Improvement of a method to reproducibly immortalize human T cells by oncogene transfection

Shahabuddin Alam, Yoshinori Katakura, Hiroshi Yoshida, Eun Ho Kim, Sanetaka Shirahata

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Abstract

The method to immortalize human T cells efficiently and reproducibly by oncogene transfection was improved. T cells were first grown selectively from peripheral blood lymphocytes population of healthy donors and atopic asthma patients, and from lymph node lymphocytes population of lung cancer patients by activating with mitogens (phytohemagglutinin and concanavalin A) and recombinant human interleukin-2 (rhIL-2) for five days. Plasmids expressing oncogenes, such as c-Ha-ras, c-myc, c-fos, v-myb and v-jun under the control of human cytomegalovirus promoter, were then introduced into these stimulated lymphocytes either separately or in various combinations by electropolation. After culturing these transfected lymphocytes for recovery for 1 day, they were fed every 3-4 days. Although all the control cells died within one month, oncogene-transfected lymphocytes continued to proliferate actively even for more than several months, indicating that oncogene-transfected lymphocytes were successfully immortalized. Flowcytometric analyses revealed that most of the immortalized lymphocytes were T cells expressing CD3+ surface antigen. The ratios of CD4+ and CD8+ subpopulations in immortalized T cells derived from healthy donors varied, depending on the kinds of oncogenes used. However, CD8+ subpopulation in immortalized T cells derived from cancer patients and atopic asthma patients were dominant, independent of the kinds of oncogenes. These immortalized T cells showed different proliferative responses in the presence or absence of exogenous human rhIL-2, depending on their origin of donors. Furthermore, immortalized T cells derived from healthy donors showed stronger cytotoxicity against K562 cells, suggesting that MHC-nonrestricted killer T cells in T cell population were also immortalized. Immortalized T cell lines, which proliferate continuously without stimulation of a mitogen or antigen in medium containing a low concentration of rhIL-2, have been maintained for more than 2 years without any growth rate decrease.

Original languageEnglish
Pages (from-to)71-81
Number of pages11
JournalCytotechnology
Volume33
Issue number1-3
DOIs
Publication statusPublished - 2000

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Clinical Biochemistry
  • Cell Biology

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