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BAFF-R, a Newly Identified TNF Receptor That Specifically Interacts with BAFF

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Science  14 Sep 2001:
Vol. 293, Issue 5537, pp. 2108-2111
DOI: 10.1126/science.1061965

Abstract

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

The TNF family ligand BAFF, also known as TALL-1, THANK, BLyS, and zTNF4 (1–5), enhances B cell survival in vitro (6) and has recently emerged as a key regulator of peripheral B cell populations in vivo. Mice overexpressing BAFF display mature B cell hyperplasia and symptoms of systemic lupus erythaematosus (SLE) (7). Likewise, some SLE patients have significantly increased levels of BAFF in their serum (8). It has, therefore, been proposed that abnormally high levels of this ligand may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells (6).

BAFF, a type II membrane protein, is produced by cells of myeloid origin (1, 4) and is expressed either on the cell surface or in a soluble form (1). Two TNF receptor family members, BCMA and TACI, have been shown to interact with BAFF (5, 9–13). APRIL, a TNF ligand with sequence homology to BAFF (14), also binds to these two receptors (11, 13,15).

Initial studies using the BJAB B cell line suggested the existence of a third BAFF receptor: the cells bound high levels of BAFF, but surface BCMA was not detected and mRNA levels for TACI were low. Screening a BJAB expression library with BAFF yielded a cDNA (16) encoding a previously unknown 184–amino acid transmembrane protein that we have named BAFF-R (Fig. 1). The human BAFF-R gene was localized to chromosome 22q13.1-13.31 by homology to a bacterial artificial chromosome (BAC) clone (GenBank Z99716). BAFF-R is a type III transmembrane protein, like BCMA (17) and TACI (18). BAFF-R contains only four cysteine residues in its extracellular or ligand binding domain, making it the smallest cysteine-rich domain (CRD) in the TNF receptor family. TNF receptors are typically organized into multiple CRDs, each composed of six cysteine residues and three disulfide bonds (19). Distinct structural modules within the CRDs have been described (20). The spacing of the four cysteines in BAFF-R most closely resembles the C2 module found in BCMA, TACI, and TNFR (17). A murine BAFF-R cDNA was isolated from a B cell lymphoma library (16), and the encoded protein was found to be 56% identical to the human sequence, with the cysteine residues conserved in number and spacing (Fig. 1). The highest level of homology is in the COOH-terminal region of the proteins, where the sequence identity over 25 consecutive amino acids suggests a conserved signaling motif. BCMA also has some homology to BAFF-R in this region (16).

Figure 1

Aligned amino acid sequences of human and murine BAFF-R. Cysteine residues are in bold; the predicted transmembrane domain is underlined. The vertical lines indicate identical residues, and the dots, similar residues. The GenBank accession numbers are AF373846 for human BAFF-R and AF373847 for murine BAFF-R.

Northern blot analysis on human tissues revealed that BAFF-R is expressed as a 4.5-kb mRNA in the secondary lymphoid organs. High levels of BAFF-R mRNA were detected in the spleen and lymph nodes, lower levels were detected in the peripheral blood leukocytes and thymus, and none was detected in the bone marrow or fetal liver (Fig. 2, A and B). Similarly, murine BAFF-R was expressed at high levels in the spleen and at low levels in the lung and thymus (Fig. 2C). The murine BAFF-R mRNA is approximately 1.9 kb—significantly shorter than the human BAFF-R mRNA.

Figure 2

Expression analysis of BAFF-R. Northern blot analysis of human BAFF-R is shown for (A) human multi-tissue blot and (B) immune system II blot (Clontech, Palo Alto, California). The filters were hybridized at 68°C with the use of a32P labeled Eco NI fragment from the BAFF-R cDNA in ExpressHyb buffer (Clontech). Filters were washed per manufacturer's protocol (Clontech) and exposed to film for 4 days. (C) A mouse tissue Northern blot (Ambion, Austin, Texas) was probed with the use of a labeled fragment from the mBAFF-R cDNA (excised with Pst I and Bgl I). The blot was hybridized, washed as above, and exposed for 1 day. An actin probe revealed similar mRNA levels in each lane.

