Transcriptional profile of ILC3s upon IL-12 and IL-15 culture is reminiscent of early differentiated NK cells. Tonsil (To)- and spleen (Sp)-derived NKp44⁺ ILC3s were expanded for 3 weeks with IL-2 and IL-7 or IL-12 and IL-15 and together with freshly sorted cells were subjected to RNA-seq. (A) Clustering of samples using the first 2 principal components (PC). Sp_uncultured, N = 13 samples (10 HFL donors, 4-15 mice per donor); Sp_2+7, N = 6 (3 HFL donors, 4 mice per donor); Sp_12+15, N = 9 (6 HFL donors, 4-5 mice per donor); To_uncultured N = 11; To_2+7, N = 7; To_12+15, N = 6. (B-D) Heatmaps depict differential expression of selected genes encoding (B) cytokines/chemokines and cytokine receptors, (C) cytotoxic effector molecules, and (D) activating and inhibitory receptors and signaling molecules. Treatment conditions are color-coded on top: uncultured cells (red), IL-2 and IL-7 (blue), and IL-12 and IL-15 (green). Red text indicates genes of interest. (E) Protein expression of selected markers in IL-2 plus IL-7 and IL-12 plus IL-15 cultures of splenic ILC3s. For quantification (mean ± SD) of NKG2A, NKG2C, and CD2, N = 4 (3 HFL donors, 4 mice per donor); for CD56, N = 11 (7 HFL donors, 2-8 mice per donor). ns, P > .05; *P < .05; **P < .01 using paired Student t test. Fe, feeders alone (pooled from several expansions); MFI, geometric mean fluorescence intensity.