Delivery of granzyme B to target cells, but not cell-cell conjugation, is impaired in LAMP1 RNAi cells. YTS or ex vivo isolated NK cells were untransduced (UT; YTS cells), mock-transduced (mock; NK cells), or transduced with control (CTRL) or LAMP1 RNAi. (A) Delivery of granzyme B to target cells. The 721.221 target cells were labeled with TFL-4 and a fluorogenic substrate of granzyme B and then mixed with NK cells for 30 minutes at 37°C. The increase of substrate fluorescence in TFL-4–positive target cells, indicating the activity of granzyme B, was monitored by flow cytometry. The dot plots show gating strategies used and illustrate an example of the results for primary NK cells. Graphs (right) show mean values + SD determined from 5 (NK) or 3 (YTS) experiments. *P < .05; **P < .01; 1-way ANOVA. (B) Levels of granzyme B. Granzyme B transcript levels for YTS and primary NK cells were analyzed by real-time PCR. Relative expression of granzyme B, normalized to actin, is shown as mean + SD from 3 (NK) or 6 (YTS) experiments. Granzyme B protein level in YTS cells was visualized by western blotting; actin was used as a loading control. (C) Granzyme B activity. YTS or primary NK cells were lysed, and the proteolytic activity of granzyme B in total cell lysates was determined by measuring the hydrolysis of the peptide substrate. The graphs show mean values + SD from 5 (NK) or 3 (YTS) experiments. Granzyme B–negative 721.221 cells served as a control. (D) Cell conjugation. YTS or primary NK cells were stained with 5-chloromethylfluorescein diacetate (CMFDA) and mixed with TFL-4–labeled 721.221 target cells for 30 minutes at 37°C. Cells were then fixed and analyzed using flow cytometry. Conjugates of NK and target cells were determined by measuring the percentage of CMFDA+TFL-4+ double-positive cells from the total pool of live CMFDA+ cells. The mean values + SD were determined from 4 experiments.