The Journal of Immunology

The Immunoregulator Soluble TACI Is Released by ADAM10 and Reflects Activation in Autoimmunity

Franziska S. Hoffmann,* Peer-Hendrik Kuhn,†,‡,x Sarah A. Laurent,* Stefanie M. Hauck,{ Kerstin Berer,‖ Simone A. Wendlinger,* Markus Krumbholz,* Mohsen Khademi,# Tomas Olsson,# Martin Dreyling,** Hans-Walter Pfister,†† Tobias Alexander,‡‡ Falk Hiepe,‡‡ Tania Kumpfel,*€ Howard C. Crawford,xx Hartmut Wekerle,‖ Reinhard Hohlfeld,*,{{ Stefan F. Lichtenthaler,†,‡,{{ and Edgar Meinl*

BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF–APRIL receptor TACI, was applied in clinical trials. However, disease activity in unexpectedly increased, whereas in systemic erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subse- quently cleaved by g-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-kB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF–APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and g-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation. The Journal of Immunology, 2015, 194: 542–552.

cells play a significant role in the pathogenesis of auto- a proportion of SLE patients (3, 4). In multiple sclerosis (MS), an immunity and B cell modulating therapies are promising in organ-specific autoimmune disease characterized by local Ig pro- B the treatment of a variety of autoimmune diseases (1). duction with long-term persistence of B cells in the CNS (5, 6), BAFF Regulation of B cell homeostasis involves the BAFF–APRIL sys- is upregulated in MS plaques and is produced by astrocytes (7). Al- tem that is comprised of two ligands, BAFF and a proliferation- though depletion of B cells in MS with anti-CD20 Abs is promising inducing ligand (APRIL), and three receptors, B cell maturation Ag (8), targeting the B cell survival factors BAFF and APRIL with (BCMA), transmembrane activator and CAML interactor (TACI), atacicept, a recombinant fusion protein containing the extracellular and BAFF receptor (2). ligand-binding portion of TACI linked to the Fc domain of human In systemic lupus erythematosus (SLE), an involvement of the IgG, unexpectedly increased disease activity in MS patients (9), BAFF–APRIL axis is prominent, as mice overexpressing BAFF de- whereas in SLE atacicept was beneficial at least at a high dose (10). velop an SLE-like phenotype, BAFF is elevated in the serum of SLE TACI is a type I–oriented transmembrane protein belonging to patients, and the mAb targeting BAFF is beneficial in the TNFR superfamily. It is expressed on CD27+ memory B cells,

