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. 1977 Aug 1;146(2):468–482. doi: 10.1084/jem.146.2.468

In vitro generation of tumor-specific cytotoxic lymphocytes. Secondary allogeneic mixed tumor lymphocyte culture of normal murine spleen cells

S Gillis, KA Smith
PMCID: PMC2180756  PMID: 69003

Abstract

In vivo or in vitro immunity to murine leukemia virus (MuLV)-induced leukemia cells which do not effectively produce virus, has been difficult to demonstrate. Because immunizations with allogeneic murine leukemia cells have been used to confer syngeneic tumor immunity to virus- producing cells, we attempted to generate lymphocytes, cytotoxic to syngeneic nonproducer leukemia cells, by stimulating normal murine spleen cells with allogeneic nonproducer leukemia cells in mixed tumor lymphocyte culture (MTLC) reactions in vitro. Secondary allogeneic MTLC of normal C57BL/6 or DBA/2 spleen cells effectively produced syngeneic tumor-specific cytotoxic lymphocytes. Target cells lysed in lymphocyte- mediated cytolysis (LMC) assays, included both Friend and Rauscher virus- induced syngeneic murine leukemia cells and chemically-induced hematopoietic tumor cells. Syngeneic tumor cells were lysed regardless of whether they produced infectious MuLV or expressed viral antigens gp-71, p-30, or p-12 at the cell surface. Syngeneic normal cells (thymus, lymph node, or Concanavalin A-stimulated spleen cells) used as targets in LMC assays were uneffected by lymphocytes harvested from secondary allogeneic MTLC. Several other in vitro culture treatments including secondary syngeneic MTLC and repetitive mixed lymphocyte culture stimulations were incapable of generating tumor-specific cytotoxic lymphocytes. Based upon these results, we propose that secondary MTLC stimulation of normal spleen cells with allogeneic nonproducer leukemia cells selects for the proliferation of two subpopulations of antigen-specific cytotoxic lymphocytes. The population capable of effecting syngeneic tumor cell lysis is directed against tumor-associated cell surface antigens which may be distinct from viral structural proteins or glycoproteins. The growth of these tumor-specific cytotoxic lymphocytes may be enhanced by a soluble allogeneic effect factor produced by the proliferation of the second subpopulation of lymphocytes generated in repetitive allogeneic MTLC, namely those lymphocytes with specificities directed against differing histocompatibility antigens.

