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Originally published In Press as doi:10.1074/jbc.M306852200 on July 16, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38220-38228, October 3, 2003
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{Delta}BAFF, an Alternate Splice Isoform That Regulates Receptor Binding and Biopresentation of the B Cell Survival Cytokine, BAFF*

Amanda L Gavin {ddagger} §, Djemel Aït-Azzouzene {ddagger}, Carl F. Ware ¶ and David Nemazee {ddagger}

From the {ddagger}Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 and the Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121

Received for publication, June 26, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor necrosis family member BAFF is limiting for the survival of follicular B lymphocytes, but excessive BAFF signaling can lead to autoimmunity, suggesting that its activity must be tightly regulated. We have identified a conserved alternate splice isoform of BAFF, called {Delta}BAFF, which lacks 57 nt encoding the A–A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse {Delta}BAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. {Delta}BAFF can associate with BAFF in heteromultimers and diminish BAFF bioactivity and release. Thus, alternative splicing of the BAFF gene suppresses BAFF B cell stimulatory function in several ways, and {Delta}BAFF may promote other functions as well.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulation of lymphocyte survival and differentiation is of central importance to immunity and the homeostasis of the immune system. BAFF1 (also known as BLYS, TALL-1, THANK, ZTNF4, TNFSF13B) is an important TNF-related molecule with critical effects on B cells (1–6). Produced predominantly by myeloid cells, BAFF is a B cell survival factor and a costimulator of B cell proliferation and antibody secretion (7, 8). The human BAFF homotrimer crystal structure, typical of the TNF family, has been determined by three different groups (9–11). The basic "jellyroll {beta}-sandwich" of the TNF monomer fold is retained in BAFF. The monomer-monomer interactions are mediated by conserved hydrophobic residues, forming three grooves on the sides of the trimer that can interact with receptors.

TNF family polypeptides are type II transmembrane proteins biologically active when presented in the membrane-bound position or in the soluble phase following release from the membrane by specific proteolysis in the membraneproximal "stalk" region (8, 12–15). In the case of TNF, membrane release requires the cis-acting metalloproteinase TACE (TNF-{alpha} converting enzyme) (16). BAFF has been found both on cell surfaces and in solution, but the proteases responsible for its release have not been identified. However, the basic cleavage site in the stalk region of BAFF is consistent with a furin-like convertase (8).

BAFF binds to three different receptors, BAFF-R, TACI, and BCMA. All of these receptors are expressed on B cells, although their expression levels change with maturation (17). In addition, TACI is also expressed on activated T cells (18). Mice functionally deficient in BAFF-R (19) are severely depleted in follicular and marginal zone B cells (20–22). BAFF-deficient mice have a very similar phenotype (23), suggesting that the BAFF-R has a nonredundant function in maintaining B cell survival. In contrast, the TACI knockout is defective in responses to T cell-independent antigens but not in B cell survival. On the contrary, B cell numbers are increased 2-fold in mice lacking TACI (24), and these mice develop autoimmunity and lymphomas with age (25). The role of the BAFF/BCMA interaction is less well understood, as mouse BAFF binds mouse BCMA poorly and mice deficient in BCMA have no obvious phenotype (23, 26). In addition, APRIL, the TNF-family ligand most closely related to BAFF, is able to bind to TACI and BCMA but not to the BAFF-R (26, 27). It is known that human BAFF can form heteromultimers with APRIL, and this formation seems to be up-regulated in patients with systemic autoimmune diseases (28).

BAFF overexpression in vivo promotes autoimmune lupus-like disease and potentiates antibody responses (23, 29–31). Because of the appeal of BAFF as a therapeutic target in systemic autoantibody diseases, the BAFF/BAFF-R interaction has been blocked experimentally. Importantly, treatment of lupus-prone mice or a mouse model of collagen-induced arthritis with soluble Fc fusion proteins of TACI, which binds to BAFF, can reduce disease incidence and severity (32). On the other hand, mice deficient in BAFF or the BAFF receptor, BAFF-R, lack long-lived follicular B cells and are hyporesponsive to immunization (20, 23, 32). It is important to understand how the balance between BAFF-mediated B cell survival and autoimmunity is controlled.

We have identified a splice isoform of BAFF that exists in both mouse and human. This form, which we call {Delta}BAFF because it lacks a 57-bp exon, can assemble disulfide-linked complexes both with itself and heteromultimerizes with full-length BAFF. The 57-bp deletion affects the compartmentalization of BAFF and receptor binding specificity. This study identifies {Delta}BAFF as a novel regulator of B cell survival.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Anti-FLAG M2 antibody-coupled agarose, anti-FLAG M2 antibody horseradish peroxidase (HRP), anti-polyhistidine (IgG2a clone HIS-1), and human IgG were purchased from Sigma. Goat anti-IgG2a HRP (Southern Biotechnology Assoc.), goat anti-rabbit Ig HRP (BD-Pharmingen), rabbit IgG anti-BAFF (ProSci Inc.), and anti-Myc HRP (Invitrogen) were also used. Human TACI-Fc fusion protein was a kind gift from Dr. M. Cancro (University of Pennsylvania, Philadelphia).