*Institute of Clinical Neuroimmunology, Ludwig Maximilian University, Munich F.S.H. performed experiments, was involved in study design, and wrote the paper; D-81377, Germany; †Neuroproteomics, Technical University Munich and Hospital P.-H.K, S.A.L., S.M.H., and K.B. performed experiments; M. Krumbholz was in- Rechts der Isar, Munich D-81675, Germany; ‡German Center for Neurodegenerative volved in study design; M. Khademi, T.O., M.D., H.-W.P., T.A., F.H., and T.K. Diseases, Bonn D-53175, Germany; xInstitute for Advanced Study, Technical provided clinical samples; H.C.C. provided ADAM10 conditional knockout mice; University, Munich D-85748, Germany; {Research Unit Protein Science, Helmholtz H.W., R.H., S.F.L., and E.M. designed the study and wrote the paper. All authors Center Munich, German Research Center for Environmental Health, Neuherberg discussed results and commented on the manuscript. D-85764, Germany; ‖Department of Neuroimmunology, Max Planck Institute of Address correspondence and reprint requests to Prof. Edgar Meinl, Institute of Neurobiology, Martinsried D-82152, Germany; #Division of Clinical Neuroscience, Clinical Neuroimmunology, Ludwig Maximilian University, Marchioninistrasse 15, Karolinska University Hospital, Stockholm 14186, Sweden; **Department of D-81377 Munich, Germany. E-mail address: [email protected] Medicine III, Ludwig Maximilian University, Munich D-81377, Germany; ††Department of Neurology, Ludwig Maximilian University, Munich D-81377, The online version of this article contains supplemental material. Germany; ‡‡Department of Rheumatology and Clinical Immunology, Charite´– xx Abbreviations used in this article: ADAM, a disintegrin and metalloproteinase; University Medicine Berlin, Berlin D-10117, Germany; Department of Cancer {{ APRIL, a proliferation-inducing ligand; BCMA, B cell maturation Ag; CIS, clini- Biology, Mayo Clinic Florida, Jacksonville, FL 32224; and Munich Cluster for cally isolated syndrome; CRD, cysteine-rich domain; CSF, cerebrospinal fluid; CTF, Systems Neurology (SyNergy), Munich D-81377, Germany C-terminal fragment; IP, immunoprecipitation; MS, multiple sclerosis; NB, neuro- Received for publication August 13, 2014. Accepted for publication October 30, borreliosis; NP-40, Nonidet P-40; OND, other neurologic disease; PVDF, polyvinyl- 2014. idene difluoride; RR-MS, relapsing-remitting multiple sclerosis; shRNA, short hairpin RNA; SLE, systemic lupus erythematosus; SP-MS, secondary progressive This work was supported by the Deutsche Forschungsgemeinschaft (SFB TR128), multiple sclerosis; sTACI, soluble TACI; TACI, transmembrane activator and CAML the Bundesministerium fur€ Bildung, Wissenschaft, Forschung und Technologie interactor; TACI–D-Ecto, TACI lacking the ectodomain; TACI-FL, full-length human (“Krankheitsbezogenes Kompetenznetz Multiple Sklerose” and “Kompetenznetz TACI; TACI–N-FLAG, TACI with an N-terminal FLAG tag; TACI–N-HA, TACI Degenerative Demenzen”), the Munich Cluster for Systems Neurology (ExC 1010 with an N-terminal hemagglutinin tag. SyNergy), the Verein zur Therapieforschung fur€ Multiple-Sklerose-Kranke, National Institutes of Health Grant CA159222 (to H.C.C.), and the Friedrich-Baur-Stiftung. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402070 The Journal of Immunology 543 plasma cells and a subpopulation of CD272 B cells and is induced Protease inhibitors early upon B cell activation (11). The ectodomain of TACI con- DAPT (Merck Calbiochem), TAPI-1 (Merck Calbiochem), C3 (Merck tains two cysteine-rich domains (CRDs). The first CRD is in- Calbiochem), and GI254023X (kindly provided by Dr. Andreas Ludwig, volved in ligand-independent assembly of TACI into multimeric University of Erlangen, Erlangen, Germany) were used. complexes, whereas the second CRD is required for binding of Cell culture BAFF and APRIL (12). Ligand binding to TACI recruits signaling molecules to the intracellular domain of TACI, which leads to Raji cells were cultured in RPMI 1640 (Sigma-Aldrich)/10% FBS (Bio- activation of NFAT and NF-kB (13, 14). Studies of TACI2/2 mice chrom AG)/100 U/ml penicillin plus 100 mg/ml streptomycin (Life Technologies, Invitrogen)/1% nonessential amino acids (Life Technolo- showed that this receptor is both a positive and negative regulator gies, Invitrogen)/1 mM sodium pyruvate (Life Technologies, Invitrogen)/ of B cell responses (15–17). Mutations in TACI are a cause of common 2mML-glutamine (Pan Biotech). HEK293T cells were cultured in variable immunodeficiency and IgA deficiency (18, 19). However, DMEM (Sigma-Aldrich)/10% FBS/100 U/ml penicillin plus 100 mg/ml some of these patients in addition develop signs of autoimmunity and streptomycin. lymphoproliferation (19). Transient transfection Importantly, the functions of some transmembrane receptors HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen). extend beyond signal transmission, as they can be processed into sTACI containing and control supernatants were generated by transfection soluble receptors (20). In this regard, proteases of the a dis- of 10 mg TACI in pCMV-XL4 or an empty vector. After 3 d, supernatants integrin and metalloproteinase (ADAM) family are involved in were harvested and centrifuged two times at 400 3 g for 7 min and one ectodomain shedding of a variety of membrane proteins (21). time at 20,000 3 g for 30 min. This can modulate signaling activity either by downregulation Human B cell culture and stimulation of membrane-bound receptors or by the release of soluble receptors like soluble TNFR1 (22) or soluble IL-6R (23). In the PBMCs were isolated by density gradient separation using Pancoll (Pan Biotech). Human B cells were isolated by negative selection using the case of type I transmembrane proteins, the g-secretase complex EasySep Human B Cell Enrichment Kit (StemCell Technologies). Human may further cleave the remaining fragment within the plasma B cells were seeded at 8 3 105 cells/ml and activated using ODN 2006 membrane (24) in a process called regulated intramembrane (2.5 mg/ml; Invivogen), anti-IgM (10 mg/ml Jackson ImmunoResearch proteolysis (25). Laboratories), R848 (1 mg/ml; Sigma-Aldrich), recombinant human IL-2 (25 ng/ml; R&D Systems), coculture with CD40L expressing mouse L cells In this study, we show that the TACI extracellular domain is shed (5 3 104 cells/ml), and recombinant human IL-21 (50 ng/ml; eBioscience). from activated B cells by ADAM10, giving rise to soluble TACI Supernatants were collected after 4 d. sTACI production and IgG produc- (sTACI). The remaining C-terminal fragment (CTF) is cleaved by tion were measured. TACI surface expression was determined using a PE g-secretase. sTACI assembles homotypically; it binds BAFF and mouse anti-human TACI Ab (5 mg/ml; clone FAB1741P; R&D Systems) APRIL to block NF-kB activation and B cell survival. In systemic and the corresponding PE mouse IgG1 isotype control (5 mg/ml; clone X40; BD Biosciences). (SLE) and compartmentalized (MS) immunopathologies, we detec- ted elevated levels of sTACI establishing sTACI as a potential Murine B cell culture and survival assay biomarker. For murine B cell cultivation, spleens from C57BL/6 mice (8–12 wk of age) were passed over a 40-mm cell strainer. Red cell lysis was performed Materials and Methods using ACK buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) Patients for 2 min on ice. B cells were isolated using the EasySep Mouse B Cell Isolation Kit (StemCell Technologies) by negative selection. The mu- All patient samples were collected following written informed consent rine B cells were seeded at a concentration of 3 3 106 cells/ml on cell- according to local ethics policy guidelines in Stockholm, Berlin, and culture plates coated with anti-murine IgM Abs (2.5 mg/ml) overnight Munich and the Declaration of Helsinki. We analyzed the following at room temperature. Cells were treated with the indicated concentrations samples: cerebrospinal fluid (CSF) from 37 untreated MS patients (clini- of HEK293T cell supernatants containing sTACI and control supernatants cally isolated syndrome [CIS]: n = 10; relapsing-remitting multiple generated as described above. Additionally, TACI-Fc was added (R&D sclerosis [RR-MS]: n = 20; and secondary progressive multiple sclerosis Systems), and cells were stimulated with BAFF (100 ng/ml; R&D Sys- [SP-MS]: n = 7) and from 20 untreated patients with other neurologic tems) and APRIL (100 ng/ml; AdipoGen). After 2 d of cultivation, mouse diseases (ONDs) (sensory symptoms: n = 7; cerebrovascular disease: n =1; B cells were analyzed by flow cytometry using a PE anti-mouse CD19 Ab migraine: n = 1; vertigo: n = 1; syringomyelia: n = 1; spinal stenosis: n =1; (1 mg/ml; clone MB19-1; eBioscience) and APC TO-PRO-3 Iodide neurasthenia: n = 1; alcohol-related spastic paraparesis: n = 1; hearing (250 nM; Life Technologies). PE-positive and allophycocyanin-negative deficit: n = 1; depression and idiopathic pain: n = 1; fatigue: n = 1; bipolar cells were determined. disorder: n = 1; schizophrenia: n = 1; and diplopia: n = 1); plasma from 57 untreated MS patients (CIS: n = 18; RR-MS: n = 23; and SP-MS: n = 16) Western blotting and immunoprecipitation and 18 untreated patients with ONDs; CSF from 25 MS patients before and To analyze the role of g-secretase, TACI with a C-terminal FLAG-tag was 12 mo after treatment with natalizumab; serum from 17 untreated SLE transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). In- patients and 22 treated SLE patients (treatment included prednisolone, creasing amounts of DAPT were added. After 48 h, cells were lysed in Nonidet chloroquine, azathioprine, cyclophosphamide, mycophenolate mofetil, and P-40 (NP-40) buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 1% NP-40, and methotrexate); serum samples of 10 patients prior and after treatment with complete protease inhibitor mixture; Roche Applied Science). Proteins were