Summary
A simple method was devised for the establishment of continuous lymphoblastoid, cell lines (LCL). Unfractionated mononuclear cells collected from healthy donors were infected in vitro by Epstein-Barr Virus (EBV) (strain B95-8) under specific, conditions: an immunosuppressive drug, Cyclosporin-A (CS-A, Sandoz), associated with the use of a feeder layer (MRC-5) led to 100% efficiency of LCL establishment. A bank of 400 LCL was set up for completion of genetic studies. Regression and kinetics of virus-induced transformation were monitored and related to donors' EBV immune status. Mean time of LCL establishment and probability of regression among seropositive donors were not linked to any given value titer of antibodies against Viral Capsid Antigen (VCA) or against Epstein-Barr Nuclear Antigen (EBNA). However, when the anti-VCA:anti-EBNA ratio was considered, this parameter, seemed to be linked to the kinetics of transformation but not to the probability of regression. Once LCL are established, large quantities of human cells can be produced. The complete cellular DNA is available so that any part of it can be scrutinized. Moreover, some of the phenotypic characteristics of these B cells be used for a wide range of investigations.
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Anderson, M. A.; Gusella, J. F. Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines. In Vitro 20:856–858; 1984.
Bejarano, M. T.; Masucci, M. G.; Ernberg, I., et al. Effect of cyclosporin A (CS-A) on the ability of T lymphocyte subsets to inhibit the proliferation of autologous EBV-transformed B cells. Int. J. Cancer 35: 327–333; 1985.
Berkel, A. I.; Henle, W.; Henle, G., et al. Epstein-Barr virusrelated patterns in ataxia-telangiectasia. Clin. Exp. Immunol. 35: 196–201; 1979.
Bird, A. G.; McLachlan, S. M.; Britton, S. Cyclosporin A promotes spontaneous outgrowthin vitro of Epstein-Barr virus-induced B-cell lines. Nature 289: 300–301; 1981.
Boyum, A. Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (suppl.): 97: 7R; 1968.
Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. 21 (suppl.): 97: 77–89; 1968.
Gaston, J. S. H. Epstein-Barr virus specific cytotoxic T lymphocytes as probes of HLA polymorphisms. Heterogeneity of T-cell restricting determinants associated with the serologically defined HLA-A2 antigen. J. Exp. Med. 158: 280–293; 1983.
Gusella, J. F.; Wexler, N. S.; Conneally, P. M., et al. A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306: 234–238; 1983.
Harada, S.; Sakamoto, K.; Seeley, J. K., et al. Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects. J. Immunol. 129: 2532–2535; 1982.
Henderson, E.; Miller, G.; Robinson, J., et al. Efficiency of transformation of lymphocytes by Epstein-Barr virus. Virology 76: 152–163; 1977.
Henle, G.; Henle, W. Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91: 1248–1256; 1966.
Jacobs, J. P.; Jones, C. M.; Baille, J. P. Characteristics of a human diploid cell designated MRC-5. Nature 227: 168–170; 1970.
Klein, E.; Massucci, M. G. Cell-mediated immunity against Epstein-Barr virus infected B lymphocytes. Springer Semin. Immunopathol. 5: 63–74; 1982.
Lange, B.; Arbetter, J., et al. Longitudinal study of Epstein-Barr virus antibody titers and excretion in pediatric patients with Hodgkin's disease. Int. J. Cancer 22: 521–527; 1978.
Lombard, Y.; Hartmann, D.; Coupin, G., et al. Human B-like lymphoblastoid cell lines obtained by long-term culture of normal spleen leukocytes. Biol. Cell. 50: 295–298; 1984.
Masucci, M. G.; Bejarano, M. T.; Masucci, G., et al. Large granular lymphocytes inhibit the in vitro growth of autologous Epstein-Barr virus-infected B cells. Cell. Immunol. 76: 311–321; 1983.
Miller, G.; Ender, J. F.; Lisco, H., et al. Establishment of lines from normal human blood leukocytes by cocultivation with a leukocyte line derived from a leukemic child (34189). Proc. Soc. Exp. Biol. Med. 132: 247–252; 1969.
Miller, G.; Lisco, H.; Kohn, H. I., et al. Establishment of cell lines from normal adult blood leukocytes by exposure to Epstein-Barr virus and neutralization by human sera with Epstein-Barr virus antibody (35810). Proc. Soc. Exp. Biol. Med. 137: 1459–1467; 1971.
Miller, G.; Lipman, M. Relesase of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci. USA 70: 190–194; 1973.
Misko, I. S.; Moss, D. J.; Pope, J. H. HLA antigen-related restriction of T lymphocyte cytotoxicity to Epstein-Barr virus. Proc. Natl. Acad. Sci. USA 77: 4247–4250; 1980.
