Abstract
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.
Highlights
It is believed that the proliferation capacity of normal differentiated human cells is limited in vivo and in vitro
Our results suggest that stimulation of B cells with CD40L/IL-4 may induce a state of immortalization, and that this is true for B cell cultures from a majority of healthy adult donors
We describe that peripheral human B cells from healthy donors were capable of very extended proliferation after specific external activation, justifying these cells’ description as being conditionally immortalized
Summary
It is believed that the proliferation capacity of normal differentiated human cells is limited in vivo and in vitro. Telomere shortening is counteracted by telomerase, which adds telomeric repeats to the chromosomes’ ends and is expressed in germline cells, but can be induced in certain somatic cells such as activated lymphocytes [3,4,5,6]. Cellular immortalization requires a mechanism to maintain telomeres and usually involves up-regulation of telomerase activity [1]. Human embryonic stem cell clones constitutively display strong telomerase activity and are immortalized in vitro without requiring genetic manipulation [7]. For human fibroblasts and T lymphocytes, transduction with telomerase was necessary and sufficient to stabilize telomeres and to achieve immortalization in vitro [8,9,10]. The ectopic expression of telomerase was not sufficient for their immortalization; inactivation of the Rb/p16 pathway was required [12]. The spontaneous in vitro immortalization of human somatic cells has so far been observed only in experiments with single human donors or in cells derived from patients with inherited genetic disorders predisposing to cancer [13,14]
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