Selective immortalization of tumor-specific T cells to establish long-term T-cell lines maintaining primary cell characteristics

Methods Mol Biol. 2009;511:143-58. doi: 10.1007/978-1-59745-447-6_6.


Antigen-specific T cells play a key role in cellular immune response against cancer. The ability to isolate, maintain, and characterize tumor-specific T cells is a prerequisite to studying anticancer immune response and developing novel strategies for cancer immunotherapy. However, the life span of human T cells in vitro is usually short and is limited by the onset of cellular senescence. To establish long-term, antigen-specific T-cell lines and clones, we selectively immortalized antigen-responsive T cells from human peripheral blood mononuclear cells (PBMCs). PBMCs were stimulated with antigens, and then infected with a murine leukemia virus-based retroviral vector carrying an immortalizing gene, the human telomerase-reverse transcriptase gene. Since such vectors can only integrate in dividing cells, only antigen-activated T cells are efficiently transduced. Using this approach, we generated immortalized T-cell lines that maintained strictly IL-2-dependent growth and MHC-restricted, antigen-specific responsiveness, some of which have been in continuous culture for longer than 1 year, far in excess of the survival of parallel control nonimmortalized cultures. These lines showed antigen-specific proliferation with induced cytokine and chemokine production, and, in the case of CD8+ T-cell lines, antigen-specific cytolytic activity. When applied to the tumor antigen-specific T cells, the approach provides a convenient, reproducible means for generating a stable, continuously renewable source of antigen-specific T lymphocytes for a variety of studies on anticancer immunity.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Culture Techniques*
  • Cell Line, Tumor*
  • Cell Separation / methods
  • Cell Transformation, Neoplastic
  • Humans
  • Neoplasms* / immunology
  • Neoplasms* / pathology
  • Retroviridae / genetics
  • Retroviridae / metabolism
  • T-Lymphocytes / cytology*
  • T-Lymphocytes / immunology
  • Telomerase / genetics
  • Telomerase / metabolism


  • Telomerase