Cell Culture—The cells lines J774, WEHI-3, and Pu5-1.8 were cultured in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal bovine serum, 25 mM HEPES, 1 mM sodium pyruvate, 55 µM 2-mercaptoethanol, penicillin, and streptomycin (Invitrogen). The cell lines HL-60, THP-1, and U937 were cultured in RPMI 1640 supplemented as for IMDM. Bone marrow-derived macrophages were cultured essentially as described (33), although IMDM was used instead of RPMI 1640. In some instances, phorbol 12-myristate 13-acetate (10 ng/ml) or lipopolysaccharide (1 µg/ml) was added to the cultures for 8 h prior to harvest. 293 EBNA cells were maintained in IMDM as above but supplemented with 0.25 mg/ml Geneticin (Invitrogen). Stably transfected 293 EBNA cells expressing recombinant soluble BAFF were maintained in IMDM as above and supplemented with 0.25 mg/ml Geneticin and 0.5 µg/ml puromycin (Sigma).

Cloning Mouse BAFF—Total RNA was harvested from the cells of mouse (WEHI-3, J774, Pu5-1.8, B10.D2 bone marrow-derived macrophages, and B10.D2 splenocytes) or human (HL-60, U937, THP-1) origin using TRIzol (Invitrogen). CDNA was generated from 5 µg of total RNA using oligo(dT) and SuperScript first strand synthesis system for reverse transcriptase-polymerase chain reaction (Invitrogen) according to manufacturers instructions. 1–2 µl of cDNA reaction was used as template for the amplification of BAFF utilizing PLATINUM Pfx DNA polymerase enzyme from Invitrogen according to manufacturer's instructions. Soluble mouse BAFF was amplified from cDNA using the primers 5'-ACTGTGCTAGCTCAGGGACCAGAGGAAAC-3' and 5'-TCTCGGATCCTGGATCACGCACTCCAGCAAG-3'. Full-length BAFF cDNA was amplified using 5'-CGGGCGGATCCCATGGATGAGTCTGCAAAGACC-3' and 5'-TCTCCTCGAGGTCGACGGTATCGATAAGCTTGATA-3'. Human BAFF was amplified using 5'-ACTGTGCTAGCTCAGGGTCCAGAAGAAACA-3' and 5'-TCTCGGATCCTAATAGCTACAGACATGGTGTAAGTA-3'.

The amplification products were electrophoresed on 4% agarose gels and then gel-purified and cloned into pBluescript for DNA sequencing (Applied Biosystems). The sequences for mouse and human {Delta}BAFF cDNA have been deposited in GenBankTM (accession numbers AY290823 [GenBank] and AY302751 [GenBank] , respectively). Full-length mouse BAFF cDNA containing an upstream Myc tag on the N terminus was cloned into the bicistronic retroviral vector, pMXI-IRES-EGFP (34). The full-length mouse {Delta}BAFF cDNA with an N-terminal FLAG tag was cloned into pMX1-IRES-TAC, where the EGFP cDNA from pMXI-IRES-EGFP was replaced with the cDNA encoding human CD25 as a downstream marker gene.

Expression of BAFF Isoforms—S17 cells, a bone marrow stromal cell line, were infected by retroviruses encoding the mouse BAFF isoforms. Briefly, retroviruses were generated by the transfection of the constructs into Phoenix cells by CaPO4 precipitation, and the supernatant was harvested 48 h post-transfection. The supernatants containing the retroviruses and 1% (v/v) dioleoyltrimethylammoniumpropane (DOTAP, Roche Applied Science), were then incubated with monolayers of S17 cells for 2 h while being centrifuged at 2000 x g at 30 °C. The supernatant was replaced with fresh IMDM growth medium, and positively infected cells were sorted by flow cytometry based on their expression of EGFP, human CD25, or both. In some cases, full-length transmembrane BAFF isoforms were expressed transiently in 293T cells using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions.

Recombinant sBAFF—Recombinant mouse soluble BAFF (sBAFF) and soluble delta BAFF (s{Delta}BAFF) with either N-terminal polyhistidine or FLAG epitope tags, were subcloned into pCEP.Pu (pCEP4 containing a puromycin resistance cassette) or pCMV-Script (Stratagene). The pCep.Pu constructs were transfected into 293 EBNA cells (Invitrogen), and stable transfectants were selected for use with Geneticin (0.25 mg/ml) and puromycin (0.5 µg/ml). Recombinant soluble BAFF proteins were purified from culture supernatant using anti-FLAG-agarose chromatography according to the manufacturer's instructions (Sigma).

