Cathepsin C is not required for granzyme B activation and cytolytic function in IL-2-activated NK cells. (A) Cathepsin C (CTSC) activity in unstimulated and IL-2-stimulated NK cells from the PLS patients (P1 and P2), heterozygote (Het), and healthy control. Hydrolysis of the colorimetric substrate Gly-Phe-pNA¹⁴  is expressed as arbitrary units of activity (based on absorbance at 405 nm). The activity observed in the patient samples is equivalent to the background absorbance of lysate alone. Each reaction contained lysate from 6 × 10⁵ cells and were performed in triplicate; standard deviation is shown. (B) Flow cytometric analysis of CD3^neg, CD56^dim (dim), and CD56^bright (br) NK cell subsets²⁰  in PLS patients 1 and 2, the heterozygote, and a healthy control. (C) Cytolytic activity of IL-2-activated NK cells from PLS patient 1, heterozygote, and healthy control. CellTracker Green-labeled K562 target cells were cocultured with IL-2-activated NK cells at an E/T ratio of 2:1 (2 × 10⁵ cells in 200 μL) for 5 hours at 37°C. Propidium iodide (PI) staining is shown at 0 hours and after 5 hours of coculture. Zero-hour time points were incubated at the same cell density but were not mixed until the 5 hours' time point. Dead or dying cells are detected as a PI+, CellTracker Green+ population by flow cytometry. The percentage of target cells in this gate is shown. This experiment is representative of 3 independent experiments and has been confirmed in patient 2 (data not shown). (D) Granzyme B activity in IL-2-activated NK cells from PLS patients, heterozygote, and healthy control. Activity was measured by hydrolysis of the colorimetric substrate Ac-IEPD-pNA as described.¹⁵  Each reaction contained lysate derived from 6 × 10⁵ cells and was performed in triplicate (standard deviation shown); activity is indicated in arbitrary units. Hydrolysis of the substrate was unaffected by the caspase inhibitor z-VAD-fmk (data not shown). (E) Activation of the caspase cascade in K562 cells by IL-2-activated NK cells. The assay was performed as in Figure 1C except that targets were labeled with CellTracker Orange and cocultured with effector cells for 2 hours. Cells were then stained with PhiPhiLux according to the manufacturer and as described.¹⁹  Caspase activation results in cleavage of a peptide spacer in PhiPhiLux and increased fluorescence.¹⁹  The percentage of cells with increased caspase activity is shown. This experiment is representative of 2 separate experiments and was confirmed using patient 2 (data not shown).