Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes

TCR engagement of immature CD4(+)CD8(+) thymocytes induces clonal maturatio n (positive selection) as well as clonal deletion (negative selection) in t he thymus, However, the cell death execution events of thymocytes during th e negative selection process remain obscure. Using a cell-free system, we i dentified two different DNase activities in the cytosol of in vivo anti-TCR -stimulated murine thymocytes: one that induced chromosomal DNA fragmentati on, which was inhibited by an inhibitor of caspase-activated DNase, and ano ther that induced plasmid DNA degradation, which was not inhibited by an in hibitor of caspase-activated DNase, We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulate d thymocytes and found that it is identical with cyclophilin B (Cyp B), whi ch was reported to locate in endoplasmic reticulum, Ab against Cyp B specif ically inhibited the DNA degradation activity in the cytosol of anti-CD3-st imulated thymocytes, Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation, Fi nally, we demonstrated that TCR engagement of a murine T cell line (EL4) wi th anti-CD3/CD28 resulted in the release of Cyp B from the microsome fracti on to the cytosol/nuclear fraction. Our data strongly suggest that both act ive caspase-activated DNase and Cyp B may participate in the induction of c hromosomal DNA degradation during cell death execution of TCR-stimulated th ymocytes.