Human BAFF Research Articles

Soluble Bcma in Myeloma Serum Binds Its Ligands BAFF and Prevents Normal Antibody Production in Multiple Myeloma Patients

Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels.Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on C57 Bl/6 and SCID mice implanted with the human MM xenografts LAGλ-1, LAGk-1A or LAGk-2. Human BCMA and mouse BAFF, IgM, IgA and IgG levels were measured with ELISA (R&D Systems & Bethyl Laboratories). The Raji B-cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients.Results: We determined if mBAFF formed complexes with human BCMA (hBCMA) in the plasma from SCID mice implanted with LAGλ-1, LAGκ-2 or LAGκ-1A, and were able to identify mBAFF-hBCMA complexes in plasma samples from these mice. To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl/6 mice, and plasma IgA, IgM and IgG levels were measured. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control group (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. For plasma IgG levels, a marked decrease was observed on day 6 following rhBCMA-Fc administration when compared to its baseline levels (P = 0.0023), and also when compared to the Ig-Fc control protein (P = 0.0014) and the untreated control (P = 0.0129) groups. We then set out to determine if sera from MM patients contained BCMA-BAFF complexes, using ELISA plates coated with an anti-human BAFF antibody followed by exposure to a polyclonal anti-human BCMA antibody. A strong absorbance indicating the presence of BCMA-BAFF complexes was detected in serum samples from MM patients, whereas no antibody cross reactivity was observed in control samples. We also determined whether human MM serum or rhBCMA-Fc blocked BAFF from binding to Raji B-cells. Raji cells (B-cell line) were incubated with serum from a MM patient containing hBCMA (0.75μg/ml) or rhBCMA-Fc (3 μg/ml) in the presence of rhBAFF (500 ng/ml). Myeloma serum and rhBCMA-Fc decreased rhBAFF binding to Raji cells by 71% (from 96.8 to 25.6 %) and 74% (from 96.8 to 22.9 %), respectively. Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (> 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (< 100 ng/ml) correlated with normal uninvolved levels (P < 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P < 0.0001) MM.Conclusions: Our laboratory previously has reported that serum levels of BCMA are increased in the serum of MM patients, and correlates with response to treatment and predicts survival. We now demonstrate 1) the formation of complexes of BCMA with its B-cell ligand BAFF in the plasma of MM xenografts and sera of MM patients, 2) show that rhBCMA and MM patient serum blocks the binding of BAFF to human B-cells, 3) administration of rhBCMA to normal mice results in marked reductions in their antibody levels, and 4) show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results, in part, from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. DisclosuresNo relevant conflicts of interest to declare.

Rapamycin inhibits BAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells.

B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin's suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin's effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Signal transduction via CD103 and BAFF-receptor cooperatively play a role in the abnormal activation of T cells. (CCR3P.216)

Abstract Background and Purpose: It was reported that the expression of CD103 (αEβ7) was elevated in infiltrated T cells in lachrymal glands of patients with Sjögren’s syndrome (SS), suggesting that abnormal expression of CD103 in T cells is involved in the pathogenesis of SS. Our previous study suggests that BAFF plays a role in the development of SS. In this study, we investigated a possible interaction between CD103 and BAFF in T cells. Materials and Methods: Jurkat, a human T cell leukemia cell line, was transfected with both human αE and β7 genes. Stable transfectants were stimulated with anti-CD3 to induce production of IL-2. The cells were further stimulated with anti-CD103, recombinant human soluble BAFF (rhsBAFF) or both. Results and Discussion: It may be noteworthy that the IL-2 production by T cells upon stimulation with ant-CD3 was elevated by the overexpression of CD103. The data suggest that CD103 plays a role in the activation of T cells. Anti-CD103 and rhsBAFF significantly enhanced the effect of anti-CD3 possibly in an additive manner. FACS and quantitative RT-PCR revealed that the expression level of a BAFF receptor (BR3) was increased in the transfectants, suggesting that the effect of CD103 on the response of the cells to rhsBAFF is mediated through the enhancement of BR3 expression. The data collectively suggest that abnormal expression of CD103 in T cells is involved in the development of SS by affecting BAFF signaling pathway.

