Baff Binds to the Tumor Necrosis Factor Receptor–Like Molecule B Cell Maturation Antigen and Is Important for Maintaining the Peripheral B Cell Population

Many members of the TNF family fulfill important roles related to the organization, function, and/or homeostasis of the immune system 1. The recently identified TNF family member B cell activating factor (BAFF) (TNF and apoptosis ligand–related leukocyte-expressed ligand 1 [TALL-1]/TNF homologue that activates apoptosis, nuclear factor κB, and c-Jun NH₂-terminal kinase [THANK]/B lymphocyte stimulator [BlyS]; references 2,3,4,5) is expressed in monocytes, macrophages, and dendritic cells 2,3,5 and has been shown to bind to B cells and increase the proliferation of B cells in combination with an anti–B cell receptor antibody in vitro 2,5. BAFF is a type II membrane protein and may act as either a membrane-bound or soluble form, the latter being generated by proteolytic cleavage at a furin consensus site 2,5. Transgenic mice overexpressing BAFF have a greatly elevated number of mature B cells and an increased number of effector T cells in their spleen and mesenteric lymph nodes 6. These mice display autoimmune-like manifestations including high levels of rheumatoid factors, circulating immune complexes, anti-DNA autoantibodies, and Ig deposition in the kidney 6. However, splenic B cells from transgenic or control mice were found to proliferate at the same rate 6, suggesting that BAFF, in addition to its ability to costimulate B cell proliferation in vitro, may have alternative functions.

B cell maturation antigen (BCMA) was first identified as part of a translocation event in a malignant T cell lymphoma patient 7. Characterization of human BCMA identified it as a type I membrane protein primarily expressed in immune organs and mature B cell lines 7,8. BCMA protein was localized to the Golgi apparatus in the U266 plasmacytoma cell line, which expresses high levels of BCMA 9. Subsequent identification of the mouse BCMA gene and further motif analysis led to the prediction that BCMA is a member of the TNF receptor superfamily 10.

Here, we show that BAFF is capable of interacting with the orphan TNF receptor, BCMA. We characterize the BAFF–BCMA interaction and show that injection of soluble BCMA-Ig fusion protein into mice leads to a dramatic reduction in the total number of B cells in the peripheral immune organs. This may be due to a B cell survival function of BAFF. Modulation of the peripheral B cell population using BCMA-Ig treatment may be efficacious in treating B cell–mediated disorders.

Human embryonic 293 cells carrying the EBNA-1 gene were cultured in DMEM plus 10% FCS, glutamine, pen-strep, and 250 μg/ml G418. Raji cells were obtained from American Type Culture Collection and were cultured as indicated. The human BCMA-hIgG1 (BCMA-Ig) expression vector was created using a murine κ light chain signal sequence, a PCR fragment generated to BCMA amino acids 1–48 (second Met), and a hIgG1 SalI + NotI fragment 11. The fragments were cloned into pCEP4 (Invitrogen) derivative. A COOH-terminal 6×His-tagged full-length human BCMA expression vector was generated by PCR. BCMA-Ig obtained by transient transfection of 293E cells using Lipofectamine (Life Technologies) was purified by protein A column chromatography, and contained <1 endotoxin U/mg of protein. The half-life of this protein in mice is ∼3.5 d. The control Ig, nonbinding LFA3-Ig, was prepared from a Chinese hamster ovary (CHO) cell line as described 12. Murine lymphotoxin (LT)βR-Ig 13 and human LTβR-Ig 14 were made as described. Receptor-Ig fusion proteins and Flag-tagged soluble ligands were constructed and expressed as described using the following amino acid sequences: hTNF-α (85–263), mBAFF (127–309), hBAFF (136–285 or 83–285), human receptor activator of nuclear factor κB ligand (hRANKL [151–316]), hCD30L (63–234), hCD40L (116–261), homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes (hLIGHT [89–240]), hTNFR2 (1–257), hTNF-related apoptosis-inducing ligand (hTRAIL)R2 (1–212), hCD30 (1–380), hCD40 (1–193), hHVEM (1–200), and mRANK (1–200) 2,15. Unless indicated, BAFF in 15-fold concentrated serum-free Optimem medium (Life Technologies) was used 2.

Rabbit anti-hBCMA antibody, Rb 1593, was generated against purified hBCMA-Ig. Affinity-purified anti-BCMA antibodies and control affinity-purified anti–glutathione S-transferase (GST) antibodies originating from the same rabbit were used for the U266 cells and tonsillar B cell staining. These antibodies have been described 9.

