Molecular Biotechnology

, Volume 15, Issue 2, pp 133–142

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes

  • Isabelle Rivière
  • Humilidad F. Gallardo
  • Andrea B. Hagani
  • Michel Sadelain


Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells’ progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral modiated gene transfer efficiency in primary T-lymphocytes, in particular T cell activation status, viral receptor expression, and culture conditions.

Index Entries

Lymphocyte retroviral vector transduction gene transfer 


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  1. 1.
    Rivière, I., and Sadelain, M. Methods for the construction of retroviral vectors and the generation of high-titer producers. Methods in Molecular Medicine, Gene Therapy Protocols. (1997) Edited by: P. Robbins Humana Press Inc. Totowa, NJ. 59–78.Google Scholar
  2. 2.
    Bunnell, B.A., Muul, L.M., Donahue, R.E., Blaese, R.M., and Morgan, R.A. (1995) High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes. Proc. Natl. Acad. Sci. USA 92, 7739–7743.PubMedCrossRefGoogle Scholar
  3. 3.
    Gallardo, H.F., Tan, C., Ory, D., and Sadelain, M. (1997) Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes. Blood 90, 952–957.PubMedGoogle Scholar
  4. 4.
    Sadelain, M. Methods for retroviral-mediated gene transfer in murine primary T-lymphocytes. (1997) In: Gene Therapy Protocols: Methods in Molecular Biology. ed. by: P. Robbins Humana Press Inc. Totowa, NJ. 241–248.Google Scholar
  5. 5.
    Hagani, A.B., Rivière, I., Tan, C., Krause, A., and Sadelain, M. (1999) Activation conditions determine susceptibility of murine primary T lymphocytes to retroviral infection. J. Gen. Medicine. 1, 341–351.CrossRefGoogle Scholar
  6. 6.
    Van Parijs, L., Refaeli, Y., Lord, J. D., Nelson, B. H., Abbas, A. K., and Baltimore, D. (1999) Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death. Immunity 11, 281–288PubMedCrossRefGoogle Scholar
  7. 7.
    Miller, D.G., Adam, M.A., and Miller A.D. (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol. 10, 4239–42.PubMedGoogle Scholar
  8. 8.
    Roe, T.Y., Reynolds, T.C., Yu, G., and Brown, P.O. (1993) Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 12, 2099–2108.PubMedGoogle Scholar
  9. 9.
    Hajihosseini, M., Iavachev, L. and Price, J. (1993) Evidence that retroviruses integrate into post-replication host DNA. EMBO J. 12, 4969–4974.PubMedGoogle Scholar
  10. 10.
    Miller, A.D. Cell-surface receptors for retroviruses and implications for gene transfer. (1996) Proc. Natl. Acad. Sci. USA 93, 11407–11413.PubMedCrossRefGoogle Scholar
  11. 11.
    Amado, R.G., and Chen, I.S. (1999) Lentiviral vectors—the promise of gene therapy within reach? Science 285, 674–676.PubMedCrossRefGoogle Scholar
  12. 12.
    Koehne, G., Gallardo, H.F., Sadelain, M., and O’Reilly, R.J. Rapidselection of antigen specific T Lymphocytes by retroviral transduction Blood, in press.Google Scholar
  13. 13.
    Kim J.W., Closs, E.I., Albritton, L.M., and Cunningham JM. (1991) Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature 352, 725–728.PubMedCrossRefGoogle Scholar
  14. 14.
    Wang, H., Kavanaugh, M.P., North, R.A., and Kabat, D. (1991) Cell-surface receptor for ectropic murine retroviruses is a basic amino-acid transporter. Nature 352, 729–731.PubMedCrossRefGoogle Scholar
