Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity

B ymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) superfamily. BLyS stimulates proliferation of, and immunoglobulin production by, B cells. However, the relative importance of BLyS in physiological B cell activation is unclear. We identified a B cell receptor for BLyS through expression cloning as TACI, an orphan TNF receptor homologue of unknown function. Binding of BLyS to TACI activated signaling by nuclear factor-B (NF-B). In vitro soluble TACI-Fc fusion protein blocked BLyS-induced NF-B activation in B lymphoma cells and IgM production in peripheral blood B cells. In vivo treatment of immunized mice with TACI-Fc inhibited production of antigen-specific IgM and IgG1 antibodies and abolished splenic germinal center (GC) formation. Thus, BLyS activity must play a critical role in the humoral immune response.

The tumor necrosis factor (TNF) ligand superfamily consists of more than 15 members that regulate a variety of cellular functions, including proliferation, differentiation and apoptosis¹. TNF and most of its homologues are Type II transmembrane proteins which can be released by cells as soluble homotrimeric molecules. These ligands interact with specific members of the TNF receptor (TNFR) superfamily, which contains more than 20 proteins^{2, 3}. Several TNF superfamily ligands, including TNF (TNFSF2), LT (TNFSF3), CD40L (TNFSF5) and FasL (TNFSF6), are important modulators of immunity and play essential roles in lymphoid or thymic development, peripheral deletion, T cell-mediated immune responses, T cell-dependent help for B cells, or humoral B cell activity¹.

BLyS (TNSF13B) a newly identified TNF homologue, is implicated in the regulation of B cell function^{4, 5, 6, 7}. BLyS is expressed on T cells, dendritic cells, monocytes and macrophages^{4, 5, 6, 7}, and binding sites for BLyS are found primarily on B cells^{4, 7}. In a similar manner to CD40L¹, BLyS promotes B cell proliferation in vitro during costimulation by antibody to IgM⁷. Short-term administration of soluble BLyS to mice disrupts the architecture of B and T cell zones in the spleen and elevates serum IgM levels⁶. Transgenic mice expressing the BLyS gene systemically show global B cell activation and develop autoimmune disorders^{8, 9}. These findings suggest that BLyS may be a positive regulator of B cell function, however, the molecular basis of BLyS function and the relative contribution of this ligand to humoral immunity is unknown. To address these questions, we looked for a receptor for BLyS in B cells. Through expression cloning, we identified such a BlyS receptor. It was a TNFR homologue which had no previously known ligand. We found that BLyS stimulation of this receptor in vitro led to activation of nuclear factor-B (NF-B) and induced immunoglobulin production by B cells. To investigate the importance of BLyS in the development of a primary immune response, we used the TACI receptor's extracellular domain as a soluble inhibitor to block BLyS activity after antigen challenge in vivo. Our results indicate that BLyS plays an important role in the induction of immunoglobulin production by B cells and is essential for formation of splenic germinal centers (GCs), the sites of antibody affinity maturation and memory B cell formation.

Figure 1. Interaction of TACI with BlyS on cells. (a) Monkey COS-7 cells were transfected with human TACI (A−D) or vector (E), incubated with conditioned medium from Chinese hamster ovary cells transfected with human AP-BLyS (A,B,C,E) or AP-TNF (E), and stained for alkaline phosphatase activity in situ. Where indicated, cells were preincubated with 2 g/ml of purified soluble human Flag-LIGHT or Flag-BLyS. (b) COS-7 cells transfected with full-length BLyS (A,B) or TNF (C,D) were incubated with 1 g/ml of human TACI-Fc (A,C) or TNFR1-Fc (B,D) and stained with biotinylated goat anti-human Fc antibody and Cy3-streptavidin. Full Figure and legend (37K)

Figure 2. Interaction of TACI and BLyS in solution. Purified human TACI-Fc, HVEM-Fc, DR3-Fc, or DR6-Fc (1 g/ml) was incubated with purified human Flag-BLyS (1 g/ml) in duplicate. (a) One set of reactions was subjected to immunoprecipitation (IP) through the receptor-Fc fusion with protein-A-agarose; for comparison, the input amount of Flag-BLyS was loaded directly in the right hand lane. (b) A second set of reactions was subjected to IP through the ligand with antibody to Flag (anti-Flag). The samples were analyzed by western blot (WB) with either: (a) anti-Flag to detect the ligand or (b) antibody to human Fc to detect Fc-fused receptors. Full Figure and legend (10K)

BLyS stimulates NF-B activity and IgM production

Many TNFR family members signal activation of the transcription factor NF-B^{2, 3} Upon transfection into human embryonic kidney 293 cells, full-length human TACI induced NF-B activation in a plasmid-dosage dependent manner, as determined in an NF-B−specific reporter gene assay (data not shown). At 0.1 ng DNA, TACI induced only marginal activation of NF-B (Fig. 3); and addition of purified human Flag-BLyS (Fig. 3a) or contransfection with full-length human BLyS (Fig. 3b) greatly enhanced NF-B activation. Treatment of non transfected IM-9 cells with human Flag-BLyS also resulted in activation of NF-B, as determined in a gel mobility shift assay (Fig. 3c). This activation was blocked by human TACI-Fc, but not by a control Fc fusion protein. Therefore, binding of BLyS to TACI activates NF-B.