All experiments were performed at 25°C with a 10 μl/min flow rate using a BIAcore 2000™. HBS buffer (10 mM Hepes, 150 mM NaCl, 0.005% P20 surfactant, pH 7.4) was used both as the running buffer and as the sample diluent. The CM5 chip (BIAcore) surface was first activated with N-hydroxysuccinimide/N-ethyl-N9-(3-diethylaminopropyl)-carbodiimide hydrochloride. 25 μl of BCMA-Ig or hLTβR-Ig was diluted to 30 μg/ml in 10 mM acetic acid (pH 5), then injected. The unreacted groups of the chip's dextran matrix were blocked twice with ethanolamine-HCl (pH 8.5). For the experiment, 100 μl of Flag-hBAFF, Flag-mBAFF, or hLTα₁β₂ at 30 μg/ml in diluent buffer were injected over the surface of the chip. The surface was regenerated between experiments by washing with 1 mM formic acid.

Flag-tagged human BAFF at 200 ng/ml was pre-incubated for 30 min on ice in FACS^® buffer (PBS, 10% FCS, 0.05% azide) with either hBCMA-Ig or hLTβR-Ig at twofold dilutions ranging from 20 μg/ml to 39 ng/ml, or with BAFF alone. The mixture was added to Raji cells for 30 min on ice. Bound BAFF was detected using 5 μg/ml anti-Flag antibody M2 (Sigma-Aldrich) followed by PE-conjugated donkey anti–mouse IgG (Jackson ImmunoResearch Laboratories). The samples were read using a FACSCalibur™ flow cytometer (Becton Dickinson).

293E cells were transfected with either vector alone or His-tagged full-length BCMA and a green fluorescent protein transfection control plasmid (gift of N. Horikoshi, Beth Israel Deaconess Medical Center, Boston, MA) using Lipofectamine 2000. After 48 h, the cells were detached in PBS containing 5 mM EDTA, washed, and stained for 30 min on ice with anti-hBCMA Rb 1593 (1:1,000) or preimmune sera (1:500) followed by PE-conjugated donkey anti–rabbit Ig (1:100; Jackson ImmunoResearch Laboratories). Alternatively, cells were stained plus or minus Flag-BAFF (2 μg/ml) and detected using M2 as described above.

Human mononuclear cells were obtained from palatine tonsils by gentle dissociation with forceps. T cells were removed by one cycle of rosette formation and further depleted with anti-CD2 magnetic beads (Dynabeads M-450; Dynal 16). The remaining B cell populations were 93 ± 4% CD19⁺, 59 ± 6% IgD⁺, 56 ± 5% IgM⁺, 81 ± 16% CD44⁺, 21 ± 6% CD38⁺, 42 ± 1% CD95⁺, and ≤ 1% CD14⁺, CD3⁺, and DRC1⁺ cells. Cells were preincubated with 10% human AB serum and stained plus or minus Flag-BAFF (amino acids 83–285) at 1 μg/ml followed by M2 (7.5 μg/ml) and PE-conjugated goat anti–mouse IgG (Immunotech). Anti-BCMA staining was done using 20 μg/ml antibody and FITC-conjugated goat anti–rabbit IgG (Immunotech).

6-wk-old female BALB/c mice (four per group; The Jackson Laboratory) received intraperitoneal injections of either 400 μg of BCMA-Ig or 400 μg of LFA3-Ig (control Ig) on days 1, 4, 8, and 11. On day 19, the mice were killed. Blood was collected via cardiac puncture into tubes containing EDTA. Single cell suspensions were prepared from spleens and mesenteric lymph nodes, and RBCs were lysed in a hypotonic buffer. Flow cytometry was performed using PE-, FITC-, or CyChrome-conjugated anti-CD45R/B220 (B cells) and anti-CD4, anti-CD8 (T cells), or anti-Gr1/anti–Mac-1 (neutrophils and macrophages). All antibodies were purchased from BD PharMingen. In brief, Fc receptors were blocked with 10 μg/ml FcBlock (BD PharMingen) for 15 min on ice followed by addition of the conjugated mAbs, and were incubated on ice for 30 min. Cells were washed twice, suspended in 0.5% paraformaldehyde, and analyzed by flow cytometry.