  15. 15.
    Kavanaugh, M.P., Miller, D.G., Zhang, W., Law, W., Kozak, S.L., Kabat, D., and Miller, A.D. (1994) Cellsurface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc. Natl. Acad. Sci. USA 91, 7071–7075.PubMedCrossRefGoogle Scholar
  16. 16.
    Wilson, C.A., Eiden, M.V., Anderson, W.B., Lehel, C., and Olah, Z. (1995) The dual-function hamster receptor for amphotropic murine leukemia virus (MuLV), 10A1 MuLV, and gibbon ape leukemia virus is a phosphate symporter. J. Virol. 69, 534–7.PubMedGoogle Scholar
  17. 17.
    Wyatt, R., and Sodroski J. (1998) The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280, 1884–1888.PubMedCrossRefGoogle Scholar
  18. 18.
    Albritton, L.M., Tseng, L., Scadden, D., and Cunningham, J.M. (1991) A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57, 659–666.CrossRefGoogle Scholar
  19. 19.
    Scott-Taylor, T., Gallardo, H.F., Gansbacher, B. and Sadelain, M. (1998) Adenovirus-facilitated infection of human cells with ecotropic retrovirus. Gene Ther. 5, 621–629.PubMedCrossRefGoogle Scholar
  20. 20.
    Lam, J. S., Reeves, M. E., Cowherd, R., Rosenberg, S. A. and Hwu, P. (1996) Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope. Hum. Gene Ther. 7, 1415–1422.PubMedGoogle Scholar
  21. 21.
    Rudoll, T., Phillips, K., Lee, S-W., Hull, S., Gaspar, O., Sucgang, N., Gilboa, E., and Smith, C. (1996) High-effiency retroviral vector mediated gene transfer into human peripheral CD4+ T lymphocytes. Gene Ther. 3, 695–705.PubMedGoogle Scholar
  22. 22.
    Schlegel, R., Tralka, T.S., Willingham, M.C., and Pastan, I. (1983) Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site? Cell 32, 639–46.PubMedCrossRefGoogle Scholar
  23. 23.
    Mastromarino, P., Conti, C., Goldoni, P., Hauttecoeur, B., and Orsi, N. (1987) Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH. J. Gen. Virol. 68, 2359–2369.PubMedGoogle Scholar
  24. 24.
    Burns, J.C., Friedmann, T., Direver, W., Burrascano, M., and Yee, J.K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: Concentratior to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033–8037.PubMedCrossRefGoogle Scholar
  25. 25.
    Kurre, P., Kiem, H.P., Morris, J., Heyward, S., Battini, J.L., and Miller, A.D. (1999) Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor. J. Virol. 73, 495–500.PubMedGoogle Scholar
  26. 26.
    Orlic, D., Girard, L.J., Jordan, C.T., Anderson, S.M., Cline, A.P., and Bodine, D.M. (1996) The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc. Natl. Acad. Sci. USA 93, 11097–11102.PubMedCrossRefGoogle Scholar
  27. 27.
    Chu, P., Lutzko, C., Stewart, A.K., and Dube, I.D. (1998) Retrovirus-mediated gene transfer into human hematopoietic stem cells. J. Mol. Med. 76, 184–192.PubMedCrossRefGoogle Scholar
  28. 28.
    Best, S., Le Tissier, P.R., and Stoye, J.P. (1997) Endogenous retroviruses and the evolution of resistance to retroviral infection. Trends Microbiol. 5, 313–318.PubMedCrossRefGoogle Scholar
  29. 29.
    Rousseau, V., Cremer, I., Lauret, E., Rivière, I., Aguet, M., and De Maeyer, E. (1995) Antiviral activity of autocrine interferon-beta requires the presence of a functional interferon type I receptor. J. Interferon Cytokine Res. 15, 785–789.PubMedCrossRefGoogle Scholar
  30. 30.
