Single-Cell Profiling Reveals Functional Heterogeneity and Serial Killing in Human Peripheral and Ex Vivo-Generated CD34+ Progenitor-Derived Natural Killer Cells

Increasing evidence suggests that natural killer (NK) cells are composed of distinct functional subsets. This multifunctional role has made them an attractive choice for anticancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in a heterogeneous NK cell population with distinct capabilities responding to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at the single-cell level and in real-time. A variable response of single NK cells toward different target cells is observed, and a distinct population of NK cells (serial killers) capable of inducing multiple target lysis is identified. By assessing the cytotoxic dynamics, it is shown that single umbilical cord blood-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior antitumor cytotoxicity. With an integrated analysis of cytotoxicity and cytokine secretion, it is shown that target cell interactions augment cytotoxic as well as secretory behavior of NK cells. By providing an integrated assessment of NK cell functions by microfluidics, this study paves the way to further functionally characterize NK cells ultimately aimed to improve cancer immunotherapy.