Establishment of Cytotoxic and Non-cytotoxic NK-92 Cell Clones – Evaluation of Their Function as Target Cells

Background/Aim: Previous studies have demonstrated that NK cells present in PBMCs might explain why clinical trials conducted with NK-92 as well as CAR modified NK-92 cells have to a large extent failed. Two NK-92 clones with different NK target cell properties have been established and are described here. Materials and Methods: Two NK-92 cell clones, NK-92 clone 1 and clone 2, were established using the limiting dilution technique. A time-resolved fluorometric assay (TDA-labeled NK-92 clone 1, 2 or K562 as target cells) was used for measuring their sensitivities to NK cell-mediated cytolysis and their NKG2D expression was identified with immunoblotting. Results: A striking difference between the NK-92 clones in their cytotoxic capacity against K562 cells was observed. A clear correlation was noticed between these NK-92 clones when used as target cells and their ability to kill K562 cells. A 50:1 effector:target ratio (PBMCs:NK-92 clone 1) gave 6.50±5.44% lysis whereas the corresponding value was 39.9±10.0% with NK-92 clone 2 as target cells. Interestingly, incubating PBMCs in medium for longer times slightly potentiated their NK activity also against the NK-92 clone 1 (E:T ratio 50:1), from 2.5±0.88% lysis (24 h pre-incubation time) to 13.7±9.04% (48 h) and 13.8±6.89% (72 h). Immunoblotting with anti-NKG2D antibodies stained an approximately 34 kDa protein band in lysates prepared from NK-92 clone 1 cells, which corresponds to the NKG2D antigen. A very faint band of the same size was observed in lysates prepared from NK-92 clone 2 cells. Conclusion: The NK-92 clones 1 and 2, established and described here, might turn out to be very useful for finding possible solutions for using NK-92 and CAR NK-92 cells in future treatments of human malignant diseases.