    Balzarini, J., and De Clercq, E. Analysis of inhibition of retroviral reverse transcriptase. (1996) Methods Enzymol. 275, 472–502.PubMedGoogle Scholar
  31. 31.
    Vieillard, V., Lauret, E., Rousseau, V., and De Maeyer, E. (1994) Blocking of retroviral infection at a step prior to reverse transcription in cells transformed to constitutively express interferon beta. Proc. Natl. Acad. Sci. USA 91, 2689–2693.PubMedCrossRefGoogle Scholar
  32. 32.
    Walker, C.M., and Levy, J.A. (1989) A diffusible lymphokine produced by CD8+ T lymphocytes suppresses HIV replication. Immunology 66, 628–630.PubMedGoogle Scholar
  33. 33.
    Cocchi, F., DeVico, A.L., Garzino-Demo, A., Arya, S.K., Gallo, R.C., and Lusso, P. (1995) Identification of RANTES, MIP-1a, and MIP-1b as the major HIV-suppressive factors produced by CD8+ T cells. Science 270, 1811–1815.PubMedCrossRefGoogle Scholar
  34. 34.
    Brinchmann, J.E., Gaudernack, G., and Vartdal, F. (1990) CD8+ T cells inhibit HIV replication in naturally infected CD4+ T cells. J Immunol. 144, 2961–2966.PubMedGoogle Scholar
  35. 35.
    Korin, Y. D., and Zack, J. A. (1999) Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes. J. Virol. 73, 6526–32.PubMedGoogle Scholar
  36. 36.
    Zack, J. A., Arrigo, S. J., Weitsman, S. R., Go, A. S., Haislip, A. and Chen, I. S. (1990) HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61, 213–22.PubMedCrossRefGoogle Scholar
  37. 37.
    Cooper, N.R., Jensen, F.C., Welsh, R.M. Jr., and Oldstone, M.B.A. (1976) Lysis of RNA tumor viruses by human serum: direct antibody-independent triggering of the classical complement pathway. J. Exp. Med. 144, 970–982.PubMedCrossRefGoogle Scholar
  38. 38.
    Zinkernagel, R.M. (1993) Immunity to viruses, in “Fundamental Immunology”, third edition, Raven Press, New York, NY, 1211–1250.Google Scholar
  39. 39.
    Bunnell, B.A., Metzger, M., Byrne, E., Morgan, R.A., and Donahue, R.E. (1997) Efficient in vivo marking of primary CD4+ T lymphocytes in nonhuman primates using a gibbon ape leukemia virus-derived retroviral vector. Blood 89, 1987–1995.PubMedGoogle Scholar
  40. 40.
    Traunecker, A., Luke, W., and Karjalainen, K. (1988) Soluble CD4 molecules neutralize human immunodeficiency virus type 1. Nature 331, 84–86.PubMedCrossRefGoogle Scholar
  41. 41.
    Fathman, C.G., Fitch, F.W., Denis, K.A., and Witte, O.N. (1989) Long term culture of immunocompetent cells, in “Fundamental Immunology”, second edition, Raven Press, New York, NY, 803–815.Google Scholar
  42. 42.
    Yamada, G., Kitamura, Y., Sonoda, H., Harada, H., Taki, S., Mulligan, R.C., Osawa, H., Diamantstein, T., Yokoyama, S. and Taniguchi, T. (1987) Retroviral expression of the human IL-2 gene in a murine T cell line results in cell growth autonomy and tumorigenicity. EMBO J. 6, 2705–2709.PubMedGoogle Scholar
  43. 43.
    Rosenberg, S.A., Aegersold, P., Cornetta, K., Kasid, A., Morgan, R., Moen, R., Karson, E.M., Lotze, M.T., Yang, J.C., Toplian, S.L., Merino, M.J., Culver, K., Miller, A.D., Blaese, R.M. and Anderson, W.F. (1990) Gene transfer into humans: immunotherapy of patients with advanced melanoma using tumor infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 323, 570–578.PubMedCrossRefGoogle Scholar
  44. 44.
    Rivière, I., Brose, K., and Mulligan, R.C. (1995) Effects of retroviral design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc. Natl. Acad. Sci. USA 92, 6733–6737.PubMedCrossRefGoogle Scholar