Mouse spleens were collected under sterile conditions and homogenized in RPMI medium plus 10% FCS using SuperFrost glass slides (Menzel-Glaser). Cells were filtered through a 70-μm nylon cell strainer (Falcon) before centrifugation at 1,000 rpm (300 g) for 5 min. The pellet was resuspended in 2 ml/spleen RBC lysis solution and incubated on ice for 5 min. The remaining white blood cells were collected by centrifugation and resuspended in media. Splenocytes were incubated at a concentration of 3 × 10⁶ cells/ml in media for 72 h. The splenocytes were either untreated or were in the presence of 2 μg/ml human Flag-BAFF (amino acids 83–285) alone or with 20 μg/ml BCMA-Ig or 20 μg/ml murine LTβR-Ig. The percent live cells remaining was determined using the annexin V–FITC Apoptosis Detection Kit 1 (BD PharMingen). Cells were washed twice and then analyzed using a FACSCalibur™ machine (Becton Dickinson).

TNF receptor family members are generally type I proteins with a leader sequence and a cysteine-rich extracellular domain. The extracellular domains are organized as a series of alternating A and B modules, which are stabilized by internal disulfide bridges 17. A single C module, not involved in ligand binding, is found in the fourth cysteine repeat of TNFRI 17. In a previous study, BAFF failed to bind to 16 members of the TNF receptor family 2. Subsequently, an additional candidate receptor for BAFF was identified, termed B cell maturation antigen 10. BCMA appears to be distantly related to the TNF receptor family, because it is devoid of a signal sequence and contains a single A and one C module instead of multiple A and B modules 10. However, the expression pattern of this atypical receptor in mature B cells prompted us to examine its interaction with BAFF 8,9. To this end, the extracellular domain of human BCMA was expressed as a human IgG1 fusion protein and was targeted to the secretory pathway with an added signal peptide. BCMA-hIgG1 (BCMA-Ig) fusion protein immunoprecipitated both recombinant human and murine BAFF very efficiently, but not five other TNF ligands (CD40L, RANKL, CD30L, LIGHT, and TNF-α) which, however, interacted with their respective receptors in control immunoprecipitations (Fig. 1 A). When the interaction was tested in another format using surface plasmon resonance (BIAcore) analysis, similar results were obtained, and both murine and human BAFF exhibited a significant affinity for human BCMA-Ig but not for LTβR-Ig (Fig. 1 B). In addition, BCMA-Ig interacted with membrane-bound BAFF expressed in 293 cells (Fig. 1 C), and in a dose-dependent manner specifically blocked the interaction of BAFF with Raji B cells (Fig. 1 D). Thus, the short extracellular domain of BCMA is sufficient for high-affinity binding to BAFF, and there is no species barrier for the murine BAFF–human BCMA interaction. The BAFF–BCMA interaction is presently unique in the family, as it involves only two modules, one predicted to be of the C type. In the two cases where the crystal structure of the trimeric ligand–receptor complex is known (LTα–TNFR1 and TRAIL–TRAILR2), the contacts involved in the interaction have been shown to involve one B and two A modules 18,19,20.

Cell surface expression of BCMA is a likely prerequisite for BAFF binding. Indeed, most 293E cells transfected with a construct encoding full-length BCMA with no added signal peptide were positive for surface staining with anti-BCMA antibodies and to a lesser extent with Flag-BAFF, whereas untransfected cells or cells transfected with a control plasmid were not (Fig. 2 A). We also found that tonsillar B cells were stained with both anti-BCMA and Flag-BAFF (Fig. 2 B), suggesting that surface expression of BCMA also occurs in primary cells and is not an artifact of overexpression. Therefore, in the absence of a signal sequence, the transmembrane domain of BCMA must dictate the topology of membrane insertion for correct expression at the cell surface. In a previous study, BCMA was located exclusively in the Golgi apparatus of the plasmocytic cell line U266, and not at the cell surface, despite the fact that these cells express high levels of the protein 9. Consistent with this observation, we find that U266 cells were not stained with either the anti-BCMA antibody or Flag-BAFF (Fig. 2 B). These results suggest that surface expression of BCMA may be a tightly controlled, cell type–dependent mechanism. Intracellular pools of the cytotoxic receptor Fas can be mobilized for surface expression in response to p53 activation, a mechanism that probably contributes to the elimination of damaged cells 21. It is an intriguing possibility that cells may release intracellular BCMA at the cell surface in response to specific stimuli, and therefore become sensitive to BAFF effects.

