Natural killer cell repertoires are functionally diversified as a result of differentiation, homeostatic receptor-ligand interactions and adaptive responses to viral infections. However, the regulatory gene-circuits that define the manifold cell states and drive NK cell differentiation have not been clearly resolved. Here, we performed single-cell RNA sequencing of 26,506 cells derived from sorted phenotypically-defined human NK cell subsets to delineate a tightly coordinated differentiation process from a small population of CD56bright precursors to adaptive NKG2C+ CD56dim NK cells. RNA velocity analysis identified a clear directionality in the transition from CD56bright to CD56dim NK cells, which was dominated by genes involved in transcription and translation as well as acquisition of NK cell effector function. Gene expression trends mapped to pseudotime, defined by increasing entropy, identified three distinct transcriptional checkpoints, reflecting important changes in regulatory gene-circuits. The CD56bright NK cell population dominated pseudotime with two distinct checkpoints separating precursors from intermediate states that gradually took on transcriptional signatures similar to CD56dim NK cells. The final checkpoint occurred during late terminal differentiation of CD56dim NK cells and was associated with unique divergent gene-expression trends. Furthermore, we utilized this single-cell RNA sequencing resource to decipher the regulation of genes involved in lysosomal biogenesis and found a coordinated gradual increase in the RAB4 and BLOC1S gene families with differentiation into CD56dim NK cells. These results identify important gene programs driving functional diversification and specialization during NK cell differentiation and hold potential to guide new strategies for NK cell-based cancer immunotherapy.

A Temporal Transcriptional Map of Human Natural Killer Cell Differentiation