Fig 2: scRNA-seq analysis reveals shifted NK heterogeneity along with the formation of cytokine-induced trained immunity. (A) Scheme of the overall design of the scRNA-seq experiment. PBMCs were isolated from fresh peripheral blood. One-quarter of the PBMCs were used to enrich day 0 NK cells, and the remaining PBMCs were preactivated with IL-15/12/18 for 16 h, then washed and cultured in medium containing a low dose of IL-15 for 1 week. The cells were restimulated with IL-12/18 for 5 h on day 7 and then enriched in day 7 NK cells. The enriched day 0 or day 7 NK cells were labeled with 10 TotalSeq™ antibodies, sequenced and analyzed. The table on the right lists the 10 TotalSeq™ antibodies examined: CD3, CD16, CD56, and CD336 for T and NK lineage definition; CD11b and CD27 for NK cell differentiation; and CD45RA, CD45RO, CD27, and CD62L for T cell memory. (B) t-SNE projection of a total of 14,765 NK cells, showing the distribution of day 0 (D0) and day 7 (D7) NK cells. Red indicates day 0, and blue indicates day 7. (C) Volcano plot of differentially expressed genes in NK cells on day 0 and day 7. All genes passing the P-value and fold difference thresholds are labeled with colored gene names. (D) 9 main NK clusters were numbered and are displayed with a t-SNE plot (left), and details on the composition and cell number for these 9 clusters are shown in the table (right). Day 0 clusters were #0, #2, #3, #4, and #5. Day 7 clusters were #1, #6, #7, and #8. (E) The top 10 upregulated differentially expressed genes (ranked by log fold change) of each cluster were plotted with a heatmap. (F) Overlaid histogram showing the expression profile of the cell surface antigens labeled by 10 TotalSeq™ antibodies in the 9 defined NK cell clusters. CD16, CD11b, and CD45RA were higher in all day 0 clusters; CD3 and CD45RO were higher in all day 7 clusters; CD56 and CD335 were higher in cluster 4# of day 0 and clusters #6 and #7 of day 7; CD27 and CD127 were higher in all day 7 clusters and in cluster #4 of day 0; and CD62 L was very high in cluster #4 of day 0 NK cells.