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

The method to immortalize human T cells efficiently and reproducibly by onc ogene 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 patien ts 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 contr ol of human cytomegalovirus promoter, were then introduced into these stimu lated lymphocytes either separately or in various combinations by electropo lation. After culturing these transfected lymphocytes for recovery for 1 da y, they were fed every 3-4 days. Although all the control cells died within one month, oncogene-transfected lymphocytes continued to proliferate activ ely even for more than several months, indicating that oncogene-transfected lymphocytes were successfully immortalized. Flowcytometric analyses reveal ed that most of the immortalized lymphocytes were T cells expressing CD3(+) surface antigen. The ratios of CD4(+) and CD8(+) subpopulations in immorta lized T cells derived from healthy donors varied, depending on the kinds of oncogenes used. However, CD8(+) subpopulation in immortalized T cells deri ved from cancer patients and atopic asthma patients were dominant, independ ent 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 d erived 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 continuous ly without stimulation of a mitogen or antigen in medium containing a low c oncentration of rhIL-2, have been maintained for more than 2 years without any growth rate decrease.