Human Plasmacytoid Dendritic Cells Display and Shed B Cell Maturation Antigen upon TLR Engagement.

The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs.

Circulating Follicular Regulatory T Cells Are Defective in Multiple Sclerosis.

Follicular regulatory T cells (TFR) have been extensively characterized in mice and participate in germinal center responses by regulating the maturation of B cells and production of (auto)antibodies. We report that circulating TFR are phenotypically distinct from tonsil-derived TFR in humans. They have a lower expression of follicular markers, and display a memory phenotype and lack of high expression of B cell lymphoma 6 and ICOS. However, the suppressive function, expression of regulatory markers, and FOXP3 methylation status of blood TFR is comparable with tonsil-derived TFR. Moreover, we show that circulating TFR frequencies increase after influenza vaccination and correlate with anti-flu Ab responses, indicating a fully functional population. Multiple sclerosis (MS) was used as a model for autoimmune disease to investigate alterations in circulating TFR. MS patients had a significantly lower frequency of circulating TFR compared with healthy control subjects. Furthermore, the circulating TFR compartment of MS patients displayed an increased proportion of Th17-like TFR. Finally, TFR of MS patients had a strongly reduced suppressive function compared with healthy control subjects. We conclude that circulating TFR are a circulating memory population derived from lymphoid resident TFR, making them a valid alternative to investigate alterations in germinal center responses in the context of autoimmune diseases, and TFR impairment is prominent in MS.

Adipocytes as immune cells: differential expression of TWEAK, BAFF, and APRIL and their receptors (Fn14, BAFF-R, TACI, and BCMA) at different stages of normal and pathological adipose tissue development.

Adipose tissue represents a rich source of multipotent stem cells. Mesenchymal cells, isolated from this source, can differentiate to other cell types in vitro and therefore can be used for a number of regenerative therapies. Our view of adipose tissue has recently changed, establishing adipocytes as new members of the immune system, as they produce a number of proinflammatory cytokines (such as IL-6 and TNFalpha and chemokines, in addition to adipokines (leptin, adiponectin, resistin) and molecules associated with the innate immune system. In this paper, we report the differential expression of TNF-superfamily members B cell activating factor of the TNF Family (BAFF), a proliferation inducing ligand (APRIL), and TNF-like weak inducer of apoptosis (TWEAK) in immature-appearing and mature adipocytes and in benign and malignant adipose tissue-derived tumors. These ligands act through their cognitive receptors, BAFF receptor, transmembrane activator and calcium signal-modulating cyclophilic ligand (TACI), B cell maturation Ag (BCMA), and fibroblast growth factor-inducible 14 (Fn14), which are also expressed in these cells. We further report the existence of functional BCMA, TACI, and Fn14 receptors and their ligands BAFF, APRIL, and TWEAK on adipose tissue-derived mesenchymal cells, their interaction modifying the rate of adipogenesis. Our data integrate BAFF, APRIL, and TWEAK and their receptors BCMA, TACI, and Fn14 as novel potential mediators of adipogenesis, in addition to their specific role in immunity, and define immature and mature adipocytes as source of immune mediators.

The activation of BAFF/APRIL system in spleen and lymph nodes of Plasmodium falciparum infected patients.

Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.

Identification of new splice variants of the genes BAFF and BCMA.

The TNF superfamily ligands BAFF and APRIL and receptors BCMA, TACI and BAFF-R play an important role in the regulation of B cell immunity. A number of functionally important splice isoforms have already been characterized for these molecules, stimulating the search for new transcript variants (TVs). Here we report two new BAFF TVs and three BCMA TVs, all potentially codifying new proteins. BAFF TVs were expressed in peripheral blood mononuclear cells (PBMC) of nearly all the individuals studied, decreasing in level when PBMC were activated by PMA and ionomycin. They were also detected in PBMC cytoplasmic RNA. Low levels of the BAFF TVs in all lymphocyte subpopulations analyzed suggest that their main source in PBMC are monocytes. BCMA TVs were observed only in some CD19+ cell samples. Functional studies concerning interaction between isoforms of BAFF, APRIL and their receptors are needed for elucidation of their significance in the immune response.

Tumor-Infiltrating Plasma Cells Are Associated with Tertiary Lymphoid Structures, Cytolytic T-Cell Responses, and Superior Prognosis in Ovarian Cancer.

