekamp, W. G.: Construction of a fusion gene that confers resistance against Hygromycin B to mammalian cells in culture. Exp. Cell Res. 158: 237–243, 1985
Boon, T.: Antigenic tumor cell variants obtained with mutagens. Adv. Cancer Res. 39: 121–151, 1983
Boon, T. and Kellermann, O.: Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line. Proc. Natl. Acad. Sci. U.S.A. 74: 272–275, 1977
Boon, T., Van Snick, J., Van Pel, A., Uyttenhove, C., and Marchand, M.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. II. Lymphocyte-mediated cytolysis. J. Exp. Med. 152: 1184–1193, 1980
Brady, G., Funk, A., Mattern, J., Schütz, G., and Brown, R.: Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences. EMBO J. 4: 2583–2588, 1985
Chang, A. C. Y. and Cohen, S. N.: Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134: 1141–1166, 1978
Corsaro, C. M. and Pearson, M. L.: Enhancing the efficiency of DNAmediated gene transfer in mammalian cells. Somatic Cell Genet. 7: 603–616, 1981
Davis, R. W., Botstein, D., and Roth, J. R.: A Manual for Genetic Engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980
Frost, P., Kerbel, R. S., Bauer, E., Tartamella-Biondo, R., and Cefalu, W.: Mutagen treatments as a means for selecting immunogenic variants from otherwise poorly immunogenic malignant murine tumors. Cancer Res. 43: 125–132, 1983
Frost, P., Liteplo, R. G., Donaghue, T. P., and Kerbel, R. S.: Selection of strongly immunogenic “tum−” variants from tumors at high frequency using 5-azacytidine. J. Exp. Med. 159: 1491–1501, 1984
Graff, R. C.: Minor histocompatibility genes and their antigens. Transplant. Proc. 10: 701–705, 1978
Graham, F. L. and Van der Eb, A. J.: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456–467, 1973
Heidman, O. and Rougeon, F.: Multiple sequences related to a constantregion kappa light chain gene in the rabbit genome. Cell 28: 507–513, 1982
Hérin, M., Lemoine, C., Weynants, P., Vessière, F., Van Pel, A., Knuth, A., Devos, R., and Boon, T.: Production of stable cytolytic T cell clones directed against autologous human melanoma. Int. J. Cancer 39: 390–396, 1987
Kavathas, P. and Herzenberg, L. A.: Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and β 2-microglobulin: Selection by fluorescence-activated cell sorting. Proc. Natl. Acad. Sci. U.S.A. 80: 524–528, 1983
Kerbel, R. S., Waghorne, C., Man, M. S., Elliott, B., and Breitman, M. L.: Alteration of the tumorigenic and metastatic properties of neoplastic cells is associated with the process of calcium phosphatemediated DNA transfection. Proc. Natl. Acad. Sci. U.S.A. 84: 1263–1267, 1987
Klein, J.: H-2 mutations: Their genetics and effect on immune functions. Adv. Immunol. 26: 56–145, 1978
Kühn, L. C., McClelland, A., and Ruddle, F. H.: Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 37: 95–103, 1984
Lambotte, P., Van Snick, J., and Boon, T.: Partial purification of a membrane glycoprotein antigen by high-pressure size-exclusion chromatography without loss of antigenicity. J. Chromatogr. 297: 139–145, 1984
Lebkowski, J. S., DuBridge, R. B., Antell, E. A., Geisen, K. S., and Calos, M. P.: Transfected DNA is mutated in monkey, mouse and human cells. Mol. Cell. Biol. 4: 1951–1960, 1984
Littman, D. R., Thomas, Y., Maddon, P. J., Chess, L., and Axel, R.: The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytes. Cell 40: 237–246, 1985
Loveland, B. and Simpson, E.: The non-MHC transplantation antigens: Neither weak nor minor. Immunol. Today 7: 223–229, 1986
Maniatis, T., Fritsch, E., and Sambrook, J.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982
Maryanski, J. L. and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variantspecific antigens by selection of antigen-loss variants with cytolytic T cell clones. Eur. J. Immunol. 12: 406–412, 1982
