Decreased perforin protein levels and cytolytic activity in patients carrying PRF1 mutations. (A) Cytoplasmic extracts from T cells of patients with aplastic anemia with PRF1 mutations (n = 5) and healthy controls (n = 8) were analyzed for perforin protein expression by immunoblot. All 5 patients carrying PRF1 mutations revealed decreased or absent perforin protein levels. All samples were run side by side. The upper panel (from left to right) shows the perforin protein levels from a healthy person and patients D, E, A, B, and C. The lower panel shows the immunoblot results from chronologically different samples obtained from the same patients (from left to right a healthy control, patient C, patient A, and patient B, and 3 patients with no mutations). Patients without PRF1 mutations revealed perforin protein levels comparable to that from healthy volunteers. (B) Decreased perforin granules in cytotoxic cells from patients with aplastic anemia with PRF1 mutations. CD8⁺ T cells from patients carrying PRF1 mutations and healthy controls were double stained in parallel with anti-cathepsin D (red, first column) and anti-perforin (green, second column) antibodies in at least 3 different experiments. Perforin and cathepsin D showed the expected pattern of colocalization in cytotoxic granules of control cells. Staining of the patients' cytotoxic cells revealed complete absence of perforin granules in patients A, B, and D (Figure 4); patient C showed slightly decreased staining of perforin granules, but they were abnormal in size. (Differences between perforin protein levels and perforin granule staining in patients A, B, and C, who have the same PRF1 mutation may be explained by different genetic background or additional unshared genetic alterations). The frequency of cathepsin-D in CD8⁺ T cells was similar in patients' and controls' samples. (C) Decreased cytolytic activity in patients with aplastic anemia. Natural killer cells were isolated from patients D and E, and their killing efficiency was determined in a standard Cr⁵¹-release assay against K562 target cells in comparison to cells from healthy donors. Patients carrying PRF1 mutations showed markedly decreased cytolytic activity compared with controls. A patient (not carrying any mutation or polymorphism) also showed decreased cytolytic activity but not as low as that observed in the patients with PRF1 mutations.