cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF13) gene in bat (Vespertilio superans Thomas)

A proliferation-inducing ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the full-length cDNA of APRIL (designated bAPRIL) from bat was cloned using RT-PCR and its biological activities have been characterized. The open reading frame (ORF) of this cDNA consists of 753 bases, encoding a protein of 250 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known APRIL homologs. Real-time quantitative PCR (qPCR) analysis indicated that bAPRIL mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsAPRIL was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that bsAPRIL could bind to its receptors on B cells. In vitro, MTT assays indicated that bsAPRIL could promote the survival/proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that bsAPRIL plays an important role in the survival and proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.     

Crystal structure of extracellular human BAFF, a TNF family member that stimulates B lymphocytes

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family, plays a critical role in regulating survival and activation of peripheral B cell populations and has been associated with autoimmune disease. BAFF is known to interact with three receptors, BCMA, TACI and BAFF-R, that have distant similarities with other receptors of the TNF family. We have determined the crystal structure of the TNF-homologous domain of BAFF at 2.8 A resolution. The structure reveals significant differences when compared to other TNF family members, including an unusually long D-E loop that participates in the formation of a deep, concave and negatively charged region in the putative receptor binding site. The BAFF structure was further used to generate a homology model of APRIL, a closely related TNF family ligand that also binds to BCMA and TACI, but not BAFF-R. Analysis of the putative receptor binding sites of BAFF and APRIL suggests that differences in the D-E loop structure and electrostatic surface potentials may be important for determining binding specificities for BCMA, TACI and BAFF-R.     

sBAFF mutants induce neutralizing antibodies against BAFF

B cell activating factor belonging to the TNF family (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family and plays an important role in B lymphocyte maturation and survival. Overexpression of BAFF is closely involved in the pathogenesis and progression of many kinds of autoimmune disorders; therefore, BAFF has been considered as an ideal therapeutic target for these conditions. In this study, we generated several candidate immune inhibitors of human BAFF by conjugating foreign immunodominant T-helper cell (Th) epitopes to the N- or C-terminus of five BAFF mutants. The recombined proteins were successfully expressed in Escherichia coli (E. coli) and purified by Ni-NTA chromatography. BALB/c mice immunized with the recombinant proteins produced high levels of anti-BAFF antibodies, and their sera inhibited the lymphocyte proliferation-inducing activity of recombinant soluble BAFF and natural soluble BAFF. Moreover, antibodies cross-reactive with BAFF were detected in sera from hu-SCID mice immunized with the recombinant proteins. These results indicated that the recombinant BAFF mutants modified with Th epitopes could induce neutralizing antibodies against BAFF in vivo. This study may provide a valuable strategy for treating BAFF-associated autoimmune diseases.     

Multiple Novel Classes of APRIL-specific Receptor-blocking Peptides Isolated by Phage Display

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC₅₀ values of 0.49-27 μM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.     

Expression,characterization of recombinant human soluble BAFF secreted from CHO cell

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 μg/ml to 0.55 μg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.     

The BAFF/APRIL system: Emerging functions beyond B cell biology and autoimmunity

The BAFF system plays a key role in the development of autoimmunity, especially in systemic lupus erythematosus (SLE). This often leads to the assumption that BAFF is mostly a B cell factor with a specific role in autoimmunity. Focus on BAFF and autoimmunity, driven by pharmaceutical successes with the recent approval of a novel targeted therapy Belimumab, has relegated other potential roles of BAFF to the background. Far from being SLE-specific, the BAFF system has a much broader relevance in infection, cancer and allergy. In this review, we provide the latest views on additional roles of the BAFF system in health and diseases, as well as an update on BAFF and autoimmunity, with particular focus on current clinical trials.     

Construction and characterization of a bi-functional EGFP/sBAFF fusion protein

A fusion between gene encoding fluoresce-enhanced green fluorescent protein variant (EGFP) and soluble domain of human B-cell-activating factor of the TNF family (sBAFF) was constructed and expressed in Escherichia coli. The EGFP/sBAFF had an apparent molecular weight of 45 kDa and was detected with anti-hsBAFF and anti-His(6) monoclonal antibodies. After being purified by immobilized metal affinity chromatography (IMAC), the fusion protein retained similar fluorescence spectra to those of EGFP. Biological activity assays showed the EGFP/sBAFF as well as sBAFF could co-stimulated human B lymphocyte proliferation in vitro. In addition, EGFP/sBAFF has shown specific binding to BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active sBAFF that may prove useful in further studies on BAFF and its receptors.     

The function of BAFF on T helper cells in autoimmunity

B cell-activating factor belonging to the TNF family (BAFF) exerts its pathogenic role in supporting the survival and proliferation of B cells, regulating class switch recombination as well as the selection of autoreactive B cells. Overexpression of BAFF induces a dramatic expansion of activated B cells, particularly marginal zone B cells, as well as hypergammaglobulinemia, autoantibody production and immune complex deposition. However, in addition to its effect on B cells, recent work has also demonstrated that BAFF can promote T cell activation, proliferation and differentiation. In this review, we have discussed the recent progress on the function and role of BAFF on T cells and T cell-mediated diseases.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus)

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753 bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.     

Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection

Background

It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.

Methods

Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.

Results

CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.

Conclusions

Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0201-y) contains supplementary material, which is available to authorized users.     

