The proteoglycan (heparan sulfate proteoglycan) - The FASEB Journal

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The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization and cross-linking Fiona C. Kimberley,*,1 Liesbeth van Bostelen,*,1 Katherine Cameron,* Gijs Hardenberg,*,2 J. Arnoud Marquart,† Michael Hahne,‡ and Jan Paul Medema*,3 *Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, and †Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and ‡Institut de Ge´ne´tique Mole´culaire de Montpellier, Centre National de la Recherche Scientifique UMR5535, Montpellier, France A proliferation-inducing ligand (APRIL) (also known as TALL-2 and TRDL-1) is a member of the tumor necrosis factor (TNF) superfamily that has tumorigenic properties but is also important for the induction of humoral immune responses. APRIL binds two TNF receptors: transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) as well as heparan sulfate proteoglycans (HSPGs). The aim of this study was to clarify the role of the HSPG interaction in canonical APRIL signaling, because it has been proposed to act as a docking site and also to play a role in direct signaling. In this study, we generated point mutants of soluble APRIL that lack either the capacity to bind HSPGs or TACI and BCMA and then tested the function of these mutants in mouse B-cell assays. In contrast to previous reports, we found that APRIL alone is sufficient to costimulate B-cell proliferation and drive IgA production and does not require artificial antibody cross-linking. We found no evidence that APRIL requires signaling through HSPGs but, notably, were able to show that binding of APRIL to HSPGs is crucial for mediating natural APRIL cross-linking to allow for optimal activation of murine B cells.—Kimberley, F. C., van Bostelen, L., Cameron, K., Hardenberg, G., Marquart, J. A., Hahne, M., Medema, J. P. The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization and cross-linking. FASEB J. 23, 1584 –1595 (2009)

ABSTRACT

Key Words: proliferation 䡠 B cells 䡠 signaling 䡠 tumors 䡠 immunoglobulins

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) superfamily and was named according to its ability to drive tumor-cell growth (1). APRIL is expressed as a type II transmembrane protein but is cleaved intracellularly by a furin convertase to release the active soluble form (2). This form is a noncovalent homotrimer, with high structural 1584

homology to several other TNF family ligands (3). APRIL is expressed by a subset of immune cells and some nonimmune cells such as epithelial cells and osteoclasts. Significantly, APRIL is overexpressed by several solid tumors and lymphoid malignancies and enhances their proliferation and survival (4-10). APRIL binds two TNF receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a property that it also shares with another TNF ligand, B-cell activating factor (BAFF) (also known as B-lymphocyte stimulator) (11-14). BAFF binds a third distinct receptor, BAFF receptor (BAFF-R or BR3), which is highly expressed by peripheral B cells and not bound by APRIL (15). Through BAFF-R, BAFF is crucial for B-cell homeostasis (16-19). Although APRIL drives proliferation and survival of several different cell types (20), APRIL knockout mice reveal no gross abnormalities in lymphoid homeostasis or activation (21). Nonetheless, APRIL was shown to regulate immunoglobulin class switch recombination (CSR) during T-cell-independent B-cell responses, and this is likely to occur through TACI, which was shown to mediate APRIL-induced IgG and IgA CSR and antibody production in both mouse and human B cells (22-26). In the intestine, dendritic and intestinal epithelial cells appear to be the source of APRIL. In response to signals from commensal bacteria, these cells produce APRIL and mediate the class switch from IgA1 to IgA2 (27). APRIL, along with BAFF, has recently also been shown to mediate BCMA-dependent survival of longlived plasmablasts in the bone marrow (28-30). In addition, APRIL can up-regulate cofactors such as 1

These authors contributed equally to this work. Current address: Department of Surgery, Jack Bell Research Centre (JBRC), The University of British Columbia, 2660 Oak St., Vancouver, BC, Canada V6H 3Z6. 3 Correspondence: LEXOR, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] doi: 10.1096/fj.08-124669 2

0892-6638/09/0023-1584 © FASEB

CD40, major histocompatability class (MHC)-II, and B7.1 and B7.2 on B cells, which may facilitate antigen presentation to T cells (31). Combined, the current data indicate that APRIL produced by different cells can enhance B-cell CSR, proliferation, and survival. However, both the receptor through which these signals are given and the form of APRIL that mediates these effects remain ill-defined. In vitro, most effects observed on the addition of APRIL depend on artificial cross-linking of the recombinant protein through antibodies directed at the Flag tag. This would be in agreement with the reported requirement for ligand multimerization to obtain effective TACI signaling (32). Nevertheless, the question remains as to how a similar effect might be induced under physiological conditions, especially because APRIL is a secreted ligand and is not reported to form larger aggregates (2). APRIL also binds to heparan sulfate proteoglycans (HSPGs) on the surface of cells as well as in the extracellular matrix (ECM) (33, 34). The region that mediates HSPG binding does not overlap with the TNF receptor interaction site; indeed, APRIL can interact with HSPGs and its TNF receptors at the same time, which suggests that APRIL could be positioned by HSPGs to activate the TNF receptors. However, a recent study concluded that direct signaling via HSPGs on the surface of the B cells, in collaboration with the TACIderived signal, was necessary for effective APRIL-driven IgA production (35), indicating that a complex consisting of APRIL, HSPGs, and TACI is required to obtain an effective signal. However, this conclusion may have been influenced by the fact that TACI is also capable of binding HSPGs. Although the exact role for this interaction remains enigmatic, under in vitro conditions it has been shown that HSPGs can activate TACI (36). Therefore, in this study we sought to define the role of the HSPG interaction in the context of APRIL signaling through its TNF receptors and to address the question of APRIL multimerization. Using various mutant forms of APRIL and several readouts for APRIL activity, we found that APRIL requires a functional HSPG-binding domain to effectively deliver a signal. In contrast to previous findings, we clearly show that wild-type secreted APRIL is active and has no need for further cross-linking. More important, loss of HSPG binding abolished APRIL activity, but this activity could be restored by addition of a cross-linking antibody. This result indicates that, in the context of the murine system studied here, the HSPG domain does not mediate an independent signal but rather provides a natural form of ligand cross-linking both in vitro and in vivo.

