Perforin maturation, expression, cytotoxicity, and granule exocytosis assays using PBMCs from patients and controls. (A) Lysates were prepared from PHA blast lymphocytes from patient 2 (P2) and control subject (Co). Perforin was detected by immunoblotting with 2d4-perforin antibody under nonreducing conditions. One band corresponding to the mature form of perforin is seen in both subjects. (B) Perforin expression in the PBMCs of patients 1 (P1) and 2 (P2) compared with control PBMCs (Co) and PBMCs from a patient with a nonsense mutation in the perforin gene (P0); percent of gated cells in heavy type and MFI in normal type are indicated in each quadrant. (C) Cytotoxic activity of T lymphocytes from the 3 patients with a perforin defect (P1, P2, and P0) is defective compared with that of T cells from 20 age-matched controls (shaded area). The effector-to–target cell ratio (x-axis) reflects the ratio of CD8⁺ T cells to target cells. Results are expressed as percentage of specific lysis (y-axis) as measured by ⁵¹Cr release. (D) Secretion of granzyme A into the cell culture supernatant of CD3-activated CD8⁺ T cells from P1 and Co was measured to quantify granule exocytosis. Cell supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for serine esterase. Data are expressed as the mean percentage ± SD specific release (test/total release) for triplicate samples.