Biochemical Analysis of BAFF Isoforms—Culture supernatants from stably transfected 293 EBNA cells were used as a source of sBAFF and s{Delta}BAFF. Recombinant BAFF was immunoprecipitated from culture supernatant using either anti-His or anti-FLAG-agarose beads. The beads were washed with phosphate-buffered saline, in some cases boiled in the presence of 0.5% SDS and 1% {beta}-mercaptoethanol, and then incubated with peptide N-glycosidase F (PNGase F) buffer and 1% Nonidet P-40; to half of the samples, 1000 units of PNGase F (New England Biolabs) was added. After incubation at 37 °C for 1 h, samples were incubated with sample buffer (reducing and nonreducing) at 56 °C for 10 mins and then boiled for 5 min. Iodoacetamide was added, and the samples were electrophoresed on 15% acrylamide SDS-PAGE. Nonreducing samples were treated in the same manner but were not initially denatured with SDS and {beta}-mercaptoethanol. In some cases, cell lysate and culture supernatant from S17 cells infected with various retroviruses expressing full-length BAFF isoform constructs was used. Western blotting was performed, and the BAFF isoforms were detected using anti-BAFF rabbit polyclonal, anti-His, anti-Myc, and anti-FLAG antibodies and enhanced chemiluminescent detection. In some cases, 1 x 107 S17 cells transduced with retroviruses were surface-iodinated using lactoperoxidase (Sigma) and hydrogen peroxide for 4 min before lysis with Nonidet P-40 buffer containing 1% Nonidet P-40, 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Anti-FLAG-agarose beads were added to the lysates and incubated at 4 °C for 2 h with rotation, and then the samples were washed five times with lysis buffer prior to analysis by SDS-PAGE under reducing conditions and autoradiography.

Receptor Binding Assay—50 µg of TACI-Fc fusion protein was iodinated using Iodogen-coated tubes and Na125I according to the manufacturer's instructions (Pierce Endogen). Various concentrations of 125I-TACI-Fc was incubated with S17 cells alone or with retrovirally transduced S17 cells at 5 x 106 cells/ml for 3 h on ice prior to centrifugation through phthalate oils (3:2 (v/v) dibutyl phthalate/dioctyl phthalate; Aldrich) and bound 125I-TACI-Fc (cellular pellet) and free 125I-TACI-Fc (supernatant) were determined. Ratios of bound and free fractions were calculated, and Scatchard analysis was performed. In some cases, TACI-Fc and human IgG were incubated with lysates from the transduced S17 cell lines, and bound material was immunoprecipitated with protein A-agarose beads before the samples were analyzed by SDS-PAGE and immunoblotting with rabbit anti-BAFF polyclonal antibody.

BAFF Biological Assay—B cells were purified from spleen and lymph nodes from B10.D2 mice using CD43 or B220 MACS beads and columns (Miltenyi Biotec). CD43-B cells were cultured at 1 x 106 cells/ml with 10 µg/ml goat F(ab)2 anti-mouse IgM (Southern Biotechnology), plated on monolayers of various dilutions of irradiated S17 cells, and incubated for 72–96 h. Cell viability was assessed by forward and side scatter analysis performed on a FACScan (BD Biosciences). In some cases, B220+ B cells were incubated with titrations of recombinant purified soluble BAFF forms in medium containing 25 µg/ml polymyxin B and 10 µg/ml anti-IgM as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of {Delta}BAFF Transcripts in Mouse and Human Myeloid Cells—In the course of reverse transcription and polymerase chain reaction cloning of BAFF from cDNA generated from the mouse myeloid cell line WEHI-3, we observed that half of the clones had a slightly smaller than expected size. Sequencing analysis revealed that the smaller transcript, herein known as {Delta}BAFF, was identical to the published BAFF sequence but lacked 57 bp encoding the predicted A to A1 strands and the intervening loop (Fig. 1A) (9). The {Delta}BAFF isoform lacks Ile156 to Lys184, and Gly185 is substituted with an arginine. The loss of these residues maintains the reading frame, leading to the generation of a new N-linked glycosylation site at Asn155 in {Delta}BAFF, as threonine is now the second residue downstream. Fig. 1 illustrates the gene structures for both human (Fig. 1B) and mouse BAFF (Fig. 1C). The exons skipped in the {Delta}BAFF transcripts of human and mouse, respectively, are exons 3 and 4. The conservation of reading frames and homologies between mouse and human BAFF gene structures indicated a possible function for {Delta}BAFF.



