A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.

To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.

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A fourth member of the emerging TRAIL receptor family, TRAIL-R4, has been cloned and characterized. TRAIL-R4 encodes a 386-amino acid protein with an extracellular domain showing 58%-70% identity to those of TRAIL-R1, TRAIL-R2, and TRAIL-R3. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-kappaB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one third of a consensus death domain motif. Transient overexpression of TRAIL-R4 in cells normally sensitive to TRAIL-mediated killing confers complete protection, suggesting that one function of TRAIL-R4 may be inhibition of TRAIL cytotoxicity. Like TRAIL-R1 and TRAIL-R2, this receptor shows widespread tissue expression. The human TRAIL-R4 gene has been mapped to chromosome 8p22-21, clustered with three other TRAIL receptors.

Suml'l'lary A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T ceils resulted in the expression of Fas-L mILNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T ceils is mediated by Fas/Fas-L interactions. Mature peripheral T cells generally undergo activation . and proliferation when stimulated through the CD3/ TCR complex. Under certain circumstances, however, thymocytes, T cell hybridomas, and both CD4 + and CD8 + T cell clones (TCC) 1 undergo cell death when stimulated through the TCR with CD3 antibody in the absence of APC (1-6). This process is rapid and exhibits classic characteristics of apoptosis such as membrane blebbing, chromatin condensation, and the formation of DNA fragments of ",,200 bp. Deletion of T cells by apoptosis appears to be important not only in regulating autoreactive T cells in the thymus, but also in regulating the peripheral T cell pool (7,8). Little is known, however, about the mechanism that mediates the lytic process that has been termed activation-induced cell death (AICD).Fas/APO-1 (CD95) is a protein expressed on the surface of a variety of transformed cell lines and chronically stimulated T cells that can mediate apoptosis after ligation with a Fas-specific antibody (9-12). Under appropriate conditions Fas also transduces a stimulatory signal to certain B cell lines (13) and to .freshly isolated human peripheral blood T cells and thymocytes (14). To investigate a possible relationship between CD3-stimulated AICD and Fas-mediated T cell apoptosis, we have used a mAb directed against human Fas (Fas M3). Immobilized Fas M3 mAb is able to lyse Fas-expressing tumor cell lines in a manner analogous to Fas-ligand (Fas-L) or the prototypic Fas mAb, CH-11, whereas soluble Fas M3 blocks Fas-mediated killing (15).1 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas-ligand; SEB, staphylococcal enterotoxin B; TCC, T cell done, Materials and MethodsT Cell Lines and Clones. The alloreactive TCC used in this study were generated by establishing MLC in bulk culture for 7 d followed by limit dilution cloning in 96-well round-bottomed plates in the presence of 10 s irradiated allogeneic PBMC and 10 ng/mt of Ib2. TCC were maintained by stimulation with irradiated PBMC and soluble CD3 antibody (10 ng/ml) approximately every 2 wk and maintenance in IL-2 (10 ng/ml) between stimulations. Shortterm staphylococcal enterotoxin B (SEB)-specific T cell lines were established by stimulation of PBMC (106) with 5 /~g/ml SEB (Sigma Chemical Co., St. Loui...