We determined that BAFF-R specifically bound BAFF with an affinity capable of blocking its function in vitro. When the isolated BAFF-R cDNA was transfected into cells, an antibody to human BAFF-R (anti-hBAFF-R) readily detected surface BAFF-R expression (Fig. 3A). A similar profile was obtained when BAFF was bound to these cells (Fig. 3A). Of note, BAFF bound to BAFF-R– and TACI-transfected cells with similar affinities (16). Further, BAFF binding to various B cell lines correlated strongly with the surface expression of BAFF-R, less so with TACI, and not at all with BCMA expression (16). Because several TNF family receptors have been shown to interact with more than one ligand (21), we tested the ability of 21 TNF ligands to bind to the extracellular domain of BAFF-R. Using BAFF-R:Fc fusion protein [the extracellular domain of BAFF-R fused to the Fc domain of human immunoglobulin G1 (hIgG1)], we found that human BAFF-R interacted with human and murine BAFF but did not bind to any other TNF ligand (Fig. 3B) (22). Most important, APRIL did not bind to BAFF-R:Fc, but it was shown to specifically bind to the TACI and BCMA fusion proteins (Fig. 3B). The functional importance of the BAFF–BAFF-R interaction was established when BAFF-R:Fc was used to block B cell proliferation and BAFF binding assays. Similar to BCMA:Fc, BAFF-R:Fc was able to completely inhibit BAFF-mediated co-stimulation of B cell proliferation (1, 4) at a concentration of 10 μg/ml (Fig. 3C) (23). It also effectively blocked BAFF binding to the BJAB cell line (16).

Figure 3

Specific binding of BAFF to BAFF-R and inhibition with the use of BAFF-R:Fc. (A) BAFF-R is detected on the surface of 293E cells co-transfected with green fluorescence protein (GFP). Cells were stained with 1:100 dilution of preimmune sera or anti-BAFF-R peptide (aa 2–18) antibody (Rb 97) and detected with PE-conjugated antibody to rabbit IgG (Jackson ImmunoResearch). BAFF-R transfected cells were also stained with either no protein or biotinylated BAFF (1 μg/ml) and were detected with PE-streptavidin. (B) A panel of Flag-tagged TNF ligands was bound to BAFF-R:Fc, BCMA:Fc, or TACI:Fc coated plates and detected with the use of the HRP-M2 (Sigma). (C) The ability of BAFF-R:Fc (▪), BCMA:Fc (•) or control IgG (▴) to block an in vitro co-stimulation assay is shown. BAFF plus antibody to IgM alone (⧫). When various amounts of BAFF-R:Fc, BCMA:Fc or TACI:Fc were titrated into the proliferation assay, all the receptors showed half-maximal blocking around 250 ng/ml (35).

A functional role of BAFF-R in the BAFF pathway was next examined with the use of A/WySnJ mice. The phenotype of this well studied mutant mouse line closely resembles that of BAFF knockout mice (24–27). In both mouse lines, the number of mature peripheral B cells is significantly reduced, although bone marrow B lymphopoiesis and peritoneal B1 cells are intact (28). The genomic defect in the A/WySnJ mice was recently localized to chromosome 15 between 27 and 56 centimorgan (29). We localized the murine BAFF-R gene to this region, approximately 48 cM on chromosome 15, by synteny with the use of sequence information from the human BAC that contains BAFF-R (30). The chromosomal location of the BAFF-R gene and the phenotype of A/WySnJ mice prompted us to look for a mutation in the A/WySnJ BAFF-R gene. Northern blot analysis on spleen and lymph node RNA from homozygous A/WySnJ mice and A/J parental mice indicated that the A/WySnJ BAFF-R mRNA was significantly shorter than the parental mRNA (Fig. 4A). Polymerase chain reaction (PCR) analysis of the A/WySnJ genomic DNA failed to detect an intact exon 3, which encodes the intracellular signaling domain of the protein (Fig. 4B) (31). The results of restriction enzyme analysis on the parental and mutant genomic DNA were not consistent with simple exon 3 deletion but rather with a more complex event(s) that could involve insertion, duplication, or rearrangement (16).

