The positive feedback loop of furin and TGFß1 enhances the immune responses of Tregs to hepatocellular carcinoma cells and hepatitis B virus in vitro.

Regulatory T cells (Tregs) can exert immunosuppressive activity. Furin can regulate Treg functions, hepatitis B virus (HBV) persistent infection, and hepatocellular carcinoma (HCC) development. However, it remains unknown whether furin can regulate the immune responses of Tregs to HBV and HCC cells. Here, coculture systems of HBV1.3P-HepG2.3P-HepG2 cells and Tregs transduced with or without lentiviral particles that could overexpress furin or knockdown furin/transforming growth factor ß1 (TGFß1) were established to investigate the regulatory relationship between furin and TGFß1 and the effect of furin/TGFß1 on Treg activity. Also, the effects of furin overexpression or furin/TGFß1 knockdown in Tregs on the immunological activity of effector T cells (Teffs)/cytotoxic T lymphocytes (CTLs) and HBV replication/expression were explored in the coculture system of Teff/CTL, Treg, and HBV1.3P-HepG2 cells. Our results showed that furin expression and TGFß1 secretion were notably increased in Tregs, and Furin and TGFß1 formed a positive feedback loop to activate Tregs in the coculture system of Tregs and HBV1.3P-HepG2 cells. Furin or TGFß1 knockdown in Tregs promoted Teff cell proliferation, stimulated interleukin-2 and interferon-γ secretion, and inhibited HBV replication/gene expression in the coculture system of Teff, Treg, and HBV1.3P-HepG2 cells. Moreover, furin or TGFß1 depletion in Tregs enhanced the killing activity of CTLs against HBV1.3P-HepG2 cells and curbed HBV replication/gene expression in the coculture system of Tregs, CTLs, and HBV1.3P-HepG2 cells. In conclusion, the positive feedback loop of furin and TGFß1 enhanced the immune responses of Tregs to HCC cells and HBV in vitro.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Harnessing proteases for T regulatory cell immunotherapy.

The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.

Inhibition of Membrane-Bound BAFF by the Anti-BAFF Antibody Belimumab.

B cell activating factor of the TNF family (BAFF, also known as BLyS), a cytokine that regulates homeostasis of peripheral B cells, is elevated in the circulation of patients with autoimmune diseases such as systemic lupus erythematosus (SLE). BAFF is synthetized as a membrane-bound protein that can be processed to a soluble form after cleavage at a furin consensus sequence, a site that in principle can be recognized by any of the several proteases of the pro-protein convertase family. Belimumab is a human antibody approved for the treatment of SLE, often cited as specific for the soluble form of BAFF. Here we show in different experimental systems, including in a monocytic cell line (U937) that naturally expresses BAFF, that belimumab binds to membrane-bound BAFF with similar EC50 as the positive control atacicept, which is a decoy receptor for both BAFF and the related cytokine APRIL (a proliferation inducing ligand). In U937 cells, binding of both reagents was only detectable in furin-deficient U937 cells, showing that furin is the main BAFF processing protease in these cells. In CHO cells expressing membrane-bound BAFF lacking the stalk region, belimumab inhibited the activity of membrane-bound BAFF less efficiently than atacicept, while in furin-deficient U937 cells, belimumab inhibited membrane-bound BAFF and residual soluble BAFF as efficiently as atacicept. These reagents did not activate complement or antibody-dependent cell cytotoxicity upon binding to membrane-bound BAFF in vitro. In conclusion, our data show that belimumab can inhibit membrane-bound BAFF, and that BAFF in U937 cells is processed by furin.

STAT6 and Furin Are Successive Triggers for the Production of TGF-ß by T Cells.

Production of TGF-ß by T cells is key to various aspects of immune homeostasis, with defects in this process causing or aggravating immune-mediated disorders. The molecular mechanisms that lead to TGF-ß generation by T cells remain largely unknown. To address this issue, we take advantage of the fact that intestinal helminths stimulate Th2 cells besides triggering TGF-ß generation by T lymphocytes and regulate immune-mediated disorders. We show that the Th2 cell-inducing transcription factor STAT6 is necessary and sufficient for the expression of TGF-ß propeptide in T cells. STAT6 is also necessary for several helminth-triggered events in mice, such as TGF-ß-dependent suppression of alloreactive inflammation in graft-versus-host disease. Besides STAT6, helminth-induced secretion of active TGF-ß requires cleavage of propeptide by the endopeptidase furin. Thus, for the immune regulatory pathway necessary for TGF-ß production by T cells, our results support a two-step model, composed of STAT6 and furin.

Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. We show here that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. We also demonstrate that Furin1 is a negative regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway, which is implicated in stress responses in the fly. In summary, our data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila-Aittomäki, S., Valanne, S., Lehtinen, T., Matikainen, S., Nyman, T. A., Rämet, M., Pesu, M. Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model.

Gelsolin amyloidosis is a dominantly inherited, incurable type of amyloidosis. A single point mutation in the gelsolin gene (G654A is most common) results in the loss of a Ca2+ binding site in the second gelsolin domain. Consequently, this domain partly unfolds and exposes an otherwise buried furin cleavage site at the surface. During secretion of mutant plasma gelsolin consecutive cleavage by furin and MT1-MMP results in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies that are able to (partly) inhibit furin or MT1-MMP proteolysis have previously been reported. In this study, the nanobodies have been combined into a single bispecific format able to simultaneously shield mutant plasma gelsolin from intracellular furin and extracellular MT1-MMP activity. We report the successful in vivo expression of this bispecific nanobody following adeno-associated virus serotype 9 gene therapy in gelsolin amyloidosis mice. Using SPECT/CT and immunohistochemistry, a reduction in gelsolin amyloid burden was detected which translated into improved muscle contractile properties. We conclude that a nanobody-based gene therapy using adeno-associated viruses shows great potential as a novel strategy in gelsolin amyloidosis and potentially other amyloid diseases.

