Dissecting the overlapping and distinct phenotypic features between immature stage 3 NK cells and ILC3. (A-C) Multiparameter FACS analysis of cells collected at each week during 4-week in vitro culture. (A) UMAP from multiparameter FACS of combined cell surface markers and intracellular TFs expression showing 4 sequential stages in NK cell trajectory as in Figure 1, with stage 3 phenotype further divided based on bifurcated NKp44 expression. (B) UMAP generated as in panel A showing expression levels of ILC-associated TFs: TBET, EOMES, and RORγt with annotated CD34⁺, CD161⁺, and CD56⁺ clusters in gated regions. (C) Comparisons of levels of NK receptors (DNAM-1 and NKG2D), and ILC3-associated markers (CD117 and CD161) between RORγt⁻ (as S3b NK) and RORγt⁺ (as ILC3) within the CD56⁺CD94⁻NKp44⁺ population by intracellular and cell surface FACS staining after 4 weeks of in vitro culture. Numbers indicate percentages of total CD56⁺CD94⁻NKp44⁺ cells in each quadrant, data obtained from 3 donors.