Mechanism of A20 protection from NK cell–mediated cell death. A20 expression protects ECs from NK cell–mediated cell death by blocking caspase 8 and 3 activation and inhibiting BID cleavage. (A) FACS analysis of Fas surface expression in PAECs and porcine PBMCs shows the absence of Fas expression in PAECs whether or not they were treated with TNF, while Fas is readily expressed on the surface of PBMCs collected from the same pig strain. (B) NI PAECs and PAECs infected with rAd.β-gal or rAdA20 were labeled with calcein and cocultured with NK 92 cells at an E/T ratio of 1:1, 10:1, and 30:1 for 4 hours. NK-induced cytotoxicity was measured by calcein release. Expression of A20 significantly protected PAECs from NK cell–mediated cytotoxicity. Data shown are representative of 6 experiments performed in triplicate. Caspase 8 (C) and caspase 3 (D) activities were analyzed in the PAEC/NK cocultures by means of a colorimetric assay based on spectrophotometric detection of the chromophore pNA after cleavage from the caspase-specific–labeled substrates. Expression of A20 significantly inhibited NK-induced activation of caspases 8 and 3. Data are expressed as mean ± SD of triplicate values. Results shown are representative of 4 independent experiments. (E) Western blot analysis of BID cleavage in NI PAECs and PAECs infected with rAdA20 or rAdβ-gal 6 hours following coculture with NK cells. A20 expression in PAECs inhibited NK-induced cleavage of BID (24 kD). Data shown are representative of 3 independent experiments. Equal loading was confirmed by β-tubulin expression.