Fig E2.

Degranulation ability of LYST-deficient NK cells. A, Delivery of granzyme B to target cells. 721.221 target cells were labeled with TFL-4 and a fluorogenic substrate of granzyme B and then mixed with EV or LYST CRISPR NK92mi cells. The increase in substrate fluorescence in TFL-4–positive target cells, indicating the activity of granzyme B, was monitored by using flow cytometry. Dot plots show gating strategies used and illustrate an example of the result. B, Assessment of degranulation. NK92mi cells, either untransduced (UT) or transduced with either EV or LYST CRISPR, were incubated alone or with 721.221 target cells for 60 minutes at 37°C. Cells were stained with anti-CD56 and anti-CD107a antibodies, and cell-surface expression of CD107a was determined by using flow cytometry. Change in CD107a cell-surface expression (ΔCD107a) was calculated as the percentage of CD107a-positive activated NK92mi cells after subtraction of the percentage of CD107a-positive NK92mi cells from the nonstimulated population. The graph shows mean values + SDs, as determined from 5 separate experiments. **P < .01 and ***P < .001, 1-way ANOVA. C, Intracellular levels of lysosomal proteins. NK92mi cells, either untransduced or transduced with EV or LYST CRISPR constructs, were fixed, permeabilized, and then stained with either anti–LAMP1–Alexa Fluor 647, anti-LAMP2–fluorescein isothiocyanate (FITC), anti-perforin–FITC, or anti–granzyme B–Alexa Fluor 647 antibodies. Cells were then analyzed by using flow cytometry for total levels of the indicated proteins. Histograms show a typical result from 2 independent experiments. Numbers in parentheses indicate median fluorescence intensity values for indicated protein staining.