A number of murine T-cell hybridomas undergo apoptosis within a few hours of activation by specific antigens, mitogens, antibodies against the T-cell antigen receptor, or a combination of phorbol ester and calcium ionophore. This phenomenon has been extensively studied as a model for clonal deletion in the immune system, in which potentially autoreactive T cells eliminate themselves by apoptosis after activation, either in the thymus or in the periphery. Here we show that the Fas/CD95 receptor, which can transduce a potent apoptotic signal when ligand, is rapidly expressed following activation of T-cell hybridomas, as is its functional, membrane-bound ligand. Interference with the ensuing Fas/Fas-ligand interaction inhibits activation-induced apoptosis. Because T-cell receptor ligation can induce apoptosis in a single T hybridoma cell, we suggest that the Fas/Fas-ligand interaction can induce cell death in a cell-autonomous manner.

The binding of heterotrimeric lymphotoxin, LT␣ 1 ␤ 2 , to the LT␤ receptor (LT␤R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor B (NF-B) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT␣ 1 ␤ 2 or agonistic LT␤R antibodies causes rapid recruitment of TNFRassociated factor 3 (TRAF3) to the LT␤R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LT␤R-mediated cell death. The inhibition is specific for LT␤R cell death signaling, since NF-B activation by LT␣ 1 ␤ 2 and Fasmediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LT␤R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LT␤R death signaling complex and indicate that at least two independent signaling pathways are initiated by LT␤R ligation.

Ligation of the lymphotoxin-␤ receptor (LT␤R) recruits tumor necrosis factor receptor-associated factor-3 (TRAF3) and initiates cell death in HT29 adenocarcinoma cells. The minimal receptor binding domain (TRAF-C) defined by two hybrid analyses is not sufficient for direct recruitment to the ligated receptor. A series of TRAF3 deletion mutants reveal that a subregion of the coiled coil motif is required for efficient recruitment to the LT␤R. Furthermore, the ability of TRAF3 to self-associate maps to an adjacent subregion. A TRAF3 deletion mutant that lacks the N-terminal zinc RING and zinc finger motifs, but retains the coiled coil and TRAF-C motifs, competitively displaces endogenous TRAF3 from the LT␤R. A second TRAF3 mutant that lacks the receptor binding domain, yet contains the TRAF3 self-association domain, prevents TRAF3 homodimers from being recruited to the LT␤R. Both of these mutants have a dominant negative effect on cell death and demonstrate that the recruitment of TRAF3 oligomers is necessary to initiate signal transduction that activates the cell death pathway.