Misko, I. S.; Kane, R. G.; Pope, J. H. Generationin vitro of HLA-restricted EB virus-specific cytotoxic human T cells by autologous lymphoblastoid cell lines: the role of previous EB virus infection and foetal calf serum. Int. J. Cancer 29: 41–48; 1982.
Misko, I. S.; Pope, J. H.; Hutter, R., et al. HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies. Int. J. Cancer 33: 239–243; 1984.
Moss, D. J.; Pope, J. H. Assay of the infectivity of Epstein-Barr virus by transformation of human leukocytesin vitro. J. Gen. Virol. 17: 233–236; 1972.
Moss, D. J.; Pope, J. H. E. B. Virus-associated nuclear antigen production and cell proliferation in adult peripheral blood leukocytes inoculated with the QIMR-WIL strain of E. B. Virus. Int. J. Cancer 15: 503–511; 1975.
Moss, D. J.; Scott, W.; Pope, J. H. An immunological basis for inhibition of transformation of human lymphocytes by EB virus. Int. J. Cancer 15:503–511; 1975.
Moss, D. J.; Rickinson, A. B.; Pope, J. H. Long-term T-cell mediated immunity to Epstein-Barr virus in man: I. Complete regression of virus-induced transformation in cultures of seropositive donor leukocytes. Int. J. Cancer 22:662–668; 1978.
Moss, D. J.; Rickinson, A. B.; Pope, J. H. Long-term T-cell mediated immunity to Epstein-Barr virus in man: III Activation of cytotoxic T-cells in, virus infected leukocyte cultures. Int. J. Cancer 23:618–625; 1979.
Nilsson, K.; Klein, G.; Henle, W., et al. The establishment, of lymphoblastoid, lines from adult, and fetal human lymphoid tissues and its dependence of EBV. Int. J. Cancer 8:443–450; 1971.
Pope, J. H. Tranformation by the virusin vitro. In: Epstein, M. A.; Achong, B. G., eds. The Esptein-Barr virus. Berlin: Springer-Verlag 1979:206–223.
Reedman, B. M.; Klein, G. Cellular localization of an EBV-associated complement fixing antigen in producer and nonproducer lymphoblastoid cell lines. Int. J. Cancer 11:499–520; 1973.
Rickinson, A. B.; Moss, D. J.; Pope, J. H. Long-term T-cell mediated immunity to Epstein-Barr virus in man. II. Components necessary for regression in virus-infected leukocyte cultures. Int. J. Cancer 23:610–617; 1979.
Rickinson, A. B.; Wallace, L. E.; Epstien, M. A. HLA-restricted T-cell recognition of Epstein-Barr virus-infected B cells. Nature 283:865–868; 1980.
Rickinson, A. B.; Moss, D. J. Epstein-Barr virus-induced transformation: immunological aspects. In: Klein, G., ed. Advances in vival oncology, vol. 3. New York: Raven Press; 1983:213–238.
Rickinson, A. B. T-cell control of Herpes virus infections: Lessons from the Epstein-Barr virus. Prog. Brain Res. 59:189–199; 1983.
Rickinson, A. B.; Rowe, M.; Hart, I. J., et al. T-cell mediated regression of “spontaneous” and of Epstein-Barr virus-induced B cell transformation in vitro: Studies with cyclosporin-A. Cell. Immunol. 87:646–658; 1984.
Schooley, R. T.; Arbit, D. I.; Henle, W., et al. T-lymphocyte subset interactions in the cell-mediated immune response to Epstein-Barr virus. Cell. Immunol. 86:402–412; 1984.
Ten Napel, C. H. H.; The, T. H.; Van Egten-Bijker, J., et al. Discordance of Epstein-Barr virus (EBV) specific humoral and cellular immunity in patients with malignant lymphomas: elevated antibody titers and lowered in vitro lymphocytes reactivity. Clin. Exp. Immunol. 34:338–346; 1978.
Thorley-Lawson, D. A.; Chess, L.; Strominger, J. L. Suppression ofin-vitro Epstein-Barr virus infection. A new role for adult human T lymphocytes. J. Exp. Med. 146:495–508; 1977.
White, R.; Leppert, M.; Bishop, D. T., et al. Construction of linkage maps with DNA markers for human chromosomes. Nature 313:101–105; 1985.
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The work was supported by Fondation de la Recherche Medicale (France) for Professor J. Dausset (Paris) and by the International Agency for Research on Cancer (Lyon).
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Pelloquin, F., Lamelin, J.P. & Lenoir, G.M. Human blymphocytes immortalization by epstein-barr virus in the presence of cyclosporin a. In Vitro Cell Dev Biol 22, 689–694 (1986). https://doi.org/10.1007/BF02621085
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DOI: https://doi.org/10.1007/BF02621085