A high-efficiency system of natural killer cell cloning

Abstract

The culture of human natural killer (NK) cell clones has traditionally been a long, laborious process with an efficiency of only 1–2%. Recently, a stem cell growth medium (SCGM) has been described to expand preferentially polyclonal NK cells from peripheral blood. We have tested SCGM in a single cell sorting system and shown a 4–5 fold increase in the number of proliferating NK clones compared to standard RPMI media. The cloning efficiency was further enhanced by the provision of irradiated feeder cells derived from multiple donors combined with the addition of the anti-CD3 antibody, OKT3. The combination of SCGM, single cell sorting and these multiple optimisations enhanced NK cloning efficiency by more than tenfold to greater than 20% for short-term cultures when deriving 10⁵ cells and as high as 10% for longer term cultures when deriving more than 2 × 10⁶ cells. This novel system thus facilitates the generation of NK clones and allows larger scale studies of NK function that were beyond the scope of previous methodology.

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.