Wiesner Martina, Zentz Caroline, Mayr Christine, Wimmer Rainer, Hammerschmidt Wolfgang, Zeidler Reinhard, Moosmann Andreas
Clinical Cooperative Group Molecular Oncology, GSF - National Research Center, Ludwig-Maximilians-Universität, Munich, Germany.
PLoS One. 2008 Jan 23;3(1):e1464. doi: 10.1371/journal.pone.0001464.
It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.
一般认为，人类分化细胞在体内的寿命和增殖能力有限，基因修饰是其在体外永生化的先决条件。在此，我们重新审视这一问题，研究人类B细胞的长期增殖潜力。此前研究表明，通过在白细胞介素-4存在的情况下刺激其受体CD40，来自健康供体外周血的人类B细胞可在体外有效诱导增殖长达十周。当我们在改变细胞数量和培养规模的条件下应用相同刺激时，我们惊讶地发现，我们的处理诱导B细胞在目前长达1650天的观察期内持续增殖，代表超过370次群体倍增，这表明这些B细胞在体外被永生化。大多数健康成年人类供体的B细胞都能建立长期CD40刺激的培养物。这些B细胞具有恒定的表型，无EB病毒，且仍然依赖于CD40连接。它们具有组成型端粒酶活性并稳定端粒长度。此外，它们易被Toll样受体9配体激活，可用于在体外扩增抗原特异性细胞毒性T细胞。我们的结果表明，人类体细胞可以逃避衰老，仅通过外部刺激即可被条件性永生化，而无需基因操作或致癌病毒感染。条件性永生化的人类B细胞是免疫治疗以及B细胞肿瘤发生、激活和功能研究的新工具。