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  		FIG. 1.
Predicted amino acid sequence of mouse BAFF (M) and the splice isoform delta BAFF ({triangleup}) and a comparison of the mouse and human BAFF gene structures. A, the intracellular, transmembrane, stalk, and predicted membrane cleavage sites are indicated. The strand and loop designations given to the extracellular domains are according to the crystal structure of human BAFF (9). Residues not encoded by the {Delta}BAFF transcript are shown as dashed lines. The number symbol (#) indicates the generation of a new predicted N-linked glycosylation site, and asterisks indicate the other predicted N-linked glycosylation sites. The bold letters indicate a change in the predicted amino acid sequences. B and C, schematic diagrams of the structure of human (B) and mouse (C) BAFF genes. The exons are drawn as boxes, expressed regions are shown in black and untranslated regions in white, and the intronic sizes are listed. The alternatively spliced exon missing in {Delta}BAFF is indicated.

 

To assess whether {Delta}BAFF mRNA was expressed in other myeloid cells, reverse transcriptase-PCR was performed on a range of mouse and human cell lines (Fig. 2). {Delta}BAFF was identified in all of the myeloid cell lines investigated, although the ratio of BAFF to {Delta}BAFF varied between 1:1 and 3:1 in mouse cell lines. Primary mouse bone marrow-derived macrophages also expressed the alternatively spliced isoform (Fig 2A, lane 15). The {Delta}BAFF isoform was expressed in the human cell lines tested, although it was only a minor species. A larger transcript (BAFF-{varphi}) was identified in the human cell lines, but sequencing revealed this transcript to be nonfunctional because of incomplete splicing of intronic sequences leading to premature stop codons. The smaller transcripts identified in HL-60, J774, Pu5-1.8, and B10.D2 macrophages were gel-purified and sequenced, revealing that they were identical to {Delta}BAFF, which was identified initially from WEHI-3 cDNA. These results indicate that {Delta}BAFF is expressed along with BAFF in many mouse and human myeloid cells and cell lines.



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  		FIG. 2.
Expression of {Delta}BAFF transcripts. A and B, reverse transcriptase-PCR analysis of BAFF and {Delta}BAFF transcripts from mouse (A) and human (B) sources. Cells used as sources of RNA are listed; some were stimulated with phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS) as indicated. The amplifications were performed on either cDNA reaction mixtures (designated +RT) or controls lacking reverse transcriptase (designated–RT). BMM{phi}, bone marrow-derived macrophages. Ratios of BAFF:{Delta}BAFF as determined by densitometry are indicated.

 

{Delta}BAFF Has an Additional Glycosylation Site and Forms Disulfide-bonded Multimers—The biochemical properties of {Delta}BAFF were analyzed by generating peptide-tagged and truncated soluble BAFF and {Delta}BAFF (sBAFF and s{Delta}BAFF, respectively) in 293 EBNA cells (Fig. 3A). Under reducing conditions, the s{Delta}BAFF form (lane 5) had a higher apparent molecular weight than sBAFF (lane 2). Incubation with PNGase F revealed that the slowed mobility of s{Delta}BAFF was the result of additional N-linked glycosylation at the newly generated site. The addition of PNGase F and reducing reagents led to the predicted mobility of s{Delta}BAFF (Fig. 3A, lane 4) relative to sBAFF (lane 1). The electrophoretic mobility difference was more easily observed when both forms were co-expressed by 293 EBNA cells (lanes 7–9). Interestingly, under nonreducing conditions some multimers of sBAFF were observed (lane 3), but s{Delta}BAFF appeared mostly as high molecular weight multimers (lanes 6 and 9), implying a role for disulfide bonds in multimer stabilization. We conclude that {Delta}BAFF mRNA encodes a protein with similarities to BAFF, including immunoreactivity and the ability to covalently homomultimerize.



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  		FIG. 3.
Biochemical characterization of {Delta}BAFF. A, recombinant soluble mouse BAFF isoforms were immunoprecipitated from the supernatant from 293 EBNA cells transfected with constructs encoding histidine-tagged sBAFF, FLAG-tagged s{Delta}BAFF, or both. Recombinant soluble forms of BAFF were immunoprecipitated with either anti-His or anti-FLAG-agarose, and the samples were electrophoresed by SDS-PAGE under either reducing or nonreducing conditions. Prior to SDS-PAGE, some samples were incubated with PNGase F. B, full-length BAFF isoforms were expressed by transiently transfecting 293T cells, and either whole cell lysates (left panel) or concentrated culture supernatant (right panel) were electrophoresed by SDS-PAGE under reducing conditions. Mock indicates 293T cells transiently transfected with empty expression vector. All of the samples (A and B) were then analyzed by Western blotting using a polyclonal rabbit anti-BAFF antibody.

