Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1992 Aug 1;176(2):449–457. doi: 10.1084/jem.176.2.449

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes

PMCID: PMC2119332  PMID: 1380062

Abstract

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H- 2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.

Full Text

The Full Text of this article is available as a PDF (941.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebischer T., Moskophidis D., Rohrer U. H., Zinkernagel R. M., Hengartner H. In vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11047–11051. doi: 10.1073/pnas.88.24.11047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bataillon G., Pross H., Klein G. Comparative in vitro sensitivity of twom methylcholanthrene-induced murine sarcoma lines to humoral and cellular immune cytotoxicity. Int J Cancer. 1975 Aug 15;16(2):255–265. doi: 10.1002/ijc.2910160208. [DOI] [PubMed] [Google Scholar]
  3. Bernards R., Schrier P. I., Houweling A., Bos J. L., van der Eb A. J., Zijlstra M., Melief C. J. Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T-cell immunity. 1983 Oct 27-Nov 2Nature. 305(5937):776–779. doi: 10.1038/305776a0. [DOI] [PubMed] [Google Scholar]
  4. Boon T., Van Pel A., De Plaen E., Chomez P., Lurquin C., Szikora J. P., Sibille C., Mariamé B., Van den Eynde B., Lethé B. Genes coding for T-cell-defined tum transplantation antigens: point mutations, antigenic peptides, and subgenic expression. Cold Spring Harb Symp Quant Biol. 1989;54(Pt 1):587–596. doi: 10.1101/sqb.1989.054.01.070. [DOI] [PubMed] [Google Scholar]
  5. De Plaen E., Lurquin C., Van Pel A., Mariamé B., Szikora J. P., Wölfel T., Sibille C., Chomez P., 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 U S A. 1988 Apr;85(7):2274–2278. doi: 10.1073/pnas.85.7.2274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deckhut A. M., Lippolis J. D., Tevethia S. S. Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen. J Virol. 1992 Jan;66(1):440–447. doi: 10.1128/jvi.66.1.440-447.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fidler I. J., Bucana C. Mechanism of tumor cell resistance to lysis by syngeneic lymphocytes. Cancer Res. 1977 Nov;37(11):3945–3956. [PubMed] [Google Scholar]
  8. Flyer D. C., Pretell J., Campbell A. E., Liao W. S., Tevethia M. J., Taylor J. M., Tevethia S. S. Biology of simian virus 40 (SV40) transplantation antigen (TrAg). X. Tumorigenic potential of mouse cells transformed by SV40 in high responder C57BL/6 mice and correlation with the persistence of SV40 TrAg, early proteins and sequences. Virology. 1983 Nov;131(1):207–220. doi: 10.1016/0042-6822(83)90546-9. [DOI] [PubMed] [Google Scholar]
  9. Gooding L. R. Characterization of a progressive tumor from C3H fibroblasts transformed in vitro with SV40 virus. Immunoresistance in vivo correlates with phenotypic loss of H-2Kk. J Immunol. 1982 Sep;129(3):1306–1312. [PubMed] [Google Scholar]
  10. Greenberg P. D. Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells. Adv Immunol. 1991;49:281–355. doi: 10.1016/s0065-2776(08)60778-6. [DOI] [PubMed] [Google Scholar]
  11. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. HABEL K., SILVERBERG R. J. Relationship of polyoma virus and tumor in vivo. Virology. 1960 Nov;12:463–476. doi: 10.1016/0042-6822(60)90167-7. [DOI] [PubMed] [Google Scholar]
  13. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hui K., Grosveld F., Festenstein H. Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation. Nature. 1984 Oct 25;311(5988):750–752. doi: 10.1038/311750a0. [DOI] [PubMed] [Google Scholar]
  15. Jung S., Schluesener H. J. Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins. J Exp Med. 1991 Jan 1;173(1):273–276. doi: 10.1084/jem.173.1.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karjalainen H. E., Tevethia M. J., Tevethia S. S. Abrogation of simian virus 40 DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure to a simian virus 40-specific cytotoxic T-lymphocyte clone. J Virol. 1985 Nov;56(2):373–377. doi: 10.1128/jvi.56.2.373-377.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kast W. M., Offringa R., Peters P. J., Voordouw A. C., Meloen R. H., van der Eb A. J., Melief C. J. Eradication of adenovirus E1-induced tumors by E1A-specific cytotoxic T lymphocytes. Cell. 1989 Nov 17;59(4):603–614. doi: 10.1016/0092-8674(89)90006-8. [DOI] [PubMed] [Google Scholar]