PURPOSE: CD8(+) tumor-infiltrating lymphocytes (TIL) are key mediators of antitumor immunity and are strongly associated with survival in virtually all solid tumors. However, the prognostic effect of CD8(+) TIL is markedly higher in the presence of CD20(+) B cells, suggesting that cooperative interactions between these lymphocyte subsets lead to more potent antitumor immunity. EXPERIMENTAL DESIGN: We assessed the colocalization patterns, phenotypes, and gene expression profiles of tumor-associated T- and B-lineage cells in high-grade serous ovarian cancer (HGSC) by multicolor IHC, flow cytometry, and bioinformatic analysis of gene expression data from The Cancer Genome Atlas. RESULTS: T cells and B cells colocalized in four types of lymphoid aggregate, ranging from small, diffuse clusters to large, well-organized tertiary lymphoid structures (TLS) resembling activated lymph nodes. TLS were frequently surrounded by dense infiltrates of plasma cells (PC), which comprised up to 90% of tumor stroma. PCs expressed mature, oligoclonal IgG transcripts, indicative of antigen-specific responses. PCs were associated with the highest levels of CD8(+), CD4(+), and CD20(+) TIL, as well as numerous cytotoxicity-related gene products. CD8(+) TIL carried prognostic benefit only in the presence of PCs and these other TIL subsets. PCs were independent of mutation load, BRCA1/2 status, and differentiation antigens but positively associated with cancer-testis antigens. CONCLUSIONS: PCs are associated with the most robust, prognostically favorable CD8(+) TIL responses in HGSC. We propose that TLS facilitate coordinated antitumor responses involving the combined actions of cytolytic T cells and antibody-producing PCs. Clin Cancer Res; 22(12); 3005-15. ©2016 AACR.

Expression of BAFF receptors in muscle tissue of myositis patients with anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies.

INTRODUCTION: Anti-Jo-1 and anti-Ro52 autoantibodies are common in patients with myositis, but the mechanisms behind their production are not known. Survival of autoantibody-producing cells is dependent on B-cell-activating factor of the tumour necrosis factor family (BAFF). BAFF levels are elevated in serum of anti-Jo-1-positive myositis patients and are influenced by type-I interferon (IFN). IFN-producing cells and BAFF mRNA expression are present in myositis muscle. We investigated expression of the receptors for BAFF in muscle tissue in relation to anti-Jo-1 and anti-Ro52/anti-Ro60 autoantibodies and type-I IFN markers. METHODS: Muscle biopsies from 23 patients with myositis selected based on autoantibody profile and 7 healthy controls were investigated for expression of BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Nineteen samples were assessed for plasma (CD138) and B-cell (CD19) markers. The numbers of positive cells per area were compared with the expression of plasmacytoid dendritic cell (pDC) marker blood dendritic cell antigen-2 (BDCA-2) and IFNα/ß-inducible myxovirus resistance-1 protein (MX-1). RESULTS: BAFF-R, BCMA and TACI were expressed in five, seven and seven patients, respectively, and more frequently in anti-Jo-1-positive and/or anti-Ro52/anti-Ro60-positive patients compared to controls and to patients without these autoantibodies (P = BAFF-R: 0.007, BCMA: 0.03 and TACI: 0.07). A local association of receptors with B and plasma cells was confirmed by confocal microscopy. The numbers of CD138-positive and BCMA-positive cells were correlated (r = 0.79; P = 0.001). Expression of BDCA-2 correlated with numbers of CD138-positive cells and marginally with BCMA-positive cells (r = 0.54 and 0.42, respectively; P = 0.04 and 0.06, respectively). There was a borderline correlation between the numbers of positively stained TACI cells and MX-1 areas (r = 0.38, P = 0.08). CONCLUSIONS: The expression pattern of receptors for BAFF on B and plasma cells in muscle suggests a local role for BAFF in autoantibody production in muscle tissues of patients with myositis who have anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies. BAFF production could be influenced by type-I IFN produced by pDCs. Thus, B-cell-related molecular pathways may participate in the pathogenesis of myositis in this subset of patients.

Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.

A PRoliferation-Inducing Ligand (APRIL) is a secreted cytokine member of the tumor necrosis factor family. It is a B-cell survival factor that also induces class switch recombination (CSR) toward immunoglobulin A (IgA), independent of T cells. It is therefore an important contributor to the maintenance of the mucosal immunological barrier, which has been linked to a putative extrafollicular inductive phase of the IgA response in lamina propria. By immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) on microdissected tissue from normal human gut, we observed APRIL expression, together with TACI (transmembrane activator and CAML interactor) and BCMA (B-cell maturation antigen), in gut-associated lymphoid tissue (GALT), lamina propria, and in the epithelium of stomach, small and large intestine, and rectum. However, no activation-induced cytidine deaminase (AID) expression (an absolute requirement for class switching) was detected in lamina propria by IHC or qRT-PCR. APRIL and its receptors were only observed alongside AID in GALT, showing that GALT contains the apparatus to support both T-independent and T-dependent routes to IgA CSR.