MATERIALS AND METHODS Cell culture All cell lines were maintained at 37°C with 5% CO2. Human embryonic kidney cells (293T) were cultured in Iscove’s NATURAL MULTIMERIZATION OF APRIL

modified Dulbecco’s medium (Invitrogen, Breda, The Netherlands) supplemented with 8% FCS with 2 mM glutamine. Mouse splenocytes and purified B cells were grown in RPMI 1640 (Invitrogen) supplemented with 8% FCS, 2 mM glutamine, and ␤-mercaptoethanol at 50 ␮M. Antibodies and reagents Commercial reagents used were as follows: monoclonal antiFlag-M2, monoclonal anti-Flag-M2-Bio, monoclonal antiFlag-M2 peroxidase conjugate, and anti-Flag-M2 Sepharose were purchased from Sigma-Aldrich Chemie BV (Zwijndrecht, The Netherlands); heparin Sepharose Fast Flow 6 was purchased from Amersham Biosciences (Diegem, Belgium); heparin was obtained from the hospital pharmacy (Leiden University Medical Center, Leiden, The Netherlands); mouse interleukin (IL)-4 and mouse IL-6 were obtained from PeproTech (London, UK); mouse IgG1 was generated in-house; and soluble Flag-TRAIL was purchased from Alexis (Axxora, Raamsdonksveer, The Netherlands). Soluble Flag-tagged APRIL and the various APRIL mutants, BCMA-Fc and TACIFc, were produced and purified in-house. Conditioned medium containing soluble OPG-Flag was generated by transient transfection. Antibodies used for flow cytometry were as follows: anti-mouse MHC class II (I-Ab), anti-mouse B220FITC, anti-mouse CD43-Biotin, anti-mouse CD43-PE, and streptavidin-APC were purchased from BD Biosciences (Alphen a/d Rijn, The Netherlands); anti-mouse BCMA-ATTO 647N (Vicky-2) was purchased from Axxora; anti-mouse TACI-PE was purchased from R&D Systems (Abingdon, UK); and 10E4 was purchased from Seikagaku Corporation (Falmouth, MA, USA). Dextran sulfate was purchased from Sigma-Aldrich Chemie BV (derived from Leuconostoc spp., average MW ⬎ 500,000). Cloning and constructs The APRIL-HSPG mutant was generated previously (33). APRIL-wild type (WT)-triple was generated using Quick Change site-directed mutagenesis (Stratagene, Huissen, The Netherlands). The APRIL R231A point mutation was generated using a two-stage polymerase chain reaction. Plasmids expressing human TACI and BCMA full-length with an internal C-terminal Flag tag and BCMA and TACI human IgG-Fc were generated previously. The numbering of amino acids in the APRIL constructs is based on the peptide sequence given by the UniProtKB/TrEMBL entry Q8NFH7. Note that the nomenclature in other publications may differ, starting with a second methionine before the transmembrane domain. See Supplemental Data for primer sequences. Protein expression and purification For purification, proteins (Flag-tagged APRIL and BCMA and TACI human IgG Fc) were transiently expressed in 293T cells using calcium phosphate precipitation. Soluble APRIL was purified from the resulting supernatant using anti-Flag-M2 Sepharose (Sigma-Aldrich), according to the manufacturer’s guidelines. Conditioned medium containing soluble APRIL was produced by transient transfection of 293T cells in six-well plates; control medium was generated by a mock transfection. BCMA- and TACI-IgG Fc were purified by running over a protein A-Sepharose column, according to the manufacturer’s guidelines (Sigma-Aldrich). Because of the high hydrophobicity of APRIL, we advise researchers to take care when freeze-thawing APRIL, both purified and in conditioned medium, as the concentration may differ from the initial measurements. 1585

Flow cytometry

RESULTS

All flow cytometry was carried out on a FACSCalibur system (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). To measure receptor binding, 293T cells were plated at 60% confluence in a 6-well plate and cotransfected with full-length BCMA or TACI plus green fluorescent protein (GFP) at a ratio of 10:1, with a total DNA content of 1 ␮g/well. Transfected 293T cells were harvested in PBS after 48 h, washed, and stained in PBS-1% BSA with the various forms of soluble APRIL, plus or minus heparin (4 IU/ml). Bound APRIL was detected with anti-Flag-M2-Bio followed by streptavidin-APC, and cells were gated on GFP.

