Figure 2
Fas expression and caspase-10 activity in subjects carrying the TNFRSF6 and CASP10 mutation. a) Fas expression was evaluated in T cell lines obtained by activating PBMC with PHA (1 μg/ml) and cultured for 6 days in RPMI 1640 +10% FCS+rIL-2 (2 U/mL). Before activation (day 0), and at day 3 and day 6 of culture, cells were stained with a FITC-conjugated anti-Fas mAb and analyzed with a cytofluorimeter. The upper panel shows the cytofluorimetric staining of cells from Pt.1, Pt.2, and a control donor after 6 days of culture. The lower panel shows the MFI ratio calculated for each subject at different times of culture. Control data are the medians ± interquartile ranges (25–75% range) from 5 control donors; their 5th percentile value at day 6 was MFI-R = 6.48. b) Caspase-10 activity was evaluated in PHA-activated T cells cultured for 12 days (see Methods) in RPMI 1640 +10% FCS+rIL-2 (10 U/mL) and then treated or not with an anti-Fas mAb for 3 hours. Results are expressed as relative caspase activity % calculated as follows: (result displayed by each subject/mean of the results displayed by the 2 controls run in the same experiment) × 100; 100% indicates the mean of the results obtained with the 2 control donors run in parallel with the patient samples in each experiment; the dotted horizontal lines indicate the 5th percentile of the activity displayed by all normal controls. The color code is the same in all panels.