POU2AF1, an amplification target at 11q23, promotes growth of multiple myeloma cells by directly regulating expression of a B-cell maturation factor, TNFRSF17.

Multiple myeloma (MM), a progressive hematological neoplasm, is thought to result from multiple genetic events affecting the terminal plasma cell. However, genetic aberrations related to MM are seldom reported. Using our in-house array-based comparative genomic hybridization system to locate candidate target genes with following their expression analysis, we identified POU2AF1 at 11q23.1 as a probable amplification target in MM cell lines. POU2AF1 is a B-cell-specific transcriptional co-activator, which interacts with octamer-binding transcription factors Oct-1 and Oct-2, and augments their function. Downregulation of POU2AF1 expression by specific small-interfering RNA (siRNA) inhibited MM cell growth, whereas ectopic expression of POU2AF1 promoted growth of MM cells. Among putative transcriptional targets for POU2AF1, B-cell maturation factor, TNFRSF17, enhanced its transcription by POU2AF1, and POU2AF1 directly bound to an octamer site within the 5' region of TNFRSF17. Expression level of TNFRSF17 was closely correlated with that of POU2AF1 in cell lines and primary samples of MM, and decreasing TNFRSF17 expression by means of TNFRSF17 siRNA inhibited MM cell growth. Taken together, our results suggest that POU2AF1, when activated by amplification or other mechanisms, may contribute to progression of MM by accelerating growth of MM cells through direct transactivation of one of its target genes, TNFRSF17.

Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL.

Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.

Regulated expression of BAFF-binding receptors during human B cell differentiation.

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27(neg) B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.

BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-kappaB pathway.

Chronic lymphocytic leukemia (CLL) B cells express BR3, the specific receptor for the B cell-activating factor of tumor necrosis factor family (BAFF). CLL cells also express 2 other receptors for BAFF, namely B-cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand-interactor (TACI), which also bind a proliferation-inducing ligand (APRIL). We found that signaling through BR3, but not BCMA or TACI, activated the alternative nuclear factor of kappa B (NF-kappaB) pathway in CLL cells, whereas signaling through BCMA/TACI induced activation of the canonical NF-kappaB pathway. Blocking BR3 did not inhibit the capacity of BAFF to support CLL cell survival in vitro. On the other hand, specifically blocking the canonical NF-kappaB pathway with UTC, an inhibitor of IkappaB kinase beta (IKKbeta), or transfection of CLL cells with the IkappaBalpha super-repressor, blocked the capacity of BAFF and APRIL to promote CLL cell survival in vitro. This contrasts what is found with normal blood B cells, which apparently depend on activation of the alternative NF-kappaB pathway for BAFF-enhanced survival. These findings suggest that inhibitors of protein kinase IKKbeta, which is required for activation of the canonical NF-kappaB pathway, might have a therapeutic role in this disease.

Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL.

Hodgkin lymphoma (HL) originates from the clonal expansion of malignant Hodgkin and Reed-Sternberg (HRS) cells. These B-cell-derived elements constitute less than 10% of the tumoral mass. The remaining tissue is comprised of an inflammatory infiltrate that includes myeloid cells. Myeloid cells activate B cells by producing BAFF and APRIL, which engage TACI, BCMA, and BAFF-R receptors on the B cells. Here, we studied the role of BAFF and APRIL in HL. Inflammatory and HRS cells from HL tumors expressed BAFF and APRIL. Unlike their putative germinal center B-cell precursors, HRS cells lacked BAFF-R, but expressed TACI and BCMA, a phenotype similar to that of plasmacytoid B cells. BAFF and APRIL enhanced HRS cell survival and proliferation by delivering nonredundant signals via TACI and BCMA receptors through both autocrine and paracrine pathways. These signals caused NF-kappaB activation; Bcl-2, Bcl-xL, and c-Myc up-regulation; and Bax down-regulation, and were amplified by APRIL-binding proteoglycans on HRS cells. Interruption of BAFF and APRIL signaling by TACI-Ig decoy receptor, which binds to and neutralizes BAFF and APRIL, or by small-interfering RNAs targeting BAFF, APRIL, TACI, and BCMA inhibited HRS cell accumulation in vitro and might attenuate HL expansion in vivo.

[Cloning, soluble expression and characterization of human sBCMA].

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.