Introduction: NK cells offer an attractive option for treatment of AML and other hematological malignancies because of an excellent graft-vs-leukemia (GVL) effect with low graft-vs-host-disease (GVHD). A critical step in the NK cell therapy is the in-vitro expansion of the NK cells. In this study, we attempted to standardize the NK cell expansion and the assay for in-vitro assessment of NK cell cytotoxicity.
Materials and Methods: Ten ml EDTA blood sample was collected from healthy volunteers (after assessing HLA and KIR genotyping) by venipuncture. For the effector cells, 106 NK cells were isolated from ~ 107-108 PBMCs using the NK MACS isolation kit (Miltenyi Biotech, Singapore) and expanded till 14 days in either RPMI 1640 or NK MACS media with heat inactivated FBS or AB serum and supplemented with IL2 or IL2+IL15. The purity of NK cells was evaluated by flow cytometry using CD45, CD3, CD16 and CD56 markers. On day 14, NK cytotoxicity was set up against K562 at different Effector:Target (E:T) ratios (6.25:1, 12.5:1, 25:1, 50:1 and 100:1). The NK cell subsets (CD56, CD16) were evaluated by flow cytometry on Day 0, Day7, and Day14.
Results: The purity of isolated NK cells was >85%. NK cells expanded best (34 fold) when cultured in NK MACS media with heat inactivated AB serum and supplemented with IL2 (500U/mL) with higher number of mature NK cells (NKG2Dhigh CD69high). At D14, >80% cytotoxicity (E:T ratio 25:1 and 50:1) was observed against K562 cell line.
Discussion: In this study we have optimized NK cell expansion using NK MACS media with AB serum supplemented with IL2. We also show that optimal E:T ratio for cytotoxicity against K562 cell line is 25:1 to 50:1. These results are expected to be of clinical relevance while preparing clinical grade NK cells for NK cell therapy.