Impaired degranulation and cytotoxicity of patient NK cells. (A) Ex vivo degranulation of NK cells (CD3^–CD56⁺) from the patient (Pat) and a healthy donor control (Ctr) after incubation with medium (left panel) or with K562 cells (middle panel) as assessed by flow cytometric analysis of CD107a surface expression. The right panel shows the difference in CD107a expression between unstimulated and stimulated cells from the patient (solid circle) and a day control (open square) relative to healthy controls (gray shaded area). The dashed line represents the level below which NK-cell degranulation has the best positive and negative predictive value for a mutation in a gene relevant for cytotoxicity in an unfiltered cohort of patients with HLH. (B) Degranulation of NK cells prestimulated with IL-2 and PHA for 48 hours. (C) Cytotoxicity of patient and control PBMCs on K562 target cells without (black symbols) or after overnight pre-incubation with IL-2 (gray symbols). (D) T-cell degranulation. Cultured T cells from the patient and a healthy control were incubated in medium or stimulated with 3 µg/mL of plate-bound anti-CD3. The increase in CD107a expression upon stimulation is shown as an overlay of histograms of unstimulated (dotted line) and stimulated (solid line) cells.