CAML Interactor Research Articles

Novel Mutations in TACI (TNFRSF13B) Causing Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production. The disorder is also characterized by co-occurrence of autoimmune, lymphoproliferative, and granulomatous diseases. Mutations in the gene encoding TACI (Transmembrane Activator and CAML Interactor, TNFRSF13B) were previously found to be associated with CVID. We therefore sequenced TNFRSF13B gene in a cohort of 48 Iranian CVID patients. Expression of TACI and binding of A proliferation-inducing ligand (APRIL) were tested by FACS. We identified one patient with a homozygous G to T substitution in the TNFRSF13B gene at the splice site of intron 1 (c.61+1G>T), which abolished expression of the TACI molecule and binding capacity of APRIL. This represents the second CVID patient in the world with a complete absence of TACI expression. B cell lines from family members carrying the same mutation in a heterozygous form showed a reduced level of TACI expression and APRIL-binding capacity, suggesting a gene dosage effect. In addition, we found the previously recognized C104R and C172Y mutations in a heterozygous form in two patients with CVID and one, novel, heterozygous P42T mutation. TACI mutations were observed in Iran CVID patients in a similar frequency as in other Caucasian populations. The novel mutations identified in this study support the notion of a crucial role for TACI in B cell differentiation.

B Lymphocyte Stimulator Regulates Adaptive Immune Responses by Directly Promoting Dendritic Cell Maturation

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.

Evidence of a role for B cell–activating factor of the TNF family in the pathogenesis of chronic rhinosinusitis with nasal polyps

The polypoid form of chronic rhinosinusitis (chronic rhinosinusitis with nasal polyps [CRSwNP]) is a highly prevalent disease that often requires surgical intervention for treatment. Nasal polyps contain large quantities of B lymphocytes and immunoglobulin as well as eosinophils. The objective of this study was to investigate the expression of B cell-activating factor of the TNF family (BAFF), an important regulator of class-switch recombination and immunoglobulin production, in patients with chronic rhinosinusitis (CRS). We collected nasal tissue and nasal lavage fluid from patients with CRS and control subjects. We assayed mRNA for BAFF and B-lymphocyte markers, CD20 and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, by using real-time PCR, and assayed BAFF protein by using ELISA and immunohistochemistry. BAFF mRNA was significantly increased in nasal polyps from patients with CRSwNP (P < .001) compared with inferior turbinate tissue from patients with CRS or healthy subjects. BAFF protein was also elevated in polypoid tissue and nasal lavage from patients with CRSwNP. Immunohistochemistry showed considerable BAFF staining in mucosal epithelial cells in nasal polyps along with unidentified cells in the lamina propria. Expression of mRNA for BAFF in sinonasal tissue was significantly correlated with CD20 and transmembrane activator and CAML interactor in sinus tissue. IgA, an immunoglobulin isotype known to activate eosinophils, was also significantly elevated in the polypoid tissue. Overproduction of BAFF in nasal polyps may contribute to the pathogenesis of CRSwNP via the local induction of IgA and activation of eosinophils.

Pharmacokinetics and Biological Activity of Atacicept in Patients With Rheumatoid Arthritis

Atacicept is a recombinant fusion protein containing the extracellular ligand-binding portion of the transmembrane activator and CAML interactor (TACI, CD267) receptor and inhibits B lymphocyte stimulator (BLyS, CD257) and a proliferation-inducing ligand (APRIL, CD256), both potent stimulators of B cell maturation, proliferation, and survival. Atacicept pharmacokinetics and pharmacodynamics were assessed in a double-blind, placebo-controlled, phase I study in patients with active, moderate to severe rheumatoid arthritis receiving atacicept either as a single subcutaneous or repeated, every other week dose. Pharmacokinetic profiles were determined by measuring serum concentrations of free atacicept and its complex with BLyS. Nonspecific immunoglobulin (Ig)M, IgG, and IgA; IgM-RF (rheumatoid factor), IgG-RF, and IgA-RF antibody levels; and B cell profiles provided markers of biological activity. Pharmacokinetic, biological activity, and relationships between atacicept dose and Ig antibody response were evaluated. Pharmacokinetic profiles of atacicept were nonlinear, influenced by saturable binding with its ligands, but were consistent and predictable. Atacicept treatment reduced Ig and RF serum concentration. IgM antibody levels were most sensitive to atacicept, followed by IgA and IgG, underlining the biological activity of atacicept in patients with rheumatoid arthritis. These findings can be used to explore dosing regimen design scenarios in future studies.