Measurements of B-Cell Activating Factor (BAFF) of Tumor Necrosis Factor Family in Patients with Allergic Bronchopulmonary Aspergillosis (ABPA) and Asthma

RationaleBAFF is a TNF superfamily member known for its role in the survival and maturation of B cells. Part of the BAFF transmembrane protein is shed and detectable in human serum. We explored whether serum BAFF was increased in patients with ABPA (ABPA pool), patients with asthma (Asthma pool) who displayed 3+ or 4+ skin prick reactivity to Aspergillus fumigatus, and normal controls without Aspergillus hypersensitivity.MethodsPatients experiencing exacerbations of ABPA, quiescent ABPA, and asthma with Aspergillus hypersensitivity were identified from the Allergy-Immunology service. Serum samples were analyzed by an ELISA kit for human BAFF (R&D Systems, Minneapolis, MN) according to manufacturer's protocol with range of detection from 6.6 to 4000 pg/mL. Total serum IgE concentrations were measured by ImmunoCAP (Phadia AB).ResultsThe concentrations of serum BAFF and total IgE, respectively are as follows: ABPA pool (416 pg/mL, 2642 kU/L), Asthma pool (466 pg/mL, 1134 kU/L), normal control #1 (254 pg/mL, 10 kU/L), normal control #2 (235 pg/mL, 105 kU/L). ABPA Patient #1: exacerbation after partial treatment with prednisone (421 pg/mL, 3261 kU/L) and quiescent ABPA (378 pg/mL, 1980 kU/L). ABPA Patient #2: exacerbation (324 pg/mL, 3261 kU/L) and quiescent ABPA (518 pg/mL, 254 kU/L).ConclusionsSerum BAFF concentrations in patients with ABPA and patients with asthma who displayed Aspergillus hypersensitivity are elevated numerically when compared with normal controls without Aspergillus hypersensitivity. In 2 patients experiencing an exacerbation of ABPA, there were no large increases in serum BAFF, opposite of total serum IgE. RationaleBAFF is a TNF superfamily member known for its role in the survival and maturation of B cells. Part of the BAFF transmembrane protein is shed and detectable in human serum. We explored whether serum BAFF was increased in patients with ABPA (ABPA pool), patients with asthma (Asthma pool) who displayed 3+ or 4+ skin prick reactivity to Aspergillus fumigatus, and normal controls without Aspergillus hypersensitivity. BAFF is a TNF superfamily member known for its role in the survival and maturation of B cells. Part of the BAFF transmembrane protein is shed and detectable in human serum. We explored whether serum BAFF was increased in patients with ABPA (ABPA pool), patients with asthma (Asthma pool) who displayed 3+ or 4+ skin prick reactivity to Aspergillus fumigatus, and normal controls without Aspergillus hypersensitivity. MethodsPatients experiencing exacerbations of ABPA, quiescent ABPA, and asthma with Aspergillus hypersensitivity were identified from the Allergy-Immunology service. Serum samples were analyzed by an ELISA kit for human BAFF (R&D Systems, Minneapolis, MN) according to manufacturer's protocol with range of detection from 6.6 to 4000 pg/mL. Total serum IgE concentrations were measured by ImmunoCAP (Phadia AB). Patients experiencing exacerbations of ABPA, quiescent ABPA, and asthma with Aspergillus hypersensitivity were identified from the Allergy-Immunology service. Serum samples were analyzed by an ELISA kit for human BAFF (R&D Systems, Minneapolis, MN) according to manufacturer's protocol with range of detection from 6.6 to 4000 pg/mL. Total serum IgE concentrations were measured by ImmunoCAP (Phadia AB). ResultsThe concentrations of serum BAFF and total IgE, respectively are as follows: ABPA pool (416 pg/mL, 2642 kU/L), Asthma pool (466 pg/mL, 1134 kU/L), normal control #1 (254 pg/mL, 10 kU/L), normal control #2 (235 pg/mL, 105 kU/L). ABPA Patient #1: exacerbation after partial treatment with prednisone (421 pg/mL, 3261 kU/L) and quiescent ABPA (378 pg/mL, 1980 kU/L). ABPA Patient #2: exacerbation (324 pg/mL, 3261 kU/L) and quiescent ABPA (518 pg/mL, 254 kU/L). The concentrations of serum BAFF and total IgE, respectively are as follows: ABPA pool (416 pg/mL, 2642 kU/L), Asthma pool (466 pg/mL, 1134 kU/L), normal control #1 (254 pg/mL, 10 kU/L), normal control #2 (235 pg/mL, 105 kU/L). ABPA Patient #1: exacerbation after partial treatment with prednisone (421 pg/mL, 3261 kU/L) and quiescent ABPA (378 pg/mL, 1980 kU/L). ABPA Patient #2: exacerbation (324 pg/mL, 3261 kU/L) and quiescent ABPA (518 pg/mL, 254 kU/L). ConclusionsSerum BAFF concentrations in patients with ABPA and patients with asthma who displayed Aspergillus hypersensitivity are elevated numerically when compared with normal controls without Aspergillus hypersensitivity. In 2 patients experiencing an exacerbation of ABPA, there were no large increases in serum BAFF, opposite of total serum IgE. Serum BAFF concentrations in patients with ABPA and patients with asthma who displayed Aspergillus hypersensitivity are elevated numerically when compared with normal controls without Aspergillus hypersensitivity. In 2 patients experiencing an exacerbation of ABPA, there were no large increases in serum BAFF, opposite of total serum IgE.

