TRAIL (also called Apo2L) belongs to the tumor necrosis factor family, activates rapid apoptosis in tumor cells, and binds to the death-signaling receptor DR4. Two additional TRAIL receptors were identified. The receptor designated death receptor 5 (DR5) contained a cytoplasmic death domain and induced apoptosis much like DR4. The receptor designated decoy receptor 1 (DcR1) displayed properties of a glycophospholipid-anchored cell surface protein. DcR1 acted as a decoy receptor that inhibited TRAIL signaling. Thus, a cell surface mechanism exists for the regulation of cellular responsiveness to pro-apoptotic stimuli.

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.

Objective. To assess the overexpression of B lymphocyte stimulator (BLyS) over time in patients with systemic lupus erythematosus (SLE).Methods. Sixty-eight SLE patients were followed up longitudinally for a median 369 days. At each physician encounter, disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index, and blood was collected for determination of the serum BLyS level, blood BLyS messenger RNA (mRNA) level, and cell surface BLyS expression. Twenty normal control subjects underwent similar laboratory evaluations.Results. In contrast to the uniformly normal serum BLyS and blood BLyS mRNA phenotypes in control subjects, SLE patients displayed marked heterogeneity, with 50% and 61% of patients manifesting persistently or intermittently elevated serum BLyS and blood BLyS mRNA phenotypes, respectively. Surface BLyS expression by SLE peripheral blood mononuclear cells was also often increased. Treatment of patients who had elevated serum BLyS levels with intensive courses of high-dose corticosteroids resulted in marked reductions in serum BLyS levels, and tapering of the corticosteroid dosage often resulted in increases in serum BLyS levels. Serum BLyS levels generally correlated with anti-double-stranded DNA (anti-dsDNA) titers (in those with detectable anti-dsDNA titers), but changes in serum BLyS levels did not correlate with changes in disease activity in individual patients. Serum BLyS phenotype did not associate with specific organ system involvement.Conclusion. Dysregulation of BLyS over extended periods of time is common in patients with SLE. Neutralization of BLyS activity with an appropriate BLyS antagonist may be therapeutically beneficial.

Objective. To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases.Methods. A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys.Results. LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers.

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...

The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival [1] [2] [3]. We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein [4]. The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand [5]. The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs [1] [6]. We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2) [7] and NF-kappaB-inducing kinase (NIK) [8]. Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.