Abstract
Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1(-/-) mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1(-/-) thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1(-/-) thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1(+/-) thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1(-/-) thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.
Highlights
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Our results showed that clonal deletion in these systems was normally executed in apoptotic protease-activating factor 1 (Apaf1)-deficient thymocytes, demonstrating that Apaf1-dependent apoptotic pathway is dispensable for programmed cell death (PCD) during the negative selection process
Since PCD plays a critical role in thymocyte selection, we examined the role of Apaf1, a central element in the mitochondria-dependent apoptotic pathway, in the development of thymocytes
Summary
PCD, programmed cell death; Apaf, apoptotic protease-activating factor 1; cyto c, cytochrome c; DiOC6(3), 3,3Ј-diehexyloxadicarbocyanine iodide; DP, double-positive; E, embryonic day; FTOC, fetal thymic organ culture; PI, propidium iodide; SEB, staphylococcal enterotoxin B; SP, single-positive; Tg, transgene (transgenic); ⌬⌿m, mitochondrial transmembrane potential. Selfreactive T cells as well as Ag-stimulated mature T cells are deleted by a mechanism of activation-induced cell death, which is mainly mediated by Fas-mediated apoptosis (6). Our results showed that clonal deletion in these systems was normally executed in Apaf1-deficient thymocytes, demonstrating that Apaf1-dependent apoptotic pathway is dispensable for PCD during the negative selection process. We showed that Apaf1-deficient thymocytes are more resistant to the peptide-induced cell death in vitro, implicating Apaf1-mediated apoptotic pathway in the negative selection of thymocytes. We demonstrate that Apaf1-independent caspase activation and cell death that were not inhibited by a broad spectrum caspase inhibitor, z-VAD-fmk, occurred during the peptide-induced cell death in vitro Taken together, these data indicate that the cell death mechanisms of negative selection are composed of several pathways, which presumably play synergistic and mutually compensatory roles
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