Programmed cell death, or apoptosis, is important in homeostasis of the immune system: for example, non-functional or autoreactive lymphocytes are eliminated through apoptosis. One member of the tumour necrosis factor receptor (TNFR) family, Fas (also known as CD95 or Apo-1), can trigger cell death and is essential for lymphocyte homeostasis. FADD/Mort1 is a Fas-associated protein that is thought to mediate apoptosis by recruiting the protease caspase-8. A dominant-negative mutant of FADD inhibits apoptosis initiated by Fas and other TNFR family members. Other proteins, notably Daxx, also bind Fas and presumably mediate a FADD-independent apoptotic pathway. Here we investigate the role of FADD in vivo by generating FADD-deficient mice. As homozygous mice die in utero, we generated FADD-/- embryonic stem cells and FADD-/- chimaeras in a background devoid of the recombination activating gene RAG-1, which activates rearrangement of the immunoglobulin and T-cell receptor genes. We found that thymocyte subpopulations were apparently normal in newborn chimaeras. Fas-induced apoptosis was completely blocked, indicating that there are no redundant Fas apoptotic pathways. As these mice age, their thymocytes decrease to an undetectable level, although peripheral T cells are present in all older FADD-/- chimaeras. Unexpectedly, activation-induced proliferation is impaired in these FADD-/- T cells, despite production of the cytokine interleukin (IL)-2. These results and the similarities between FADD-/- mice and mice lacking the beta-subunit of the IL-2 receptor suggest that there is an unexpected connection between cell proliferation and apoptosis.

FADD͞Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAILreceptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD ؊/؊ T cells from RAG-1 ؊/؊ reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADDdeficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4 ؊ CD8 ؊ stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development. Fas-associated death domain (FADD)͞Mort1 was initially isolated as a protein that interacts with the cytoplasmic tail of the Fas receptor in a yeast two-hybrid system (1-3). FADD consists of a ''death effector domain'' at its amino terminus and a ''death domain'' at its carboxyl terminus. The death domain was shown to serve as a protein-protein interaction region with several death-domain containing members of the tumor necrosis factor receptor (TNFR) family. The FADD death effector domain recruits caspase-8 (4, 5). Upon interaction of Fas by FasL, for example, FADD, and subsequently caspase-8, are brought to the ''death-inducing signaling complex'' at the membrane (3, 6, 7). It was suggested that proximity among caspase-8 molecules within the death-inducing signaling complex leads to intermolecular processing and activation of caspase-8 (8, 9). This eventually leads to cleavage of the downstream targets and apoptosis. In addition to the Fas pathway, FADD also is required for signaling of cell death initiated by TNFR-I, DR3, and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. FADD-deficient MEFs or FADD Ϫ/Ϫ Jurkat T cells are completely resistant to apoptosis mediated by anti-Fas agonist antibody, TNF␣,or TRAIL (10 -15).Apart from its role in apoptosis, analysis of FADD Ϫ/Ϫ mice showed that FADD is also involved in other biological processes (10, 11). FADD-deficient embryos die of heart defects at day 10 of gestation. This could be caused by either a requirement of a death-domain containing receptor in heart development or an involvement of FADD in other unknown processes during differentiation (10, 11). Additionally, FADD appears to play a role in T cell proliferation and͞or lymphocyte development. In The impaired T cell development obser ved in FADD Ϫ/Ϫ 3RAG-1 Ϫ/Ϫ chimeras could be caused by the early effect of the FADD mutation in lymphoid progenitor cells or later during T cell development. To explore thi...

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.