DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.

We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-B responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.Members of the tumor necrosis factor superfamily of cytokines play diverse roles in the regulation of cell proliferation, differentiation, and survival. Notably, several members of this family play key roles in the regulation of the immune system (1). We and others have previously identified a novel TNF 1 -related ligand, BLyS (also known as BAFF, TALL-1, THANK, TNFSF20, and zTNF4) which is expressed on monocytes and induces B-cell proliferation and immunoglobulin secretion in vitro and in vivo (2-6). Like many members of the TNF family, BLyS has activity in vitro as a 152-amino acid soluble molecule and as a 258-amino acid transmembrane form (3). However, the biological significance of these two forms and their relative contributions in vivo remain to be resolved. More recently, transgenic mice that ectopically overexpress BLyS were shown to develop autoimmune-like phenotypes reminiscent of those observed in systemic lupus erythematosus (7-9). These findings suggest that BLyS plays an important role in the regulation of B-cell growth and humoral immunity.In order to understand the precise mechanism by which BLyS activates B-cells, the range of cell types BLyS may affect, and the potential role of BLyS as a therapeutic agent or target, we have used expression cloning to identify the receptor for BLyS. We have identified the orphan receptor TACI (10), previously characterized as being present on B-cells and a subset of T-cells, as the receptor for BLyS and show that this receptor is capable of mediating NF-B signaling in response to ligand binding. We also show that TACI interacts with another TNF family member, APRIL, which is closely related to BLyS. Parallel work by others has recently shown that TACI and a second TNFR family member, BCMA, are BLyS receptors (9, 11-14). EXPERIMENTAL PROCEDURESCell Culture and Media-HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and transfected using LipofectAMINE Plus (Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. For expression cloning screens, cells were attached to plates with poly-D-lysine.Flow Cytometry-Cells were stained with monoclonal antibodies raised against BLyS at the indicated protein concentrations, with biotinylated BLyS as described previously (2), with recombinant TACI-Fc fusion protein or with recombinant Flag-tagged proteins which were subsequently detected by the M2 anti-Flag monoclonal antibody (Sigma). Flow cytometry was performed using a FACScan instrument and associated CellQuest software (Becton Dickinson, San Jose, CA).Library Preparation, Screening, and Other DNA Manipulations-All common DNA...

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibodysecreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

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