Single-cell sequencing of healthy peripheral blood NK cells. (A) Flow cytometry plots of negatively isolated human blood NK cells from both donors, showing the gating of live singlet cells and then showing the expression of CD14 (monocyte marker). On CD14⁻ cells, expression of CD56 (NK cell marker), CD3 (T-cell marker), and CD19 (B-cell marker) is shown. (B) Total percentage of CD107a⁺ cells on negatively isolated bulk NK cells after a 5-hour culture, alone (NK only) or with the B lymphoblast cell line Daudi; Daudi cells transfected to express major histocompatibility (MHC) class I–related chain A (Daudi-MICA) or β₂m (Daudi-β₂m); or rituximab-coated Daudi (Daudi-RTX) or NK cells alone stimulated with phorbol myristate acetate/ionomycin (PMA/IONO). NK cells from both donors are shown separately, with and without IL-2 treatment. (C) tSNE, 2-dimensional plot of 8462 individual NK cells, with clusters (0-9) identified using unsupervised hierarchical clustering. (D) Heat map of top 20 markers distinguishing each NK cell cluster (n = 120 unique genes), identified by differential expression analysis and showing a maximum of 500 genes per cluster, excluding clusters 5, 7, and 9. Cells are plotted in columns, and genes are shown in rows. Gene expression is color coded, using a scale based on z-score distribution. (E) Same tSNE plot as shown in panel C (but excluding clusters 5, 7, and 9), showing the expression of the top 2 markers which distinguish each cluster. Expression is color coded from blue (low) to red (high) and cells positively expressing a marker are brought toward the front of the plot.