APRIL-induced B-cell proliferation, but not IgA production, is enhanced by antibody-mediated ligand cross-linking

Mouse B-cell isolation, B-cell proliferation, IgA production assay, and MHC class II up-regulation Splenic mouse B cells were isolated using magnetic activated cell separation (MACS) columns with CD45R/B220 MACS beads (Miltenyi Biotec, Utrecht, The Netherlands). After purification, cells were cultured in 96-well round-bottomed microtiter plates at a density of 2 ⫻ 105/well in a final volume of 200 ␮l. When indicated, plates were coated with dextran sulfate (SigmaAldrich) overnight in PBS. To measure proliferation, cells were treated with anti-IgM (Jackson ImmunoResearch, Newmarket, UK), plus soluble APRIL in either conditioned medium or as purified protein at a final concentration of 1 ␮g/ml; anti-Flag monoclonal antibody was added at a final concentration of 1 ␮g/ml, and cells were incubated at 37°C. Proliferation was assayed using tritiated thymidine (Amersham Biosciences). To measure IgA production, mouse B cells were cultured and treated with APRIL as above (but without anti-IgM). After 6 days, supernatant was collected and assayed for IgA by ELISA. To measure MHC class II expression, splenic mouse B cells were cultured and treated with APRIL as above, in the presence of mouse IL-4 and IL-6 at a concentration of 0.4 ng/ml. The cells were stained for MHC class II 48 h later and analyzed by flow cytometry. See Supplemental Data for more details. Immunoblotting and immunoprecipitations Conditioned medium containing the various forms of soluble APRIL was quantified by Western blotting and normalized for subsequent assays by comparing the intensity of resulting bands. To compare the binding of the various forms of soluble APRIL to heparin, quantified supernatant was immunoprecipitated by mixing with heparin Sepharose (SigmaAldrich) and incubated at room temperature for 1 h. The beads were then washed 3 times with 0.5 M NaCl and 3 times with PBS, boiled in reducing sample buffer and subjected to Western blotting, and then visualized using anti-Flag-M2HRP. To compare binding of APRIL with that of TACI and BCMA, purified receptor Fc-fusion proteins were incubated with protein A plus APRIL supernatants and processed as above. Surface plasmon resonance Surface plasmon resonance measurements were carried out on a BIAcore 2000 instrument (BIAcore, Uppsala, Sweden) using PBS at 25°C. The binding curves were analyzed using ClampXP Biosensor Data Analysis software (37). See Supplemental Data for a more detailed description. 1586

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The mechanism by which APRIL stimulates B cells remains controversial. Most data suggested that artificial antibody-mediated cross-linking of APRIL using the Flag tag is crucial to observe signaling in vitro (32, 34). However, anti-Flag-mediated cross-linking does not occur under physiological conditions, suggesting that other mechanisms of cross-linking are in place. It has been proposed by us and others that the interaction of APRIL with HSPGs could serve such a function (33, 34). However, Sakurai et al. (35) suggested that APRIL binding to HSPGs and TACI elicited independent signals in B cells that cooperated to provide an effective signal. We therefore first tested whether the requirement for APRIL cross-linking is absolute when B cells are costimulated. In our proliferation experiments, mouse splenic B cells were stimulated via the B-cell receptor (BCR) with anti-IgM and costimulated with supernatant containing N-terminal Flag-tagged soluble APRIL (amino acids 105-250), with and without cross-linking antibody. It is important to note that the form of APRIL used is the naturally processed and secreted form of APRIL and thus contains full HSPG-binding capacity (2). Surprisingly, we did observe a costimulatory effect of APRIL, and this was evident over a range of APRIL (Fig. 1A) and anti-IgM concentrations (Supplemental Fig. 1A). In contrast to previous observations, we concluded that APRIL can costimulate B cells without the need for anti-Flag-induced cross-linking. Costimulation was diluted out at lower concentrations of APRIL but was reinforced by the addition of monoclonal anti-Flag (Fig. 1A). This was a specific consequence of ligand cross-linking, because an isotype-matched control antibody (mouse IgG1) had no effect (Supplemental Fig. 1B). Combined, these data clearly reveal that APRIL alone can costimulate B cells, without the absolute requirement for antibody-mediated cross-linking. Because different biological effects require different thresholds for signaling, we also examined the effect of APRIL on IgA-dependent CSR. In the absence of BCR triggering, APRIL has been shown to stimulate IgA secretion in B cells. In human B cells, this appears to be due to the induction of CSR in vitro (25). In murine B cells, the picture may be more complicated, and enhanced IgA secretion may be the result of enhanced IgA production, with survival of IgA-producing B cells as well as CSR. This process appears to be mediated exclusively via TACI (22, 23). In our hands, the stimulation of mouse splenic B cells with soluble APRIL alone was sufficient to enhance IgA production. The effect of adding anti-Flag was minimal and only observed at the lower dilutions of APRIL-containing supernatant, again indicating that antibody-induced oligomerization is not a requirement

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Figure 1. APRIL-induced B-cell proliferation is enhanced by antibody-mediated ligand cross-linking. A) Mouse splenic B cells were stimulated with anti-IgM (to activate BCR) at a concentration of 5 ␮g/ml and costimulated with conditioned medium containing soluble APRIL, plus or minus cross-linking anti-Flag monoclonal antibody. Proliferation was measured using tritiated thymidine. All samples were performed in triplicate. B) Mouse splenic B cells were treated with conditioned medium containing soluble APRIL plus or minus cross-linking anti-Flag monoclonal antibody. Cell supernatant was assayed for IgA content by ELISA. For both assays, the starting dilution of the supernatant used to treat the cells was actually a final dilution of 1 in 3. All samples were analyzed in triplicate; error bars ⫽ sd.

