Reduced degranulation and cytotoxicity of MADD-deficient patient cells and knockout cells. (A-C) Patient cells compared with healthy control cells. (A-B) Degranulation of fresh NK cells after stimulation with K562 cells (A) and of d3 PHA/IL-2–activated CD8 T cells, and γδ T cells stimulated with anti-CD3/CD28 beads (B) as detected by upregulation of CD107a. (C) Defective cytotoxicity of patient d12 PHA/IL-2 blasts from the patient, a patient with RAB27A deficiency and 2 healthy donors on L1210 targets in a 51Cr redirected lysis assay. The gray area indicates mean ± SD of a cohort of 25 healthy donors. Assays with patient cells were repeated twice with similar result. (D-F) NK-92mi MADD knockout cells vs NK-92mi wild-type cells. (D) Representative plot of reduced degranulation after stimulation with K562 or PMA/ionomycin. (E) Significantly lower proportion of degranulated NK-92mi MADD knockout cells after stimulation than that of wild-type cells (3 test series with triplicates each, P < .0001). (F) Defective cytotoxicity in a flow cytometric assay, reported as the proportion of propidium iodide (PI)–positive cells.