Thymus‐independent class switch recombination is affected by APRIL

The tumour necrosis factor (TNF) family member a proliferation-inducing ligand (APRIL) is implicated in various B-cell processes, such as class switch recombination, plasma cell differentiation and plasma cell survival. This was suggested from initial studies analysing B-cell responses in APRIL-deficient and transgenic mice, and mice deficient for the TNF receptors of APRIL, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen. Here, we present additional evidence for the importance of APRIL in thymus-independent (TI) B-cell responses, using APRIL-deficient and transgenic mice. APRIL-deficient mice show an impaired immunoglobulin A (IgA) response towards TI B-cell antigens, whereas APRIL transgenic mice show exaggerated TI B-cell responses. Moreover, antibody titres to TI antigens were sustained in APRIL transgenic mice for a long time and even increased up to 75 days in the case of IgA against 4-hydroxy-nitrophenacetyl-lipopolysaccharide.

Atacicept (TACI-Ig) inhibits growth of TACIhigh primary myeloma cells in SCID-hu mice and in coculture with osteoclasts

APRIL (a proliferation-inducing Ligand) and BLyS/BAFF (B-lymphocyte stimulator/B-cell-activating factor of the TNF (tumor necrosis factor) family have been shown to be the survival factors for certain myeloma cells in vitro. BAFF binds to the TNF-related receptors such as B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) and BAFFR, whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as syndecan-1. TACI gene expression in myeloma reportedly can distinguish tumors with a signature of microenvironment dependence (TACI(high)) versus a plasmablastic signature (TACI(low)). We tested the effect of atacicept (formerly TACI-Ig, which blocks APRIL and BAFF) and BAFFR-Ig (which blocks BAFF only) on primary myeloma growth in the SCID-hu model and in coculture with osteoclasts. With only few exceptions, atacicept and to a lesser extent BAFFR-Ig, inhibited growth of TACI(high) but not TACI(low) myeloma samples in vivo and ex vivo, and the response rate was inversely correlated with TACI expression. Most TACI(high) myeloma cells were molecularly classified as being low risk with our recently described 70-gene model. APRIL and BAFF were highly expressed by osteoclasts and were upregulated in myeloma cells after coculture with osteoclasts. Our findings suggest that APRIL plays an essential role in the survival of TACI(high) bone marrow-dependent myeloma cells and TACI gene expression may be a useful predictive marker for patients who could benefit from atacicept treatment.

APRIL-TACI Interactions Mediate Non-Hodgkin Lymphoma B Cell Proliferation through Akt Regulated Cyclin D1 and P21.

B cell non-Hodgkin lymphoma (NHL) is a serious and frequently fatal illness. Some of the genetic changes that lead to lymphoma have been discovered, but many of the other contributing mechanistic details underlying transformation events are not yet known. Members of the TNF super-family have been shown to be key mediators in the formation and regulation of both normal and malignant cell growth. APRIL (A Proliferation Inducing-ligand), a TNF family member expressed by monocytes, macrophages, and tumor cells, stimulates B-cell NHL growth and transgenic over-expression of APRIL in mice results in B cell hyperplasia and lymphoma. The ability of APRIL to bind to TNFR related receptors, BCMA (B Cell Maturation Antigen) and TACI (Transmembrane Activator and CAML Interactor), and with lower affinity to heparan sulfate proteoglycans (HSPG) has complicated studies to better understand APRIL mediated signaling. It has been shown that NHL cells variably express TACI and BCMA, with TACI being most predominant, however, the specific mechanisms of how APRIL induces NHL cell growth remain to be fully defined. Therefore, we sought to specifically characterize APRIL-mediated signaling events through TACI to better understand the role of this receptor-ligand interaction in B cell NHL biology. Using a TACI-expressing NHL cell line (Karpas) we found that APRIL consistently increased NHL cell proliferation, but had very little effect on cell survival. Since the PI-3K/Akt pathway has been shown to be important in regulating cell cycle progression, we explored the possibility that APRIL-TACI interactions resulted in activation of this pathway. We found that stimulation of TACI-expressing NHL cells with APRIL results in phosphorylation of Akt but had no effect on NF-kB classical pathway activation. Because APRIL can also bind to cells via proteoglycans, we also tested the ability of APRIL to activate Akt in the presence of heparin and found that APRIL-mediated Akt phosphorylation was still detectable. These results suggest that APRIL-mediated signaling occurs independent of proteoglycan binding. In addition to activating Akt, we also found that stimulation of NHL cells with APRIL resulted in up-regulation of cyclin D1 and p21, but not c-myc and p27. Inhibition of the PI3K/AKT signal transduction pathway with the pharmacological inhibitor wortmannin prevented APRIL-induced proliferation as well as up-regulation of p21 and cyclin D1 levels suggesting that PI-3K kinase is required. To further substantiate the contribution of TACI in APRIL-mediated signaling, we selectively targeted TACI expression using a siRNA approach. When TACI expression was inhibited, APRIL no longer induced NHL-B cell proliferation, Akt phosphorylation or up-regulation of p21 and cyclin D1. Taken together, these studies suggest that APRIL-TACI interactions result in activation of the PI3K/Akt pathway and up-regulation of cyclin D1 and p21, and help define the mechanism by which APRIL enhances B cell NHL growth. The inhibition of APRIL by blocking APRIL/receptor or interruption of downstream signaling may provide potential therapeutic option for patients with B-cell non-Hodgkin's Lymphoma.

Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID)

B cells from patients with common variable immunodeficiency (CVID) who are heterozygous for transmembrane activator and CAML interactor (TACI) mutation C104R, which abolishes ligand binding, fail to produce Igs in response to TACI ligand. It is not known whether this is due to haploinsufficiency or dominant interference. Using in vitro transfection assays, here we demonstrate that C104R and the corresponding murine TACI mutant, C76R, which also does not bind ligand, dominantly interfere with TACI signaling. This effect was dependent on preassociation of the mutants with WT TACI in the absence of ligand. The mutants did not interfere with ligand binding by WT TACI, suggesting that they may act by disrupting ligand-induced receptor rearrangement and signaling. This work demonstrates that TACI preassembles as an oligomeric complex prior to ligand binding and provides a mechanistic insight into how the heterozygous C104R TACI mutation can potentially lead to CVID.

Sequence Analysis of TNFRSF13b, Encoding TACI, in Patients with Systemic Lupus Erythematosus

B cell activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL), and their receptors BAFF receptor (BAFFR), B cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI) are involved in the regulation of B cell homeostasis and differentiation. BAFF overexpression leads to systemic lupus erythematosus (SLE) in mice and elevated BAFF levels have been observed in human SLE and mouse models for SLE. Furthermore, genetic inactivation of TACI in mice results in a SLE-like phenotype. Based on our recent finding that TACI is mutated in patients with common variable immunodeficiency, of whom more than 30% suffer from autoimmune conditions, we analyzed TACI in humans with SLE. Sequence analysis of TNFRSF13b/TACI in 119 unrelated SLE patients revealed four variants: R20C in exon 1, R72H in exon 3, the silent variation c.327 G > A in exon 3, and A181E in exon 4. No significant association with any of these variants was found, when compared to the frequencies of the variants in a healthy control cohort. Furthermore, the mutated alleles R20C and R72H did not segregate with the SLE phenotype in familial cases of SLE. Thus, our evaluation of the coding region of TNFRSF13b/TACI did not reveal any deleterious or disease-associated mutations.

Functional Implication of BAFF Synthesis and Release in Gmix-stimulated Microglia (B174)

Abstract BAFF (B-cell activating factor) is a recently identified member of TNF ligand superfamily that plays a critical role in B cell differentiation, survival, and regulation of immunoglobulin production. It exerts its effect by binding to three receptors: BAFF-R (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B-cell maturation antigen) which was known to be primarily expressed in B-lineage cells. In this present study, we examined whether or not BAFF is also expressed in microglia and the synthesis and release of BAFF is regulated by stimuli. BAFF is expressed and released in primary rat microglia as well as BV-2 cells, mouse microglial cell line and the expression and release of BAFF is increased by Gmix-stimuli in microglia. The expression and release of BAFF is regulated by JAK-STAT, especially STAT1 and STAT3-dependent signaling pathway. Interestingly, SP600125, and SB203580, inhibitors of p38 and JNK, respectively, did not inhibit BAFF expression, but inhibit the release of BAFF by soluble form. It suggested that p38 and JNK signaling pathway regulate the release, not the synthesis of BAFF in microglia. In BV-2 cells, only BAFF-R is expressed in cytoplasm and cell-surface. However, in primary rat microglia of microglia, BAFF-R and TACI are expressed in cytoplasm and cell-surface. Recombinant BAFF increases cytokines release, especially, IL-6 and IL-10 in primary rat microglia as well as BV-2 cells. It was suggested that BAFF, secreted by microglia may play important roles in CNS inflammation through regulating microglia as well as infiltrated B cells.

BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas‐mediated cell death

Microorganisms with pathogen-associated molecular patterns (PAMP) activate B cells directly by binding to TLR and also indirectly by inducing APC to release cytokines such as BAFF that promote B cell survival. We found that murine B cells activated concomitantly with LPS (TLR-4 ligand) and BAFF are protected from spontaneous apoptosis, but are more susceptible to Fas/CD95-mediated cell death. This increased susceptibility to Fas-induced apoptosis is associated with a dramatic coordinated up-regulation of Fas/CD95 and IRF-4 expression through a mechanism mediated, at least in part, by inhibition of the MEK/ERK pathway. Up-regulation of Fas/CD95 by BAFF is restricted to B cells activated through TLR-4, but not through TLR-9, BCR or CD40. TLR ligands differ in the BAFF family receptors (R) they induce on B cells: BAFF-R is increased by the TLR4 ligand, LPS, but not by the TLR9 ligand, CpG-containing oligodeoxynucleotides, which, in contrast, strongly up-regulates transmembrane activator and CAML interactor (TACI). This suggests the up-regulation of Fas by BAFF is mediated by BAFF-R and not by TACI. Consistently, APRIL, which binds to TACI and B cell maturation antigen but not BAFF-R, did not enhance Fas expression on LPS-activated B cells. Increased susceptibility to Fas-mediated killing of B cells activated with LPS and BAFF may be a fail-safe mechanism to avoid overexpansion of nonspecific or autoreactive B cells.

The role of BLyS/BLyS receptors in anti-chromatin B cell regulation

B lymphocyte stimulator (BLyS), also known as B cell-activating factor, is a key positive regulator of B cell homeostasis, and elevated levels of BLyS have been observed in systemic lupus erythematosus (SLE) patients. Given that anti-chromatin auto-antibodies are one of the hallmarks of SLE, we examined the role of BLyS and its receptors in the regulation of anti-chromatin B cells. We demonstrate that exogenous BLyS treatment leads to an increase in B cell numbers, particularly anti-chromatin B cells; yet, their localization in the spleen and auto-antibody production remain unaffected. We also examined transmembrane activator and CAML interactor (TACI), BLyS receptor 3 (BR3) and B cell maturation antigen expression on anti-chromatin B cells before and after receiving T cell help. Interestingly, in the absence of T cell help, TACI expression is greater on immature anti-chromatin B cells compared with immature Tg(-) B cells, whereas BR3 levels are comparable. After receiving T cell help, the anti-chromatin B cells that have differentiated into short-lived plasma cells no longer express BR3 but retain TACI. These data suggest a novel role for TACI in anti-chromatin B cell homeostasis and differentiation.

Atacicept (TACI-Ig) Inhibits Growth of TACIhigh Primary Myeloma Cells in SCID-Hu Mice.