for APRIL to activate B cells. Using Western blotting we estimated the concentration of APRIL in conditioned medium generated from confluent 293T cells to be in the range of 0.5 to 5 ␮g/ml (Supplemental Fig. 2). From our experiments, we can conclude that the effect of soluble APRIL alone is observed at ⬍50 ng/ml for IgA and at ⬍250 ng/ml for B-cell proliferation (Fig. 1). These values are well within the range of what dendritic cells produce in vitro, which we have previously shown to be ⬃0.5 ␮g/ml of APRIL (38). APRIL serum levels in healthy donors are, on average, 10 ng/ml, which suggests that the local concentration of APRIL at the site of production is also high enough to observe direct stimulation of B cells (39). The physiological relevance of these findings is further supported by the observation that APRIL is required for T-cell-independent IgA responses in mice and thus is capable of signaling B-cell activation in vivo (22). In summary, using assays similar to those reported previously (34, 35), we observed pronounced stimulation by the naturally processed form of APRIL, in the absence of cross-linking antibody. The HSPG domain of APRIL is required for effective production of IgA Because both APRIL-induced IgA production and proliferation are in vivo effects of APRIL, it is important to consider how different forms of ligand presentation might occur. It is possible that natural oligomerization might occur as a side effect of high concentrations of APRIL produced locally. In the case of BAFF, the formation of large, soluble, virus-like assemblies has been reported (32), but to date no higher-order forms of APRIL have been identified. Alternatively, TWEPRIL, the membrane-bound splice variant of APRIL, NATURAL MULTIMERIZATION OF APRIL

may direct oligomerization at the membrane (40). Although this hypothesis might provide an explanation for the effects in vivo, it fails to explain why soluble recombinant APRIL is capable of enhancing IgA production in vitro. A more likely possibility is that oligomerization of soluble APRIL may occur via the HSPG-binding domain. This domain consists of two regions: 1) a hydrophobic motif at the N terminus (Q109KQKKQ114) of the secreted ligand and 2) three basic amino acids on the side of the molecule opposite to the TACI/BCMA binding site: R146, R189, and H220 (33, 34). Notably, this domain was also suggested to trigger signaling via HSPGs expressed on the surface of B cells. To test the role of this domain in APRIL signaling, we used site-directed multimutagenesis to generate several mutant clones of APRIL: 1) APRIL-triple (designated WT-triple), containing 3 point mutations: R146S, R189S, and H220E; 2) APRIL-HSPG (designated HSPG), containing three point mutations in the hydrophobic motif; and 3) APRIL-HSPG-triple (designated HSPG-triple), in which all 6 amino acids were mutated at both these sites. Finally, as a control, we generated a form of APRIL capable of binding HSPGs but lacking the ability to bind either TACI or BCMA (Fig. 2). Based on the crystal structure of the TACI-D2-APRIL complex (41), we mutated a key arginine to alanine within the proposed receptor-binding region of human APRIL, generating APRIL-R231A. To validate these proteins, we first examined their binding capacity using immunoprecipitation (Fig. 3A). Conditioned supernatants containing the various forms of soluble APRIL were incubated with protein A beads plus either TACI- or BCMA-Fc or with heparin Sepharose to test HSPG binding. Immunoprecipitation with receptor-Fc indicates that all mutant forms of APRIL, 1587

Figure 2. APRIL-HSPG binding. A) A schematic diagram to illustrate the point mutations made in the various APRIL constructs and the terminology used throughout the article. The numbering of amino acids in the APRIL constructs is based on the peptide sequence given by the UniProtKB/TrEMBL entry Q8NFH7, which represents the sequence of the immature unprocessed form. B) Structure of the APRIL-BCMA complex (visualized using Cn3D and PDB coordinate 1XU2) with the specific mutations highlighted. The position of the N terminus is highlighted at K113, as only this part of the hydrophobic motif (QKQKK113Q) is present in the structure. The R231A mutation within the BCMA and TACI binding pocket is also highlighted.

except R231A, retain the ability to bind both BCMA and TACI. The R231A mutant showed complete loss of binding to both receptors but retained its ability to bind HSPGs, as indicated by effective immunoprecipitation

with heparin Sepharose. In agreement with data published previously, mutations in the N-terminal region of APRIL (Q109KQKKQ114) (as in HSPG and HSPG-triple), lead to complete loss of heparin binding (33). We

Figure 3. The HSPG domain of APRIL is required for effective production of IgA, but loss of HSPG-binding capacity does not affect binding to either TACI or BCMA. A) Western blot after immunoprecipitation of the various forms of APRIL to validate HSPG and TNF receptor binding. Conditioned supernatant-containing soluble APRIL was incubated with either BCMA- or TACI-Fc fusion protein plus protein A or with heparin Sepharose beads directly. Samples were then washed and run on an acrylamide gel. Bound APRIL was visualized using anti-Flag-HRP. To confirm normalization of the APRIL supernatants used in immunoprecipitation, these were blotted directly with anti-Flag (bottom panel). B) Mouse splenic B cells were treated with conditioned medium containing the various soluble forms of APRIL. Supernatant was analyzed for IgA content by ELISA after a 6-day incubation. Each sample was analyzed in triplicate. *P ⬍ 0.05, **P ⬍ 0.001; Student’s t test. C) 293T cells transiently transfected with TACI and BCMA were stained with various forms of soluble purified APRIL, with and without heparin, and analyzed by flow cytometry. Solid lines, staining with APRIL; dashed lines, APRIL plus heparin; filled gray lines, unstained control; gray dotted line, unstained control plus heparin. All samples were analyzed in triplicate; error bars ⫽ sd. D) Comparative affinities of the receptors for the WT and HSPG-triple mutant form of APRIL were measured using BIAcore. Affinities given are in nanomolar concentrations and represent calculated KD values based on a simultaneous fit for data from 3 separate measurements. 1588

