Molecular Medicine

, Volume 17, Issue 11–12, pp 1223–1232 | Cite as

Retroviral Insertional Mutagenesis Can Contribute to Immortalization of Mature T Lymphocytes

  • Sebastian Newrzela
  • Kerstin Cornils
  • Tim Heinrich
  • Julia Schläger
  • Ji-Hee Yi
  • Olga Lysenko
  • Janine Kimpel
  • Boris Fehse
  • Dorothee von Laer
Research Article


Several cases of T-cell leukemia caused by gammaretroviral insertional mutagenesis in children treated for x-linked severe combined immunodeficiency (SCID) by transplantation of autologous gene-modified stem cells were reported. In a comparative analysis, we recently showed that mature T cells, on the contrary, are highly resistant to transformation by gammaretroviral gene transfer. In the present study, we observed immortalization of a single T-cell clone in vitro after gammaretroviral transduction of the T-cell protooncogene LMO2. This clone was CD4/CD8 double-negative, but expressed a single rearranged T-cell receptor. The clone was able to overgrow nonmanipulated competitor T-cell populations in vitro, but no tumor formation was observed after transplantation into Rag-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptors were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. This study demonstrates that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results show that signaling of the IL-2 receptor and the protooncogene LMO2 can act synergistically in maligniant transformation of mature T lymphocytes.



The authors would like to thank Marianne Hartmann for technical assistance and Felix Hermann for discussions. This study was supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany; LA1135/9-1 within the SPP1230 and FE568/11-1).

Supplementary material

10020_2011_17111223_MOESM1_ESM.pdf (451 kb)
Supplementary material, approximately 450 KB.


