Expansion and activation of NK cells supported by accessory cells. Phenotypic and functional characterization

Natural Killer cells (NK) are cytotoxic lymphocytes from the innate immune system that recognize and eliminate virally infected and tumor cells. Accordingly, manipulation of NK cells has been the focus of several immunotherapy protocols aimed at eradicating cancer cells. Allogeneic NK cell therapy was initially described over two decades ago, emphasizing KIR-mismatch's importance in preventing NK cell inhibition and promoting cytotoxicity and tumor elimination without inducing graft-versus-host disease (GvHD). While unstimulated NK cells have shown limited antitumoral activity in adoptive cell therapy, various activation and expansion protocols have been proposed to enhance their cytotoxic potential. Activated and expanded allogeneic NK cells, especially with the rise of chimeric antigen receptor (CAR) therapies, have attracted significant attention from academic and commercial sectors. Protocols typically involve using cytokines and stimulatory cells, such as Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell lines (LCLs) or K562 leukemic cells, before or after NK cell enrichment. Here we present two different standardized protocols for NK cell activation and expansion, offering insights into NK cell-based immunotherapies for cancer treatment. We also present a comprehensive methodology for assessing NK cell-mediated cytotoxicity against Neuroblastoma cell lines in both 2D and 3D cultures. The comprehensive methodology presented here lays the foundation for further research in the field, driving advancements in NK cell-based therapies against malignancies.