Herpesvirus saimiri Transformation of Human T Lymphocytes

Bernhard Fleckenstein1, Armin Ensser1

1 Institut für Klinische und Molekulare Virologie, Friedrich‐Alexander‐Universität Erlangen‐Nürnberg, Erlangen
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.21
DOI:  10.1002/0471142735.im0721s63
Online Posting Date:  November, 2004
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Abstract

Viral transformation of T cells is an effective method for obtaining large numbers of T cells that are easily maintained in the laboratory. This unit describes a method for generating antigen‐independent, virally‐transformed T cells using a T‐lymphotropic primate gamma‐2 herpesvirus, Herpesvirus saimiri (HVS; strain C488). Support protocols for preparing and titrating HSV C488 stocks and testing the functional status of transformed T cells are also included.

Keywords: herpesvirus; tumor virus; T cell transformation; T cell lines; immortalization

     
 
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Table of Contents

  • Basic Protocol 1: Generation of HVS‐Transformed T Cell Lines
  • Support Protocol 1: Culture of Permissive OMK Cells and Generation of HVS C488 Virus Stock
  • Support Protocol 2: Titration of Virus Stock
  • Support Protocol 3: HVS‐Transformed T Cell Lines: Verification of Nonproducer Status and Functional Testing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of HVS‐Transformed T Cell Lines

  Materials
  • Cord blood, peripheral blood, or cultured T cell line (see unit 7.19 for culture of T cells)
  • Lymphocyte growth medium (LGM; see recipe) with and without IL‐2
  • Phytohemagglutinin (PHA; e.g., Burroughs Wellcome, Difco, or Murex)
  • Human recombinant interleukin‐2: hIL‐2, recombinant (E. coli) from Roche Diagnostics or Aldesleukin (Proleukin, Chiron)
  • HVS C488 virus stock (≥105 TCIP/ml; see Support Protocols protocol 21 and protocol 32)
  • 25‐cm2 tissue culture flasks
  • Additional reagents and equipment for Ficoll‐Hypaque gradient centrifugation (unit 7.1) and counting viable cells ( appendix 3B)

Support Protocol 1: Culture of Permissive OMK Cells and Generation of HVS C488 Virus Stock

  Materials
  • OMK cell line (ATCC #CRL‐1556)
  • Complete medium for OMK cells: Eagle minimal essential medium (MEM) or Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; appendix 2A) and (optional) appropriate antibiotics/antimycotics (these complete media are referred to as MEM‐10 or DMEM‐10, respectively)
  • Phosphate‐buffered saline (PBS; appendix 2A) or culture medium without serum
  • 0.25% (w/v) trypsin/0.1% (w/v) EDTA, sterile, mycoplasma screened (prepare fresh)
  • HVS strain C488 (ATCC #VR‐1414 or ATCC #VR‐1396; Desrosiers and Falk, )
  • 75‐cm2 tissue culture flasks
  • Centrifuge
  • Additional reagents and equipment for counting viable cells ( appendix 3B) and titration of HVS ( protocol 3)

Support Protocol 2: Titration of Virus Stock

  • HVS stock ( protocol 2)
  • 96‐well flat‐bottom tissue culture plates
  • 15‐ml polypropylene tubes, sterile
  • Aerosol‐barrier pipet tips
  • Inverted microscope
CAUTION: The virus stocks are a biohazard and must be handled with care. However, HVS is rapidly inactivated by heat, UV, or common disinfectants.

Support Protocol 3: HVS‐Transformed T Cell Lines: Verification of Nonproducer Status and Functional Testing

  • HVS‐transformed human T cells (see protocol 1)
  • 25‐cm2 tissue culture flasks
  • Additional reagents and equipment for PCR (unit 10.20)
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Figures