Circulating Bcma Binding to Its Ligand BAFF Prevents Normal Antibody Production in Multiple Myeloma Patients

Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels.Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on SCID mice implanted with the human xenografts LAGλ-1, LAGk-1A or LAGk-2 following BCMA treatment. Human BCMA and mouse BAFF, IgM and IgA levels were measured with ELISA (R&D Systems & Bethyl Laboratories). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients.Results: Recombinant mouse BAFF (rmBAFF) was mixed with recombinant human BCMA (rhBCMA) and incubated on plates coated with anti-mouse BAFF antibody (Ab). An anti-BCMA detection Ab was added, and BCMA-BAFF complexes were easily detected at concentrations of human BCMA and BAFF present in MM pts. We determined if mBAFF from SCID mice would also bind rhBCMA. SCID mice were bled; their plasma isolated and incubated with rhBCMA, and showed that rhBCMA formed complexes with mBAFF.To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl and Balb/c mice, and plasma IgA and IgM levels were measured. Following rhBCMA-Fc injection, a marked decrease in both antibody classes was observed in both mouse strains. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control groups (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. To determine if rhBCMA-mBAFF complexes formed in vivo, an ELISA was performed. Plates were pre-coated with a monoclonal mouse anti-BAFF capture Ab. Plasma samples were incubated and an anti-human-BCMA detection Ab was added. rhBCMA-mBAFF complexes were detected at high levels in plasma samples from mice dosed 4 and 6 days previously with BCMA-Fc whereas no complexes were found in samples in control Ig-Fc or untreated mice. We evaluated mBAFF levels in SCID mice with and without human MM. We have previously shown high levels of human BCMA in the plasma of MM tumor-bearing mice (Sanchez et al. Br J Haematol 2012). On days 14, 21 and 28 post-tumor implantation, mice implanted with LAGλ-1 had markedly lower plasma levels of mBAFF (P = 0.0143, P = 0.0002 and P = 0.0008, respectively) when compared to age and sex-matched mice not bearing any tumors. We obtained similar results using two additional human MM xenograft models (LAGk-1A and LAGk-2). Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (> 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (< 100 ng/ml) correlated with normal uninvolved levels (P < 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P < 0.0001) MM.Conclusions: Our laboratory previously reported that BCMA is present in the serum of MM patients, correlates with response to treatment and predicts survival. We now demonstrate the formation of circulating BCMA-BAFF complexes in MM, and administration of recombinant BCMA to normal mice results in marked reductions in their antibody levels. We also show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results in part from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. DisclosuresNo relevant conflicts of interest to declare.