  45. 45.
    Strair, R.K., Towle, M., Heald, P.W. and Smith, B.R. (1990) Retroviral-mediated transfer and expression of exogenous genes in primary lymphoid cells: assaying for a viral transactivator activity in normal and malignant cells. Blood 76, 1201–1208.PubMedGoogle Scholar
  46. 46.
    Ferrari, G., Rossini, S., Giavazzi, R., Maggioni, D., Nobili, N., Soldati, M., Ungers, G., Mavilio, F., Gilboa, E. and Bordignon, C. (1991) An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency. Science 251, 1363–1366.PubMedCrossRefGoogle Scholar
  47. 47.
    Culver, K., Cornetta, K., Morgan, R., Morecki, S., Aebersold, P., Kasid, A., Lotze, M., Rosenberg, S.A., Anderson, W.F. and Blaese, R.M. (1991) Lymphocytes as cellular vehicles for gene therapy in mouse and man. Proc. Natl. Acad. Sci. USA 88, 3155–3159.PubMedCrossRefGoogle Scholar
  48. 48.
    Wu, A. G., Liu, X., Mazumder, A., Bellanti, J. A., and Meehan, K. R. (1999) Improvement of gene transduction efficiency in T lymphocytes using retroviral vectors. Hum. Gene Ther. 10, 977–982.CrossRefGoogle Scholar
  49. 49.
    Pollok, K. E., Hanenberg, H., Noblitt, T. W., Schroeder, W. L., Kato, I., Emanuel, D., and Williams, D. A. (1998) High effciency gene transfer into normal and adenosine deaminase- deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments. J Vitrol. 72, 4882–4892.Google Scholar
  50. 50.
    Ayuk, F.A., Li, Z., Kühlcke, K., Lindemann, C., Schade, U.M., Eckert, H-G., Zander, A.R., and Fehse, B. (1999) Establishment of an optimized gene transfer protocol for human primary T lymphocytes according to clinical requirements. Gene Ther. 6, 1788–1792.PubMedCrossRefGoogle Scholar
  51. 51.
    Dardalhon, V., Noraz, N., Pollok, K., Rebouissou, C., Boyer, M., Bakker, A. Q., Spits, H., and Taylor, N. (1999) Green fluorescent protein as a selectable marker of fibronectin- facilitated retroviral gene transfer in primary human T lymphocytes. Hum. Gene Ther. 10, 5–14.PubMedCrossRefGoogle Scholar
  52. 52.
    Sadelain, M., and Mulligan, R.C. (1992) Efficient retroviral-mediated gene transfer into murine primary lymphocytes. 88–34, Ninth International Immunology Congress, Budapest.Google Scholar
  53. 53.
    Mavilio, F., Ferrari, G., Rossini, S., Nobili, N., Bonini, C., Casorati, G., Traversari, C., and Bordignon, C. (1994) Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. Blood 83, 1988–1997.PubMedGoogle Scholar
  54. 54.
    Cullen, B.R., Lomedico, P.T., and Ju, G. (1984) Transcriptional interference in avian retroviruses: implications for the promoter insertion model of leukemogenesis. Nature 307, 241–245.PubMedCrossRefGoogle Scholar
  55. 55.
    Bowtell, D.D., Cory, S., Johnson, G.R., and Gonda, T.J. (1988) Comparison of expression in hematopoietic cells by retroviral vectors carrying two genes. J. Virol. 62, 2464–2473.PubMedGoogle Scholar
  56. 56.
    Gallardo, H.F., Tan, C., and Sadelain, M. (1997) The internal ribosomal entry site of encephalomyocarditis virus enables very reliable coexpression of two transgene in human primary T lymphocytes. Gene Ther. 4, 1115–1119PubMedCrossRefGoogle Scholar
  57. 57.
    Onodera, M., Nelson, D. M., Yachie, A., Jagadeesh, G. J., Bunnell, B. A., Morgan, R.A., and Blaese, R. M. (1998) Development of improved adenosine deaminase retroviral vectors. J. Virol. 72, 1769–1774.PubMedGoogle Scholar