Two TNF family members known to play key roles in normal B cell biology, BLyS/BAFF (B-lymphocyte stimulator/B cell activating factor) and APRIL (A PRoliferation-Inducing Ligand), also promote the survival of various malignant B cell types, including multiple myeloma (MM). BLyS binds to 3 TNF-R-related receptors, BCMA (B-cell maturation antigen, TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF-receptor), whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as CD138. All MM cells express HSPG and one or more of these 3 receptors, and incubation of MM cells with BLyS and/or APRIL leads to enhanced survival of these malignant cells in vitro. Stromal cells and osteoclasts residing in the bone marrow (BM) produce BLyS and APRIL and thus provide a paracrine source of these survival factors MM cells. Inhibition of BLyS and APRIL in vitro using a soluble receptor, TACI-Ig, causes cultured MM cells to die rapidly. It has been shown (Moreaux et al, Blood 106:1021) that differences in TACI gene expression can distinguish tumors with a BM microenvironment dependence signature (TACIhigh) from those with a plasmablastic signature (TACIlow), suggesting that TACIhigh MM cells may be more sensitive to growth factor withdrawal. We tested the ability of atacicept (TACI-Ig) to inhibit MM growth in the SCID-hu model of MM (Yaccoby et al, Blood 92:2908). Myelomatous SCID-hu mice were constructed by implanting a human fetal bone into which primary MM cells were directly injected. Changes in levels of tumor burden were monitored by weekly measurements of serum human monotypic immunoglobulins (hIg), and confirmed by histology. We compared the ability of atacicept and BAFFR-Ig (which binds BLyS, but not APRIL) to inhibit MM cell growth in this model. Upon establishment of myeloma growth, mice were injected intraperitoneally with atacicept (5 or 10 mg/kg, 3 times a wk; n=7), BAFFR-Ig (10 mg/kg, 3 times a wk; n=5) or with vehicle (PBS; n=7) for 6 wks. Compared to controls, in which tumor burden (hIg) increased by 1007±260% (avg±SEM) from pre-treatment levels, atacicept treatment markedly reduced tumor burden in 5 experiments in which TACIhigh MM cells were used, and delayed growth or had no effect in 2 experiments using TACIlow MM cells (avg increase in tumor burden of 302±184% for all 7 experiments; p<0.01 vs. control). Inhibition of tumor infiltration was confirmed by histology. Preliminary data also indicate that atacicept can inhibit growth of bortezomib-resistant MM cells in vivo. The level of TACI gene expression by MM cells generally correlated with response to atacicept. In vitro MM cell/osteoclast co-culture experiments confirmed these results, as atacicept inhibited growth of TACIhigh MM cells, but had a minimal effect on growth of TACIlow MM cells. In contrast, BAFFR-Ig treatment in the SCID-hu model resulted in tumor reduction in 1 experiment, delayed growth in 2 experiments and no effect in 2 experiments (avg increase in hIg of 762±298 and 208±46% in control and BAFFR-Ig groups, respectively; p<0.078), suggesting that APRIL plays a non-redundant role in survival of tumor cells from certain MM patients. Our results suggest that atacicept might represent an important new treatment for MM, and that TACI gene expression may prove to be a useful predictive marker for this therapeutic intervention.

Monogenetic defects in common variable immunodeficiency: what can we learn about terminal B cell differentiation?

In human primary immunodeficiencies, more than 100 different genetic defects have been described. For the most prevalent primary immunodeficiency requiring medical attention, however, termed common variable immunodeficiency, no genetic cause had been defined until recently. In this review we will summarize the current progress in the molecular genetics of common variable immunodeficiency and put them in context with other important developments in the field. In recent years the first three monogenetic defects in the inducible costimulator, transmembrane activator and CAML interactor (TACI), and CD19 were discovered in patients with common variable immunodeficiency revealing a multifaceted genetic background for this disease. As a concise phenotype cannot be assigned to each of these genetic defects, there is a need for further development of classification systems for common variable immunodeficiency and the search of epigenetic factors influencing the course of the disease. Subgroups of common variable immunodeficiency patients with low IgM memory B cells may suffer from an increased rate of infections. Human herpes virus type 8 infections were identified as a risk factor for the development of granulomatous disease complications. The pathogenesis of common variable immunodeficiency shows a convergence on impaired terminal B cell differentiation. Recently discovered genetic defects support this view. A combined effort of genetic analysis and standardized assessment of immunological and clinical phenotypes will be necessary to further unravel the conundrum of common variable immunodeficiency.

BAFF costimulation of Toll‐like receptor‐activatedB‐1 cells

B cell-activating factor belonging to the TNF family (BAFF) and its receptor BAFF-R play critical roles in the maturation and survival of conventional peripheral B cells. However, they appeared to be dispensable for the generation and maintenance of CD5(+) B-1 cells as BAFF(-/-) and BAFF-R(-/-) mice have normal B-1 cell populations. Hence, it is presently unclear if B-1 cells are responsive to BAFF and if BAFF regulates some aspects of B-1 cell function. We show here that BAFF-R and transmembrane activator and CAML interactor (TACI) are the major receptors expressed by B-1 cells. Specifically, we show that BAFF treatment of B-1 cells leads to increased NF-kappaB p100 processing and CD21/CD35 expression. Interestingly, toll-like receptor (TLR) engagement of B-1 cells augmented the surface expression of BAFF receptors and rendered them responsive to BAFF costimulation, as evidenced by their increased proliferation, expression of cell surface activation markers and secretion of the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10. This costimulatory effect is achieved primarily through BAFF-R as BAFF failed to costimulate B-1 cells obtained from A/WySnJ mice which have defective BAFF-R signaling. Thus, as TLR are innate immune receptors and B-1 cells are "innate-like" lymphocytes, our data provide evidence that BAFF plays a role in innate immunity.

