Abstract
Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum− antigens) recognized by cytolytic T cells. The gene encoding tum− antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.
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Allen, P. M., Strydom, D., and Unanue, E.R.: Processing of lysozyme by macrophages: identification of the determinant recognized by two T cell hybridomas. Proc Natl Acad Sci USA 81: 2489–2493, 1984
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.: Current Protocols in Molecular Biology. J. Wiley, New York, 1987
Boon, T. and Van Pel, A.: T cell recognized antigenic peptides derived from the cellular genome are not protein degradation products but can be generated directly by transcription and translation of short subgenic regions. A hypothesis. Immunogenetics 29: 75–79, 1988
Boon, T., Van Snick, J., Van Pel, A., Uyttenhove, C., and Marchand, M.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. II. T lymphocyte-mediated cytolysis. J Exp Med 152: 1184–1193, 1980
Chimini, G., Pala, P., Sire, J., Jordan, B. R., and Maryanski, J. L.: Recognition of oligonucleotide-encoded T cell epitopes introduced into a gene unrelated to the original antigen. J Exp Med 169: 297–302, 1989
Colbere-Garapin, F., Chousterman, S., Horodniceanu, F., Kourilsky, P., and Garapin, A. C.: Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia Coli K-12. Proc Natl Acad Sci USA 76: 3755–3759, 1979
Davis, L. G., Dibner, M. D., and Battey, J. F.: Basic Methods in Molecular Biology: Elsevier, New York, 1986
Del Val, M., Volkmer, H., Rothbard, J. B., Jonjic, S., Messerle, M., Schickedanz, J., Reddehase, M. J., and Koszinowski, U. H.: Molecular basis for cytolytic T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89. J Virol 62: 3965–3972, 1988
Demotz, S., Grey, H. M., and Sette, A.: The minimal number of class II MHC-antigen complexes needed for T cell activation. Science 249: 1028–1030, 1990
De Plaen, E., Lurquin, C., Van Pel, A., Mariamé, B., Szikora, J.-P., Wölfel, T., Sibille, C., Chomez, P., and Boon, T.: Immunogenic (tum−) variants of mouse tumor P815: Cloning of the gene of tum− antigen P91A and identification of the tum− mutation. Proc Natl Acad Sci USA 85: 2274–2278, 1988
Espevik, T. and Nissen-Meyer, J.: A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods 95: 99–105, 1986
Falk, K., Rötzschke, O., and Rammensee, H.-G.: Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 348: 248–251, 1990
Frischauf, A.-M., Lehrach, H., Poustka, A., and Murray, N.: Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170: 827–842, 1983
Hansen, M. B., Nielsen, S. E., and Berg, K.: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119: 203–210, 1989
Heard, J.-M., Herbomel, P., Ott, M.-O., Mottura-Rollier, A., Weiss, M., and Yaniv, M.: Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system. Mol Cell Biol 7: 2425–2434, 1987
Israël, A., Kimura, A., Fournier, A., Fellous, M., and Kourilsky, P.: Interferon response sequence potentiates activity of an enhancer in the promoter region of a mouse H-2 gene. Nature 322: 743–746, 1986
Kozak, M.: An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15: 8125–8132, 1987
Lewin, B.: Units of transcription and translation: sequence components of heterogeneous nuclear RNA and messenger RNA. Cell 4: 77–93, 1975
Ljunggren, H.-G., Stam, N. J., Öhlén, C., Neefjes, J. J., Höglund, P., Heemels, M.-T., Bastin, J., Schumacher, T. N. M., Townsend, A., Kärre, K., and Ploegh, H. L.: Empty MHC class I molecules come out in the cold. Nature 346: 476–480, 1990
Lurquin, C., Van Pel, A., Mariamé, B., De Plaen, E., Szikora, J.-P., Janssens, C., Reddehase, M. J., Lejeune, J., and Boon, T.: Structure of the gene coding for tum− transplantation antigen P91A. A peptide encoded by the mutated exon is recognized with Ld by cytolytic T cells. Cell 58: 293–303, 1989
Mansour, S. L., Thomas, K. R., and Capecchi, M. R.: Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348–352, 1988
Maryanski, J. L. and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variant-specific 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
Nakamaye, K. and Eckstein, F.: Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res 14: 9679–9698, 1986
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
Peabody, D. S. and Berg, P.: Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol 6: 2695–2703, 1986
Perucho, M., Hanahan, D., and Wigler, M.: Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22: 309–317, 1980
Rötzschke, O., Falk, K., Wallny, H.-J., Faath, S., and Rammensee, H.-G.: Characterization of naturally occuring minor histocompatibility peptides including H-4 and H-Y. Science 249: 283–287, 1990
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
Sambrook, J., Fritsch, E. F., and Maniatis, T.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989
Schøller, J.: Construction of novel eukaryotic transfection-vector, pJNL-1. Nucleic Acids Res 16: 769, 1988
Sibille, C., Chomez, P., Wildmann, C., Van Pel, A., De Plaen, E., Maryanski, J. L., de Bergeyck, V., and Boon, T.: Structure of the gene of tum− transplantation antigen P198: a point mutation generates a new antigenic peptide. J Exp Med 172: 35–45, 1990
Szikora, J.-P., Van Pel, A., Brichard, V., André, M., Van Baren, N., Henry, P., De Plaen, E., and Boon, T.: Structure of the gene of tum− transplantation antigen P35B: presence of a point mutation in the antigenic allele. EMBO J 9: 1041–1050, 1990
Thomas, K. R., Folger, K. R., and Capecchi, M. R.: High frequency targeting of genes to specific sites in the mammalian genome. Cell 44: 419–428, 1986
Townsend, A. and Bodmer, H.: Antigen recognition by class I-restricted T-lymphocytes. Annu Rev Immunol 7: 601–624, 1989
Townsend, A., Rothbard, J., Gotch, F., Bahadur, G., Wraith, D., and McMichael, J.: The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44: 959–968, 1986
Townsend, A., Öhlén, C., Bastin, J., Ljunggren, H. G., Foster, L., and Kärre, K.: Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340: 443–448, 1989
Traversi, C., van den Bruggen, P., Van den Eynde, B., Hainaut, P., Lemoine, N., Ohta, N., Old, L., Boon, T.: Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes. Immunogenetics 35: 145–152, 1992
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., De Plaen, E., and Boon, T.: Selection of highly transfectable variant from mouse mastocytoma P815. Somatic Cell Genet 11: 467–475, 1985
Wagner, M. J., Sharp, J. A., and Summers, W. C.: Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci USA 78: 1441–1445, 1981
Wölfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L., and Boon, T.: Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VIII. Detection of stable transfectants expressing a tum− antigen with a cytolytic T cell stimulation assay. Immunogenetics 26: 178–187, 1987
Yewdell, J. W. and Bennink, J. R.: Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes. Science 244: 1072–1075, 1989
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Chomez, P., De Plaen, E., Van Pel, A. et al. Efficient expression of tum− antigen P91A by transfected subgenic fragments. Immunogenetics 35, 241–252 (1992). https://doi.org/10.1007/BF00166829
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DOI: https://doi.org/10.1007/BF00166829