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also observed effective binding of the WT-triple mutant to heparin Sepharose under these conditions. Although this mutant was previously shown to have reduced binding (34), it is clear that the majority of the HSPG interaction is mediated via the N-terminal region. These mutants were subsequently used to analyze the role of the HSPG interaction in APRIL-induced IgA production. Supernatant containing soluble protein was normalized for APRIL expression and added to mouse B cells. After 6 days, IgA production was assayed (Fig. 3B). The result was a significant decrease in IgA production for both HSPG and HSPG-triple, indicating that HSPG binding is crucial for APRIL to signal. Despite its capacity to bind HSPGs, the R231A mutant showed no activity above background levels, indicating that any potential signal for IgA production coming from HSPGs is ineffective without TNF receptor binding. Loss of HSPG-binding capacity does not affect binding to BCMA or TACI To dissect the role of the HSPG domain in further detail, we proceeded with purified WT-APRIL and compared this with the purified HSPG-triple mutant, which showed complete loss of HSPG binding. To exclude the possibility that the differential activity of these two forms on B cells is not due to a change in receptor affinity, which may not be detected in the immunoprecipitation experiments, we first tested the binding capacity of the purified proteins in a more physiological context: on the surface of cells using flow cytometry (Fig. 3C). Both TACI and BCMA are absent on 293T cells, but these contain cell surface HSPGs. Therefore, 293T cells were transfected with full-length BCMA or full-length TACI and then stained with the various forms of soluble APRIL. Because heparin can compete for HSPG binding, the cells were also stained in the presence of heparin to remove any HSPG interactions and reveal specific binding to BCMA or TACI. The binding of WT-APRIL to control transfected cells signifies interaction with cell surface HSPGs; this binding can be completely competed off with unfractionated heparin (Fig. 3C). Notably, TACI- and BCMAtransfected cells show enhanced binding of APRIL, and this binding was only partially competed off with heparin, indicating specific binding to the TNF receptors. The HSPG-triple mutant, as expected, shows no binding to HSPGs on control transfected cells, but distinct binding to BCMA- and TACI-transfected cells, comparable to the level of binding observed with WT-APRIL in the presence of heparin. This result thus indicates that both forms of APRIL have an affinity comparable to that of the TNF receptors. As a control, we made use of the R231A mutant and found clear binding to control transfected cells, which is HSPG-dependent and removed by the addition of heparin. However, there is no additional binding in the TACI- or BCMAtransfected cells, confirming that APRIL-R231A does not bind cell surface BCMA or TACI. Because the HSPG-triple mutant contains a total of 6 NATURAL MULTIMERIZATION OF APRIL

point mutations, it is possible that these lead to disruptions in overall protein folding, which could affect the affinity of receptor binding. The binding of WT-APRIL measured by flow cytometry is the sum of two interactions, and it is difficult to compare the affinity by which the mutant binds to the receptors. Therefore, we measured the exact affinity of the two forms of APRIL for both BCMA and TACI using BIAcore (Fig. 3D). In agreement with previously published affinities for APRIL, binding to both receptors was in the nanomolar range (Fig. 3D) (11, 13, 42, 43). Significantly, the affinity of the HSPG-triple mutant for both TACI and BCMA was comparable to that of WT-APRIL (binding to BCMA-Fc was measured as 2.7 and 1.37 nM and binding to TACI-Fc was measured as 1.18 and 1.60 nM for WT-APRIL and HSPG-triple, respectively). The inherent trimerization of the ligand is therefore also retained in the mutant, because monomeric and trimeric forms of APRIL would have significantly different affinities. The R231A mutant did not bind BCMA or TACI. Therefore, we can conclude that decreased activity of HSPG-triple is not related to reduced receptor binding affinity. Antibody-mediated ligand cross-linking of APRIL can compensate for loss of the HSPG-binding domain In a recent study it was suggested that APRIL-driven IgA production by TACI requires an additional intracellular signaling event emanating from HSPGs, triggered via the APRIL-HSPG-binding domain (35). Therefore, the non-HSPG-binding form of APRIL would not be capable of generating these additional costimulatory signals and thus would fail to enhance IgA production. In agreement, purified HSPG-triple added to splenic mouse B cells did not stimulate IgA production, whereas purified WT-APRIL induced a 2-fold increase in IgA (Fig. 4A). The same effect was observed for proliferation (Fig. 4B) and for APRIL-induced upregulation of MHC class II (Fig. 4C). In all assays the HSPG-triple form of APRIL did not stimulate above background levels when administered on its own, whereas WT-APRIL alone gave a definite response. This result again confirms that WT-APRIL does not require antibody-mediated cross-linking and shows that the HSPG-binding domain is crucial for APRIL activity. To extend this finding, we also tested the effect of adding anti-Flag (Fig. 4A–C). As observed with supernatant containing soluble APRIL (Fig. 1A), antibody cross-linking had no additional effect on the signal from purified WT-APRIL in the case of IgA production (Fig. 4A), whereas it enhanced the proliferative signal of WT-APRIL and the up-regulation of MHC class II (Fig. 4B, C). Surprisingly, anti-Flag-mediated cross-linking had a pronounced effect on the activity of HSPGtriple. In all assays, the activity of this mutant was restored to the level achieved with WT-APRIL (Fig. 4A–C). This enhancement is specifically due to APRIL cross-linking, rather than to a possible effect via the Fc-␥ receptors, because an isotype-matched control antibody (mouse IgG1) had no effect (Supplemental 1589

