Anti-CD94-induced expansion of highly functional adaptive NKG2C+NKG2A−CD57+selfKIR+ NK cells from CMV-seropositive healthy donors

Human Natural Killer (NK) cells are innate lymphocytes, short-living and providing rapid responses against viral infections and tumors. Adaptive NK cells are an NK cell subpopulation arising upon cytomegalovirus (CMV) infection, characterized by the expression of the CD94/NKG2C heterodimer (a HLA-E-specific activating receptor), by a highly mature CD57+KIR+NKG2A- phenotype and endowed with marked antitumor functions and prolonged lifespan1,2. In view of these traits, adaptive NK cells could be exploited in cell-based immunotherapies aimed at enhancing NK cell cytotoxicity against specific targets (e.g. through Chimeric Antigen Receptors (CAR)-engineering or in combination with cell engagers). To this end, we developed a method to efficiently and specifically expand adaptive NK cells starting from NK-enriched cell preparations, obtained from the peripheral blood of selected CMV-seropositive healthy donors. Our system is based on the use of specific cytokines combined with an anti-CD94 monoclonal antibody that induces the selective expansion of adaptive CD94/NKG2C+ NK cells lacking the inhibitory heterodimer CD94/NKG2A. The resulting expanded adaptive NK population consists in NKG2C+NKG2A- NK cells, mainly co-expressing CD57 and a single self-KIR, already characterizing adaptive NK cells before the expansion. Moreover, expanded NK cells do not upregulate PD-1 and other immune checkpoints, but maintain the molecular signature of CMV-induced adaptive NK cells and their high ADCC abilities. This protocol improves previous expansion methods by eliminating genetically modified cells in the culture system as stimulus and avoiding unwanted upregulation of inhibitory receptors3,4, thus providing a technically and economically advantageous method suitable for large-scale production of adaptive NK cells for immunotherapeutic purposes.