A cytolytic defect in unstimulated NK cells from PLS patients is associated with defective granzyme B processing. (A) Cytolytic activity of unstimulated NK cells derived from PLS patient 1, heterozygote, and healthy control. The killing assay was performed as in Figure 1C except that exogenous IL-2 was not used. The percentage of PI+ target cells is indicated. This experiment is representative of 3 separate killing assays performed and has been confirmed in patient 2. (B) Cytolytic activity of control, heterozygote, and PLS patient NK cells at different effector-target (E/T) ratios. Percentage killing is indicated. ○ indicates patient 1; ▪, heterozygote; ▵, control. (C) Caspase activation by unstimulated NK cells. This experiment was performed as in Figure 1E except that exogenous IL-2 was not used for NK stimulation. Percentages of cells with increased caspase activity are indicated. (D) Granzyme B activity in unstimulated NK cell samples (performed as in Figure 1D); activity is displayed in arbitrary units (standard deviation is shown). A lysate derived from IL-2-activated NK cells contained approximately 4-fold more granzyme B activity than unstimulated NK cells, as in Figure 1D (data not shown). The level of substrate hydrolysis detected in a B-cell line (BJAB), which does not express granzyme B, is similar to the level of activity found in patient 2 (data not shown). (E) Granzyme B immunoblot (using anti-granzyme B antibody clone 2C5/F5) contains lysate from 1 × 10⁶ IL-2-stimulated NK cells per lane or 2 × 10⁶ unstimulated NK cells boiled in SDS-containing loading buffer. A second, shorter exposure of the IL-2-stimulated sample is shown to clearly reveal the different species of granzyme B present in the samples. The unlabeled band at the top of the granzyme B blot highlights the small size difference between granzyme B in the patient and control samples. The mature and pro-form of granzyme B are indicated. Actin was used as a loading control. (F) Aprotinin agarose binding^16,17  of granzyme B from unstimulated NK cells and IL-2-activated NK cells from patient 2, heterozygote, and control. Lysates were mixed with aprotinin-agarose beads, the beads were pelleted, and the supernatant removed. The beads were washed and bound (B) and unbound (U) fractions analyzed by granzyme B immunoblotting (as in Figure 2E).