A TEMPORAL DEVELOPMENTAL MAP SEPARATES HUMAN NK CELLS FROM NON-CYTOTOXIC ILCS THROUGH CLONAL AND SINGLE-CELL ANALYSIS.

Natural Killer (NK) cells represent the cytotoxic member within the innate lymphoid cell (ILC) family that are important against viral infections and cancer. While the NK cell emergence from hematopoietic stem and progenitor cells through multiple intermediate stages and the underlying regulatory gene network has been extensively studied in mouse, this process is not well characterized in human. Here, using a temporal in vitro model to reconstruct the developmental trajectory of NK lineage, we identified an ILC-restricted oligo-potent Stage 3a CD34-CD117+CD161+CD45RA+CD56- progenitor population, that exclusively gave rise to CD56-expressing ILCs in vitro. We also further investigated a previously non-appreciated heterogeneity within the CD56+CD94-NKp44+ subset, phenotypically equivalent to Stage 3b population containing both group-1 ILC and RORt+ ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1hi S3b and CD161hiCD117hi ILC3 populations distinctively differed in their expression of effector molecules, cytokine secretion, and cytotoxic activity. Furthermore, analysis of lineage output using DNA-barcode tracing across these stages supported a close developmental relationship between S3b-NK and S4 (CD56+CD94+) cells, while distant to ILC3 subset. Cross-referencing gene signatures of culture derived NK cells and other non-cytotoxic ILCs with publicly available datasets validated that these in vitro stages highly resemble transcriptional profiles of respective in vivo ILC counterparts. Finally, by integrating RNA-velocity and gene-network analysis through SCENIC we unravel a network of coordinated and highly dynamic regulons driving the cytotoxic NK cell program, as a guide map for future studies on NK cell regulation.