  18. Lurquin C., Van Pel A., Mariamé B., De Plaen E., Szikora J. P., Janssens C., Reddehase M. J., Lejeune J., Boon T. Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells. Cell. 1989 Jul 28;58(2):293–303. doi: 10.1016/0092-8674(89)90844-1. [DOI] [PubMed] [Google Scholar]
  19. Manjunath R., Graziano R. F., Green W. R. The specificity of H-2-restricted cytotoxic T lymphocytes directed to AKR/Gross leukemia virus-induced tumors. III. Coordinate alterations in viral gp70 antigen expression and restoration of CTL-susceptibility to insusceptible variant tumors. J Immunol. 1986 Mar 15;136(6):2271–2279. [PubMed] [Google Scholar]
  20. Maryanski J. L., Pala P., Cerottini J. C., Corradin G. Synthetic peptides as antigens and competitors in recognition by H-2-restricted cytolytic T cells specific for HLA. J Exp Med. 1988 Apr 1;167(4):1391–1405. doi: 10.1084/jem.167.4.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maryanski J. L., Van Snick J., Cerottini J. C., Boon T. Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. III. Clonal analysis of the syngeneic cytolytic T lymphocyte response. Eur J Immunol. 1982 May;12(5):401–406. doi: 10.1002/eji.1830120508. [DOI] [PubMed] [Google Scholar]
  22. Mora P. T., Parrott C. L., Baksi K., McFarland V. Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA. J Virol. 1986 Sep;59(3):628–634. doi: 10.1128/jvi.59.3.628-634.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Myers R. M., Lerman L. S., Maniatis T. A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985 Jul 19;229(4710):242–247. doi: 10.1126/science.2990046. [DOI] [PubMed] [Google Scholar]
  24. Pala P., Bodmer H. C., Pemberton R. M., Cerottini J. C., Maryanski J. L., Askonas B. A. Competition between unrelated peptides recognized by H-2-Kd restricted T cells. J Immunol. 1988 Oct 1;141(7):2289–2294. [PubMed] [Google Scholar]
  25. Pan S., Abramczuk J., Knowles B. B. Immune control of SV40-induced tumors in mice. Int J Cancer. 1987 Jun 15;39(6):722–728. doi: 10.1002/ijc.2910390612. [DOI] [PubMed] [Google Scholar]
  26. Peace D. J., Chen W., Nelson H., Cheever M. A. T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes. J Immunol. 1991 Mar 15;146(6):2059–2065. [PubMed] [Google Scholar]
  27. Phillips R. E., Rowland-Jones S., Nixon D. F., Gotch F. M., Edwards J. P., Ogunlesi A. O., Elvin J. G., Rothbard J. A., Bangham C. R., Rizza C. R. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature. 1991 Dec 12;354(6353):453–459. doi: 10.1038/354453a0. [DOI] [PubMed] [Google Scholar]
  28. Pircher H., Moskophidis D., Rohrer U., Bürki K., Hengartner H., Zinkernagel R. M. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo. Nature. 1990 Aug 16;346(6285):629–633. doi: 10.1038/346629a0. [DOI] [PubMed] [Google Scholar]
  29. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  30. Sheen J. Y., Seed B. Electrolyte gradient gels for DNA sequencing. Biotechniques. 1988 Nov-Dec;6(10):942–944. [PubMed] [Google Scholar]
  31. Szikora J. P., Van Pel A., Brichard V., André M., Van Baren N., Henry P., De Plaen E., Boon T. Structure of the gene of tum- transplantation antigen P35B: presence of a point mutation in the antigenic allele. EMBO J. 1990 Apr;9(4):1041–1050. doi: 10.1002/j.1460-2075.1990.tb08208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tanaka K., Yoshioka T., Bieberich C., Jay G. Role of the major histocompatibility complex class I antigens in tumor growth and metastasis. Annu Rev Immunol. 1988;6:359–380. doi: 10.1146/annurev.iy.06.040188.002043. [DOI] [PubMed] [Google Scholar]