 

Unlike BAFF, {Delta}BAFF Is Not Efficiently Shed from Expressing Cells—Full-length transmembrane-anchored forms of BAFF and {Delta}BAFF cDNAs were transiently transfected into 293T cells to compare these isoforms for membrane expression and shedding into the extracellular medium. BAFF and {Delta}BAFF were detected in cell lysates, although {Delta} BAFF was expressed to a lesser extent (Fig. 3B, left panel). BAFF that had been cleaved was detected in the culture supernatant (Fig. 3B, right panel), whereas {Delta}BAFF was not detected. Interestingly, the amount of BAFF cleaved in cells co-expressing both forms of BAFF was less than BAFF alone, implying a role for {Delta}BAFF in inhibiting BAFF secretion.

To further assess the biochemical properties of the transmembrane forms of BAFF and {Delta}BAFF in a system with stable expression, pMX1 retroviral constructs were generated encoding either full-length BAFF with an N-terminal Myc tag or full-length {Delta}BAFF with an N-terminal FLAG tag (Fig. 4A). The BAFF constructs were linked to either EGFP or human CD25 via an internal ribosomal entry site (IRES) sequence, which allowed detection of the positively infected cells using flow cytometry. A mouse bone marrow stromal cell line, S17, was infected with the various retroviruses, and infected cells were sorted based on expression of EGFP, human CD25, or both (Fig. 4B). Three stably expressing S17 cell lines were generated, S17 BAFF-Myc, S17 {Delta}BAFF-FLAG, and S17 BAFF-Myc + {Delta}BAFF-FLAG, which expressed either full-length BAFF or full-length {Delta}BAFF or co-expressed both, respectively. Similar expression levels of the BAFF isoforms were detected in the cell lysates of the transduced S17 cells (Fig. 4C, lanes 2–4). Analysis of the culture supernatant, however, revealed that although BAFF could be cleaved and released into the supernatant (Fig. 4C, lane 6), nothing was detected in supernatant from cells expressing {Delta}BAFF alone (lane 7). Culture supernatant from cells expressing both forms showed some cleavage of BAFF (Fig. 4C, lane 8) although this was reduced by 40%. As expected, no BAFF was detected in either lysate or supernatant from S17 cells alone (Fig. 4C, lanes 1 and 5). These data indicate that unlike BAFF, {Delta}BAFF is not released efficiently from expressing cells and that {Delta}BAFF expression might hinder release of BAFF.



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  		FIG. 4.
Expression and localization of protein encoded by full-length {Delta}BAFF cDNA. A, diagram of pMX retroviral constructs used to generate stable expression of either BAFF with an N-terminal Myc tag or {Delta}BAFF with an N-terminal FLAG tag. The BAFF or {Delta}BAFF cDNAs are linked via an IRES to the markers EGFP or human CD25, respectively. B, expression of BAFF constructs by S17 cells. S17 cells were left untreated or transduced with retroviruses encoding BAFF-Myc, {Delta}BAFF-FLAG, or a mixture of both retroviruses. Infected cells showed either green fluorescence (EGFP) or human (Hum) CD25 staining when analyzed by flow cytometry. C, the level of BAFF and {Delta}BAFF expression by S17 cells alone or after infection with BAFF-Myc, {Delta}BAFF-FLAG, or both retroviruses was determined by Western blotting of total cell lysate and concentrated culture supernatant using anti-BAFF polyclonal antibody. D, surface expression of {Delta}BAFF. S17 cells alone or S17 infected with retroviral constructs were cell surface-iodinated, and then cell lysates were incubated with anti-FLAG-agarose beads, and the immunoprecipitated material was analyzed by SDS-PAGE and autoradiography.

 

{Delta}BAFF and BAFF Form Heteromultimers—Because the inability of {Delta}BAFF to be released from cells might have important biological consequences, the mechanism of its retention was explored further. As the predicted proteolytic cleavage site was retained in {Delta}BAFF, its inability to be shed into the supernatant implied that it was either not expressed on the cell surface or that the loss of residues around the A-strand and additional glycosylation may have inhibited cleavage. Surface iodination and immunoprecipitation from lysates of S17 cells that expressed the various BAFF forms revealed that {Delta}BAFF, when expressed alone, was present on the cell surface (Fig. 4D, lane 3). Cell surface expression was also observed when both BAFF and {Delta}BAFF were co-expressed (Fig. 4D, lane 4). This result indicated that {Delta}BAFF multimers could be expressed on the cell surface and suggested that {Delta}BAFF could have a function independent of full-length BAFF.