Figure 3. BLyS activates NF-B through TACI. (a,b) Human 293 cells were transfected with 0.25 g of ELAM-luciferase reporter gene construct, 25 ng pRL-TK, and an expression plasmid for full-length human TACI at the indicated amounts. In (a) purified human Flag-BLyS was added at the indicated concentrations 4 h after transfection; in (b) the cells were cotransfected with full-length human BLyS or TRANCE. Total amount of DNA was normalized with vector DNA. NF-B activation was determined 20−24 h later by Dual-Luciferase reporter assay (Promega, Madison, WI). (c) Human IM-9 cells were incubated with PBS buffer (lanes 1 and 5), purified human Flag-BLyS (0.3 g/ml) (lanes 2 and 6) or human Flag-BLyS in combination with 20 g/ml of human TNFR1-Fc (lanes 3 and 7) or human TACI-Fc (lanes 4 and 8). NF-B activity was measured by electrophoretic mobility shift assay using an NF-B-specific DNA oligonucleotide probe (lanes 1−4), or a control, mutated oligonucleotide (lanes 5−8)22. Full Figure and legend (16K)

Figure 4. Inhibition of BLyS activity in vitro by TACI-Fc. Human donor PBL were incubated for 72 h with either: PBS buffer; IL-4 (100 ng/ml); or human Flag-BLyS (1 g/ml) either alone or in combination with 20 g/ml human TACI-Fc or a human IgG1 control. Cell supernatant IgM levels were analyzed by ELISA (Bethyl Laboratories, Montgomery, TX). Full Figure and legend (18K)

Next we tested whether blocking BLys-TACI interactions in vivo impairs humoral immune responses. We immunized mice with hapten-conjugated chicken globulin (NP₂₃-CgG), and treated them for 14 days with control murine immunoglobulin or murine TACI-Fc. TACI-Fc substantially inhibited the production of NP-specific IgM as compared to control (Fig. 5a), indicating that BLyS function is important during the early phase of B cell activation that leads to IgM secretion. To investigate the effect of TACI-Fc treatment on IgG1 production, we determined the levels of specific antibodies to NP₂₃-CgG and to NP₂-CgG, representing total IgG1 and the high-affinity IgG1 compartment, respectively, in the serum of immunized mice. Murine TACI-Fc inhibited the total NP-specific IgG1 response, as well as the production of high-affinity anti-NP IgG1 (Fig. 5b,c). Thus, BLyS activity is important not only for IgM production, but also for immunoglobulin class-switching and affinity maturation.

Figure 5. TACI-Fc inhibits antigen-specific antibody responses in vivo. Mice (in sample groups of five) were immunized with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl-conjugated chicken globulin (NP23-CgG) and treated daily with 50 g control murine immunoglobulin (open bars) or murine TACI-Fc (filled bars). At day 14 after immunization serum was assayed for (a)NP-specific IgM (b) IgG1 that binds to NP23, representing total anti-NP IgG1 (c) or IgG1 that binds to NP2, representing high-affinity anti-NP IgG1. Full Figure and legend (14K)

Figure 6. TACI-Fc inhibits antigen-induced splenic B cell activation and GC formation. Immunohistochemical analysis of spleens from mice treated with control murine immunoglobulin (left hand panels) or murine TACI-Fc (right hand panels) during the primary immune response. Mice were immunized and treated as in Fig. 5. (a) Immunoglobulin class switching in PALS-associated B cell foci: spleen sections were prepared 10 days after immunization and stained with FITC-conjugated anti-IgG1. (b) GC formation: spleen sections were prepared 14 days after immunization and stained with FITC-conjugated anti-PNA (green fluorescence) and Texas Red-conjugated anti-IgM (red fluorescence). (c) Follicular morphology: day 14 spleen sections were stained with FITC-conjugated anti-CD4 (green fluorescence) and Texas Red-conjugated anti-B220 (red fluorescence) to visualize T and B cells, respectively. Full Figure and legend (198K)

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We have identified TACI as a receptor for BlyS. BLyS activation of TACI in vitro stimulated NF-B and induced IgM production in B cells. Blocking BLyS activity by introducing TACI-Fc during primary immunization in vivo inhibited several aspects of the B cell response. These were: the early phase of extrafollicular B cell activation that leads to antigen-specific IgM production; differentiation of B cells that leads to immunoglobulin class switching; and formation of splenic GCs, where affinity maturation occurs and memory B cells are generated. Although GC formation was blocked completely by TACI-Fc, some residual IgM and IgG1 production and affinity maturation occurred. In other systems it has been observed that attenuated antibody responses can proceed despite the absence of GCs^{26, 27, 28}, and it is possible that other factors besides BLyS and TACI mediate the remaining antibody production. Alternatively, TACI-Fc treatment in vivo may not have sufficed to prevent all BLyS-TACI binding events. It is also possible that BLyS interacts with some other, yet to be identified receptor(s), which can not be blocked by TACI-Fc.