  58. 58.
    Unutmaz, D., KewalRamani, V. N., Marmon, S., and Littman, D. R. (1999) Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J. Exp. Med. 189, 1735–1746.PubMedCrossRefGoogle Scholar
  59. 59.
    Douglas, J., Kelly, P., Evans, J. T. and Garcia, J. V. (1999) Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virusbased gene transfer vectors. Hum. Gene. Ther. 10, 935–945.PubMedCrossRefGoogle Scholar
  60. 60.
    Farina, A. R., Davis-Smyth, T., Gardner, K., and Levens, D. (1993) An early response of an AP1-junD complex during T-cell activation. J. Biol. Chem. 268, 26466–26475.PubMedGoogle Scholar
  61. 61.
    Sun, W., Graves, B. J., and Speck, N. A. (1995) Transactivation of the Moloney murinc leukemia virus and T-cell receptor beta-chain enhancers by cbf and ets requires intact binding sites for both proteins. J. Virol. 69, 4941–4949.PubMedGoogle Scholar
  62. 62.
    Speck, N. A., Renjifo, B., and Hopkins, N. (1990) Point mutations in the Moloney murine leukemia virus enhancer identify a lymphoid-specific viral core motif and 1,3-phorbol myristate acetate- inducible element. J. Virol. 64, 543–550.PubMedGoogle Scholar
  63. 63.
    Gunther, C. V., and Graves, B. J. (1994) Identification of ETS domain proteins in murine T lymphocytes that interact with the Moloney murine leukemia virus enhancer. Mol. Cell. Biol. 14, 7569–7580.PubMedGoogle Scholar
  64. 64.
    Hannibal, M. C., Markovitz, D. M., Clark, N., and Nabel, G. J. (1993) Differential activation of human immunodeficiency virus type 1 and 2 transcription by specific T-cell activation signals. J Virol. 67, 5035–5040.PubMedGoogle Scholar
  65. 65.
    Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., and Nolan, G. P. (1997) The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity 6, 235–244.PubMedCrossRefGoogle Scholar
  66. 66.
    Pollok, K. E., van der Loo, J. C., Cooper, R. J., Kennedy, L., and Williams, D. A. (1999) Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus. Hum. Gene Ther. 10, 2221–36PubMedCrossRefGoogle Scholar
  67. 67.
    Danos, O., and Mulligan, R.C. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host range. Proc. Natl. Acad. Sci. USA 85, 6460–6464.PubMedCrossRefGoogle Scholar
  68. 68.
    Miller, A.D., Garcia, J.V., von Suhr, N., Lynch, C.M., Wilson, C., and Eidon, M.V. (1990) Constraction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65, 2220–2224.Google Scholar
  69. 69.
    Fassati, A., Wells, D. J., Walsh, F. S., and Dickson, G. (1995) Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice. Hum. Gene Ther. 6, 1177–1183.PubMedCrossRefGoogle Scholar
  70. 70.
    Farson D., McGuinness, R.,, Dull T., Limoli, K., Lazar, R., Jalali, S., Sridhar Reddy, Pennathur-Das, R., Broad, D., and Finer, M. (1999) Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols. The Journal of Gene Medicine 1, 195–209.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2000

Authors and Affiliations

  • Isabelle Rivière
    • 1
  • Humilidad F. Gallardo
  • Andrea B. Hagani
  • Michel Sadelain
  1. 1.Gene Transfer and Somatic Cell Engineering Facility, Department of Human GeneticsMemorial Sloan-Kettering Cancer CenterNew York

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