Because some members of the TNF superfamily can form heterotrimers, the possibility that {Delta}BAFF associates with BAFF was tested in a co-precipitation assay using epitope-tagged versions of these molecules. Immunoprecipitations were performed from lysates of S17 cells expressing either {Delta}BAFF alone (Fig. 5A, lane 3, upper panel) or co-expressing {Delta}BAFF with BAFF (lane 4, upper panel). BAFF (carrying a Myc tag) was co-precipitated with {Delta}BAFF from cells expressing both forms (Fig. 5A, lane 4, lower panel), indicating the formation of heteromultimers between BAFF and {Delta}BAFF. This association was also demonstrated by co-precipitation of soluble BAFF (sBAFF-His) with the immunoprecipitation of soluble {Delta}BAFF (s{Delta}BAFF-FLAG) from the supernatants of transfected 293EBNA cells (Fig. 5B, lane 3). Thus, heterotrimers form in cells co-expressing BAFF and {Delta}BAFF.



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  		FIG. 5.
Analysis of the association of {Delta}BAFF with BAFF. A, cell lysates from either S17 alone or S17 expressing full-length BAFF (Myc tag), {Delta}BAFF (FLAG tag), or both were incubated with anti-FLAG-agarose, and the immunoprecipitated material (IP) was analyzed by Western blotting with either anti-FLAG- or anti-Myc-specific antibodies. B, supernatants from 293 EBNA cells expressing soluble BAFF with a histidine tag, soluble {Delta}BAFF with a FLAG tag, or both forms were incubated with anti-FLAG-agarose beads, washed, and analyzed by Western blotting with anti-FLAG- and anti-His-specific antibodies

 

{Delta}BAFF Fails to Bind to TACI and Suppresses BAFF B Cell Bioactivity—As {Delta}BAFF is truncated in the A–A1 loop, which in other TNF-related family members plays a crucial role in receptor interactions (35), we compared {Delta}BAFF with BAFF for its ability to recognize the TACI-Fc fusion protein in an immunoprecipitation assay. Lysates of control S17 cells or S17 cells expressing various BAFF forms were incubated with human TACI-Fc fusion protein or control human IgG, immunoprecipitated with protein A-agarose beads, and analyzed by immunoblotting (Fig. 6). As expected, BAFF bound TACI-Fc but not control IgG (Fig. 6, lanes 2 and 6, respectively); in contrast, {Delta}BAFF failed to bind detectably to TACI-Fc (lane 3). BAFF was also precipitated from lysates of cells co-expressing both forms, although slightly less material was observed (Fig. 6, lane 4). This result was confirmed in cell binding assays in which iodinated TACI-Fc was incubated with control or experimental S17 cells expressing the various BAFF forms (Fig. 7A). As expected, BAFF-expressing S17 cells could bind 125I-TACI-Fc, whereas S17 cells expressing {Delta}BAFF could not. Interestingly, although S17 cells co-expressing both forms bound 125I-TACI-Fc with a similar affinity to cells expressing BAFF alone, the number of sites/cell was only 60% that of BAFF alone. Analysis of the levels of BAFF expression in both cell lines (as determined by the bicistronic gene marker, EGFP) revealed that both cells lines had high EGFP expression, and cells co-expressing both BAFF isoforms had equal or slightly higher mean fluorescence (Fig. 7B). As others have shown that the marker expression correlates well with the expression of the input gene, we conclude that although similar amounts of BAFF are being made, the co-expression of {Delta}BAFF, and presumably the co-association in heterotrimers, limits the amount of functional BAFF on the cell surface.



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  		FIG. 6.
{Delta}BAFF does not bind to the receptor, TACI. Cell lysates from S17 cells alone or S17 cells infected with BAFF and {Delta}BAFF retroviruses were incubated with TACI-Fc fusion protein or human IgG and then with protein A-Sepharose beads. The immunoprecipitated material (IP) was analyzed by SDS-PAGE and Western blotting with anti-BAFF polyclonal antibody. The BAFF moiety is indicated with an arrow.

 


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  		FIG. 7.
{Delta}BAFF modifies the surface expression of functional BAFF. A, Scatchard plots of 125I-TACI-Fc binding to S17 cells alone or S17 cells infected with retroviruses. The Kd and B-max values are indicated. ND, not determined. B, flow cytometry histograms of the EGFP fluorescence of uninfected S17 cells (dotted line) or S17 cells expressing BAFF-Myc (left) or S17 cells expressing both {Delta}BAFF-FLAG and BAFF-Myc (bold line). The percentage of cells expressing EGFP and the geometric mean fluorescence are indicated.