Cooperation between T and B cells is critical for GC formation; however, the precise signals and microenvironment that induce GC formation are not known. Previous studies indicate that CD40L-CD40²⁹ and CD86−CD28−CTLA-4^{30, 31} interactions are important for entry of extrafollicular B cells into GC areas and for GC establishment. Inhibition of these interactions through gene knockouts or by treatment with blocking antibodies or receptor-Fc fusion proteins diminishes antibody production and blocks GC formation^{32, 33, 34, 35}. There are some striking similarities between the BLyS-TACI and CD40L-CD40 systems. Both ligands are related to TNF and are expressed on activated T cells^{4, 29}, and both receptors are TNFR homologues that signal through NF-B and are expressed on B cells^{5, 29}. Hence, the interaction between BLyS and TACI may mediate T-cell help of B cells in a similar fashion to the interaction between CD40L and CD40^{30, 31, 32, 33, 34, 35}. BLyS also may contribute to the activation of B cells by dendritic cells, which do express this ligand⁴. In addition, as TACI is also expressed on activated T cells⁵, BLyS may modulate directly T cell functions that support GC formation through TACI. Unlike CD40L and CD40 knockout mice, which exhibit impaired IgG but normal IgM responses, and unlike CD40L-deficient patients with hyper-IgM syndrome^{36, 37, 38, 39, 40, 41, 42}, TACI-Fc-treated mice showed a marked deficit in both IgM and IgG production. Thus, it is possible that BLyS and TACI operate early on in B cell activation, and blocking their action impairs all phases of the humoral response. In contrast, CD40L and CD40 may operate later in B cell activation, such that their blockade impairs only late phases of the antibody response. Alternatively, modulation of B cells by activated T cells in extrafollicular areas through CD40L-CD40 interactions may commit B cells to differentiate further to form GC in a BlyS-dependent fashion. It is possible also that BlyS-TACI interactions are important for GC maintenance and for survival of B cells in the GC, both of which are essential for an effective adaptive humoral response. Further studies will be needed to examine whether there are temporal and functional relationships between the CD40L-CD40 and the BlyS-TACI ligand receptor systems. Regardless of this, our results demonstrate that the novel molecular interaction of BLyS and TACI is critical for the antigen-specific humoral response. These observations may have implications for natural immunity, as well as for autoimmune disease.

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Sequences encoding human BLyS (aa 136−285) and human placental alkaline phosphatase were amplified by PCR, fused and cloned into the expression vector pFlag-CMV1 (Sigma, St Louis, MO) with alkaline phosphatase at the NH₂-terminus of BLyS. AP-BLyS was expressed in human embryonic kidney 293 cells. Conditioned medium from transfected cells was filtered (0.45 M), stored at 4 °C in a buffer containing 20 mM HEPES (pH 7.0) and 1 mM sodium azide, and subsequently used for cell staining. Flag-BLyS was constructed by cloning the same region of BLyS into pFlag-CMV1, with the Flag epitope at the N-terminus. Flag-BLyS was purified from serum-free supernatants of transfected 293 cells using anti-Flag M2-agarose (Sigma). In all experiments involving BLyS, the human version of the ligand was used.

Expression cloning was performed as described¹¹. A cDNA expression library was constructed in pRK5 vector using PolyA+ mRNA from IM-9 cells. The primary transformants were divided into pools of 1000 colonies. For each pool, a glycerol stock was prepared. Miniprep DNA (Qiagen, Valencia, CA) from each pool was transiently transfected into COS-7 cells in six-well plates using lipofectamine (GIBCO-BRL, Rockville, MD). After 36−48 h, cells were incubated with AP-BLyS conditioned medium and stained for AP activity in situ. A positive pool was broken down successively into smaller pools until a single positive clone was identified.

Human peripheral blood mononuclear cells were isolated on a Ficol gradient (LSM media, ICN/Cappel). Peripheral blood leukocytes (PBL) were then obtained by removal of plastic-adherent cells. PBL were plated in 48-well dishes (3 10⁴ cells per well in 0.3 ml of RPMI1640 medium containing 10% FBS and incubated with ligands for 72 h at 37 °C, 5% CO₂.

Two groups of female C57BL/6 mice aged 6 to 8-weeks-old received intraperitoneal (i.p.) immunization with 100 g of NP₂₃-CgG (Biosource Technologies) precipitated in alum. The groups were treated daily with 50 g of murine IgG or TACI-Fc by i.p. injection. After 14 days, mouse sera were analyzed for NP-specific IgM, or IgG1 that binds to NP₂₃-BSA, representing total anti-NP IgG1, or IgG1 that binds to NP₂-BSA, representing high-affinity anti-NP IgG1, by ELISA. Serum anti-NP IgG1 was quantified by reference to a standard curve generated with serial dilutions of an NP-specific IgG1 monoclonal antibody pEVHC1³⁰, kindly provided by Garnett Kelsoe, Duke University Medical Center. Bound antibodies were detected with AP-conjugated goat anti-mouse IgM or IgG1 (Pharmingen, San Diego). All animal experiments were performed in compliance with institutional guidelines and approved by Genentech's IACUC committee.

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