Educated NK cells contain more granzyme B than uneducated. (A) Imaging of CTV-labeled sorted educated or uneducated NK cells (red and blue respectively) coincubated with K562 for 0 or 60 min and stained for GranToxiLux plus (orange). (B) Bar graph representing percentage of K562 positive for GranToxiLux after 1 hour challenge with either educated or uneducated NK cells (E:T) ratio of (2:1), or no NK cells. (C) Correlation between granzyme B content of NK cells measured preassay correlates with the percentage of K562 positive for GranToxiLux. (D) Correlation of GranToxiLux positive K562 cells and DCM⁺ cells. (E) Soluble granzyme B measured in the supernatant after stimulation of NK cells by CD16 crosslinking compared with unstimulated NK cells as baseline. (F) Correlation between granzyme B content of NK cells measured preassay with the amount of granzyme B lost by NK cells during the degranulation assay using K562 (n = 49). (G) Number of granules mobilized per kill in educated and uneducated NK cells (n = 49). (B-G) Self- KIR NK cells are represented in red and the nonself KIR NK cells in blue. Whiskers show the fifth to 95th percentiles. Bars show the median. One-way ANOVA tests followed by Tukey multiple comparison tests were performed in panels B and E. R is the Pearson coefficient in panels C, D, and F. Unpaired t test was performed in panel G. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; and ∗P < .05. CTV, CellTrace Violet; DCM, dead cell marker.