Figure 4. Antibody-mediated ligand cross-linking of APRIL can compensate for loss of the HSPG-binding domain. Mouse splenic B cells were treated with purified forms of APRIL (WT, HSPG-triple, and R231A) at a final concentration of 1 ␮g/ml plus or minus cross-linking anti-Flag monoclonal antibody. Lipopolysaccharide (LPS) and transforming growth factor ␤ (TGF␤) served as a positive control. All conditions were set-up in triplicate. A) Supernatants were assayed for IgA content by ELISA after a 6-day incubation. B) Cells were assayed for cell proliferation after 48 h using tritiated thymidine. C) Cells were incubated with 0.5 ␮g/ml soluble APRIL and stained for the expression of MHC class II after 48 h. All samples were analyzed in triplicate; error bars ⫽ sd. D) Mouse splenic B cells were treated with conditioned medium normalized for expression of the various APRIL mutants. R231A and HSPG-triple were mixed at a ratio of 1:1 and added at two different concentrations: 1) total amount of APRIL added (both mutants combined) was the same as that in the wells treated with only one form of APRIL, denoted as 1⫻; 2) final amount of each mutant was the same as when they were not mixed, making the total amount of APRIL twice as much as when added alone, denoted as 2⫻. All samples were analyzed in triplicate; error bars ⫽ sd.

Fig. 3). In addition, anti-Flag mediated cross-linking of two irrelevant ligands also showed no effect on IgA production (Supplemental Fig. 4). The fact that a monoclonal antibody can compensate for loss of HSPG binding argues against HSPG signaling but instead provides evidence that clustering via HSPG binding in vivo is necessary for APRIL signaling. In agreement, the non-TNF receptor-binding mutant R231A showed no activity even with antibody crosslinking, which indicates that there is no separate signal from the HSPG alone. More important, this mutant could not cooperate with HSPG-triple, indicating that stimulation of TACI/BCMA via HSPG-triple plus a separate HSPG interaction via R231A, is not sufficient to trigger IgA secretion (Fig. 4D). This finding is in contrast with the finding by Sakurai et al. (35), which suggested that two separate signals are required: one via HSPG and one via TACI. Combined these data strongly suggest that the HSPG domain of APRIL does not mediate a separate signal but instead forms a platform to mediate natural ligand cross-linking. HSPGs are predominantly present on CD43ⴙ B cells, the APRIL-responsive cells Our data point to a significant role for HSPGs in B-cell stimulation but do not provide any insight into the localization of these HSPGs. In our assays as well as in vivo, HSPGs can be present on the surface of the B cell, 1590

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within the extracellular matrix or in serum. Because APRIL has been shown to mediate CSR via TACI, we expected IgA production to be a TACI-dependent event (23). To validate the expression of this receptor we stained both naive and activated splenocytes (stimulated with anti-IgM for 2 days) for BCMA and TACI, using CD43 as an activation marker (Fig. 5A). Both BCMA and TACI were found to be expressed on both CD43⫹ and CD43⫺ B-cell subsets. Expression of TACI on adult murine primary splenic B cells is consistent with previously published data (31, 44, 45). Reports regarding the expression of BCMA on naive B cells are somewhat conflicting, which in part is explained by differences observed between mice and humans and between different B-cell compartments (31, 45, 46). However, expression of BCMA has been reported previously on T1-, T2- and T3-cell subsets (46) and on whole splenocytes (44), and, in our hands, BCMA was clearly present on both naive and activated splenocytes. Because TACI appears to be crucial for IgA production, these observations indicate that both naive and activated B cells can potentially respond to APRIL. To detect HSPGs on the B-cell subsets, we used both a direct approach with anti-heparan sulfate (10E4 monoclonal antibody) and anti-CD138 (syndecan-1) and an indirect approach using APRIL-R231A, which binds HSPGs but not the TNF receptors. In line with previous findings, both naive and anti-IgM-stimulated splenocytes were found to express HSPGs (Fig. 5C)

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Figure 5. HSPGs are predominantly present on CD43⫹ B cells, the APRIL responsive cells. A) Mouse splenocytes were stained for expression of BCMA and TACI. B cells were gated according to B220 positivity, and then the populations were separated into CD43⫺ and CD43⫹. Gray filled plots, isotype control; black line, staining with mouse anti-BCMA or -TACI. B) IgM stimulation of B cells drives cells into the activated state, as measured by an increase in CD43 expression. C) Mouse splenocytes were stained for HSPGs using APRIL-R231A, anti-CD138, and anti-heparan sulfate. B cells were gated on B220 and then separated according to CD43 expression. Specific antibody combinations are given in Supplemental Data. Gray filled plots, isotype control; black line, staining with APRIL-R231A, anti-CD138, or 10E4. D) B cells were purified from splenocytes using B220 beads. Activated B cells were then removed using CD43 beads, and both populations were treated with soluble APRIL as described previously and then assayed for proliferation and IgA production. All samples were analyzed in triplicate.

