Immortalization of Epstein-Barr virus-negative human B lymphocytes with minimal chromosomal instability

The genes required for immortalization of human B cells infected by Epstein-Barr virus are multiple, and the precise mechanism of this process remains to be elucidated. In the present study HPV16 E6 and E7 were retrovirally transduced into human primary B cells stimulated by CD40-CD40L interaction, thereby establishing an Epstein-Barr virus negative immortalized human B cell line, which continued to proliferate for more than 2 years (100 population doublings). The established cell line had a high telomerase activity from the beginning of the culture period, and no shortening of the telomere length was observed. A chromosomal analysis revealed that a large portion of the HPV16E6E7 transduced cells had retained a normal karyotype. Similar to human epithelial cells, human B lymphocytes seem to require two steps for immortalization, namely, the inactivation of the p16/Rb pathway and the activation of telomerase, the latter that can be induced by the CD40-CD40L interaction. Furthermore, using this system, it is possible to analyze the role of individual genes in human B lymphocyte immortalization without the influence of a pre-existing Epstein-Barr virus genome.