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Selected References

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  1. Abrell J. W., Gallo R. C. Purification, characterization, and comparison of the DNA polymerases from two primate RNA tumor viruses. J Virol. 1973 Sep;12(3):431–439. doi: 10.1128/jvi.12.3.431-439.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersson J., Möller G., Sjöberg O. B lymphocytes can be stimulated by concanavalin A in the presence of humoral factors released by T cells. Eur J Immunol. 1972 Feb;2(1):99–101. doi: 10.1002/eji.1830020119. [DOI] [PubMed] [Google Scholar]
  3. Armerding D., Katz D. H. Activation of T and B lymphocytes in vitro. II. Biological and biochemical properties of an allogeneic effect factor (AEF) active in triggering specific B lymphocytes. J Exp Med. 1974 Jul 1;140(1):19–37. doi: 10.1084/jem.140.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bach F. H., Bach M. L. Comparison of mitomycin C and X-irradiation as blocking agents in one-way mixed leucocyte cultures. Nat New Biol. 1972 Feb 23;235(60):243–244. doi: 10.1038/newbio235243a0. [DOI] [PubMed] [Google Scholar]
  5. Bianco A. R., Glynn J. P., Goldin A. Induction of resistance against the transplantation of leukemias induced by Rauscher virus. Cancer Res. 1966 Aug;26(8):1722–1728. [PubMed] [Google Scholar]
  6. Boyse E. A., Hubbard L., Stockert E., Lamm M. E. Improved complementation in the cytotoxic test. Transplantation. 1970 Nov;10(5):446–449. doi: 10.1097/00007890-197011000-00019. [DOI] [PubMed] [Google Scholar]
  7. Click R. E., Benck L., Alter B. J. Immune responses in vitro. I. Culture conditions for antibody synthesis. Cell Immunol. 1972 Feb;3(2):264–276. doi: 10.1016/0008-8749(72)90165-7. [DOI] [PubMed] [Google Scholar]
  8. Feldmann M., Basten A. Cell interactions in the immune response in vitro. I. Metabolic activities of T cells in a collaborative antibody response. Eur J Immunol. 1972 Jun;2(3):213–224. doi: 10.1002/eji.1830020305. [DOI] [PubMed] [Google Scholar]
  9. Feldmann M., Basten A. Cell interactions in the immune response in vitro. IV. Comparison of the effects of antigen-specific and allogeneic thymus-derived cell factors. J Exp Med. 1972 Oct 1;136(4):722–736. doi: 10.1084/jem.136.4.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feldmann M., Basten A. Specific collaboration between T and B lymphocytes across a cell impermeable membrane in vitro. Nat New Biol. 1972 May 3;237(70):13–15. doi: 10.1038/newbio237013a0. [DOI] [PubMed] [Google Scholar]
  11. Fenyö E. M., Klein G. Independence of Moloney virus-induced cell-surface antigen and membrane-associated virion antigens in immunoselected lymphoma sublines. Nature. 1976 Mar 25;260(5549):355–357. doi: 10.1038/260355a0. [DOI] [PubMed] [Google Scholar]
  12. Freedman H. A., Lilly F., Steeves R. A. Antigenic properties of cultured tumor cell lines derived from spleens of Friend virus-infected BALB/c and BALB/c-H-2b mice. J Exp Med. 1975 Dec 1;142(6):1365–1376. doi: 10.1084/jem.142.6.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenberger J. S., Stephenson J. R., Aoki T., Aaronson S. A. Cell-surface antigens of murine sarcoma-virus-transformed non-producer cells: further evidence for lack of transplantation immunity. Int J Cancer. 1974 Aug 15;14(2):145–152. doi: 10.1002/ijc.2910140202. [DOI] [PubMed] [Google Scholar]
  14. Hartley J. W., Rowe W. P. Clonal cells lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology. 1975 May;65(1):128–134. doi: 10.1016/0042-6822(75)90013-6. [DOI] [PubMed] [Google Scholar]
  15. Hunsmann G., Claviez M., Moennig V., Schwarz H., Schäfer W. Properties of mouse leukemia viruses. X. Occurrence of viral structural antigens on the cell surface as revealed by a cytotoxicity test. Virology. 1976 Jan;69(1):157–168. doi: 10.1016/0042-6822(76)90203-8. [DOI] [PubMed] [Google Scholar]
  16. Hunsmann G., Moennig V., Schäfer W. Properties of mouse leukemia viruses. IX. Active and passive immunization of mice against Friend leukemia with isolated viral GP71 glycoprotein and its corresponding antiserum. Virology. 1975 Jul;66(1):327–329. doi: 10.1016/0042-6822(75)90203-2. [DOI] [PubMed] [Google Scholar]
  17. Ikeda H., Pincus T., Yoshiki T., Strand M., August J. T., Boyse E. A., Mellors R. C. Biological expression of antigenic determinants of murine leukemia virus proteins gp69-71 and p30. J Virol. 1974 Nov;14(5):1274–1280. doi: 10.1128/jvi.14.5.1274-1280.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katz D. H., Dorf M. E., Benacerraf B. Cell interactions between histoincompatible T and B lymphocytes. VI. Cooperative responses between lymphocytes derived from mouse donor strains differing at genes in the S and D regions of the H-2 complex. J Exp Med. 1974 Jul 1;140(1):290–295. doi: 10.1084/jem.140.1.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kedar E., Unger E., Schwartzbach M. In vitro induction of cell-mediated immunity to murine leukemia cells. I. Optimization of tissue culture conditions for the generation of cytotoxic lymphocytes. J Immunol Methods. 1976;13(1):1–19. doi: 10.1016/0022-1759(76)90182-4. [DOI] [PubMed] [Google Scholar]
  20. Lilly F., Steeves R. Antigens of murine leukemia viruses. Biochim Biophys Acta. 1974 Apr 29;355(1):105–118. doi: 10.1016/0304-419x(74)90009-2. [DOI] [PubMed] [Google Scholar]
  21. McCoy J. L., Fefer A., Glynn J. P. Comparative studies on the induction of transplantation resistance in BALB-c and C57BL-6 mice in three murine leukemia systems. Cancer Res. 1967 Oct;27(10):1743–1748. [PubMed] [Google Scholar]
  22. Old L. J., Boyse E. A., Stockert E. The G (Gross) leukemia antigen. Cancer Res. 1965 Jul;25(6):813–819. [PubMed] [Google Scholar]
  23. Parks W. P., Noon M. C., Gilden R., Scolnick E. M. Serological studies with low-molecular-weight polypeptides from the Moloney strain of murine leukemia virus. J Virol. 1975 Jun;15(6):1385–1395. doi: 10.1128/jvi.15.6.1385-1395.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Plata F., Cerottini J. C., Brunner K. T. Primary and secondary in vitro generation of cytolytic T lymphocytes in the murine sarcoma virus system. Eur J Immunol. 1975 Apr;5(4):227–233. doi: 10.1002/eji.1830050402. [DOI] [PubMed] [Google Scholar]
  25. Plata R., MacDonald H. R., Engers H. D. Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors. J Immunol. 1976 Jul;117(1):52–58. [PubMed] [Google Scholar]
  26. Rowe W. P., Pugh W. E., Hartley J. W. Plaque assay techniques for murine leukemia viruses. Virology. 1970 Dec;42(4):1136–1139. doi: 10.1016/0042-6822(70)90362-4. [DOI] [PubMed] [Google Scholar]
  27. Taylor G. M., Harris R., Freeman C. B. Cell-mediated cytotoxicity as a result of immunotherapy in patients with acute myeloid leukaemia. Br J Cancer. 1976 Feb;33(2):137–143. doi: 10.1038/bjc.1976.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ting C. C., Bonnard G. D. Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses. J Immunol. 1976 May;116(5):1419–1425. [PubMed] [Google Scholar]
  29. Ting C. C., Rodgigues D., Bushar G. S., Herberman R. B. Cell-mediated immunity to Friend virus-induced leukemia. II. Characteristics of primary cell-mediated cytotoxic response. J Immunol. 1976 Jan;116(1):236–243. [PubMed] [Google Scholar]
  30. Yoshiki T., Mellors R. C., Hardy W. D., Jr, Fleissner E. Common cell surface antigen associated with mammalian C-type RNA viruses. Cell membrane-bound gs antigen. J Exp Med. 1974 Apr 1;139(4):925–942. doi: 10.1084/jem.139.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zarling J. M., Raich P. C., McKeough M., Bach F. H. Generation of cytotoxic lymphocytes in vitro against autologous human leukaemia cells. Nature. 1976 Aug 19;262(5570):691–693. doi: 10.1038/262691a0. [DOI] [PubMed] [Google Scholar]

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