Maryanski, J. L., Van Snick, J., Cerottini, J.-C., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. III. Clonal analysis of the syngeneic cytolytic T lymphocyte response. Eur. J. Immunol. 12: 401–406, 1982
Maryanski, J. L., Marchand, M., Uyttenhove, C., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VI. Occasional escape from host rejection due to antigenloss secondary variants. Int. J. Cancer 31: 119–123, 1983a
Maryanski, J. L., Szpirer, J., Szpirer, C., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VII. Dominant expression of variant antigens in somatic cell hybrids. Somatic Cell Genet. 9: 345–357, 1983b
Nicolas, J. F. and Berg, P.: Regulation of expression of genes transduced into embryonal carcinoma cells. Cold Spring Harbor Conferences Cell Prolif. 10: 469–485, 1983
Perucho, M., Hanahan, D., and Wigler, M.: Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22: 309–317, 1980a
Perucho, M., Hanahan, D., Lipsich, L., and Wigler, M.: Isolation of the chicken thymidine kinase gene by plasmid rescue. Nature 285: 207–210, 1980b
Ryser, J. E., Cerottini, J.-C., and Brunner, K. T.: Generation of cytolytic T lymphocytes in vitro. IX. Induction of secondary CTL responses in primary long-term MLC by supernatants from secondary MLC. J. Immunol. 120: 370–377, 1978
Santos, E., Premkumar Reddy, E., Pulciani, S., Feldmann, R. J., and Barbacid, M.: Spontaneous activation of a human proto-oncogene. Proc. Natl. Acad. Sci. U.S.A. 80: 4679–4683, 1983
Scangos, G. and Ruddle, R.: Mechanisms and applications of DNAmediated gene transfer in mammalian cells — a review. Gene 14: 1–10, 1981
Seed, B.: Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Res. 11: 2427–2445, 1983
Simpson, E.: The role of H-Y as a minor transplantation antigen. Immunol. Today 3: 97–106, 1982
Stein, R., Razin, A., and Cedar, H.: In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc. Natl. Acad. Sci. U.S.A. 79: 3418–3422, 1982
Uyttenhove, C., Van Snick, J., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. I. Rejection by syngeneic mice. J. Exp. Med. 152: 1175–1183, 1980
Van Pel, A., Georlette, M., and Boon, T.: Tumor cell variants obtained by mutagenesis of a Lewis lung carcinoma cell line: Immune rejection by syngeneic mice. Proc. Natl. Acad. Sci. U.S.A. 76: 5282–5285, 1979
Van Pel, A., Vessière, F., and Boon, T.: Protection against two spontaneous mouse leukemias conferred by immunogenic variants obtained by mutagenesis. J. Exp. Med. 157: 1992–2001, 1983
Van Pel, A., De Plaen, E., and Boon, T.: Selection of highly transfectable variant from mouse mastocytoma P815. Somatic Cell Genet. 11: 467–475, 1985
Van Snick, J., Uyttenhove, C., Van Pel, A., and Boon, T.: Stimulation of cytolytic T lymphocytes by azaguanine-resistant mouse tumor cells in selective HAT medium. J. Immunol. Methods 46: 321–326, 1981
Van Snick, J., Maryanski, J. L., Van Pel, A., Parmiani, G., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. V. H-2 associativity of variant-specific antigens. Eur. J. Immunol. 12: 905–908, 1982
Wigler, M., Pellicer, A., Silverstein, S., and Axel, R.: Biochemical transfer of single-copy eukaryotic genes using total cellular DNA as donor. Cell 14: 725–731, 1978
Zinkernagel, R. M. and Doherty, P. C.: Restriction of in vitro T cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248: 701–702, 1974
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wölfel, T., Van Pel, A., De Plaen, E. et al. Immunogenic (tum−) variants obtained by mutagenesis of mouse mastocytoma P815. Immunogenetics 26, 178–187 (1987). https://doi.org/10.1007/BF00365909
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00365909