Different role of Apaf-1 in positive selection, negative selection and death by neglect in foetal thymic organ culture

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1(-/-)) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4(+) CD8(+) (DP) thymocytes from Apaf-1 (-/-) mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1 (-/-)mice were crossed with the T-cell receptor (TCR)- alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha (-/-) Apaf-1 (-/-) and TCR-alpha (-/-) Apaf-1 (+/+) mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1 D- -/-(b)/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33(+) thymocytes and an increased percentage of the autoreactive CD8(+) M33(+) thymocytes in male, but not female Apaf-1 (-/-) D-b/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development