| During immune responses to conventional peptide antigens or superantigens, clonal expansion of antigen specific T cells results in a dramatic increase in their numbers. The vast majority of these activated T cells are cleared at the end of the response, a process termed activated T cell autonomous death (ACRD). We studied the roles of several Bcl-2 family proteins in this process.; Bax and Bak, two multidomain killers of the Bcl-2 family, participate in ACAD. In healthy resting T cells, Bax resides mainly in the cytosol but also on the mitochondria. Mitochondrial Bax molecules in these cells are sensitive to alkali-mediated removal, suggesting a weak mode of association between these molecules and mitochondria. After T cell activation, mitochondrial Bax becomes strongly attached to mitochondria, resistant to alkali-mediated removal. In T cells that have undergone ACAD, additional changes occur in Bax, including a conformational change that exposes its NH2-terminus and the relocalization of cytosolic Bax to mitochondria. Bak, a close homologue of Bax, is exclusively localized to mitochondria in both healthy and apoptotic T cells and is constitutively resistant to alkali-mediated removal. After T cell activation, Bak also undergoes a conformational change that exposes its NH2-terminus, but with a much earlier onset compared to Bax: it is detectable before any sign of apoptosis becomes evident. Therefore although Bax and Bak are highly homologous and both participate in ACAD, they demonstrate distinct behaviors in ACAD.; Surprisingly, T cells deficient in either Bax or Bak showed no defect in ACAD, suggesting either that they are not necessary for ACAD, or that their functions are interchangeable and the presence of either one of them is sufficient for ACAD.; We found that T cells deficient in the BH3-only killer, Bim, have profound defects in ACRD. We then studied the mechanisms of Bim-mediated killing in ACAD. It has been described in other cell types that the pro-apoptotic activity of Bim is constrained in healthy cells by sequestration to microtubules. This sequestration has been shown to be mediated by the binding of Bim to LC8, a component of the dynein motor complex. We found that despite its ability to bind LC8, most of the Bim in both healthy and apoptotic T cells is associated with mitochondria not microtubules. This is probably because in T cells LC8 is present mainly as a cytosolic protein with limited microtubule association.; In healthy resting T cells, mitochondrially located Bim is bound to the anti-apoptotic proteins, Bcl-2 and Bcl-xL. We hypothesize that these interactions inhibit the pro-apoptotic activity of Bim. During T cell activation, the level of Bim remains unchanged, whereas the level of Bcl-2 falls and that of Bcl-xL increases transiently and then falls. We hypothesize that these changes during T cell activation leave Bim unopposed, unleashing its pro-apoptotic activity.; In summary, our results suggest that ACRD is tightly regulated by the balance between the protectors and the killers of the Bcl-2 family. |