The Role BAFF and BAFF Receptors in Macrophage Induced Drug Resistance of Multiple Myeloma

Background: B-cell–activating factor (BAFF) is a member of TNF family that critical for maintenance of B-cell development and homeostasis. BAFF also modulates the proliferation, survival and drug resistance of multiple meyloma (MM) cells. BAFF can be secreted by neutrophils, monocytes,dentritic cells and macrophages. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by a cell-to-cell interaction between MM cells and macrophages. We supposed that the interaction of BAFF and BAFF receptors plays a role in macrophages induced MM resistance.Methods: First, we detected the expression levels of BAFF and its three receptors in MM cells were detected by semiquantitative real time-polymerase chain reaction (qPCR) and flow-cytometry. The concentration of BAFF in the culture supernatants of MM cell lines was detected by ELISA. Second, we collect peripheral blood monocytes form healthy donors. Monocytes were induced into macrophages by culturing with M-CSF for 7 days.BAFF expression level in macrophages was detected by qPCR, flow-cytometry and ELISA. Then MM cells were sole-cultured or coculture with macrophages for the indicated time (usually 24 h), after that bortezomib was added to the culture system. Cell viability and apoptosis of MM cells were verified by MTT and flow cytometry.At last,the recombinant human BAFF were added to MM cells, MTT and flow cytometry were used for detection of cell viability and apoptosis of MM cells.Results: Two receptors of BAFF, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA), are highly expressed in various MM cell lines evidenced by real-time PCR and flow cytometry. The expression of BAFF in PBMC-induced macrophages are heterogeneous. We verify macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, 8226 and OPM2) with PBMC-derived macrophages from healthy donors. Functional studies show that recombinant human BAFF rescues RPMI8226 myeloma cells from dexamethasone-induced apoptosis.Conclusions: Our data showed that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors. Further study will focused on the mechanism of interaction between BAFF and BAFF receptors. DisclosuresNo relevant conflicts of interest to declare.

Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34+CD117+CD41+CD151+ megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFβ, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

AB0174 Increased Proportion of CD38+Igd+ B Cells Contributes to Pathogenesis of Primary SjÖGren's Syndrome

BackgroundPrimary Sjögren's syndrome (pSS) is characterized by overproduction of autoantibodies against nuclei etc. and clinical manifestations caused by immune complexes, such as peripheral neuropathy, vasculitic lesions and hypocomplementemia. These denote...

Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc-engineered monoclonal antibody targeting the BAFF-R.

B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

BAFF activates Erk1/2 promoting cell proliferation and survival by Ca2+-CaMKII-dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca2+-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca2+-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca2+ may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Increased B Cell-Activating Factor Promotes Tumor Invasion and Metastasis in Human Pancreatic Cancer

B cell-activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) superfamily. It has been reported that BAFF is elevated in patients with autoimmune pancreatitis and contributes to the malignant potential of blood cancers and solid tumors. In this study, clinical evidence of increased BAFF levels in patients with pancreatic ductal adenocarcinoma (PDAC) was obtained, and the roles and mechanisms of BAFF in PDAC were clarified in human tissues of PDAC and from in vitro data of PDAC cell lines. Serum levels of BAFF in patients with PDAC were significantly higher than in healthy subjects (p = 0.0121). Patients with UICC stage IV PDAC (T1-4, N0-1, M1) had significantly higher levels of serum BAFF compared to patients with PDAC (p = 0.0182). BAFF was remarkably expressed in infiltrating B lymphocytes surrounding pancreatic cancer in human pancreatic tissues, suggesting that BAFF may play a role in progression of pancreatic cancer. PDAC cell lines were cultured with human recombinant BAFF, and morphology and gene expression were analyzed; pancreatic cancer cells changed to a fibroblast-like morphology, and showed altered gene expression of E-cadherin, vimentin and Snail. These BAFF-induced changes reflect enhanced cell motility and invasion. BAFF-R-overexpressing cell clones confirmed the association between these BAFF-induced changes and epithelial-mesenchymal transition (EMT)-related genes. BAFF was elevated in patients with metastatic advanced PDAC and induced alterations in PDAC cells via regulation of EMT-related genes. Elucidation of the precise role and mechanism of control of BAFF may lead to new therapeutic approaches with the aim of improving pancreatic cancer survival.

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 ~36KDa 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.

Protein kinase C stimulates human B cell activating factor gene expression through reactive oxygen species-dependent c-Fos in THP-1 pro-monocytic cells

BAFF is associated with various immunological diseases. Previously, we have reported that mouse B cell activating factor (mBAFF) expression was dependent on nuclear localization of co-activator, p300 and the activation of transcription factors including NF-κB and CREB. Here, we investigated whether transcription factor, c-Fos, regulates human (h) BAFF expression through promoter activation by PMA-induced reactive oxygen species (ROS) production. We cloned hBAFF promoter into luciferase-expressing pGL3-basic vector. The activity of 1.0kb hBAFF promoter was higher than that in 0.75, 0.5 or 0.25kb hBAFF promoter. The existence of three AP-1 binding motifs was computer-analyzed in hBAFF promoter. The stimulation with PMA and ionomycin (IOM) increased 1.0kb hBAFF promoter activity, time-dependently. PMA/IOM-stimulation rapidly enhanced c-Fos expression in THP-1 human pro-monocytic cells. Binding of c-Fos to hBAFF promoter was detected by chromatin immunoprecipitation (ChIP) assay. hBAFF expression and its promoter activity were decreased by the transfection with small interference (si) RNA of c-Fos. ROS production in THP-1 cells was increased by PMA/IOM-stimulation. In addition, hBAFF activity stimulated by PMA/IOM was reduced by N-acetyl-cysteine (NAC), a well-known ROS scavenger. Serum starvation (0.5% FBS) producing ROS and the exogenous H2O2 treatment also enhanced hBAFF promoter activity. c-Fos expression and AP-1 binding to oligonucleotide were reduced by the treatment with NAC. H2O2 was not able to induce hBAFF expression in the presence of staurosporine, PKC inhibitor. Data suggest that hBAFF expression could be regulated by promoter activation through c-Fos association, which might be dependent on PMA-induced ROS production.

Soluble BAFF Levels Inversely Correlate with Peripheral B Cell Numbers and the Expression of BAFF Receptors

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.

B-cell activating factor targeted therapy and lupus

B-cell activating factor (BAFF), a member of the family of TNF-like cytokines, supports the survival and differentiation of B cells. The successful development of belimumab, a human antibody targeting soluble BAFF, has marked an important milestone in the development of biologic therapy for treatment of systemic lupus erythematosus (SLE), although much remains unknown regarding the clinical utility of BAFF inhibition in SLE and other autoimmune diseases. In the present review, we provide an overview of the knowledge concerning BAFF's role in murine and human B-cell development and maturation, as well as the clinical and mechanistic effects of BAFF inhibition in human SLE.

