Fas/FasL-mediated cytotoxicity. (A) T-cell blasts from a healthy control, the mother, and the patient were stimulated with PMA plus ionomycin (P+I) and tested for cell-mediated FasL-induced cytolytic activity using L1210 cells transfected (L1210.Fas) or not (L1210.3) with Fas as targets at a fixed 5:1 E/T ratio in 16-hour cytotoxicity assays. (B) COS-7 cells were transfected with an expression vector containing the wild-type (WT) FASL allele or the mutant FASL allele from the patient. Forty-eight hours after transfection, transfected COS-7 cells were tested against L1210 Fas target cells at the indicated E/T ratios in 6-hour cytotoxicity assays. (C) Supernatants (ssn) from P+I-stimulated T-cell blasts from the healthy control and the patient were collected and tested for cell-free FasL-induced toxicity on Jurkat cells, as described in “Patient, materials, and methods.” (A-C) Results are the mean ± SD of at least 3 different experiments. (D) Exosomes secreted by human T-cell blasts from the healthy control and the patient after P+I stimulation were labeled with H11-FITC and analyzed by flow cytometry using a gating protocol specific for microvesicles/exosomes. Percentage of FasL⁺ exosomes in each case is shown.