Inhibition of furin results in increased growth, invasiveness and cytokine production of synoviocytes from patients with rheumatoid arthritis.

OBJECTIVES: Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis play a key role by local production of cytokines and proteolytic enzymes that degrade the extracellular matrix and cartilage. These synoviocytes acquire phenotypic characteristics commonly observed in transformed cells, like anchorage-independent growth, increased proliferation and invasiveness, and insensitivity to apoptosis. Furin is a ubiquitous proprotein convertase that is capable of cleaving precursors of a wide variety of proteins. In patients with rheumatoid arthritis, furin is reported to be highly expressed in the synovial pannus compared with healthy persons. However, the mechanisms are poorly understood. This study is to explore the effect of furin overexpression in rheumatoid synoviocytes. METHODS: In this study, RNA interference was used to knock down furin expression and to assess the resultant effects on biological behaviors of synoviocytes, such as cell proliferation, invasion, migration, cell cycle and cell apoptosis. In addition, the production of inflammatory cytokines was evaluated. RESULTS: The results showed that the inhibition of furin enhanced proliferation, invasion, and migration of synoviocytes in vitro. Cell cycle was accelerated and cell death was affected by furin knockdown. Also, the inhibition of furin increased interleukin-1ß and tumor necrosis factor-α secretion of synoviocytes. CONCLUSIONS: Inhibition of furin enhances invasive phenotype of synoviocytes from patients with rheumatoid arthritis, implying a protective role of furin. Agents targeting upregulation of furin may have therapeutic potential for rheumatoid arthritis.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

BACKGROUND: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. RESULTS: We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. CONCLUSIONS: The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways.

DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a 'handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the 'handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.

Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes.

Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.

Generation and characterization of non-competitive furin-inhibiting nanobodies.

The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.

Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

Development and characterisation of an assay for furin activity.

Furin is a serine endoprotease that is responsible for the proteolytic processing of proteins within the secretory pathway, including cytokines, hormones, integrins, other proteases, and also pathogen-derived proteins. It is likely that the level of furin activity determines the extent of processing of these substrates. Furin is ubiquitously expressed across all tissues, at low levels, but can be induced in response to environmental cues such as hypoxia and cytokine stimulation. However, all studies to date that have investigated furin expression have been limited to analysis of furin mRNA; there has been no assay sensitive enough to quantify endogenous furin. Though activity-based assays have been described for furin-like enzyme activity, we demonstrate that these assays are dominated by the activity of other enzymes and cannot be used to approximate furin activity. A sensitive and specific assay for furin activity was therefore developed and characterised, using an antibody capture step to immobilise furin from whole cell lysates. Furin activity is quantified relative to that of recombinant active furin protein, to allow estimation of active furin protein concentration. The assay has a minimum detection limit of 0.006 nM; sensitive enough to determine the furin activity of many of the cell lines tested. The specificity of the assay was demonstrated by genetic modulation of furin expression. Furthermore, the assay was used to demonstrate that the cytokine transforming growth factor beta (TGF-ß) stimulates increased furin activity in HepG2 cells, confirming and extending previous reports that TGF-ß increases furin expression, and adding to the mounting body of evidence that cellular furin activity can be modulated.

Furin-processed antigens targeted to the secretory route elicit functional TAP1-/-CD8+ T lymphocytes in vivo.

Most pathogen-derived peptides recognized by CD8+ CTL are produced by proteasomes and delivered to the endoplasmic reticulum by the TAP transporters associated with Ag processing. Alternative proteases also produce antigenic peptides, but their actual relevance is unclear. There is a need to quantify the contribution of these supplementary pathways in vitro and in vivo. A well-defined TAP-independent secretory route of Ag processing involves the trans-Golgi network protease furin. Quantitation of this route by using OVA constructs encoded by vaccinia viruses indicates that it provides approximately one-third of all surface complexes of peptide and MHC class I molecules. Generation of the epitope carboxyl terminus is a dramatic rate-limiting step, since bypassing it increased efficiency by at least 1000-fold. Notably, the secretory construct activated a similar percentage of Ag-specific CD8+ T cells in wild type as in TAP1-deficient mice, which allow only secretory routes but which have a 10- to 20-fold smaller CD8 compartment. Moreover, these TAP1(-/-) OVA-specific CD8+ T lymphocytes accomplished elimination of epitope-bearing cells in vivo. The results obtained with this experimental system underscore the potential of secretory pathways of MHC class I Ag presentation to elicit functional CD8+ T lymphocytes in vivo and support the hypothesis that noncytosolic processing mechanisms may compensate in vivo for the lack of proteasome participation in Ag processing in persons genetically deficient in TAP and thus contribute to pathogen control.

Aberrant expression of BAFF in T cells of systemic lupus erythematosus, which is recapitulated by a human T cell line, Loucy.

B cell-activating factor of the tumor necrosis factor (TNF) family, or BAFF, is mainly produced in monocytes and dendritic cells, and indispensable for proliferation, differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF), which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). In this study, we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand, the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line, Loucy (American Type Tissue Collection CRL-2629), in response to several stimuli, while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38, and that shedding of BAFF was catalyzed by a membrane-bound protease, furin.