Reduced bioactive FasL and granule secretion by WASp-deficient T cells. (A) Secreted FasL measured by ELISA in supernatants after 6 hours of stimulation of activated CD4⁺ T lymphocytes with the indicated concentrations anti-CD3 antibody. Values are the average ± SEM of data from 2 mice per group, and similar results were obtained in 3 independent experiments. (B) Supernatants from CD4⁺ T cells restimulated for 6 hour with anti-CD3 were fractionated by centrifugation through 100-kDa cutoff membranes, and FasL was quantitated in each fraction by ELISA. No FasL was detected in the < 100-kDa fraction when purified vesicular FasL was filtered through identical membranes. (C) The indicated concentrations of purified vesicular and soluble FasL were added to WEHI-279 cells, and cytotoxicity was quantitated by a luminescent cell viability assay. Anti-FasL antibody was added to demonstrate specificity of this assay for bioactive FasL. Specific cell death was quantitated as described in the methods. (D) FasL-dependent apoptosis-inducing activity of the indicated fractions of supernatants collected from cells WT and WASp-KO T cells. Supernatants from cells restimulated in C were assayed on WEHI-279 cells for apoptosis-inducing activity. Anti-FasL was added to the indicated samples to neutralize FasL activity. Asterisks mark the results of comparisons of WASp-KO with the identical WT cell supernatant fractions, and anti-FasL–treated samples compared with the same samples without anti-FasL. Results of P values from comparisons using Student unpaired t test are denoted as *P < .05, **P < .005, ***P < .001. (E) β-Hexosaminadase release from activated WASp-deficient and control T cells restimulated with the indicated concentrations of anti-CD3 mAb. The curve of percent specific release was significantly different in WASp-deficient mice for both CD4 and CD8 than controls (P < .001, 2-way analysis of variance).