BAFF and APRIL Protect CLL B Cells from Apoptosis through Activation of NF-κB Canonical Pathway.

B cell activating factor belonging to the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) are members of the TNF super family that are potent regulators of normal B cell development and function. BAFF binds to three distinct TNF receptors: BCMA (B cell maturation antigen), TACI (transmembrane activator and CAML interactor) and BAFF-R (also called BR3). Two of these receptors, TACI and BCMA, also bind APRIL. Some TNF super family proteins are known to induce activation of nuclear factor kB (NF-κB). Recent studies revealed two major pathways for NF-κB activation. One is the canonical pathway, which is regulated by the inhibitor of κB (IκB), and the alternative pathway, which is dependent upon the processing of NF-κB2/p100 to p52. The relative role of each pathway in enhancing CLL cell survival have not been well described. The aim of this study was to clarify the mechanism of CLL cell survival supported by BAFF and APRIL.CLL cells express BCMA, TACI, and BR3 by flow cytometry. CLL cells were cultured alone, with recombinant BAFF (rBAFF), or recombinant APRIL (rAPRIL). After 24 hours in culture, cytoplasmic and nuclear extracts were evaluated for p100/p52, p65, and phospho-IκBα by immunoblot analysis. When CLL cells were cultured with rBAFF or rAPRIL, the viability was higher compared to CLL cells in control cultures without such factors, indicating that BAFF or APRIL could protect CLL cells from spontaneous apoptosis. rBAFF could induce translocation of both p52 and p65 to the nucleus. On the other hand, rAPRIL could induce only p65 translocation to the nucleus, meaning that rBAFF could activate both the canonical and the alternative pathway of NF-κB, but rAPRIL could activate only the canonical pathway. These data suggest that signaling from BR3, but not from BCMA or TACI, can activate the alternative pathway. To verify this, CLL cells were cultured with rBAFF and anti-BR3, which can block BAFF binding to BR3. Anti-BR3 at 10μ M could completely inhibit p52 translocation to the nucleus induced by rBAFF. This data indicate that signaling from BR3 is necessary and sufficient to activate the alternative pathway in CLL cells. We examined the role of each pathway in the survival of CLL cells. 10μ M of anti-BR3, which can block activation of the alternative pathway, did not impair survival of CLL cells cultured with rBAFF. On the other hand, an IKKβ inhibitor, 5-(4-fluorophenyl)-2-ureido-thiophene-3 carboxylic acid amide (UTC), which can block the canonical pathway but not the alternative pathway, blocked the capacity of these factors to enhance CLL survival. UTC had less effect on survival induced by rBAFF on isolated normal B cells of healthy donors. We then transfected super repressor IkB (SR- IκB) to CLL B cells. SR- IκB transfected CLL cells underwent apoptosis more readily than control cells transfected with empty vector 24 hours after transfection. The survival of SR-IκB transfected cells could not be enhanced by rBAFF or APRIL.Collectively, BAFF and APRIL protect CLL B cells from apoptosis. Anti-apoptotic effects of these factors apparently are mediated via activation of the NF-κB canonical pathway. We speculate that inhibitors of IKKβ that inhibit the canonical pathway might have therapeutic activity in this disease.

BLyS and APRIL in rheumatoid arthritis.

The cytokines B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) enhance autoimmune disease by sustaining B cell activation. In RA, B cells contribute to the formation of 3 functionally distinct types of lymphoid microarchitectures in the inflamed synovium: ectopic GCs; T cell-B cell aggregates lacking GC reactions; and unorganized, diffuse infiltrates. We examined 72 tissues representing the 3 types of synovitis for BLyS and APRIL production and for expression of APRIL/BLyS receptors. Biologic effects of BLyS and APRIL were explored by treating human synovium-SCID mouse chimeras with the APRIL and BLyS decoy receptor transmembrane activator and CAML interactor:Fc (TACI:Fc). GC+ synovitis had the highest levels of APRIL, produced exclusively by CD83+ DCs. BLyS was present in similar levels in all tissue types and derived exclusively from CD68+ macrophages. In GC+ synovitis, treatment with TACI:Fc resulted in GC destruction and marked inhibition of IFN-gamma and Ig transcription. In contrast, inhibition of APRIL and BLyS in aggregate and diffuse synovitis left Ig levels unaffected and enhanced IFN-gamma production. These differential immunomodulatory effects correlated with the presence of TACI+ T cells in aggregate and diffuse synovitis and their absence in GC+ synovitis. We propose that BLyS and APRIL regulate B cell as well as T cell function and have pro- and antiinflammatory activities in RA.