Videos

Literature Cited

   Akari, H., Mori, K., Terao, K., Otani, I., Fukasawa, M., Mukai, R., and Yoshikawa, Y. 1996. In vitro immortalization of Old World monkey T lymphocytes with herpesvirus saimiri: Its susceptibility to infection with simian immunodeficiency viruses. Virology 218:382‐388.
   Biesinger, B., Müller‐Fleckenstein, I., Simmer, B., Lang, G., Wittmann, S., Platzer, E., Desrosiers, R.C., and Fleckenstein, B. 1992. Stable growth transformation of human T lymphocytes by herpesvirus saimiri. Proc. Natl. Acad. Sci. U.S.A. 89:3116‐3119.
   Brielmeier, M., Bechet, J.M., Falk, M.H., Pawlita, M., Polack, A., and Bornkamm, G.W. 1998. Improving stable transfection efficiency: Antioxidants dramatically improve the outgrowth of clones under dominant marker selection. Nucl. Acids Res. 26:2082‐2085.
   Daniel, M.D., Silva, D., and Ma, N. 1976. Establishment of owl monkey kidney 210 cell line for virological studies. In Vitro 12:290.
   Desrosiers, R.C. and Falk, L.A. 1982. Herpesvirus saimiri strain variability. J. Virol. 43:352‐356.
   Duboise, S.M., Guo, J., Czajak, S., Desrosiers, R.C., and Jung, J.U. 1998. STP and Tip are essential for herpesvirus saimiri oncogenicity. J. Virol. 72:1308‐1313.
   Ensser, A., Neipel, F., and Fickenscher, H. 2002. Rhadinovirus pathogenesis. In Structure‐function Relationships of Human Pathogenic Viruses (E. Bogner and A. Holzenburg, eds.) pp. 349‐429. Kluwer Academic Publishers/Plenum Publishers, New York.
   Ensser, A., Thurau, M., Wittmann, S., and Fickenscher, H. 2003. The primary structure of the herpesvirus saimiri strain C488 genome. Virology. 314:471‐487.
   Feldmann, G., Fickenscher, H., Bodemer, W., Spring, M., Nisslein, T., Hunsmann, G., and Dittmer, U. 1997. Generation of herpesvirus saimiri–transformed T‐cell lines from macaques is restricted by reactivation of simian spuma viruses. Virology 229:106‐112.
   Fickenscher, H. and Fleckenstein, B. 2001. Herpesvirus saimiri. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356:545‐567.
   Fickenscher, H., Biesinger, B., Knappe, A., Wittmann, S., and Fleckenstein, B. 1996. Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T‐cell activation genes, in growth‐transformed human T lymphocytes. J. Virol. 70:6012‐6019.
   Fickenscher, H., Bökel, C., Knappe, A., Biesinger, B., Meinl, E., Fleischer, B., Fleckenstein, B., and Bröker, B.M. 1997. Functional phenotype of transformed human αβ and γδ T cells determined by different subgroup C strains of herpesvirus saimiri. J. Virol. 71:2252‐2263.
   Glykofrydes, D., Niphuis, H., Kuhn, E.M., Rosenwirth, B., Heeney, J.L., Bruder, J., Niedobitek, G., Müller‐Fleckenstein, I., Fleckenstein, B., and Ensser, A. 2000. Herpesvirus saimiri vFLIP provides an antiapoptotic function but is not essential for viral replication, transformation, or pathogenicity. J. Virol. 74:11919‐11927.
   Kärber, G. 1931. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedebergs. Arch. Exp. Pathol. Pharmakol. 162:480‐483.
   Knappe, A., Hiller, C., Thurau, M., Wittmann, S., Hofmann, H., Fleckenstein, B., and Fickenscher, H. 1997. The superantigen‐homologous viral immediate‐early gene ie14/vsag in herpesvirus saimiri‐transformed human T cells. J. Virol. 71:9124‐9133.
   Knappe, A., Thurau, M., Niphuis, H., Hiller, C., Wittmann, S., Kuhn, E.M., Rosenwirth, B., Fleckenstein, B., Heeney, J., and Fickenscher, H. 1998. T‐cell lymphoma caused by herpesvirus saimiri C488 independently of ie14/vsag, a viral gene with superantigen homology. J. Virol. 72:3469‐3471.
   Knappe, A., Feldmann, G., Dittmer, U., Meinl, E., Nisslein, T., Wittmann, S., Mätz‐Rensing, K., Kirchner, T., Bodemer, W., and Fickenscher, H. 2000. Herpesvirus saimiri‐transformed macaque T cells are tolerated and do not cause lymphoma after autologous reinfusion. Blood 95:3256‐3261.
   Medveczky, M.M., Geck, P., Sullivan, J.L., Serbousek, D., Djeu, J.Y., and Medveczky, P.G. 1993. IL‐2 independent growth and cytotoxicity of herpesvirus saimiri–infected human CD8 cells and involvement of two open reading frame sequences of the virus. Virology 196:402‐412.
   Meinl, E., Fickenscher, H., Hoch, R.M., de Waal Malefyt, R., 't Hart, B.A., Wekerle, H., Hohlfeld, R., and Fleckenstein, B. 1997. Growth transformation of antigen‐specific T cell lines from rhesus monkeys by herpesvirus saimiri. Virology 229:175‐182.
   Mittrücker, H.W., Müller‐Fleckenstein, I., Fleckenstein, B., and Fleischer, B. 1992. CD2‐mediated autocrine growth of herpes virus saimiri–transformed human T lymphocytes. J. Exp. Med. 176:909‐913.
   Simmer, B., Alt, M., Buckreus, I., Berthold, S., Fleckenstein, B., Platzer, E., and Grassmann, R. 1991. Persistence of selectable herpesvirus saimiri in various human haematopoietic and epithelial cell lines. J. Gen. Virol. 72:1953‐1958.
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   Yasukawa, M., Inoue, Y., Kimura, N., and Fujita, S. 1995. Immortalization of human T cells expressing T‐cell receptor gamma delta by herpesvirus saimiri. J. Virol. 69:8114‐8117.
Key References
   Biesinger et al., 1992. See above.
  Primary reference for growth transformation of human T cells by Herpesvirus saimiri.
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