 

To see whether co-expression of {Delta}BAFF may regulate the biological activity of BAFF in promoting B cell survival, we performed a co-stimulation assay with purified B cells incubated with monolayers of control or BAFF isoform-expressing S17 cell lines and stimulated with anti-B cell receptor (anti-BCR) antibodies. 72 h after incubation with the S17 cells, the B cells were scored for viability by flow cytometry analysis of forward scatter and side scatter parameters. Viable and proliferating cells are marked in the R1 gate (Fig. 8A). B cells incubated on S17 cells expressing BAFF showed increased viability (% of R1 cells) when compared with cells incubated with S17 alone. B cells incubated with S17 cells expressing {Delta}BAFF alone were not co-stimulated, indicating that {Delta}BAFF either does not bind or does not generate a signal through the BAFF receptor (Fig. 8B). S17 cells co-expressing both forms, however, showed an intermediate co-stimulatory activity, implying that {Delta}BAFF can down-regulate BAFF function when co-expressed. Furthermore, recombinant sBAFF, s{Delta}BAFF, and heterotrimers (predominantly of the form s{Delta}BAFF2sBAFF1) isolated from the supernatants of transfected cells were tested for B cell binding and for bioactivity in a B cell survival assay (Fig. 8C). Only sBAFF showed significant activity, indicating that {Delta}BAFF "poisons" the B cell stimulatory properties of BAFF-containing heterotrimers not only by intracellular retention but also by direct interference with receptor stimulation.



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  		FIG. 8.
Co-expression of {Delta}BAFF modulates BAFF function. A, flow cytometry analysis of forward scatter versus side scatter plots of CD43–splenocytes after 3 days of culture on irradiated S17 cells alone or those expressing BAFF-Myc. The medium contained goat anti-mouse IgM antibody at 10 µg/ml. R1 gate designates viable B cells. B, CD43–splenocytes were cultured as described in A, on monolayers of titrated and irradiated S17 cells alone (X) or S17 expressing BAFF-Myc (filled squares) or {Delta}BAFF-FLAG (open triangles) or co-expressing both {Delta}BAFF-FLAG and BAFF-Myc (half-filled squares). The percentages of viable and proliferating cells in the R1 gate as shown in A were plotted against the plated S17 cell number. The assay was performed in duplicate. C, B220+ splenocytes were cultured with 10 µg/ml goat anti-mouse IgM antibody and titrations of purified recombinant soluble BAFF (rs-BAFF, filled squares), recombinant soluble {Delta}BAFF (rs{Delta}BAFF, open squares), recombinant soluble {Delta}BAFF/BAFF (half-filled squares), or media alone (*). The percentage of cells in the viable R1 gate (A) was plotted against the concentration of recombinant proteins.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BAFF plays a critical role in maintaining the homeostasis of peripheral B cells, but its level must be carefully regulated; too much or too little BAFF signaling causes massive changes in the B cell pool, resulting in an autoimmune syndrome (29, 30) or hypogammaglobulinemia (23), respectively. Here we have characterized {Delta}BAFF, a novel splice isoform of BAFF that appears to negatively regulate BAFF activity in three ways.

First, mouse {Delta}BAFF fails to bind to two different receptors recognized by BAFF, TACI and BAFF-R, indicating that skipping exon 4 during splicing blocks BAFF function. In addition, soluble recombinant {Delta}BAFF did not bind to cell lines expressing BAFF-R and TACI (data not shown). Second, we find that {Delta}BAFF physically associates with BAFF in disulfide-bonded heteromultimers (Fig. 5) and that soluble forms of these mixed molecules bind poorly to receptors relative to homomultimers of BAFF (Fig. 8C). Thus, {Delta}BAFF can suppress BAFF function by competitive co-association, limiting BAFF homotrimerization. Finally, {Delta}BAFF co-expression appears to regulate the ability of BAFF to appear on the cell surface and to be subsequently shed into the extracellular space. This appears to be due to the retention of {Delta}BAFF/BAFF multimers inside the cell. As the function of other family members, such as TNF, is tightly regulated by receptors being kept in intracellular stores (36), it is possible that this is the main mechanism by which {Delta}BAFF regulates BAFF function. We therefore suggest that {Delta}BAFF may play important roles in limiting BAFF function at both the post-transcriptional and post-translational levels. Analysis of primary myeloid cells and several myeloid cell lines revealed that the {Delta}BAFF transcript was prevalent in BAFF-producing cells, indicating that its pattern of expression is consistent with such a regulatory role. In addition, it is clear that under certain stimulatory conditions BAFF:{Delta}BAFF mRNA ratios can change (Fig. 2), implying a sensitive transcriptional mechanism to control the co-stimulation of B cells by BAFF.

