Generation of HTLV‐I‐Transformed T Cell Lines

Thomas B. Nutman1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.20
DOI:  10.1002/0471142735.im0720s47
Online Posting Date:  May, 2002
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Viral transformation of T cells is an effective method for obtaining large numbers of T cells that are easily maintained in the laboratory. Human T cell leukemia/lymphoma virus (HTLV‐I), a type C retrovirus clearly implicated as the etiologic agent of adult T cell leukemia, can both infect and transform normal T cells. This unit presents a protocol in which human T cells are activated and then transformed with HTLV‐I. Detection of virally transformed cells is discussed in the commentary.

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Table of Contents

  • Commentary
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Basic Protocol 1:

  • Cord blood cells, peripheral blood mononuclear cells (PBMC; unit 7.1), or T cell clones (unit 7.19)
  • Complete RPMI‐10 medium ( appendix 2A) without and with 10 to 100 U/ml IL‐2
  • Phytohemagglutinin (PHA; e.g., Burroughs Wellcome or Difco) or specific antigen
  • HTLV‐I‐transforming cell line (ATCC #HUT‐102)
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)
  • 24‐well flat‐bottom microtiter plate (Costar)
  • 137Cs irradiator (e.g., Gammacell 1000, Nordion)
  • Additional reagents and solutions for Ficoll‐Hypaque gradient centrifugation (unit 7.1) and counting cells ( appendix 3A)
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Literature Cited

Literature Cited
   Graziano, S.L., Lehr, B.M., Merl, S.A., Ehrlich, G.D., Moore, J.L., Hallinan, E.J., Hubbell, C., Davey, F.R., Vournakis, J., and Poiesz, B.J. 1987. Quantitative assay of human T‐cell leukemia/lymphoma virus transformation. Cancer Res. 47:2468‐2472.
   Hinuma, Y., Nagata, K., Hanaoka, M., Nakai, M., Matsumoto, T., Kinoshita, K.I., Shirakawa, S., and Miyoshi, I. 1981. Adult T‐cell leukemia: Antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. Natl. Acad. Sci. U.S.A. 78:6476‐6480.
   Markham, P.D., Gallo, R.C., and Salahuddin, S.Z. 1984. In vitro transformation of human cord blood and bone marrow T lymphocytes by HTLV. Kroc Found. Ser. 18:323‐335.
   Poiesz, B.J., Ruscetti, F.W., Gazdar, A.F., Bunn, P.A., Minna, J.P., and Gallo, R.C., 1980. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T‐cell lymphoma. Proc. Natl. Acad. Sci. U.S.A. 77:7414‐7419.
   Poiesz, B.J., Ruscetti, F.W., Reitz, M.S., Kalyanaraman, V.S., and Gallo, R.C. 1981. Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of a patient with Sezary T cell leukemia. Nature (Lond.) 294:268‐271.
   Popovic, M., Lange‐Wantzin, G., Sarin, P.S., Mann, D., and Gallo, R.C. 1983. Transformation of human umbilical cord blood T cells by human cell leukemia/lymphoma virus. Proc. Natl. Acad. Sci.U.S.A. 80:5402‐5406.
   Sarin, P.S. and Gallo, R.C. 1985. Cellular transformation by human T cell leukemia retroviruses. (HTLV). Pharmacol. Ther. 27:49‐62.
   Wong‐Staal, F., Ratner, L., Shaw, G., Hahn, B., Harper, M., Franchini, G., and Gallo, R. 1985. Molecular biology of human T‐lymphotropic retroviruses Cancer Res. 45:4539s‐4544s.
   Selden, R.F., Schatz, D.G., and Coen, D.M. 1989. Analysis of DNA sequences by blotting and hybridization. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.9.1‐2.9.17. Greene Publishing and Wiley‐Interscience, New York.
   Yoshida, M., Miyoshi, I., and Hinuma, Y. 1982. Isolation and characterization of retrovirus from cell lines of human adult T‐cell leukemia and its implication in the disease. Proc. Natl. Acad. Sci. U.S.A. 79:2031‐2035.
Key References
   Poiesz et al., 1980. See above.
  Primary reference for the isolation and description of the HTLV‐I retrovirus.
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