corticosteroids; and serum from 33 healthy volunteers. Intrathecal IgG electrophoresed on 10–20% Tricine gels, transferred to polyvinylidene production was calculated as: IgG-index (CSF IgG/CSF albumin)/(serum difluoride (PVDF) membranes, and blotted with an anti-FLAG Ab (clone IgG/serum albumin). M2; Sigma-Aldrich). To control for equal protein loading, membranes Plasmids were stained with anti–b-actin Ab (clone C4; Santa Cruz Biotechnology). For immunoprecipitation (IP) of sTACI from HEK293T, a monoclonal Human TACI in pCMV6-XL4, human BCMA in pCMV6-XL5, and the mouse anti-TACI Ab (clone MAB174; R&D Systems) or a mouse IgG1 control vector pCMV6-AN-DDK were purchased from Origene Technol- control (R&D Systems) was used. For IP from Raji cell supernatant, the ogies. TACI with a C-terminal FLAG-tag, TACI with an N-terminal FLAG- polyclonal goat anti-TACI Ab (clone AF174; R&D Systems) or a goat IgG tag (TACI–N-FLAG), and TACI with an N-terminal hemagglutinin tag control (R&D Systems) was applied. Abs were coupled to Dynabeads (TACI–N-HA) were added in pcDNA3.1 using standard cloning proce- Protein G (Life Technologies) and cross-linked with bis-sulfosuccinimidyl- dures. To generate TACI lacking the ectodomain (TACI–D-Ecto), the TACI suberate (Pierce). sTACI derived from HEK293T cells was eluted with sequence coding for aa 155–293 was cloned into pcDNA3.1. CRE fol- NuPAGE LDS Sample Buffer (Life Technologies), electrophoresed on lowed by the T2A peptide sequence and GFP or GFP alone were cloned 4–12% Bis-Tris gels (Life Technologies) with MES-running buffer, blotted into the retroviral vector pMSCV. The pCL-Eco packaging construct was on PVDF membranes, and stained with a monoclonal mouse anti-TACI Ab additionally used for retrovirus production. (clone MAB174; R&D Systems). Alternatively, the PVDF membrane was 544 ADAM10 RELEASES SOLUBLE TACI FROM B CELLS cut prior to staining at the height corresponding to sTACI, and N-terminal used in combination with the TaqMan PCR Core Reagent Kit (Applied sequencing was performed. sTACI derived from RAJI cells was obtained Biosystems). Cyclophilin (Applied Biosystems) expression was determined by acidic elution. For coimmunoprecipitation of TACI–N-HA and TACI– as a housekeeping gene. Samples were run in MicroAmp Optical 96-well N-FLAG, HEK293T cells were transfected with the respective expression reaction plates (Applied Biosystems) in a 7900HT Fast Real-Time PCR constructs. Forty-eight hours later, cells and supernatants were harvested. System (Applied Biosystems). Data was analyzed using SDSv2.3 software Cells were lysed in IP buffer (0.5% NP-40, 50 mM HEPES, 250 mM NaCl, (Applied Biosystems). 5 mM EDTA, and complete protease inhibitor mixture; Roche Applied Science). IP was performed using anti-FLAG magnetic beads (Sigma- Statistics Aldrich). IP eluates and pre-IP samples were subjected to ELISA. Statistical significance was assessed with Prism Software (GraphPad) by Mass spectrometry and Edman sequencing unpaired or paired, nonparametric, or parametric t test analysis or by Spearman correlation, as appropriate. The p values *p , 0.05, **p , 0.01, sTACI derived from Raji cells was digested in solution with trypsin or GluC; ***p , 0.001, and ****p , 0.0001 were considered significant and sTACI derived from HEK293T cells was digested with trypsin and chy- designated accordingly. motrypsin. Mass spectrometry (LTQ Orbitrap XL; Thermo Scientific) was performed. Results ELISA sTACI is released by activated B cells Human sTACI and IgG concentrations were determined using the Human As TACI is described as an ambivalent regulator of B cell homeo- TACI DuoSet (DY174; R&D Systems) and the Human IgG ELISA de- stasis, we speculated that this effect could be due to a soluble variant of velopment kit (Mabtech), respectively. Complex formation between sTACI TACI. Therefore, we stimulated primary human B cells with a panel of and BAFF and APRIL was determined by coating anti-FLAG Ab (M2; 5 mg/ml; Sigma-Aldrich) on ELISA plates, incubating them with BAFF- different B cell activators and determined plasma membrane ex- FLAG (Enzo Life Sciences) and APRIL-FLAG (AdipoGen) (200 ng/ml) pression of TACI (Fig. 1A) and in parallel release of sTACI (Fig 1B). and adding sTACI and TACI-Fc (R&D Systems) (25 ng/ml). Bound TACI Although B cells in human blood expressed little TACI on their was detected using the TACI DuoSet ELISA Kit. To detect TACI–N-HA surface, we noted a strong induction by TLR7/8 and TLR9 agonists coimmunoprecipitated with TACI–N-FLAG, ELISA plates were coated and by CD40L, which was further enhanced by addition of IL-21 with an anti-HA.11 (Covance) Ab (5 mg/ml). The following ELISA steps were performed using the TACI DuoSet ELISA Kit (R&D Systems). (Fig. 1A) (11). sTACI release and TACI surface expression, induced by the different stimuli, correlated strongly (Fig. 1C), suggesting that NF-kB reporter assay sTACI production by primary B cells parallels levels of TACI on the HEK293T cells were cotransfected with a firefly luciferase reporter plasmid, cell surface. In addition, we analyzed whether sTACI release is linked the internal control CMV Renilla luciferase plasmid, and the respective ex- to differentiation into Ig-secreting cells. We found that activated pression plasmids. The decoy-function of sTACI was assessed by adding B cells released sTACI without producing IgG (e.g., after CD40L and BAFF or APRIL-FLAG (100 ng/ml) to supernatants containing sTACI or to control supernatants. After incubation at 37˚C for 30 min, supernatants were R848 stimulation) (Fig. 1B). To identify stimuli that trigger sTACI added to the BCMA-transfected cells. Sixteen hours later, cells were lysed release from membrane-bound TACI, we stimulated the Burkitt with passive lysis buffer (Promega), and reporter gene activity was determined lymphoma cell line Raji, which endogenously expresses TACI, with using firefly luciferase substrate (Biozym) and Renilla luciferase substrate different TLR ligands and (polyinosinic-polycytidylic acid, (Promega), respectively. To analyze the role of g-secretase on NF-kB acti- CpG ODN 2006, TNF-a,IFN-g,andPMA).Wedidnotobserve vation, TACI–D-Ekto or full-length TACI was transfected together with the above-described luciferase plasmids. Eight hours after transfection, DAPT and a strong modulation of sTACI release (Supplemental Fig. 1A), BAFF were added. Sixteen hours later, NF-kB-activation was measured as and only PMA weakly and transiently induced TACI shedding described. (Supplemental Fig. 1B). Thus, when TACI is present on the cell Retrovirus production and transduction of B cells from surface, it is shed without the need of an additional stimulus. To- ADAM10 conditional knockout mice gether, this suggests that sTACI release reflects TACI expression on the cell surface. We isolated splenic B cells from ADAM10 conditional knockout mice (26), which contain two flox sites flanking the ADAM10 gene. Mouse B cells sTACI represents the extracellular part of TACI were stimulated for 2 d with CpG ODN 1668 20 mg/ml (Invivogen) and Next, we determined the m.w. and amino acid composition of rCD40L 2.5 mg/ml (R&D Systems). Retrovirus was produced as described previously (27) using the vector pMSCV expressing GFP or the CRE- sTACI. We immunoprecipitated sTACI from the supernatant of recombinase followed by the sequence of the self-cleaving T2A peptide HEK293T cells that had been transfected with full-length human and GFP. B cells were spin-infected as described previously (27). On day 4, TACI. sTACI had a molecular mass of ∼13 kDa in Western blot TACI expression was determined using a rat monoclonal anti-mouse TACI analysis and silver staining (Fig. 2A). For mass spectrometry, we Ab (clone FAB1041A; 5 mg/ml; R&D Systems). Knockdown of ADAM10 was determined by Western blot using a rabbit monoclonal anti-ADAM10 analyzed the band detected by silver staining and sTACI immuno- Ab (clone EPR5622; Abcam) in FACS-sorted GFP-positive cells. precipitated from Raji cell supernatant. After trypsin, chymotrypsin, and GluC digestion as well as N-terminal sequencing, we found sol- Lentiviral short hairpin RNA–mediated knockdown uble TACI to be composed of the extracellular part of TACI (Fig. 2B). Lentivirus production was performed as described previously (28). Short The sequence at the C terminus ended with K154, adjacent to L155 in hairpin RNA (shRNA) sequences are listed in Supplemental Table 1. Raji cells sTACI derived from HEK293T cells and Raji cells (Fig. 2B). K154/ 3 6 were plated at 1 10 cells/ml and transfected with conditioned medium L155 is within the juxtamembrane region and comprises a typical containing the lentiviruses. The transduced Raji cells were plated at 5 3 105 cells/ml. Twenty-four hours later, supernatants were harvested and ana- cleavage motif of metalloproteinases of the ADAM and membrane- lyzed for sTACI concentration by ELISA. TACI surface expression was de- type matrix metalloproteinases family (28). Therefore, K154/L155 termined by FACS using a mouse monoclonal anti-TACI Ab (clone MAB174; could be the naturally occurring cleavage site of TACI resulting in 1 mg/ml; R&D Systems) and the corresponding mouse IgG1 isotype control generation of sTACI. Alternatively, thissitecanalsoberecognizedby (R&D Systems). trypsin. Real-time PCR ADAM10 sheds TACI RNA from transduced Raji cells was isolated using the RNeasy Micro Kit To identify the protease that sheds TACI, we applied different protease (Qiagen). cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For real-time PCR, TaqMan assays inhibitors to Raji cells. We found that the metalloproteinase inhibitor for ADAM10 and ADAM17 (Applied Biosystems) and PrimeTime qPCR TAPI-1, as well as GI254023X (29), a specific ADAM10 inhibitor, Assays for ADAM9 and ADAM19 (Integrated DNA Technologies) were blocked sTACI release and increased surface expression of TACI The Journal of Immunology 545

FIGURE 1. sTACI is released by activated B cells and closely correlates with membrane-bound TACI. (A and B) Human purified B cells were activated using CD40L, IL-21, CD40L + IL-21, CpG (ODN2006), anti-IgM, anti-IgM + IL-21, R848, IL-2, and R848 + IL-2 for 4 d. (A) TACI surface expression was determined by FACS 4 d after activation. Filled histograms represent the isotype control; solid lines the TACI expression. (B) sTACI and IgG production were analyzed by ELISA. (C) Mean fluorescence intensity (MFI) of TACI surface expression was calculated by subtracting the isotype fluorescence signal. sTACI production and MFI of membrane-bound TACI correlated strongly (p = 0.0105, r = 0.7818 Spearman correlation). Representative data of two different donors. 546 ADAM10 RELEASES SOLUBLE TACI FROM B CELLS

FIGURE 2. sTACI is composed of the extracellular part of TACI. (A)sTACIwas immunoprecipitated from supernatant of HEK293T cells transfected with TACI-FL or an empty vector as control (IP: MAB174) followed by SDS-PAGE and Western blotting (WB). IP eluates were probed for TACI (MAB174). Silver staining was performed after IP from supernatant of HEK293T cells transfected with full-length TACI or an empty vector as control (IP: AF174). (B)AfterIPof sTACI from HEK293T cell supernatant, the band corresponding to sTACI in silver stain- ing was excised and digested with trypsin or chymotrypsin. IP of sTACI from Raji cell supernatant followed by acidic elution and digestion with trypsin or GluC was per- formed. The amino acid sequences of TACI and peptides identified by mass spectrometry after tryptic (green), chymotryptic (blue), or GluC (orange) digestion are shown. sTACI derived from HEK293T cells was addi- tionally analyzed by N-terminal sequenc- ing (Edman) (red).

(Fig. 3A, 3B). The b-secretase inhibitor C3 (30) and the g-secretase compared with GFP-negative cells after transduction with the inhibitor DAPT had no effect (Fig. 3A, 3B). A combined treatment CRE-T2A-GFP virus (Supplemental Fig. 2C–E). This shows that with TAPI-1 and GI254023X did not further decrease sTACI pro- ADAM10 also sheds TACI from primary murine B cells. duction (Fig. 3A), suggesting that besides ADAM10 no other pro- tease susceptible to inhibition by TAPI-1 is involved in TACI g-Secretase cleaves the CTF of TACI cleavage. TAPI-1 also reduced the release of sTACI from primary As ectodomain shedding of type I membrane proteins is commonly human B cells activated with CD40L and IL-21, whereas DAPT had followed by g-secretase cleavage of the CTF (25), we investigated no effect (Supplemental Fig. 1C). To confirm the role of ADAM10 in whether g-secretase is also involved in the cleavage of the TACI TACI cleavage, we lentivirally transduced Raji cells with shRNAs CTF. When we transfected TACI with a C-terminal FLAG-tag in targeting ADAM9, ADAM10, ADAM17, and ADAM19 (Fig. 3C, HEK293T cells and treated them with DAPT, we observed accumu- 3D). As knockdown of ADAM17 influenced cell survival and pro- lation of the TACI CTF (Fig. 4A), which demonstrates that the CTF of liferation, we corrected sTACI levels for cell number. Knockdown TACI is cleaved by g-secretase. As a next step, we investigated po- was confirmed by quantitative PCR (Fig. 3E). We found that only tential functional consequences of TACI-cleavage by g-secretase. We a knockdown of ADAM10 led to a decrease of sTACI production used an NF-kB reporter assay in HEK293T cells transfected with (Fig. 3D), paralleled by an increase of TACI surface expression either full-length human TACI (TACI-FL) or TACI–D-Ecto resem- (Fig. 3C). None of the other applied shRNAs influenced membrane- bling the TACI CTF. Both TACI-FL and TACI–D-Ecto exhibited bound TACI or sTACI production (Fig. 3C, 3D). The effect medi- constitutive NF-kB activation in the absence of a ligand (Fig. 4B). ated by ADAM10 shRNA was similar to that of TAPI-1 in ELISA Spontaneous dimerization via the CRD1 (12) is believed to confer and FACS (Fig. 3A, 3B). To confirm the role of ADAM10 in TACI constitutive activity to TACI. TACI–D-Ecto lacks the CRD1. The CTF shedding in primary B cells, we used murine B cells from condi- might therefore dimerize independent of the CRD1 due to high tional ADAM10 KO mice that contain two flox sites flanking the amounts of TACI present in this overexpression system. Additionally, ADAM10 gene (26). To achieve the ADAM10 knockout, B cells we stimulated the transfected cells with BAFF. As expected, BAFF were transduced with retroviral particles expressing the CRE- increased NF-kBactivationincellstransfectedwithTACI-FL,butdid recombinase followed by the self-cleaving T2A peptide (31) and not activate TACI–D-Ecto, which lacks the ligand binding domain GFP (CRE-T2A-GFP), which resulted in efficient knockout of (Fig. 4B). DAPT treatment increased NF-kB signaling mediated by ADAM10 as confirmed by Western blot (Supplemental Fig. 2A). As TACI–D-Ecto, indicating that cleavage of the CTF of TACI by a control, a retrovirus expressing GFP was used. TACI expression g-secretase reduces NF-kB activation. This suggests that g-secretase was determined by FACS. Knockout of ADAM10 led to an increase cleavage of TACI CTF might be relevant to limit NF-kB activation of TACI surface expression when comparing cells transduced with after TACI ectodomain shedding. DAPT treatment had no effect on the control virus to cells transduced with the CRE-T2A-GFP virus cells transfected with TACI-FL presumably because the amount of (Supplemental Fig. 2B) and also when GFP-positive cells were TACIcleavedbyADAM10comparedwithTACI-FLwastoolow. The Journal of Immunology 547

FIGURE 3. ADAM10 sheds TACI. (A and B) Raji cells were treated for 24 h with the indicated concentrations of DAPT, C3, TAPI-1, GI354023X, and TAPI-1 + GI354023X. (A) sTACI release was determined by ELISA (combined data of four independent experiments [mean 6 SEM]). (B) TACI surface expression was analyzed by FACS. Only the high inhibitor concentrations with corresponding vehicle control and isotype controls are depicted (repre- sentative data of four independent experiments). (C–E) Raji cells were lentivirally transduced with two different shRNAs targeting ADAM9, ADAM10, ADAM17, and ADAM19, respectively, or a nontargeting shRNA (Con). For comparison, cells were treated with TAPI-1. (C) TACI surface expression was analyzed by FACS; representative data of two independent experiments. (D) sTACI release was determined by ELISA and controlled for cell number (two- tailed, paired t test, including two technical replicates of the respective experiments); combined data of two independent experiments (mean 6 SEM). (E) Knockdown of ADAM9, ADAM10, ADAM17, and ADAM19 was determined by quantitative PCR; 1 and 2 represent two different shRNAs targeting the same ADAM; combined data of two independent experiments (mean 6 SEM). **p , 0.01, ***p , 0.001.

sTACI assembles in homotypic interaction sTACI binds BAFF and APRIL, blocking B cell survival and To establish whether sTACI exists as a monomeric or an olig- NF-kB activation omeric form, we coexpressed TACI–N-HA or TACI–N-FLAG in To explore the function of sTACI generated after ADAM10 cleavage, HEK293T cells. We found that sTACI–N-HA could be coimmu- we determined binding of sTACI to BAFF and APRIL. We estab- noprecipitated with sTACI–N-FLAG, which argues for a homotypic lished an ELISA in which sTACI was captured by BAFF-FLAG or interaction of sTACI monomers in the absence of ligand (Fig. 5A). APRIL-FLAG immobilized on an anti-FLAG–coated ELISA plate. As a positive control, full-length TACI–N-HA derived from cell We found that sTACI as well as TACI-Fc bound both BAFF and lysates was coimmunoprecipitated with full-length TACI–N-FLAG, APRIL (Fig. 6A). In the following, we evaluated the functional because membrane-bound TACI was reported previously to interact consequences of this binding and determined the influence of sTACI homotypically (12) (Fig. 5B). on BCMA-mediated NF-kB activation in the presence of BAFF and 548 ADAM10 RELEASES SOLUBLE TACI FROM B CELLS

FIGURE 4. g-Secretase degrades the CTF of TACI shutting down NF-kB activation. (A) HEK293T cells were transfected with TACI- FL with a C-terminal FLAG-tag and treated with the indicated concentrations of DAPT or vehicle control. After 48 h, lysates were ob- tained for SDS-PAGE and Western blotting. Lysates were probed for expression of tagged TACI demonstrating TACI-FL and the CTF of TACI (representative data of three inde- pendent experiments). Relative intensities are depicted below the bands. (B) HEK293T cells weretransfectedwith1ngofTACI-FLor TACI–D-Ecto and a luciferase-based NF-kB reporter. DAPT (1 mM) and BAFF (100 ng/ml) were added as indicated, and NF-kB activation was determined (two-tailed, paired t tests); combined data of five independent experiments (mean 6 SEM). *p , 0.05, **p , 0.01.

APRIL in HEK293T cells. In our assay, transfection of BCMA blocked NF-kB activation (Fig. 6B). BAFF-mediated NF-kB leads to lower constitutive activity and higher activation after stim- activation could be blocked slightly more efficiently (Fig. 6B). ulation compared with TACI transfection and was therefore more Recombinant TACI-Fc was used as a control and exhibited com- suitable for testing a possible decoy function of sTACI. Addition of parable blocking capacity as sTACI (Fig. 6B). To further support sTACI to APRIL and BAFF-stimulated cells dose-dependently the functional relevance of sTACI, we tested the impact of sTACI

FIGURE 5. sTACI assembles homotypically independent of a ligand. (A and B) HEK293T cells were transfected with expression plasmids containing TACI with an N-terminal FLAG-tag or HA-tag as indicated. Forty-eight hours after transfection, supernatants (A) were harvested, and cell lysates (B) were prepared. A part of the supernatants (A) and lysates (B) prior to IP were stored; the remaining supernatants and lysates were subjected to FLAG-IP, and the IP eluates plus the supernatants and lysates stored prior to IP were measured by ELISA. An anti-HA Ab was used for coating, and an anti-TACI Ab was applied for detection; combined data of three independent experiments (mean 6 SEM). The Journal of Immunology 549

FIGURE 6. sTACI acts as a decoy for BAFF and APRIL and blocks B cell survival. (A) ELISA plates were coated with anti-FLAG Abs (M2; 5 mg/ml); BAFF-FLAG and APRIL-FLAG were added; and sTACI (25 ng/ml) derived from supernatant of transfected HEK293T cells or concentrated supernatants of Raji cells and TACI-Fc (25 ng/ml) were used; combined data of three independent experiments (mean 6 SEM). (B) HEK293T cells were transfected with full-length BCMA (2.5 ng) and a luciferase-based NF-kB reporter. BAFF and APRIL (100 ng/ml) were added together with increasing amounts of sTACI- containing supernatant (33, 100, and 300 ng/ml), control supernatant, or TACI-Fc (300 ng/ml), and NF-kB activation was determined (two-tailed, paired t test); combined data of three independent experiments (mean 6 SEM). (C) Murine B cells were activated via anti-IgM and cultured for 2 d with APRIL or BAFF (100 ng/ml) in the presence or absence of sTACI (100, 200, and 400 ng/ml) and TACI-Fc (400 ng/ml). Survival of B cells was determined by FACS analyzing the percentage of living CD19+ cells after staining with TO-PRO-3 iodide. Viability was calculated in relation to the BAFF- and APRIL-induced survival that was assigned as 1 (two-tailed, paired t test between BAFF-treated and BAFF + sTACI/TACI-Fc–treated conditions); combined data of four to five independent experiments (mean 6 SEM). *p , 0.05, **p , 0.01, ***p , 0.001. on survival of primary B cells. We purified mouse B cells from without signs of CNS inflammation (Fig. 7A). Moreover, sTACI levels spleens and stimulated them with anti-IgM plus BAFF or APRIL correlated strongly with intrathecal IgG production (Fig. 7B). This and increasing concentrations of sTACI. BAFF increased B cell correlation was confirmed in a second cohort of 25 MS patients (p , survival more strongly than APRIL. This enhanced survival by 0.0001; r = 0.82). We investigated whether this elevation is specific both ligands could efficiently be blocked by sTACI and TACI-Fc for MS or a consequence of compartmentalized inflammation in the (Fig. 6C). These data point to a negative regulatory function of brain. Therefore, we analyzed patients with NB, which is like MS sTACI on B cell survival. characterized by intrathecal IgG production. We detected elevation of sTACIandcorrelationtointrathecal IgG production also in NB, sTACI as a potential biomarker in human immunopathologies which suggests that increased sTACI levels in the CSF are not disease We evaluated whether sTACI is detectable in: 1) inflammatory CNS specific, but the consequence of local accumulation and activation of diseases with compartmentalized IgG production (MS and neu- B cells. In contrast to CSF, in plasma, no significant difference be- roborreliosis [NB]); and 2) a systemic autoimmune disease (SLE). tween patients with MS and ONDs was detected (Supplemental Fig. We found significantly increased sTACI levels in the cerebrospinal 3A). sTACI levels in CSF did not differ among CIS, RR-MS, and SP- fluid (CSF) of MS patients compared with patients with ONDs MS patients (Supplemental Fig. 3B). Immunosuppressive treatment 550 ADAM10 RELEASES SOLUBLE TACI FROM B CELLS influenced sTACI levels, as we found decreased sTACI levels in the the cell surface and observed only little induction of shedding CSF of MS patients 12 mo after initiation of monthly i.v. natalizumab by PMA. treatment (Fig. 7C). Furthermore, sTACI levels were transiently re- TACI shedding was followed by g-secretase cleavage of the duced3daftercorticosteroidtreatmentandreturnedtobaseline remaining membrane stub representing the CTF. It is unclear levels within a period of 4 wk (Supplemental Fig. 3C). In SLE, we whether cleavage of TACI by g-secretase solely leads to the deg- found significantly increased sTACI levels in untreated patients radation of the TACI CTF (proteasome of the membrane) (34) or to compared with healthy control patients (Fig. 7D), which decreased the generation of a TACI intracellular domain with signaling func- upon treatment (Fig. 7D). Interestingly, sTACI levels in untreated tion as is the case for a few other described g-secretase substrates patients correlated with disease activity as expressed by the SLE [e.g., Notch (35)]. Inhibition of g-secretase increased ligand- Disease Activity Index (Fig. 7E). independent NF-kBactivationmediatedbyTACI–D-Ecto in trans- fected HEK293T cells. We speculate that cleavage of the TACI CTF Discussion by g-secretase also takes place in primary B cells, thereby restricting In this study, we report that the membrane-bound BAFF–APRIL TACI-mediated NF-kB activation after shedding of the ectodomain. receptor TACI undergoes ectodomain shedding by ADAM10 and However, formal proof for this hypothesis is lacking at the moment. consecutive cleavage by g-secretase. Shedding of TACI was a con- Previous work with TACI2/2 mice and common variable im- sequence of membrane expression of TACI on activated B cells. The munodeficiency patients revealed a dual role of TACI in B cell generation of soluble receptors is a common principle involving homeostasis (15–19). On the one hand, TACI promotes IgG and a variety of membrane proteins like growth factors, receptors and IgA class-switch recombination (18, 36), maintains Ab production their ligands, cytokines, and cell adhesion molecules (20, 21). We (18, 19), and increases survival of malignant B cells (37). On the identified ADAM10 as the sheddase releasing sTACI, whereas the other hand, TACI has negative regulatory effects on B cells, be- prototype of this superfamily, TNFR1, is shed by ADAM17 (32). cause TACI-deficient mice show a high number of hyperreactive ADAM10 is believed to be mainly involved in the constitutive B cells resulting in autoimmunity and lymphoma development shedding of membrane proteins, whereas ADAM17 can be activated (15–17). Mechanisms that might contribute to this negative reg- by PMA (33). Consistent with this principle, we found ADAM10- ulatory effect include: 1) TACI-mediated induction of BLIMP-1, dependent sTACI production to mainly reflect TACI expression on which orchestrates the switch from B cell proliferation to plasma

FIGURE 7. sTACI as a biomarker. (A– E) sTACI levels were determined by ELISA in CSF or serum; horizontal bars indicate the mean. (A) sTACI is elevated in the CSF of patients with CIS/MS and NB compared with ONDs (two-tailed, unpaired, and nonparametric t test). (B) sTACI in the CSF correlates with intra- thecal IgG production (MS/CIS: p , 0.0001, r = 0.75; NB: p = 0.0167, r = 0.9429; OND: NS, Spearman correla- tion); some data points of OND patients are hidden behind data points of CIS/MS and NB patients. (C) sTACI levels in CSF decrease after 12 mo of natalizumab treatment (n = 25) (two-tailed, paired t test). (D) sTACI is elevated in the serum of untreated SLE patients and less prominent also in the treated SLE cohort compared with healthy controls (HC; two-tailed, unpaired, and nonparametric t test); with treatment, sTACI levels de- crease compared with the untreated SLE patients (two-tailed, unpaired, and non- parametric t test). (E) sTACI in serum of untreated SLE patients correlates strongly with disease activity quantified by the SLE Disease Activity Index (SLEDAI; p = 0.0019; r = 0.697, Spearman correlation). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 551 cell differentiation (38); 2) involvement of TACI in activation- Acknowledgments induced cell death of marginal zone B cells (39); and 3) the role We thank H. Rubsamen€ for excellent technical assistance and Dr. Gurumoorthy of TACI in central removal of autoreactive B cells (40). We pro- Krishnamoorthy and Dr. Naoto Kawakami for critical comments on the pose that the release of endogenous sTACI contributes to these manuscript. negative regulatory features of TACI, because the decoy functions of sTACI reduce BAFF- and APRIL-mediated survival of different Disclosures B cell subpopulations. The authors have no financial conflicts of interest. 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