(47). However, expression appeared to be limited primarily to the activated CD43⫹ B cells. An increase in CD43 expression after activation with anti-IgM concurred with a dramatic increase in HSPG-positive B cells. Notably, this indicates that the CD43⫹ cells are the only B cells that can bind APRIL by both its receptor and HSPGs and are therefore likely to be the responsive cells in our assays. In agreement, we found that depletion of already activated (CD43⫹) B cells before the assay led to complete loss of IgA production (Fig. 5D). This result confirms the recent suggestion that APRIL actually signals the survival, expansion, or antibody production of preactivated IgA-producing mouse B cells (32). The effect of depleting the CD43⫹ population on APRILinduced proliferation was less pronounced. This can possibly be attributed to the fact that the CD43⫺ cells in this assay are all triggered to become CD43⫹/HSPG⫹ by anti-IgM stimulation and can thus respond to APRIL after encountering anti-IgM.

similar fashion and through the same region (33). When proliferation is analyzed, low concentrations of WT-APRIL were cross-linked by anti-Flag and thereby mediated B-cell proliferation. Notably, dextran sulfate coating also led to effective cross-linking and thereby B-cell stimulation (Fig. 6A). The addition of anti-Flag to dextran sulfate-coated wells had no additive effect, suggesting that WT-APRIL was already cross-linked sufficiently via its HSPG domain. The HSPG-triple mutant was not activated by immobilized dextran sulfate and could only be activated by the addition of anti-Flag, confirming that it is not bound and crosslinked by HSPG (Fig. 6B). This result provides biological relevance for our finding and supports our proposal that in vivo multimerization of APRIL occurs via binding to HSPG either on B cells directly or via the ECM.

A dextran sulfate matrix cross-links APRIL and can substitute for cross-linking antibody

With the use of well-characterized mutant forms of secreted APRIL, we show in this study that the HSPGbinding domain of APRIL does not signal through HSPGs but instead serves as a platform to mediate natural ligand cross-linking. We found that WT-APRIL alone was capable of inducing both proliferation and IgA production, but that the non-HSPG-binding mutant of APRIL was inactive in these assays. The activity of the mutant was completely restored with the addition of cross-linking antibody, providing evidence that the

Our data show that APRIL uses HSPGs as a platform to allow for B-cell stimulation. Because APRIL was recently suggested to be deposited on ECM via HSPGs (48), we decided to mimic the in vivo situation with plate-bound dextran sulfate to provide a platform for APRIL crosslinking. Although dextran sulfate is not an HSPG, we have shown previously that it can bind APRIL in a NATURAL MULTIMERIZATION OF APRIL

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Figure 6. A dextran sulfate matrix cross-links WT-APRIL and can substitute for anti-Flag to enhance signaling. A 96-well round-bottomed plate was coated with dextran sulfate (1 and 10 ␮g/ml) in PBS overnight and then preincubated with soluble WT-APRIL (A) or HSPG-triple (B) for 1 h before addition of B cells plus or minus anti-Flag antibody. Cells were then assayed for proliferation as described. All samples were analyzed in triplicate.

natural mechanism for effective APRIL signaling relies on binding to HSPGs. Signaling through TACI was recently shown to require ligand multimerization (32). In the case of BAFF, this was taken as evidence for a physiological function for a 60-mer form of BAFF, identified previously by crystallography (49). Because APRIL also binds TACI, it is logical to assume the same requirement for multimerization. In apparent agreement, antibody-mediated cross-linking of APRIL was shown to be necessary for APRIL-dependent B-cell activation and MHC class II up-regulation via TACI (32). However, because no higher order oligomers have been reported for soluble APRIL and antibody-mediated cross-linking does not occur under physiological conditions, how multimerization of APRIL might occur in vivo remained enigmatic. On the basis of our data, we now propose that multimerization of soluble APRIL is achieved naturally by interactions with cell surface- or ECM-expressed HSPGs. The difference between these results and previously published models, in which there was an absolute requirement for antibody-mediated cross-linking, may lie in the observation that the natural stimulatory effect of APRIL is lost at lower concentrations. The concentration used in our in vitro studies is difficult to relate back to the in vivo situation, in which local concentrations may vary widely. However, it is clear that dendritic cells can produce enough APRIL to reach such concentrations. In addition, the level of circulating APRIL in serum of healthy donors also suggests that the local concentration at the site of production is high enough to directly stimulate B cells. Another possible explanation for the difference in cross-linking dependency between these studies is the form of APRIL used. Bossen et al. (32) used a form of APRIL that begins directly at the HSPG binding consensus (QKQKKQ), which contains 4 amino acids less than that of the 1592

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naturally processed form (105-250 amino acids) and our construct. This may lead to a lower affinity for HSPGs and thus increase the dependence on crosslinking. It is important to note, though, that despite our efforts, we found no evidence to support APRIL-induced signaling through HSPGs. Whether this lack of signaling constitutes a difference between mouse and human B cells will need to be addressed. Previously, Sakurai et al. (35) showed that antibodies against human TACI and HSPGs can stimulate human B cells when used in combination. However, neither antibody was formally shown to be capable of cross-linking. More important, these observations are complicated by the fact that TACI also binds HSPGs. This latter interaction was reported to be stimulatory, so it is possible that the combination of antibodies used actually served to crosslink TACI via HSPG interactions (36). Physiologically, the proposed model of APRIL-HSPG binding provides several advantages over normal membrane-bound ligands. For instance, APRIL as a secreted molecule can act at a longer range because it can potentially diffuse away from the producing cell and form a gradient similar to the mechanism by which the HSPG-binding ligand wingless orchestrates wing formation in Drosophila (50). Alternatively, APRIL could be deposited on the ECM and remain present and active long after the producing cell has left the scene. In vivo, this was recently suggested to occur in the mucosa, wherein binding to HSPGs leads to the formation of specialized APRIL-rich niches that can provide stimulation for immunoglobulin-producing plasma cells (48). We believe that our observations further support this phenomenon and demonstrate that APRIL is a unique member of the TNF family. Despite the fact that cross-linking is not required in our hands, we do find that additional cross-linking can enhance proliferation, which has been a consistent finding in all studies published to date (32, 34). We

