Natural killer (NK) cells are a subset of peripheral blood lymphocytes that can kill a variety of target cells and are emerging as a powerful tool for cell therapy of haematological malignancies and other cancers (Kiessling et al, 1975; Farag et al, 2002; Moretta & Moretta, 2004; Passweg et al, 2004; Lanier, 2005; Miller et al, 2005; Verheyden & Demanet, 2008). In addition to their direct antitumour activity, NK cells influence immune responses by interacting with dendritic cells, macrophages, T cells and endothelial cells (Vivier et al, 2008). Thus, many have proposed NK cell strategies as a means to improve cancer immuno-therapy, transplantation of solid organs and haematopoietic stem cells, and the control of inflammatory and autoimmune disorders (Terme et al, 2008).
Unlike T and B lymphocytes, which readily respond to a variety of proliferative stimuli, NK cells are well known to be refractory to sustained proliferation in vitro. Several cytokines (such as interleukin [IL]-2, IL-12 and IL-15) have been reported to stimulate NK cells (Trinchieri et al, 1984; Perussia et al, 1987; Miller et al, 1992; Naume et al, 1992; Robertson et al, 1996; Carson et al, 1997; Guven et al, 2003), as does contact with human leucocyte antigen (HLA) class I-negative cells such as the K562 leukaemia cell line or the HFWT Wilms’ tumor cell line (Phillips & Lanier, 1985; Robertson et al, 1996; Harada et al, 2002). However, the capacity of these methods to induce long-term proliferation of CD56+ CD3− cells is generally modest and/or of short duration. Therefore, it is unclear how many cycles of cell division human NK cells can undergo while maintaining their function, and what factors limit their proliferation.
We devised a method that allows specific activation and expansion of human NK cells from peripheral blood cells (Imai et al, 2005). This method relies on co-culture with the K562 leukaemia cell line transfected with two NK stimulatory molecules, membrane-bound interleukin 15 and 4-1BB ligand, termed K562-mb15-41BBL cells. In this study, we used this method to stimulate the long-term expansion of human NK cells. We determined their replicative potential, as well as genetic integrity and function after sustained proliferation.