  1. 1.
    Li Z, et al. (2002) Murine leukemia induced by retroviral gene marking. Science. 296:497.CrossRefGoogle Scholar
  2. 2.
    Modlich U, et al. (2005) Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood. 105:4235–46.CrossRefGoogle Scholar
  3. 3.
    Stein S, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med. 16:198-204.CrossRefGoogle Scholar
  4. 4.
    Hacein-Bey-Abina S, et al. (2008) Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 118:3132–42.CrossRefGoogle Scholar
  5. 5.
    Hacein-Bey-Abina S, et al. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348:255–6.CrossRefGoogle Scholar
  6. 6.
    Hacein-Bey-Abina S, et al. (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 302:415–9.CrossRefGoogle Scholar
  7. 7.
    Howe SJ, et al. (2008) Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118:3143–50.CrossRefGoogle Scholar
  8. 8.
    Laufs S, et al. (2004) Insertion of retroviral vectors in NOD/SCID repopulating human peripheral blood progenitor cells occurs preferentially in the vicinity of transcription start regions and in introns. Mol. Ther. 10:874–81.CrossRefGoogle Scholar
  9. 9.
    Wu X, Li Y, Crise B, Burgess SM. (2003) Transcription start regions in the human genome are favored targets for MLV integration. Science. 300:1749–51.CrossRefGoogle Scholar
  10. 10.
    Baum C, von Kalle C. (2003) Gene therapy targeting hematopoietic cells: better not leave it to chance. Acta Haematol. 110:107–9.CrossRefGoogle Scholar
  11. 11.
    Kustikova OS, et al. (2009) Cell-intrinsic and vector-related properties cooperate to determine the incidence and consequences of insertional muta-genesis. Mol. Ther. 17:1537–47.CrossRefGoogle Scholar
  12. 12.
    Deeks SG, et al. (2002) A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol. Ther. 5:788–97.CrossRefGoogle Scholar
  13. 13.
    Mitsuyasu RT, et al. (2000) Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood. 96:785–93.PubMedGoogle Scholar
  14. 14.
    Recchia A, et al. (2006) Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells. Proc. Natl. Acad. Sci. U. S. A. 103:1457–62.CrossRefGoogle Scholar
  15. 15.
    Walker RE, et al. (2000) Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection. Blood. 96:467–74.PubMedGoogle Scholar
  16. 16.
    Newrzela S, et al. (2008) Resistance of mature T cells to oncogene transformation. Blood. 112:2278–86.CrossRefGoogle Scholar
  17. 17.
    Nagai T, et al. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20:87–90.CrossRefGoogle Scholar
  18. 18.
    Rizzo MA, Springer GH, Granada B, Piston DW. (2004) An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22:445–9.CrossRefGoogle Scholar
  19. 19.
    Kalamasz D, et al. (2004) Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies. J. Immunother. 27:405–18.CrossRefGoogle Scholar
  20. 20.
    Schmidt M, et al. (2001) Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum. Gene Ther. 12:743–9.CrossRefGoogle Scholar
  21. 21.
    Currier JR, Robinson MA. (2001) Spectratype/immunoscope analysis of the expressed TCR repertoire. Curr. Protoc. Immunol. Chapter 10: Unit 10.28.Google Scholar
  22. 22.
    Hintzen RQ, et al. (1993) Regulation of CD27 expression on subsets of mature T-lymphocytes. J. Immunol. 151:2426–35.PubMedGoogle Scholar
  23. 23.
    Schmitt TM, et al. (2004) Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5:410–7.CrossRefGoogle Scholar
  24. 24.
    Ambrogio C, et al. (2009) NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells. Cancer Res. 69:8611–9.CrossRefGoogle Scholar
  25. 25.
    Mathas S, et al. (2006) Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma. Nat. Immunol. 7:207–15.CrossRefGoogle Scholar
  26. 26.
    Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131:861–72.CrossRefGoogle Scholar
  27. 27.
    Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663–76.CrossRefGoogle Scholar
  28. 28.
    Woods NB, Bottero V, Schmidt M, von Kalle C, Verma IM. (2006) Gene therapy: therapeutic gene causing lymphoma. Nature. 440:1123.CrossRefGoogle Scholar
  29. 29.
    von Laer D. (2009) Peaceful coexistence or clonal dominance? Blood. 114:3507–8.CrossRefGoogle Scholar
  30. 30.
    Gilliland DG, Tallman MS. (2002) Focus on acute leukemias. Cancer Cell. 1:417–20.CrossRefGoogle Scholar
  31. 31.
    Hahn WC, Weinberg RA. (2002) Rules for making human tumor cells. N. Engl. J. Med. 347:1593–603.CrossRefGoogle Scholar
  32. 32.
    Kohn DB, Sadelain M, Glorioso JC. (2003) Occurrence of leukaemia following gene therapy of X-linked SCID. Nat. Rev. Cancer 3:477–88.CrossRefGoogle Scholar
  33. 33.
    Lamprecht B, et al. Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma. Nat. Med. 16:571-9, 1p following 579.Google Scholar
  34. 34.
    Smith KA. (1988) Interleukin-2: inception, impact, and implications. Science. 240:1169–76.CrossRefGoogle Scholar
  35. 35.
    Willerford DM, et al. (1995) Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 3:521–30.CrossRefGoogle Scholar
  36. 36.
    Lodolce JP, et al. (1998) IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 9:669–676.CrossRefGoogle Scholar
  37. 37.
    Fehniger TA, et al. (2001) Fatal leukemia in interleukin-15 transgenic mice. Blood Cells Mol. Dis. 27:223–30.CrossRefGoogle Scholar
  38. 38.
    Hsu C, et al. (2007) Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene. Blood. 109:5168–77.CrossRefGoogle Scholar
  39. 39.
    Charo J, et al. (2005) Bcl-2 overexpression enhances tumor-specific T-cell survival. Cancer Res. 65:2001–8.CrossRefGoogle Scholar

Copyright information

© The Feinstein Institute for Medical Research 2011

Authors and Affiliations

  • Sebastian Newrzela
    • 1
  • Kerstin Cornils
    • 2
  • Tim Heinrich
    • 1
  • Julia Schläger
    • 1
  • Ji-Hee Yi
    • 1
  • Olga Lysenko
    • 3
  • Janine Kimpel
    • 4
  • Boris Fehse
    • 2
  • Dorothee von Laer
    • 4
  1. 1.Senckenberg Institute of PathologyGoethe-University Hospital FrankfurtFrankfurt am MainGermany
  2. 2.Interdisciplinary Clinic and Policlinic for Stem Cell Transplantation, Research Department Cell and Gene TherapyUniversity Medical Centre Hamburg-EppendorfHamburgGermany
  3. 3.Institute for Molecular MedicineGoethe-University Hospital FrankfurtFrankfurt am MainGermany
  4. 4.Division for Virology, Institute of VirologyInnsbruck Medical UniversityInnsbruckAustria

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