We have shown that cells co-expressing both BAFF and {Delta}BAFF have less functional BAFF on the cell surface and released into the extracellular medium than cells expressing equivalent levels of BAFF alone. Although the inhibition of function of BAFF by the co-expression of {Delta}BAFF was incomplete, it is clear that in vivo even 2-fold differences in BAFF levels can have profound effects on the peripheral B cell pool. Studies in A/WySnJ, a mouse strain that has a mutant, hypoactive allele of BAFF-R (called Bcmd) revealed that peripheral B cells require continual BAFF-mediated signals for survival and that slightly reducing the BAFF signal in (Bcmd/+)F1 mice significantly reduces B cell life span (20, 22). Hence the total size of the peripheral B cell pool in (Bcmd/+)F1 mice is reduced by 50% relative to wild type (22). Similarly, heterozygous BAFF knockout mice have about half of the normal levels of serum immunoglobulins (23). These findings suggest that by limiting BAFF bioavailability {Delta}BAFF is likely to play an important regulatory role.

This study identifies alternate splicing as a method for modulating the amount of functional BAFF available to B cells. Many alternatively spliced transcripts have been identified in the TNF superfamily (37–39). The alternatively spliced fourth exon in the mouse BAFF gene encodes the A–A1 strands in the protein structure. Recent studies investigating the human BAFF/BAFF receptor interaction reveal a role for this region in receptor interactions (40, 41). In addition, the mutation of homologous stretches in related family members, including TNF, lymphotoxin, and receptor activator of nuclear factor {kappa}B ligand (RANKL), similarly leads to ablation of cognate receptor binding (42–45). It is interesting to note that in the case of human APRIL, a TNF family member closely related to BAFF, a transcript (TRDL-1{beta}) lacking the equivalent exon encoding the A–A1 strand has also been described although not functionally characterized (46). As is the case for mouse {Delta}BAFF, TRDL-1{beta} also creates an additional potential N-linked glycosylation site at the new exon:exon junction, and in light of the data presented herein, we would predict altered function for this APRIL isoform. As heterotrimers between APRIL and BAFF have been described and are found in higher amounts in humans with systemic autoimmune disease, it is interesting to postulate the role {Delta}BAFF and the APRIL isoform TRDL-1{beta} may have in tightly regulating the potency of BAFF/APRIL functions. Alternate splicing of the region critical for receptor binding may be an efficient way to regulate how much functional BAFF, or APRIL, is on the surface or is available for proteolytic cleavage.

We do not exclude the possibility that, in addition to its ability to limit BAFF function, {Delta}BAFF may have independent bioactivity. Importantly, surface iodination/immunoprecipitation studies revealed that mouse {Delta}BAFF can be found on the cell surface (Fig. 4D). This finding is apparently in contrast to the unpublished data mentioned by Mackay et al. (6) regarding the human {Delta}BAFF form, a discrepancy that may be explained by species or cell type differences. The surface expression of {Delta}BAFF indicates that although it does not bind to two of the known BAFF receptors, {Delta}BAFF may have a unique receptor specificity or function, yet to be determined.

It is possible that the tight regulation of BAFF by differential splicing that is suggested by our results is representative of a broader theme in lymphocyte homeostasis. In the case of interleukin-15, a molecule unrelated to the TNF receptor family that plays a role in the maintenance of NK (natural killer cells), NKT, and memory CD8 T lymphocytes, an alternatively spliced transcript lacking exon 5 remains in the intracellular compartment where it retains and significantly limits the bioactivity of the normally active form (47). It remains to be seen whether this is a universal property of survival-enhancing cytokine genes.


   FOOTNOTES
 
* This work was supported in part by the United States Public Health Service, National Institutes of Health Grants A13306 [GenBank] 8 (to C. F. W.) and GM44809 and AG01743 (to D. N.), and a post-doctoral fellowship from the Cancer Research Institute (to A. L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9546; Fax: 858-784-9554; E-mail: agavin{at}scripps.edu.

1 The abbreviations used are: BAFF, B cell-activating factor belonging to TNF family; TNF, tumor necrosis factor; BAFF-R, BAFF receptor; sBAFF, soluble BAFF; BCMA, B cell maturation antigen; TACI, transmembrane activator and CAML (calcium modulator and cyclophilin ligand)-interacting protein; 125I-TACI-Fc, 125I-labeled TACI-Fc; APRIL, a proliferation-inducing ligand; PNGase F, peptide N-glycosidase F; IRES, internal ribosomal entry site; EGFP, enhanced green fluorescent protein; IMDM, Iscove's modified Dulbecco's medium; HRP, horseradish peroxidase. Back


   ACKNOWLEDGMENTS
 
We thank Mike Cancro for TACI-Fc reagents and Luc Teyton and Annica Mårtensson for critical reading of the manuscript.



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