Research paperA high-efficiency system of natural killer cell cloning
Introduction
Natural killer (NK) cells were identified by their ability to kill virus-infected and tumour cells (Takasugi et al., 1973, Trinchieri and Santoli, 1978). Major advances over the last 10 years have identified many of the cell surface receptors that determine NK function. These include the constitutively expressed killer immunoglobulin-like receptors (KIR), which are specific for the highly polymorphic major histocompatibility complex (MHC) class I proteins (reviewed by Parham, 2005). NK cells also express other receptors specific for MHC class I including the leukocyte inhibitory receptor (LIR-1) and CD94/NKG2 receptors (reviewed by Lanier, 1998, Moretta et al., 2000, Moretta et al., 2001). Unlike the KIRs which are specific for particular class I allotypes, the CD94/NKG2 receptors are specific for the non-classical MHC class I molecule HLA-E (Braud et al., 1998, Lanier, 1998, Vales-Gomez et al., 1999). Individual NK cells express between 1 and 6 inhibitory and between 0 and 5 stimulatory MHC class I receptors and it is the combination of these receptors which defines the responsiveness of any particular NK cell (Valiante et al., 1997). In addition, a number of activating and inhibitory receptors that recognise other non-MHC class I ligands have been identified. These include NKG2D and the NK-specific natural cytotoxicity receptors (NCR), NKp30, NKp44, NKp46 (reviewed by Moretta and Moretta, 2004) as well as a number of receptors with shared expression on T-cells, such as the Leukocyte associated Ig-like receptors, LAIR-1 and LAIR-2 (Meyaard et al., 1997) and the inhibitory receptor protein 60 (IRp60) (Cantoni et al., 1999). In this context it is becoming more important to understand the role of each of the inhibitory and stimulatory molecules in determining the NK response.
Analysis of NK clones permits dissection of the NK response in the context of inhibitory and stimulatory receptor expression. However, current methods of NK cloning are inefficient and laborious. The principles of NK cloning have remained relatively unchanged since Yssel et al. first described a method for cloning both NK and T cells in 1984 (Yssel et al., 1984). In this method, NK cells are isolated by limiting dilution and stimulated with irradiated Epstein Barr virus-immortalised lymphoblastoid B cell lines, peripheral blood mononuclear cells (PBMC) and the polyclonal activating lectin, phytohaemaglutinnin. NK cells are then cultured in Yssel's medium, a serum free medium containing bovine serum albumin, transferrin, insulin, linoleic-oleic and palmitic acid, penicillin and streptomycin. Whilst Yssel's medium is shown to be better than RPMI for NK clone culture, NK cloning using these methods are still inefficient (Spits and Yssel, 1996). More recently, Carlens et al. (2000) demonstrated that the commercially available serum free medium, SCGM is capable of supporting the expansion of polyclonal NK cells within PBMC (Carlens et al., 2000, Carlens et al., 2001). Additionally, they demonstrated that the culture of PBMC in vitro in SCGM with the anti-CD3 specific antibody, OKT3, boosted the number of NK cells within PBMC, although they did not investigate these conditions in an NK cloning environment. Here, we report a novel system of NK cloning using SCGM and multiple optimisations, which boosts cloning efficiency by over tenfold without the use of activating lectins such as phytohaemaglutinin.
Section snippets
Cell culture media and reagents
Cellgro SCGM serum-free medium was purchased from CellGenix (Freiburg, Germany) and RPMI 1640 was purchased from Gibco (Paisley, UK). The media were supplemented with 5% heat-inactivated, human AB serum (Rhydlafa, UK), penicillin (1 × 105 IU/ml) (Gibco) and streptomycin (100 mg/ml) (Gibco). IL-2 was purchased from Cetus (Emeryville, USA) and the monoclonal antibody OKT3 grown from the J.CAM1.6 hybridoma from ATCC (Middlesex, UK).
Isolation of PBMC
Informed consent was obtained from all donors (designated D#). Blood
Culture of polyclonal NK cells in SCGM and RPMI
We first tested the ability of SCGM to maintain culture of peripheral blood-derived polyclonal NK cells. CD3+CD56− NK cells were cell sorted from three different donors and cultured in SCGM or RPMI supplemented with IL-2 (500 IU/ml) for 7 days and proliferation was measured on days 1, 3, 5 and 7. The combined mean 3H-thymidine incorporation values from all 3 donors are shown in Fig. 1A. 3H-thymidine incorporation was greatest in SCGM + IL-2 and this was significantly greater than for cells
Discussion
In this study, a novel and highly efficient system for NK cloning was developed. In contrast to current techniques, which stimulate NK clones with irradiated EBV-LCL and PHA, we used a mixture of irradiated allogeneic PBMC supplemented with IL-2 and OKT3 (Roncarolo et al., 1991, Litwin et al., 1993, Carr et al., 2002). This was sufficient to achieve cloning efficiencies of up to 10% as measured by the derivation of at least 2 × 106 cells for any specific clone. This represents a significant
Acknowledgements
This work was funded by grants from The Wellcome Trust and Medical Research Council. We are grateful to the Flow Cytometry Facility of the Central Biotechnology Service, Wales College of Medicine, Cardiff University, for provision of cell sorting and flow cytometers for benchtop analysis.
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