FADD-deficient T cells exhibit a disaccord in regulation of the cell cycle machinery.

FADD is an adapter protein that was originally isolated as a transducer of apoptotic signals for death domain-containing receptors. However, FADD-deficient mice are embryonic lethal and FADD(-/-) T cells developed from FADD(-/-) embryonic stem cells in the RAG-1(-/-) hosts lack the full potential to proliferate when stimulated through their T-cell receptor complex, suggesting that FADD protein might play a dualistic role in mediating not only cell death signaling but other non-apoptotic cellular pathways as well. Here we show that a substantial number of freshly isolated FADD(-/-) peripheral T cells are cycling but are defective in their co-stimulatory response when stimulated. Analysis of several cell cycle proteins shows normal down-regulation of p27 inhibitor but increased levels of p21, decreased levels of cyclin D2, and constitutive activation of several cyclin-dependent kinases in activated T cells. These data suggest that FADD is involved in the regulation of cell cycle machinery in T lymphocytes.

T cell-specific FADD-deficient mice: FADD is required for early T cell development.

FADD/Mort1, 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 TRAIL-receptors. 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 FADD-deficient 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.

In vivo overexpression of Dad1, the defender against apoptotic death-1, enhances T cell proliferation but does not protect against apoptosis.

The Dad1 protein has been shown to play a role in prevention of apoptosis in certain cell types. Dad1 is also a subunit of the oligosaccharyltransferase enzyme complex that initiates N-linked glycosylation. It is encoded by a gene located adjacent to the TCR alpha and delta genes on mouse chromosome 14. We have investigated the role of Dad1 during T cell development and activation. We observe that endogenous Dad1 levels are modulated during T cell development to reach maximal expression in mature thymocytes. Transgenic mice that overexpress Dad1 in both the thymus and peripheral immune system have been generated. Apoptosis of thymocytes from such mice is largely unaffected, but peripheral T cells display hyperproliferation in response to stimuli. Therefore, the linkage between the TCR and Dad1 genes may have important consequences for T cell function.

A tailless fas-FADD death-effector domain chimera is sufficient to execute Fas function in T cells but not B cells of MRL-lpr/lpr mice.

The Fas receptor delivers signals crucial for lymphocyte apoptosis through its cytoplasmic death domain. Several Fas cytoplasmic-associated proteins have been reported and studied in cell lines. So far, only Fas-associated death domain protein (FADD), another death domain-containing molecule has been shown to be essential for Fas signals in vivo. FADD is thought to function by recruiting caspase-8 through its death-effector domain. To test whether FADD is sufficient to deliver Fas signals, we generated transgenic mice expressing a chimera comprised of the Fas extracellular domain and FADD death-effector domain. Expression of this protein in lymphocytes of Fas-deficient MRL-lpr/lpr mice completely diminishes their T cell but not their B cell abnormalities. These results suggest that FADD alone is sufficient for initiation of Fas signaling in primary T cells, but other pathways may operate in B cells.

Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1.

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.

CD38-mediated ribosylation of proteins.

The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.