High Numbers of NK Cell Clones Can Be Generated by Limiting Dilution in SCGM Medium Supplemented with IL-2, IL-15 and Anti-NKp46 Antibody 9E2.
Authors: Kowald Sabine , Arendt Andreas , Schmitz Jurgen , Huppert Volker 
Published: April 2020

Abstract
NK cell clones obtained by limiting dilution have frequently been used for functional studies and as an analytical tool e.g. for assessment of alloreactivity in the context of haploidentical stem cell transplantation [Velardi et al., Blood 2007]. NK cell clones can be used in co-culture experiments with allogeneic target cells to determine the number of alloreactive clones and therefore to quantify NK cell alloreactivity. This method provides additional quantitative data, while PCR based methods for HLA and KIR typing only provide qualitative results of potential alloreactivity. Generation of NK cell clones usually is difficult and cloning efficiency is low (&lt; 5% [Yssel et al., J. Immunol. Methods 1984]). Protocols have been developed to increase cloning efficiency [Wang et al., J. Immunol. Methods 2005] We evaluated whether cloning efficiency can be further increased by optimization of cell processing prior to culture and/or of medium components. Published reference methods use media supplemented with Interleukin-2 (IL-2). These conditions also stimulate proliferation of T cells, which usually overgrow NK cells in culture. Thus depletion of T cells prior to incubation with IL-2 may facilitate identifation of proliferating NK cells. T cells can be actively depleted upon CD3 magnetic labelling, by use of an NK cell isolation kit or passively by CD56 enrichment. CD 56 enrichment efficiently depletes CD3+CD56- T cells and residual CD3+CD56+ NKT cells do not expand under the chosen culture conditions. IL-15 is capable of preventing NK cell apoptosis at low concentrations [Caligiuri et al., J. Clin. Invest. 1997; Carson et al., J Clin Invest. 1997; Puzanov et al., J Immunol. 1996)] and plays a role in proliferation and survival of NK cells. Cross linking of NKp46 leads to NK cell activation [Long et al., Blood 2005]. As simultaneous engagement of two independent activating NK cell receptors (IL-2/IL-15 receptors and NKp46) may enhance NK cell activation and proliferation, we evaluated different combinations of IL-2, IL-15 and the anti-NKp46 antibody 9E2. Optimal results were obtained when CD56 selected cells were used (compared to CD3 depletion, NK cell enrichment, KIR2D selection). Supplementation of SCGM medium (Cell Genix) with IL-2 (500u/mL), IL-15 (10ng/mL) and anti NKp46 antibody (2μg/mL, CD335, clone 9E2) increased the number of NK cell clones by a factor of 2.3–2.5 (n=3) compared to medium and IL-2 alone. 37 NK cell clones with yields of &gt; 2E6 cells per clone have been generated using this optimised method and will now be used for Killer Immunoglobulin-like receptor mRNA analyses and antibody based KIR phenotyping (CD158a/h, CD158b, CD158e, CD158i, anti KIR2D). KIR analysed NK cell clones may be used to generate novel highly specific anti KIR antibodies for flowcytometric, quantitative KIR phenotyping. Such a panel can be helpful for optimization of donor selection for allogeneic stem cell transplantation.