Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56bright and CD56dim NK cells

Natural killer (NK) cells represent a promising source for off-the-shelf cellular therapies. Typically, NK cells are expanded with cytokines and K562 feeder cells expressing 4-1BBL and membrane-bound IL-21, which induce a uniform and activated CD56 superbright phenotype. However, the developmental origin of these cells remains unclear, arising either from CD56 dim NK cells through phenotypic plasticity or from functional maturation of the CD56 bright subset. In this study, we dissected the respective contributions of CD56 bright and CD56 dim NK cells to engineered NK cell products. NK cell populations were sorted, and their activation, expansion, phenotype, and functionality were assessed. We found that CD56 bright NK cells were initially critical for proliferation and feeder clearance, but after restimulation, they exhibited comparable cytotoxicity and expansion to CD56 dim NK cells. This was further highlighted by the ability of both subsets to mediate CAR- or TCR-driven cytotoxicity. Eventually, sorted and expanded NK cells acquire the same activated phenotype, but can be distinguished by CD16 expression, predominantly maintained by CD56 dim cells, resulting in superior antibody-dependent cellular cytotoxicity. Together these findings indicate that preselection of a single subset is redundant, and that bulk isolation enables optimal NK cell engineering by leveraging the complementary strengths of CD56 bright and CD56 dim NK cells.