BAFF and APRIL induce inflammatory activation of THP-1 cells through interaction with their conventional receptors and activation of MAPK and NF-κB

BAFF and APRIL, as closely related members of the TNF superfamily, are important regulators of B-cell survival. They share two receptors, TACI and BCMA, and BAFF can stimulate an additional receptor, BAFF-R. Although these molecules have been under intense investigation in order to identify their role in immune reactions, the effect of BAFF and APRIL on macrophage function has not been tested. The human macrophage-like cell line THP-1, which expresses BAFF/APRIL and all three of their receptors, was stimulated with recombinant human BAFF or APRIL or monoclonal antibodies against the receptors and the resulting cellular responses were investigated. Treatment of the cells with these agents induced the expression of pro-inflammatory mediators such as matrix metalloproteinase (MMP)-9 and IL-8. Suppression of the expression of these receptors using specific siRNAs resulted in the blocking of the response, confirming that these responses require specific interaction between BAFF/APRIL and their receptors. Inhibitors of MAPK and NF-κB blocked the expression of IL-8. Furthermore, inhibitors of MAPK blocked the BAFF-induced specific DNA binding activity of NF-κB. These data indicate that BAFF and APRIL can induce inflammatory activation of THP-1 cells through the activation of MAPK, which leads to the subsequent activation of NF-κB.

HsBAFF regulates proliferation and response in cultured CD4 + T lymphocytes by upregulation of intracellular free Ca 2+ homeostasis

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T-cell activation. Recently, we have demonstrated that treatment of mice with human soluble BAFF (hsBAFF) causes a significant increase of percentages of splenic CD4 + T lymphocytes dose-dependently, but the CD8 + T lymphocyte percentages maintained unchanged. Here, we show that hsBAFF significantly enhanced CD4 + T lymphocyte response of cultured mouse splenic cells, and hsBAFF induced the proliferation and IL-2/IFN-γ secretion of purified CD4 + T lymphocytes suboptimally stimulated through anti-CD3. Of importance, we observed that IL-2 or IFN-γ cytokine has additive effect on the proliferation and activity of hsBAFF-stimulated CD4 + T lymphocytes. Using Flow cytometry with fluorescent probe, Fluo-3/AM, we found that hsBAFF elicited [Ca 2+] i elevation contributing to CD4 + T cell proliferation. This is evidenced by our finding that pretreatment with BAPTA/AM, an intracellular Ca 2+ chelator, significantly attenuated the proliferation of hsBAFF-stimulated CD4 + T lymphocytes. Subsequently, we revealed that hsBAFF-stimulated CD4 + T cell proliferation was markedly suppressed after pretreatment with EGTA, an extracellular Ca 2+ chelator, or with 2-APB, an inhibitor of Ca 2+ influx through CRAC channels, respectively, suggesting that extracellular Ca 2+ influx due to hsBAFF is closely associated with [Ca 2+] i elevation contributing to CD4 + T cell proliferation. In addition, we noticed that hsBAFF-treated cells conferred partial resistance to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca 2+-ATPase inhibitor. Taken together, our data indicate that hsBAFF may promote CD4 + T cell proliferation and response by upregulation of [Ca 2+] i homeostasis.

Cloning, expression and bioactivity of BAFF from Petaurus breviceps

B-cell activating factor (BAFF), belonging to the TNF (tumor necrosis factor) family, is crucial for B-cell survival and maturation. In the present study, PbBAFF cDNA was amplified from the sugar glider Petaurus breviceps by RT-PCR and RACE (rapid amplification of cDNA ends) strategies. The open reading frame (ORF) of PbBAFF cDNA encodes a protein consisting of 287-amino acid. The deduced amino acid sequence contains a predicted transmembrane domain, a putative furin protease cleavage site, a potential N-glycosylation site and conserved cysteine residues similar to that identified in other mammalian BAFF. The soluble mature part of PbBAFF (PbsBAFF) showed 88–92% sequence identity with mammalian homologs. The predicted three-dimensional (3D) structural analysis of PbsBAFF analyzed by comparative protein modeling revealed that they are very similar to the 3D structure of human BAFF. Recombinant PbsBAFF fused with His 6 tag was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, purified PbsBAFF co-stimulates the proliferation of human B-cells. These findings indicate PbBAFF, the first BAFF cloned from marsupial, plays an important role in proliferation of B-cells, and phylogenetic analyses reveal that the work is of value with respect to continued refinement of our understanding of mammalian phylogenetic relationships.