SPECIFIC ACTIVATION OF THE CYSTEINE PROTEASE CPP32 DURING THE NEGATIVE SELECTION OF T-CELLS IN THE THYMUS

Cysteine proteases of the CED-3 and ICE family have been recently prop osed as the ultimate executioners in several mammalian cell death path ways. Among them, the cysteine protease CPP32 has been shown to partic ipate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism thro ugh which developing thymocytes expressing a TcR with high affinity fo r self peptide-MHC complexes are eliminated by PCD. In order to invest igate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the abs ence or alter crosslinking with anti-CD3 mAb, specific activation of C PP32, as assessed by the extent of proteolytic cleavage of the p32 zym ogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very earl y event during apoptosis as it occurred before the exposure of phospha tidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, usin g mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice e xpressing a permissive MHC haplotype for PCC presentation (H-2k). More over, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(A DP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concep t that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also sugge st the involvement of a distinct oo CPP32-like cysteine protease in sp ontaneous apoptosis of thymocytes.