Nurselike Cells in Chronic Lymphocytic Leukemia (CLL) Express Both BAFF and APRIL that Protect CLL B Cells from Spontaneous Apoptosis by Paracrine Manner.

Large, adherent nurse-like cells (NLC) can differentiate from the blood mononuclear cells of patients with chronic lymphocytic leukemia (CLL) that, in turn, can support leukemia cell survival in vitro, and presumably in vivo. These cells differentiate in the context of CLL B cells and can be found in the secondary lymphoid tissues of patients with this disease. NLC express high levels of B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF), a factor that can support normal and leukemia B cell survival in vitro. BAFF binds to three distinct members of TNF-receptor superfamily : TACI (transmembrane activator and CAML interactor), BCMA (B cell maturation antigen) and BAFF-R, the latter two of which are expressed by CLL B cells. We found that addition of saturating amounts of a recombinant BAFF-receptor-Fc fusion protein (BAFF-R:Fc) to cultures of NLC and CLL B cells failed to inhibit the protective effect(s) of NLC on CLL B cell survival in vitro. This suggested that another factor(s) made by NLC can contribute to the survival effect(s) of BAFF on CLL B cells. A candidate for this is a proliferation-inducing ligand (APRIL), another member of the TNF family that can interact with TACI and BCMA, but not BAFF-R. We found that, in contrast to CLL B cells or blood mononuclear cells, NLC express high-levels of APRIL mRNA and protein, as assessed by quantitative RT-PCR and immunoblot analyses. Furthermore, recombinant human APRIL also could support CLL B cell survival via mechanism similar to that used by recombinant BAFF. Consistent with the fact that APRIL and BAFF each can function to support CLL B cell survival, we found that a recombinant BCMA-Fc fusion protein could significantly inhibit the protective effects of NLC on CLL B cells in vitro. These data suggest that BCMA-Fc might be better suited than BAFF-R:Fc to inhibit the interactions between NLC and CLL B cells in vivo.

Control of spontaneous B lymphocyte autoimmunity with adenovirus-encoded soluble TACI.

Serum B lymphocyte stimulator (BLyS) is increased in autoimmune diseases, both in animal models and in humans. This study examined the effect of BLyS blockade in 3 animal models of lupus. Antibodies and lupus-like disease manifestations were examined in mice after administration of a single injection of an adenoviral construct for the transmembrane activator and CAML interactor receptor (AdTACI) that produces high serum levels of TACI-Fc fusion protein. In C57BL/6 (B6) lpr/lpr mice (B6.lpr/lpr), which were used to model autoimmunity in the absence of severe disease, treatment of younger mice with AdTACI prevented the development of hypergammaglobulinemia. In contrast, use of AdTACI for BLyS blockade had only transient effects on the levels of IgG in normal B6 mice. AdTACI blocked the development of autoantibodies in younger B6.lpr/lpr mice and reversed the production of autoantibodies in older B6.lpr/lpr mice, and also reduced the numbers of splenic plasma cells. In MRL.lpr/lpr mice, which were used to examine disease manifestations, AdTACI reduced the extent of glomerulonephritis and proteinuria and improved survival, but had little effect on T cell infiltration and interstitial nephritis. However, in (NZB x NZW)F(1) mice, AdTACI induced neutralizing anti-TACI antibodies and failed to reduce the numbers of B cells. BLyS blockade has little effect on IgG levels in normal mice, but reverses the production of spontaneously produced IgM and IgG autoantibodies in the setting of established autoimmunity. Blockade of BLyS ameliorates B cell-dependent disease manifestations even in the MRL.lpr/lpr model, but its effectiveness on autonomous T cell aspects of the disease is limited. Moreover, its effectiveness is neutralized by anti-TACI antibodies when present. These results provide a basis for understanding the potential effects of BLyS blockade in human disease.

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF.

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.