A small, novel population of blood NK cells exhibits loss of ribosomal expression. (A) Same heat map of the top 20 markers distinguishing each NK cell cluster as shown in Figure 1D, highlighting the similarities (blue boxes) and dissimilarity (red box) between clusters 2 and 8. Cells are plotted in columns, and genes are shown in rows. Gene expression is color coded using a scale based on z-score distribution, ranging from low expression (purple) to high expression (yellow). (B) Dot plot (left) of 20 top markers associated with cluster 8 within unstimulated cells only (columns) across the different clusters (rows). The size of the dot represents the percentage of cells expressing the markers, whereas the color encodes the average scaled expression values. Heat map (right) of the same markers of interest within unstimulated cells only. Clusters are plotted in columns, and genes are shown in rows. The gene expression scale is as in panel A. (C) Module score analysis for each NK cell cluster at the single-cell level, defined using markers of mammalian autophagy.⁶³  Module scores were calculated for unstimulated cells only. Violin plots represent the distribution of the module scores for each cluster and the error bars represent median and interquartile range. One-way analysis of variance with Bonferroni’s multiple comparison. Nonsignificant (n.s) P > .05; *P < .03; **P < .02; ***P < .0002; ****P < .0001. (D) tSNE plot (excluding clusters 5, 7, and 9), showing the expression of selected autophagy markers within the full data set. Expression is color coded from blue (low) to red (high) and cells positively expressing a marker were brought toward the front of the plot. (E) Selected GO terms using all conserved markers downregulated within this cluster with an adjusted P < .05. No markers were upregulated within this cluster at this significance threshold. (F) tSNE, 2-dimensional plot of 1000 NK cells from the unstimulated condition (500 randomly selected from each donor). The analysis was performed using the first 15 principle components and a resolution of 0.4. The top markers positively associated with each cluster are highlighted. (G) The same tSNE plot as in panel F) but cells are color coded according to the cluster identities assigned using the full data set of 8462 cells.