Molecular mechanism of the selectivity between BAFF/APRIL and their receptors by molecular simulations

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) belonging to the tumour necrosis factor (TNF) ligand family can bind three unusual TNF receptors (BCMA, TACI and BR3) with various binding affinities. BAFF and APRIL are regarded as promising therapeutic targets for autoimmune diseases because of their pivotal roles in cell survival and immune regulation. In this work, we carried out molecular dynamics calculations to explore the structural and chemical features responsible for ligand recognition by extracellular functional segments of TNF receptors. We found that the conserved pocket D_cons of BAFF/APRIL contacted the DxL motif of TNF receptors, while the D_spe1–3 sub-domains were responsible for their different affinities, especially D_spe1 and D_spe2. The residues at position II–V of DxL motif were wrapped into the D_cons pocket via salt-bridge and hydrophobic interactions. The hydrophobic residues of strand3 and helix1 in TNF receptors provided remarkable contributions for the affinities to BAFF/APRIL. Additionally, Arg^VI of DxL motif played a key role in the binding selectivity via salt-bridge interaction with residue D275B in BAFF. Arg27 in BCMA contributed to the high affinity for APRIL so that BCMA showed a preference for APRIL. Our studies indicated that Arg84 and Gln95 in TACI2 played an important role in the selectivity of two cysteine-rich domain segments in TACI, leading to the higher binding affinities of TACI2 than those of TACI1. The primary cause of the disability to bind APRIL was the space conflict with the rigid conformation of the C-terminus coil of BR3. These thorough understanding of the molecular mechanism for BAFF/APRIL recognition by their receptors provides new insights for guiding inhibitor design.     

BAFF inhibition: a new class of drugs for the treatment of autoimmunity

BAFF (BLyS) and APRIL are TNF-like cytokines that support survival and differentiation of B cells. Recent studies have discovered a role for BAFF in augmenting both innate and adaptive immune responses as well as in collaborating with other inflammatory cytokines to promote the activation and differentiation of effector immune cells. BAFF is an important pathogenic factor in lupus mouse models and BAFF inhibition successfully delays disease onset in these mice, although the responsiveness to BAFF inhibition varies among different strains. These results have led to the development of inhibitors targeting BAFF and APRIL in humans. An anti-BAFF antibody has shown significant but modest efficacy in two Phase III clinical trials for moderately active SLE and other inhibitors are being developed or at early stages of clinical testing.     

Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells

B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.     

The current status of targeting BAFF/BLyS for autoimmune diseases

It is increasingly recognized that B cells have multiple functions that contribute to the pathogenesis of autoimmunity. Specific targeting of B cells might therefore be an appropriate therapeutic intervention. The tumor necrosis factor-like molecule BAFF (BLyS) is a key B cell survival factor and its receptors are expressed on most peripheral B cells. Several different BAFF antagonists are under development and in early clinical trials. We review here the rationale for BAFF blockade, and its predicted mechanism of action in autoimmune diseases.     

The first BAFF gene cloned from the cartilaginous fish

B-cell activating factor (BAFF), a member of the TNF family, is critical to the survival, proliferation, maturation, and differentiation of B-cells. In the present study, a CpBAFF was amplified from the white-spotted catshark (Chiloscyllium plagiosum) using RT-PCR and RACE (rapid amplification of cDNA end) techniques. To our knowledge, this is the first report of any BAFF gene being cloned from a cartilaginous fish. The open reading frame (ORF) of CpBAFF cDNA consists of 819 bases encoding a protein of 272 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other identified BAFF homologues. Sequence alignment showed that CpBAFF shares 37-57% identity with BAFF amino acid sequences reported in other vertebrates. Three-dimensional structure modeling analysis revealed a soluble mature portion of CpBAFF (CpsBAFF) with a long D-E loop specific to the BAFF gene, which has not been found in other reported TNF proteins. Phylogenetic reconstruction showed that CpBAFF is most closely related to other fish BAFFs and clusters with BAFF genes from higher vertebrates (reptiles, birds, and mammals). Real-time quantitative RT-PCR demonstrated that CpBAFF mRNA expression was high in the spleen but moderate in the kidney and branchia. Recombinant CpsBAFF fused to NusA-His(6)-tag was efficiently expressed in Escherichia coli BL21 (DE3), and a molecular weight of approximately 83 kDa was determined using SDS-PAGE and Western blotting. In vitro MTT assay indicated that the purified pET43.1a (+)-CpsBAFF protein can co-stimulate the proliferation of mammalian B-cells with anti-IgM in a dose-dependent manner. The present findings not only present novel information that may be relevant to shark immunity but also provide some new insights into the origins and evolution of immunity in all vertebrates.     

BAFF在自身免疫性疾病方面的研究进展

自身免疫性疾病的特征是B细胞耐受丧失,B细胞激活因子（B cell activating factor belonging to theTNF family,BAFF）通过与受体结合,对B细胞的增殖、存活起重要作用。BAFF转基因小鼠易出现自身免疫性疾病。因此,拮抗或抑制BAFF的表达可能是治疗自身免疫性疾病的靶点。本文主要对BAFF在自身免疫性疾病方面的研究进展进行综述。     

Serum B-cell activating factor predicts prognosis in nasal type,extranodal NK/T cell lymphoma

B-cell activating factor (BAFF) plays important roles in a variety of lymphoid malignancies. Compared with healthy adults, patients with non-Hodgkin lymphoma had higher level of serum BAFF, and it corresponded with disease severity, response for therapy and clinical outcome. Latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) which is a known agent of nasal, extranodal NK/T cell lymphoma (ENKTCL) can switch the BAFF activating promoter leading to higher expression of BAFF in EBV-related tumor cells. However, the relationship between BAFF and ENKTCL has not been reported. Here we proposed a hypothesis that BAFF might play a regulatory role in ENKTCL development and maintenance. Our results showed that serum BAFF in ENKTCL patients was significantly higher than that in control group and negatively correlated with patients’ survival. It may be a valuable prognostic factor and deserved further study.