Figure 4

Analysis of the A/WySnJ BAFF-R gene. (A) Northern blot analysis of spleen or lymph node RNA isolated from the A/J or A/WySnJ mouse lines (The Jackson Laboratory). Spleen RNA represents three different mice; the lymph node RNA was prepared from pooled tissue. Twenty micrograms of RNA was electrophoresed on a 1.2% formaldehyde gel. The gel was blotted and probed with a 32P-labeled murine BAFF-R cDNA fragment. The filter was hybridized and washed as in Fig. 2. (B) PCR analysis of the BAFF-R coding region from A/J (P), A/WySnJ (M) genomic DNA, or a cloned mBAFF-R genomic fragment (C). Shown are the results of exon 1 (aa 1–48), exon 2 (aa 49–116), and exon 3 (aa 117–175) reactions. (C) FACS plot showing A/J or A/WySnJ splenocytes stained with APC-conjugated anti-B220 and biotinylated BAFF (200 ng/ml) followed by phycoerythrin (PE)-conjugated streptavidin (lymphocyte gate). (D) FACS analysis on splenocytes stained with APC-conjugated anti-B220 and a 1:2000 dilution of antibody to murine BAFF-R (Rb 116) followed by PE-conjugated donkey antibody to rabbit Ig.

The disruption of the intracellular domain of the A/WySnJ BAFF-R protein does not eliminate expression of the extracellular and transmembrane domains located on exons 1 and 2. Fluorescence-activated cell sorting (FACS) analysis of A/WySnJ splenocytes stained with either an antibody to BAFF-R or with biotinylated BAFF revealed that surface BAFF-R was present and capable of binding BAFF, though the profiles were qualitatively and quantitatively different from wild-type B cells (Fig. 4, C and D) (32). Although the complete characterization of the A/WySnJ mutation will require further cloning and sequence analysis, the reduced number of mature B cells observed in these mice most likely results from the inability of the intracellular domain to engage in normal signal transduction.

The A/WySnJ mice exhibit a B cell phenotype similar, but not identical, to animals deficient in BAFF (28). Because the A/WySnJ mice do not have a complete null mutation, it is possible that some signaling occurs through BAFF-R or that TACI and/or BCMA compensate for the BAFF-R receptor defect, leading to a less severe phenotype than the BAFF knockout mice. In contrast, the B cell phenotypes of the BCMA- and TACI-deficient mice differ markedly from that observed in the BAFF-deficient mice (28, 33,34). Antibody responses to various antigens have also been studied in these mice. BAFF-deficient mice are impaired for both T cell–dependent and –independent responses (28), whereas A/WySnJ mice have normal T cell–independent responses, but impaired T cell–dependent responses (25) and TACI knockout mice are only deficient in the T-independent response (34).

We have identified BAFF-R, a previously unknown receptor specific for BAFF that has a role in BAFF signaling which is distinct from the two other known receptors for BAFF, BCMA, and TACI. A functional role for BCMA has not yet been defined, and even though TACI may play a role in T cell–independent responses, BAFF-R appears to be the principal receptor required for BAFF-mediated mature B cell survival and for generating an effective T cell–dependent immune response. Future studies on BAFF-R will more precisely elucidate its role in BAFF signaling.

  • * Present address: Department of Biologics Research, Merck & Co., 126 East Lincoln Avenue, RY 80Y-310, Rahway, NJ 07065, USA.

  • To whom correspondence should be addressed. E-mail: christine_ambrose{at}biogen.com

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