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believe that this cross-linking may be a hint to the formation of receptor superclusters, which could potentially form in vivo when APRIL is coated on the ECM at a high density (Fig. 7). In our study, this effect was indeed mimicked with the use of plate-bound dextran sulfate, which allowed clustering of WT-APRIL and abolished the additional effect of anti-Flag cross-linking. Apparently, the clustering is sufficiently high when exposed on a platform such as dextran sulfate. Of note is the differential sensitivity to further enhancement by antibody-mediated cross-linking in proliferation and IgA production. It was recently shown that APRIL-induced antibody production by B cells may be mediated by a combination of both TACI and BCMA, but that MHC II up-regulation was TACIdependent (32). In our study, we detected both BCMA and TACI on naive and activated splenic B cells. Our data further show that APRIL-induced IgA expression depends on the presence of preactivated HSPG⫹ B cells and therefore may be mediated through TACI and BCMA. For proliferation, the increase in CD43 positivity after IgM treatment corresponded with an increase in HSPG expression, providing a larger pool of APRILresponsive cells. Thus, both proliferation and IgA production may depend on one receptor or a combination of signals via both receptors, but, notably, both crucially depend on HSPGs. The observation that IgA production is completely lost when activated B cells are depleted before APRIL stimulation is a surprising finding that would on first glance argue against the induction of CSR and suggest that APRIL signals only the survival of preexisting IgA-producing B cells. In reality, it could also indicate that APRIL is only capable of inducing CSR in preactivated B cells, which would be in line with the HSPG expression on these cells and not on naive B cells. Because TACI also binds HSPGs, APRIL-TACI-HSPG complexes may be formed preferentially, and so it would be interesting to examine the specific require-

ments for BCMA- and TACI-driven signals separately. Although Bossen et al. (32) used splenocytes from receptor knockout mice to address this point, their results were not fully conclusive, as there seems to be overlap between the use of BCMA and TACI for the various B-cell responses and to date little is known about specific intracellular signals for each receptor. It is hoped that future experiments to study receptor signaling in isolation will address this point. However, we do know that plasmablast survival, a BCMA-specific response, also relies on HSPGs (M. Spaargaren and R. M. Reijmers, Universiteit van Amsterdam, personal communication, September 24, 2008). Also of note in this study is the observation that binding of the R231A mutant to untransfected 293T cells was marginally lower than binding of the WT. It is known that WT-APRIL can stimulate proliferation of cell lines derived from solid tumors that lack BCMA and TACI, but the mechanism for this proliferation is not yet known. It is therefore possible that a potential third TNF-like receptor, which is not bound by the R231A mutant, exists on these cells. In a discussion on multimerization, it is essential to consider the membrane-bound form of APRIL, which is generated by alternative splicing and is termed TWEPRIL (40, 51). Previously, we detected TWE-PRIL on monocytes, T cells (40, 51), and dendritic cells (unpublished results), which might suggest a balance between cell surface-expressed (TWE-PRIL), secreted, and ECMbound ligand (APRIL). That many TNF family ligands require cross-linking for an effective signal has been well described (52, 53). However, dependence of the soluble ligand on cross-linking in vitro is often attributed to the fact that signaling through the membranebound form is dominant in vivo. Differences in the requirement for ligand presentation can distinguish between the role of soluble and membrane-bound forms of the same ligand and result in different receptor specificities, as exemplified by TNF (54, 55). The

Figure 7. Schematic diagram to illustrate natural cross-linking of APRIL by HSPGs on the extracellular matrix. A) In vitro cross-linking by monoclonal antibody can mimic that of natural HSPGs on the extracellular matrix. To deliver an effective signal, WT-APRIL needs to bind receptors in functional signaling complexes, leading to recruitment of internal adaptor proteins and downstream signaling. B) Physiologically, HSPGs cluster APRIL and achieve this multimeric assembly at the cell surface. This can potentially occur in a cis (same cell) or trans (different cell) fashion. Further antibody cross-linking of WT-APRIL could also lead to “superclustering” and hence enhanced signaling. NATURAL MULTIMERIZATION OF APRIL

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suggestion by Bossen et al. (32) that TACI requires multimerized ligands would suggest a predominance of TWE-PRIL, at least when cell-cell interactions are considered, unless soluble APRIL is bound in a rigid formation by HSPGs on the cell surface (32). Future experiments should therefore be focused on understanding the biological difference between these two forms of APRIL ligand. In conclusion, because APRIL and possibly also TWEPRIL are crucial for the IgA class switch in vivo and involved in survival of long-lived plasma cells, we believe our data provide an important framework to explain how these signals are conveyed to responding B cells under physiologically relevant conditions. We thank the animal caretakers at the Academic Medical Center, University of Amsterdam. The work in this study was supported by TI-Pharma, Dutch Cancer Society grants 20032812 and 2007-3750, and Stichting Vanderes. M.H. is supported by the Fondation de France and Association pour la Recherche sur le Cancer. The authors declare no competing financial interests.

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