Because BIAcore studies revealed that murine BAFF was able to effectively bind human BCMA-Ig, we used the fusion protein to examine the effect of disrupting the BAFF signal in normal mice. Four administrations of BCMA-Ig at intervals of 3–4 d dramatically reduced the total number of B cells in the blood and peripheral lymphoid organs examined. The B220⁺ B cell population in the spleen of BCMA-Ig–treated animals was reduced by 50% compared with the control Ig–treated mice. In contrast, populations of neutrophils, macrophages, CD4⁺ T cells, and CD8⁺ T cells were not significantly affected by BCMA-Ig (Fig. 3 and Table). The decrease in absolute B cell number corresponded to the decrease in absolute total cell number (Table), indicating that the reduction in the B cell population was not due to a concordant increase in other cell types. Therefore, the BCMA-Ig treatment specifically affected B cells. Similar results were observed in the blood and mesenteric lymph nodes, where approximately half of the B220⁺ B cells were lost (data not shown). As B cells do not express BAFF 2,5, and because human BCMA-Ig does not bind to mouse splenic B cells (data not shown), we can exclude antibody-dependent cell-mediated cytotoxicity as the mechanism of action. Although BAFF is thought to be primarily secreted, staining of splenocytes using BCMA-Ig revealed that BAFF is found on the surface of Mac-1⁺ cells (data not shown).

BCMA-Ig treatment given over the course of 11 d may reduce the peripheral B cell pool by inhibiting production or release of immature B cells from the bone marrow, by blocking maturation of immature B cells, or by sequestering a factor required for B cell survival. At least 20 × 10⁶ immature B cells are generated in the bone marrow each day, and from this population an estimated 2–3 × 10⁶ B cells enter the periphery 22. It has been shown that maintenance of the peripheral B cell pool requires a minimum continuous input of newly formed cells 23, but that less than half of the B cells that reach the spleen progress to the mature phenotype 24. Although the percentage of long-lived and short-lived B cells in the periphery is controversial 22, the average half-life of the B220⁺ cells in the spleen is 4.3 wk, indicating that the majority of the B cells are long-lived mature cells 24. As we see a 50% decrease in the total B cell population in the periphery, we may be affecting the repopulation of the spleen with mature B cells or the survival of the existing mature B cells. One possible mechanism for sustaining B cell survival is to upregulate the antiapoptotic protein Bcl-2. Interestingly, elevated levels of Bcl-2 have been reported in the PBL B cells isolated from BAFF transgenic mice 6. To delineate the peripheral B cell subpopulations modulated by BCMA-Ig, the mechanism of action, as well as any potential effect on B cell development in the bone marrow, a more comprehensive study is in progress.

The in vivo analysis, taken together with the finding that BAFF transgenic mice have an increased number of mature B cells and Bcl-2 expression, indicates that BAFF may promote B cell survival. We next examined the direct effect of BAFF on the survival of murine splenocytes in vitro. Splenocytes were cultured for 72 h in the presence or absence of BAFF and either BCMA-Ig or LTβR-Ig (Fig. 4). BAFF alone caused an increase in the percentage of cells that survived, and this effect was blocked with BCMA-Ig. The increase in cell numbers was not due to the increase in proliferation, as this has not been observed in the absence of B cell receptor stimulation 2,3. The majority of the surviving cells, ∼80%, were B220⁺ B cells, although a small percentage of T cells also survived (data not shown). Thus, BAFF is capable of directly enhancing B cell survival.

In conclusion, we have identified BCMA as a receptor for BAFF. Soluble BCMA antagonizes BAFF action and leads to a rapid, significant B cell decline in vivo, suggesting that BAFF is necessary for homeostasis of the peripheral B cell pool. The increase in mature B cell numbers observed in the BAFF transgenic mice and the ability of BAFF to sustain B cells in vitro indicate that BAFF functions as a survival factor. Modulation of the peripheral B cell population using soluble BCMA may have applications in treating B cell–mediated autoimmune diseases, plasma cell disorders, and B cell cancers. A very recent report has demonstrated that blocking the BAFF pathway in two models of SLE using a soluble version of another receptor for BAFF, transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI)-Ig, eliminates proteinurea and prolongs the life span of the mice 25.

We thank Sukumari Mohan and Akos Szilvasi for excellent technical assistance, Apinya Ngam-ek for modifying the green fluorescent protein plasmid, and Paul Rennert for helpful advice.