  33. Tanaka Y., Anderson R. W., Maloy W. L., Tevethia S. S. Localization of an immunorecessive epitope on SV40 T antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide. Virology. 1989 Jul;171(1):205–213. doi: 10.1016/0042-6822(89)90527-8. [DOI] [PubMed] [Google Scholar]
  34. Tanaka Y., Tevethia M. J., Kalderon D., Smith A. E., Tevethia S. S. Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen. Virology. 1988 Feb;162(2):427–436. doi: 10.1016/0042-6822(88)90483-7. [DOI] [PubMed] [Google Scholar]
  35. Tanaka Y., Tevethia S. S. In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones. J Immunol. 1988 Jun 15;140(12):4348–4354. [PubMed] [Google Scholar]
  36. Tanaka Y., Tevethia S. S. Loss of immunorecessive cytotoxic T lymphocyte determinant V on SV40 T antigen following cocultivation with site-specific cytotoxic T lymphocyte clone Y-5. Intervirology. 1990;31(2-4):197–202. doi: 10.1159/000150154. [DOI] [PubMed] [Google Scholar]
  37. Tevethia M. J. Immortalization of primary mouse embryo fibroblasts with SV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitative assay. Virology. 1984 Sep;137(2):414–421. doi: 10.1016/0042-6822(84)90234-4. [DOI] [PubMed] [Google Scholar]
  38. Tevethia S. S., Blasecki J. W., Waneck G., Goldstein A. L. Requirement of thymus-derived theta-positive lymphocytes for rejection of DNA virus (SV 40) tumors in mice. J Immunol. 1974 Nov;113(5):1417–1423. [PubMed] [Google Scholar]
  39. Tevethia S. S., Lewis M., Tanaka Y., Milici J., Knowles B., Maloy W. L., Anderson R. Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants. J Virol. 1990 Mar;64(3):1192–1200. doi: 10.1128/jvi.64.3.1192-1200.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tornow J., Polvino-Bodnar M., Santangelo G., Cole C. N. Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis. J Virol. 1985 Feb;53(2):415–424. doi: 10.1128/jvi.53.2.415-424.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Townsend A. R., Rothbard J., Gotch F. M., Bahadur G., Wraith D., McMichael A. J. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986 Mar 28;44(6):959–968. doi: 10.1016/0092-8674(86)90019-x. [DOI] [PubMed] [Google Scholar]
  42. Urban J. L., Burton R. C., Holland J. M., Kripke M. L., Schreiber H. Mechanisms of syngeneic tumor rejection. Susceptibility of host-selected progressor variants to various immunological effector cells. J Exp Med. 1982 Feb 1;155(2):557–573. doi: 10.1084/jem.155.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Uyttenhove C., Maryanski J., Boon T. Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen-loss variants rather than immunosuppression. J Exp Med. 1983 Mar 1;157(3):1040–1052. doi: 10.1084/jem.157.3.1040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Uyttenhove C., Van Snick J., Boon T. Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. I. Rejection by syngeneic mice. J Exp Med. 1980 Nov 1;152(5):1175–1183. doi: 10.1084/jem.152.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vasmel W. L., Sijts E. J., Leupers C. J., Matthews E. A., Melief C. J. Primary virus-induced lymphomas evade T cell immunity by failure to express viral antigens. J Exp Med. 1989 Apr 1;169(4):1233–1254. doi: 10.1084/jem.169.4.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wallich R., Bulbuc N., Hämmerling G. J., Katzav S., Segal S., Feldman M. Abrogation of metastatic properties of tumour cells by de novo expression of H-2K antigens following H-2 gene transfection. Nature. 1985 May 23;315(6017):301–305. doi: 10.1038/315301a0. [DOI] [PubMed] [Google Scholar]
  47. Wortzel R. D., Urban J. L., Schreiber H. Malignant growth in the normal host after variant selection in vitro with cytolytic T-cell lines. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2186–2190. doi: 10.1073/pnas.81.7.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. van der Hoorn F. A., Lahaye T., Müller V., Ogle M. A., Engers H. D. Characterization of gP85gag as an antigen recognized by Moloney leukemia virus-specific cytolytic T cell clones that function in vivo. J Exp Med. 1985 Jul 1;162(1):128–144. doi: 10.1084/jem.162.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES