IL-10 Production B Activation and Κ BAFF-R-Mediated NF

TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-κB Activation and IL-10 Production This information is current as of September 26, 2021. Liang-Guo Xu and Hong-Bing Shu J Immunol 2002; 169:6883-6889; ; doi: 10.4049/jimmunol.169.12.6883 http://www.jimmunol.org/content/169/12/6883 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-␬B Activation and IL-10 Production

Liang-Guo Xu* and Hong-Bing Shu2*†

TALL-1 is a member of the TNF family that is critically involved in B cell survival, maturation, and progression of lupus-like autoimmune diseases. TALL-1 has three receptors, including BCMA, TACI, and BAFF-R, which are mostly expressed by B lymphocytes. Gene knockout studies have indicated that BAFF-R is the major stimulatory receptor for TALL-1 signaling and is required for normal B cell development. The intracellular signaling mechanisms of BAFF-R are not known. In this report, we attempted to identify BAFF-R-associated downstream proteins by yeast two-hybrid screening. This effort identified TNFR-associated factor (TRAF)3 as a protein specifically interacting with BAFF-R in yeast two-hybrid assays. Coimmunoprecipitation Downloaded from experiments indicated that BAFF-R interacts with TRAF3 in B lymphoma cells and this interaction is stimulated by TALL-1 treatment. Domain mapping experiments indicated that both a 6-aa membrane proximal region and the C-terminal 35 aa of BAFF-R are required for its interaction with TRAF3. Moreover, overexpression of TRAF3 inhibits BAFF-R-mediated NF-␬B activation and IL-10 production. Taken together, our findings suggest that TRAF3 is a negative regulator of BAFF-R-mediated NF-␬B activation and IL-10 production. The Journal of Immunology, 2002, 169: 6883–6889. http://www.jimmunol.org/ ALL-1, also called BAFF, Blys, THANK, and zTNF4, is uria, and glomerulonephritis, phenotypes that mimic those of sys- a member of the TNF family of ligands identified by us temic lupus erythemia (3, 5, 14, 29, 30). Conversely, it has been T and others (1Ð5). TALL-1 is specifically and constitu- shown that recombinant soluble TACI-Ig fusion proteins can sig- tively expressed by monocytes, macrophages, and dendritic cells nificantly inhibit progression of lupus-like autoimmune syndrome (1Ð3, 6), and is induced by IFN-␥ and IL-10 (3, 6). Like most and collagen-induced arthritis in animal models (27, 31). Recently, members of the TNF family, the extracellular domain of TALL-1 gene knockout studies further confirmed that TALL-1 is required can be cleaved to form a soluble cytokine (2). Crystal structure for normal B cell development (26). Surprisingly, gene inactiva- studies suggest that soluble TALL-1 (sTALL-1)3 contains an tion studies also indicated that BAFF-R, but not TACI and BCMA, unique “flap” region that is important for its virus-like assembly, is required for TALL-1-triggered B cell development (26, 32Ð34). by guest on September 26, 2021 receptor binding, and biological activities (7Ð9). Taken together, these studies suggest that the TALL-1/BAFF-R TALL-1 signals through three receptors, including BCMA, signaling plays critical roles in regulation of B cell function and TACI, and BAFF-R, which are members of the TNFR family (5, autoimmune diseases. 10Ð24). All three receptors are mainly expressed by B lympho- The signal transduction pathways triggered by BCMA, TACI, cytes, while TACI is also induced in a subset of T cells following and BAFF-R are poorly characterized. Like many other members their activation (17Ð24). Early studies suggest that sTALL-1 can of the TNFR family, BCMA and TACI can bind to TNFR-asso- potently stimulate B lymphocyte proliferation in vitro, either alone ciated factor (TRAF) proteins and activate the transcription factor or in synergy with anti-IgM (2, 3, 10Ð13). It is now believed that NF-␬B and the serine/threonine protein kinase JNK (10Ð13, 16, sTALL-1 promotes either differentiation of B cells or survival of 35). In addition, it has also been shown that TACI can activate the mature B cells (25Ð28). Administration of recombinant sTALL-1 transcription factor NF-AT (36). or overexpression of sTALL-1 in mice leads to increased numbers The intracellular signaling pathways and downstream effectors of mature B lymphocytes, splenomegaly, anti-DNA Abs, protein- triggered by BAFF-R are not known. In this study, we identified TRAF3 as a BAFF-R-associated protein. Our findings suggest that TRAF3 negatively regulates BAFF-R-mediated NF-␬B activation *Department of Immunology, National Jewish Medical and Research Center, Uni- versity of Colorado Health Sciences Center, Denver, CO 80206; and †Department of and IL-10 production. Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, People’s Republic of China Received for publication July 12, 2002. Accepted for publication October 11, 2002. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Reagents and cell culture charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Mammalian-derived recombinant Flag-tagged human sTALL-1 (10) and 1 This work was supported by grants from the Ellison Medical Foundation, National Escherichia coli-derived recombinant His6-tagged human sTALL-1 (7) Institutes of Health (1RO1 AI49992-01), Arthritis Foundation, National Natural Sci- were previously described. The human embryonic kidney 293 (Dr. Z. Cao, ence Foundation of China (39925016), Chinese “863” Program (2001AA221281), Tularik, South San Francisco, CA), the B lymphoma Bjab (Dr. J. Hagman, and Special Funds for Major State Basic Research of China (G19990539). National Jewish Medical and Research Center, Denver, CO) and RPMI 2 Address correspondence and reprint requests to Dr. Hong-Bing Shu, Department of 8226 (American Type Culture Collection (ATCC), Manassas, VA) cells, Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, the mAbs against Flag (Sigma-Aldrich, St. Louis, MO) and hemagglutinin K516c, Denver, CO 80206. E-mail address: [email protected] (HA) (Covance, Berkeley, CA) epitopes, and the rabbit polyclonal anti- 3 Abbreviations used in this paper: sTALL-1, soluble TALL-1; T2, transitional type II; TRAF3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) were obtained LT␤R, lymphotoxin-␤ receptor; TRAF, TNFR-associated factor; HA, hemagglutinin. from the indicated sources.

Bjab and RPMI 8226 cells were cultured in RPMI 1640 medium con- minescent kit (Tropix, Medford, MA). Luciferase activities were normal- taining 10% FBS. 293 cells were maintained in high glucose DMEM con- ized on the basis of ␤-galactosidase expression levels. taining 10% FCS. ELISA Yeast two-hybrid screening Human IL-10 ELISA were performed using the human IL-10 ELISA To construct a BAFF-R bait vector, a cDNA fragment encoding for aa Ready-Set-Go kit (eBioscience, San Diego, CA) by following procedures 97Ð184 of BAFF-R was inserted in-frame into the Gal4 DNA-binding do- recommended by the manufacturer. main vector pGBT (Clontech Laboratories, Palo Alto, CA). The human B cell cDNA library (ATCC) was screened as described (37, 38). Results Identification of TRAF3 as a specific BAFF-R-interacting Coimmunoprecipitation and Western blot protein 293 cells (5 ϫ 106) were transfected with the indicated plasmids. Twenty- four hours after transfection, cells were lysed in 1 ml lysis buffer (20 mM The intracellular signaling pathways mediated by BAFF-R are un- Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 ␮g/ml aprotinin, known. Similar to other members of the TNFR family, BAFF-R 10 ␮g/ml leupeptin, 1 mM PMSF). For each immunoprecipitation, 0.4 ml does not have intrinsic enzymatic activity and is believed to trans- aliquot of lysates was incubated with 0.5 ␮g of the indicated mAb or duce signals through physical interaction with downstream signal- ␮ control mouse IgG, and 25 l of a 1:1 slurry of GammaBind G Plus Sepha- ing proteins. To identify BAFF-R-associated signaling proteins, rose (Amersham Pharmacia Biotech, Piscataway, NJ) for at least 1 h. The Sepharose beads were washed three times with 1 ml lysis buffer containing we used the yeast two-hybrid system to screen a human B cell 500 mM NaCl. The precipitates were fractionated on SDS-PAGE and sub- cDNA library with the intracellular domain of BAFF-R as bait. We sequent Western blot analyses were performed as described (37, 38). screened a total of 5 million independent clones and obtained 78 ␤ Downloaded from Vectors -galactosidase-positive clones. Sequence analysis of 20 clones indicated that most clones encode a poorly characterized protein To construct C-terminal HA-tagged expression plasmids for BCMA, p14.5 (40). Subsequent transient transfection and coimmunopre- TACI, BAFF-R, and BAFF-R mutants, cDNAs for these proteins were amplified by PCR from a B cell cDNA library and inserted into a C- cipitation experiments indicated that BAFF-R did not interact with terminal HA-tagged pcDNA3 plasmid (39). p14.5 in 293 cells (data not shown). One of the sequenced clones To make retroviral vectors for TRAF3 and its TRAF domain (aa 258Ð encodes for TRAF3.

568) mutant (TRAF3-C), cDNAs encoding for full-length TRAF3 and aa Because TRAF proteins are involved in signaling by many http://www.jimmunol.org/ 258Ð568 were ampliﬁed by PCR and inserted into the MSCV2.2IRES- members of the TNFR family (41Ð43), we decided to investigate GFP␣ retroviral vector (provided by Dr. P. Marrack, National Jewish Med- ical and Research Center). a potential role for TRAF3 in BAFF-R signaling. To determine Expression plasmids for TRAF1, 2, 3, 5, 6 (Dr. D. Goeddel, Tularik) and whether TRAF3 is associated with BAFF-R in mammalian cells, NF-␬B-luciferase reporter plasmid (Dr. G. Johnson, University of Colo- expression plasmids for C-terminal HA-tagged BAFF-R and N- rado Health Sciences Center, Denver, CO) were provided by the indicated terminal Flag-tagged TRAF3 were transfected into 293 cells. Co- investigators. immunoprecipitation experiments indicated that BAFF-R inter- Establishment of BCMA, TACI, and BAFF-R stable cell lines acted with TRAF3 (Fig. 1A). In the same experiments, BAFF-R did not interact with other cytoplasmic TRAF proteins, including

Expression plasmids for C-terminal HA-tagged BCMA, TACI, and by guest on September 26, 2021 BAFF-R were linearized and transfected into RPMI 8226 cells or Bjab TRAF1, TRAF2, TRAF5, and TRAF6 (Fig. 1A). TRAF4 was not cells by electroporation. The transfected cells were selected by G418 (1 included in these experiments because it is primarily localized in mg/ml) for 2 wk. Cells with high-level receptor expression were sorted the nucleus in mammalian cells (41Ð43). These data suggest that with Flag-tagged sTALL-1 by flow cytometry. Overexpression of the receptors in the stable lines were confirmed by Western blot with BAFF-R specially interacts with TRAF3. anti-HA Ab. Domain mapping of the interaction between BAFF-R and Establishment of TRAF3 and TRAF3-C-terminal stable cell lines TRAF3 Retroviral plasmid (15 ␮g) for wild-type TRAF3 or TRAF3-C was trans- TRAF proteins interact with members of the TNFR family through fected into the packaging cell line 293Ð10A1 (ϳ2 ϫ 106) by calcium phos- their TRAF domains (41Ð43). We determined whether the TRAF phate precipitation. Eighteen hours after transfection, the cells were domain of TRAF3 (TRAF3-C) is sufficient to interact with washed with PBS and cultured in 5 ml of fresh medium for 24 h. The recombinant retrovirus-containing medium was collected and centrifuged. BAFF-R. As shown in Fig. 1B, transient transfection and coim- The supernatant, supplemented with 4 ␮g/ml of polybrene, was used to munoprecipitation experiments suggest that the TRAF domain of infect RPMI 8226 or Bjab cells (ϳ2 ϫ 106). Two days after infection, TRAF3 is sufficient for interaction with BAFF-R. green fluorescent protein-positive cells were isolated using a cell sorter. Previously, conserved TRAF-binding motifs, such as (P/S/A/ Flow cytometry analysis T)x(Q/E)E and PxQxxD, have been identified in the cytoplasmic domains of some TNFR family members (41Ð44). However, these RPMI 8226 or Bjab cells were incubated in staining buffer (PBS/2% FBS) conserved TRAF-binding motifs are not easily recognizable in the in the absence or presence of Flag-sTALL-1 (100 ng/ml) for 40 min. Cell staining was performed by sequential incubation (each 40 min) with anti- cytoplasmic domain of BAFF-R. To determine the regions of Flag mAb (1 ␮g/ml) and R-PE-conjugated goat anti-mouse IgG (1/200 BAFF-R that are required for interaction with TRAF3, we con- dilution) in staining buffer. Cells were washed two times with staining structed a series of C-terminal HA-tagged deletion mutants of the buffer following each incubation. Cells with high-level Flag-sTALL-1 BAFF-R (Fig. 2A). Transient transfection and coimmunoprecipi- binding were isolated by a cell sorter. tation experiments suggest that six aa (PDGDKD) at the mem- Reporter gene assays brane proximal region (aa 117Ð122) and the C-terminal tail (aa Bjab stable cell lines (ϳ2 ϫ 105) were seeded on 6-well (35-mm) dishes 150Ð184) are both required for interaction with TRAF3 (Fig. 2, A and were transfected with 1.0 ␮gofNF-␬B-luciferase reporter plasmid by and B). Lipofectamine 2000 (Invitrogen, Carlsbad, CA). To normalize for transfection efficiency and protein amount, 0.5 ␮g of RSV-␤-galactosidase plas- TRAF3 is recruited to BAFF-R by TALL-1 stimulation mid was added to all transfections. Fourteen hours after transfection, cells Under physiological conditions, BAFF-R is specifically expressed were treated with His-sTALL-1 (200 ng/ml) or untreated for 6 h. Lucif- erase reporter assays were performed using a luciferase assay kit (BD in B lymphocytes. We next determined whether BAFF-R interacts PharMingen, San Diego, CA) and following the manufacturer’s protocols. with TRAF3 in B lymphoma cells and whether this interaction is ␤-Galactosidase activity was measured using the Galacto-Light chemilu- affected by TALL-1 stimulation. The Journal of Immunology 6885

enhanced the interaction between BAFF-R and TRAF3. The weak interaction between BAFF-R and TRAF3 in the absence of sTALL-1 is probably due to the fact that overexpression of BAFF-R mimics TALL-1 stimulation of endogenous BAFF-R. It is possible that the association of TRAF3 with BAFF-R is dependent on TALL-1 stimulation in untransfected cells. In these experiments, TRAF3 was also recruited to TACI, but not to BCMA, in a ligand-dependent manner (Fig. 3). These data are consistent with previous observations that TRAF3 interacts with TACI, but not BCMA, in mammalian overexpression systems (10, 13).

Inhibition of BAFF-R-mediated NF-␬B activation by TRAF3 Many TNFR family members, including TALL-1 receptors BCMA and TACI, can activate the transcription factor NF-␬B. Previously, we have shown that TALL-1 can induce NF-␬B activation in the B lymphoma RPMI 8226 cells (39). To determine whether BAFF-R can activate NF-␬B, we transfected a NF-␬B- luciferase reporter plasmid into Bjab cells overexpressing

BAFF-R. Luciferase assays indicated that overexpression of Downloaded from BAFF-R could signiﬁcantly induce NF-␬B activation, and this effect was enhanced by sTALL-1 stimulation (Fig. 4). Moreover, overexpression of TRAF3 and TRAF3-C both inhibited BAFF-R- mediated NF-␬B activation (Fig. 4).

Inhibition of BAFF-R-mediated IL-10 expression by TRAF3 http://www.jimmunol.org/ Previously, we showed that TALL-1 stimulation or overexpression of BAFF-R in RPMI 8226 cells induced IL-10 production (39). To determine whether TRAF3 is involved in BAFF-R-induced IL-10 production, we transfected TRAF3 or its TRAF domain into BAFF-R-overexpressing RPMI 8226 cells by retroviral-mediated gene transfer and measured IL-10 levels by ELISA. The results indicated that both wild-type TRAF3 and TRAF3-C inhibited BAFF-R-induced IL-10 production (Fig. 5). by guest on September 26, 2021 Discussion After migrating from the bone marrow to the spleen, immature B cells undergo two transitional stages, transitional type I and II (T2), before differentiating into naive mature B cells. In vitro ex- FIGURE 1. BAFF-R interacts with TRAF3 in 293 cells. A, BAFF-R periments suggest that sTALL-1 specifically promotes the survival interacts with TRAF3 but not TRAF1, 2, 5, and 6. 293 cells were trans- of T2 B cells and their differentiation into mature B cells (25, 27, fected with expression plasmids for C-terminal HA-tagged BAFF-R and 28). Consistent with these observations, inhibition of TALL-1 sig- the indicated Flag-tagged TRAF proteins. Cell lysates were immunopre- naling by administration of TACI-Ig decoy receptor or gene cipitated with anti-HA Ab or control mouse IgG. The immunoprecipitates knockout of TALL-1 causes deficiency of T2 and mature B cells were analyzed by Western blot with anti-Flag Ab (upper panel). Expres- (26, 27, 31). These studies point to a crucial role for TALL-1 in B sion of BAFF-R and TRAF proteins was confirmed by Western blots with anti-HA (lower panel) and anti-Flag (middle panel) Abs, respectively. B, cell survival and maturation. The TRAF domain of TRAF3 is sufficient for interaction with BAFF-R. Among the three TALL-1 receptors, BCMA and TACI are not 293 cells were transfected with expression plasmids for C-terminal HA- important for B cell development because normal B cell matura- tagged BAFF-R and wild-type TRAF3 or its TRAF domain mutant. Cell tion is found in BCMA- and TACI-deficient mice (32Ð34). In con- lysates were immunoprecipitated with anti-HA Ab or control mouse IgG. trast, inactivation of BAFF-R in mice causes loss of T2 and mature The immunoprecipitates were analyzed by Western blot with anti-TRAF3 B cells (26). These studies suggest that BAFF-R signaling is es- Ab, which recognizes the C-terminal TRAF domain of TRAF3 (upper sential for TALL-1-triggered B cell survival and maturation. panel). Expression of BAFF-R, TRAF3, and its mutant was confirmed by The downstream effector molecules responsible for TALL-1/ Western blots with anti-HA (lower panel) and anti-TRAF3 (middle panel) BAFF-R-triggered B cell survival and maturation are not clear. It Abs, respectively. is possible that TALL-1 signaling initiates the anti-apoptotic activity that is associated with the activation of NF-␬B. Previously, it has been shown that disruption of NF-␬B activation pathways, To do this, the Bjab lymphoma cells were stably transfected such as by gene knockout of IKK␣, Rel, and RelA, impairs B cell with C-terminal HA-tagged BAFF-R, BCMA, and TACI. To de- maturation (45Ð47). In this context, we found in this study that termine whether BAFF-R is associated with endogenous TRAF3 BAFF-R could also activate NF-␬B. in these cells, we performed coimmunoprecipitation experiments Both BCMA and TACI can activate NF-␬B but are not important with anti-HA Ab and the immunoprecipitates were analyzed by for TALL-1-triggered B cell survival, suggesting that activation of Western blots with anti-TRAF3 Ab. These experiments indicated NF-␬B is not sufficient for TALL-1-triggered B cell survival. Re- that BAFF-R could weakly interact with endogenous TRAF3 in cently, we identified multiple downstream genes transcriptionally in- Bjab cells (Fig. 3). Moreover, sTALL-1 stimulation significantly duced by TALL-1, including the cytokine IL-10, the chemokine 6886 TRAF3 IS INVOLVED IN BAFF-R SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Domain mapping of BAFF-R interaction with TRAF3. A, A schematic presentation of the BAFF-R deletion mutants and their interaction with TRAF3. ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; ϩ, interaction detected; Ϫ, no interaction. B, Coim- munoprecipitation between TRAF3 and BAFF-R mutants. 293 cells were transfected with expression plasmids for Flag-tagged TRAF3 and the indicated C-terminal HA-tagged BAFF-R mutants. Cell lysates were immunoprecipitated with anti-HA Ab and the immunoprecipitates were analyzed by Western blot with anti-Flag Ab (upper panel). Expression of BAFF-R mutants and TRAF3 was conﬁrmed by Western blots with anti-HA (middle panel) and anti-Flag (lower panel) Abs, respectively.

LAG-1, and GCP-2, the secreted protein pre-B cell colony enhancing volved in signaling by many members of the TNFR family (41Ð factor, among others (39). Among the genes induced by TALL-1, 43). TRAF3 was first identified as a molecule that binds to the IL-10 is particularly interesting. It has been shown that IL-10 can cytoplasmic tails of CD40 and EBV-transforming protein LMP1 suppress cytokine production and several accessory cell functions by (51Ð54). Signaling through CD40 in B cells causes rescue from Th1 cells, macrophages, and NK cells and is regarded as a potent apoptosis, proliferation, differentiation, Ig production, class suppressor of the effector functions of these cells (48). Conversely, switching, and expression of costimulatory molecules. Overex- IL-10 is a potent stimulator of B cell proliferation and differentiation pression of TRAF3 inhibits CD40-mediated Ab secretion (54). The and is critically involved in regulating autoantibody-secreting B cell TRAF domain of TRAF3 is sufficient to bind to CD40 and also activities in systemic lupus erythemia (49, 50). Our previous studies inhibits CD40-mediated Ab secretion when overexpressed, sug- suggest that BAFF-R, but not TACI and BCMA, can dramatically gesting that the physical association of TRAF3 with CD40 medi- up-regulate IL-10 expression in primary and transformed B cells (39), ates its negative regulatory function (55). TRAF3 is also recruited pointing to the possibility that IL-10 is an important effector molecule in a ligand-dependent manner to lymphotoxin-␤ receptor (LT␤R) for TALL-1-triggered B cell survival and maturation. and has an inhibitory effect on LT␤R-mediated NF-␬B activation In an attempt to decipher the intracellular signaling pathways (56, 57). In this context, it is interesting that both TRAF3 and its mediated by BAFF-R, we identified TRAF3 as a cytoplasmic pro- TRAF domain inhibit BAFF-R-mediated downstream effects, such tein physically binding to the cytoplasmic domain of BAFF-R. as NF-␬B activation and IL-10 production. Taken together, these TRAF3 is a member of the TRAF protein family, which is in- studies suggest that TRAF3 is involved in negative regulation of The Journal of Immunology 6887

FIGURE 3. BAFF-R interacts with endogenous TRAF3 in B lymphoma cells. Bjab cells stably transfected with HA-tagged BCMA, TACI, BAFF-R, or a control vector were treated with sTALL-1 (200 ng/ml) (ϩ) or left untreated for 5 min. Cells were then lysed and the lysates were immunoprecipitated with anti-HA Ab. The immunoprecipitates were ana-

lyzed by Western blot with anti-TRAF3 Ab (upper panel). Expression of Downloaded from BCMA, TACI, and BAFF-R was conﬁrmed by Western blot with anti-HA Ab (middle panel). Expression of endogenous TRAF3 was conﬁrmed by Western blot with anti-TRAF3 Ab (lower panel).

signaling by several TNFR family members, including BAFF-R,

CD40, and LT␤R. http://www.jimmunol.org/ The cytoplasmic domain of BAFF-R is not conserved with those of other TNFR family members. The major conserved TRAF-binding motif, (P/S/A/T)x(Q/E)E (44), is not easily recognizable in the cytoplasmic domain of BAFF-R. A 6-aa sequence at position 117 of BAFF-R, PDGDKD, is weakly similar to the minor consensus TRAF-binding motif PxQxxD found in the EBV LMP1 protein and the TRAF-interacting protein I-TRAF/TANK (44). Previ- ously, it has been shown that TRAF3 binds to LT␤R through amino acids PEEGDPG at position 389 of LT␤R (57). This site is by guest on September 26, 2021 also not found in BAFF-R. We made a series of deletion mutants of BAFF-R and examined their interaction with TRAF3 in coim- FIGURE 5. Effects of TRAF3 on TALL-1- and BAFF-R-induced IL-10 munoprecipitation experiments. These studies suggest that the production. A, TRAF3 inhibits TALL-1-induced IL-10 production in RPMI PDGDKD motif at position 117 and the C-terminal 35 aa are both 8226 cells. RPMI 8226 cells stably transfected with empty control or TRAF3 plasmid were treated with sTALL-1 (100 ng/ml) for 24 h (ϩ)or left untreated Ϫ. IL-10 levels in the culture supernatants were measured by ELISA. B, TRAF3 inhibits BAFF-R-mediated IL-10 production. IL-10 levels in culture supernatants of RPMI 8226 cells stably transfected with BAFF-R, or BAFF-R plus TRAF3 or TRAF3-C were measured by ELISA. Data shown are averages and SDs of three independent experiments.

required for BAFF-R’s association with TRAF3. The simplest ex- planation for this observation is that these amino acids form a novel spatial structure for BAFF-R binding to TRAF3. In this context, it has previously been shown by crystal structure studies that binding of TRAF3, but not TRAF2, to CD40 is inﬂuenced by both the PXQXT motif and residues distal to this site (58Ð60). Alter- natively, it is also possible that TRAF3 binds only to one site in the cytoplasm of BAFF-R in vivo and the results from our mapping experiments are due to altered folds caused by deletion. In conclusion, we have identiﬁed TRAF3 as a BAFF-R-associated signaling protein. TRAF3 can inhibit BAFF-R-mediated ␬ FIGURE 4. TRAF3 inhibits BAFF-R-mediated NF- B activation. Bjab NF-␬B activation and IL-10 production, suggesting a negative reg- cells stably overexpressing BAFF-R, or BAFF-R plus TRAF3 or ulatory role for TRAF3 in TALL-1-triggered B cell survival and TRAF3-C, were transfected with NF-␬B-luciferase and RSV-␤-galactosidase reporter plasmids. Fourteen hours after transfection, cells were treated maturation. with sTALL-1 (200 ng/ml) (f) or left untreated (Ⅺ) for 6 h. Luciferase activities were measured and normalized based on ␤-galactosidase levels. Acknowledgments Data shown are averages and SDs of one representative experiment in We thank Drs. Zhaodan Cao, David Goeddel, Gary Johnson, and Philippa which each transfection was performed in triplicate. Marrack for reagents. 6888 TRAF3 IS INVOLVED IN BAFF-R SIGNALING

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