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The Journal of Immunology, 2007, 179, 7276 -7286
Copyright © 2007 by The American Association of Immunologists, Inc.
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Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation1

Jaime R. Darce, Bonnie K. Arendt, Xiaosheng Wu and Diane F. Jelinek2

Department of Immunology, Mayo Clinic College of Medicine, Mayo Graduate School, Rochester MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 CD27neg 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The B cell-activating factor belonging to the TNF family, BAFF3 (also known as BLyS), is required for the development and homeostasis of mature B cells. This was first demonstrated using BAFF-deficient mice, which exhibited a block in B cell maturation evidenced by a lack of transitional 2 and marginal zone B cells (1, 2), and BAFF-transgenic mice, which developed B cell hyperplasia and accompanying autoimmunity (3, 4). The signals that drive BAFF-dependent B cell maturation in mice appear to be mediated exclusively through the BAFF receptor (BAFF-R), which binds BAFF with high affinity (2, 5). Thus, mice bearing a mutated form of BAFF-R (6, 7) or BAFF-R null mice (8, 9) are comparable in phenotype to BAFF-deficient mice. Still, those murine models only provide insights into the possible role of BAFF and BAFF-R in human B cell biology. In addition, these models do not allow an assessment of the involvement of the BAFF-BAFF-R interaction during later stages of B cell maturation.

Despite lack of a clear understanding of the effects of BAFF in later stages of B cell differentiation, there is accumulating evidence that BAFF is involved in murine and human B cell differentiation as revealed by BAFF’s ability to enhance Ig gene diversification and augment B cell differentiation into Ig-secreting cells (ISCs). However, how BAFF precisely influences these events remains elusive since it has been demonstrated that in addition to BAFF-R, BAFF can also bind two other receptors, transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) (10, 11, 12) and B cell maturation Ag (BCMA) (1, 13), which bind BAFF with intermediate and low affinity, respectively (5, 14).

To date, investigation into the functions of TACI and BCMA during late-stage B cell biology have been largely conducted in the murine system. For example, it has been suggested that TACI plays an important role in negatively regulating mature B cell homeostasis. This was proposed after discovering that mature B cell numbers increased in TACI-deficient mice, resulting in splenomegaly and in one case, a B cell lymphoma-like phenotype (15, 16). Although these data suggest that TACI may down-regulate mature B cell proliferation, a recent study demonstrated that when syndecan-2 binds to TACI, B cell proliferation is augmented suggesting that the negative regulatory properties of TACI could be ligand specific (17). In regard to B cell differentiation, it was discovered that TACI also plays a pivotal role in class switch recombination (CSR), since B cells in TACI-deficient mice were unable to class switch even in the presence of BAFF (18). Similarly, studies of patients suffering from common variable immunodeficiency suggested that the absence in Ig H chain diversity observed is due to a defective form of TACI present in B cells from these individuals (19, 20). Most recently, it has been proposed that TACI expression is required for ISC differentiation, at least in the context of T cell-independent types 1 and 2 B cell responses (21, 22). Although the requirement of TACI expression was not evaluated, Castigli et al. (23) also demonstrated that signals through TACI promoted T cell-dependent ISC differentiation, contending a previous report claiming otherwise (24). Unfortunately, much less is presently known about BCMA, other than it is expressed in normal and malignant plasma cells (PCs) (25, 26) and that it is critical in maintaining the survival of bone marrow (BM) long-lived PCs (27). In addition, one report demonstrated that BCMA is induced upon T cell-dependent activation of blood MB cells, which may promote their survival in response to BAFF (28).

In an effort to understand the specific roles of these receptors during later stages of B cell differentiation, it is necessary to first understand their expression in B-lineage cells at various stages of maturation. Expression of BAFF-R has been found on most B-lineage cells, with low levels of expression on germinal center (GC) B cells (26, 29, 30). Furthermore, there is some evidence to suggest that TACI and BCMA are expressed at later stages of B cell development. Still, the precise developmental stage(s) at which B cells express these two receptors remains controversial. For instance, Zhang et al. (30) reported that TACI is only expressed by tonsillar MB cells, while Ng et al. (29) found TACI expressed in mature naive and MB B cells, but lacking in GC B cells. Most recently, Chiu et al. (26) demonstrated that TACI is expressed by all tonsillar B-lineage cells, ranging from naive to PC. Similarly, there are also inconsistent reports of BCMA expression, including those that show that BCMA is expressed in tonsillar GC B cells and PCs (26), GC and memory B cells (29), or not expressed at all in tonsillar B-lineage cells (30). Thus, the lack of clarity concerning BAFF-binding receptor (BBR) expression at various stages of B cell development has made it difficult to understand the specific roles these receptors play in human B cell biology.

Furthermore, studies describing the specific signaling events that regulate the expression of BBRs during B cell activation through various polyclonal stimuli are limited in number. A recent report revealed that activation of murine B cells through TLR4 or TLR9 promotes the induction of TACI through a MyD88-dependent mechanism (31). These investigators also demonstrated that TACI expression in response to TLR4 signals requires components of the NF-{kappa}B pathway (c-Rel and p50), which are not required by TLR9 to augment TACI expression. The signaling pathway(s) involved in TACI induction mediated through TLR9 was not further investigated and thus remains unknown. In contrast, much less is known about the signals that regulate BAFF-R and BCMA. Early reports proposed that signals through the BCR enhanced expression of BAFF-R, although a specific signaling pathway(s) responsible for its expression remains undefined. To date, the regulation of BCMA expression has yet to be examined.

Our study was predicated on the knowledge that there are multiple receptors that can independently bind BAFF and they are likely to be variably coexpressed on B cells in a development/differentiation stage-specific manner. To better understand the function of each receptor in B cell biology, it is essential to first characterize the precise BBR profile as well as the stimuli that regulate individual receptor expression in B cells. In this study, we describe our comprehensive analysis of BBR expression in normal human B-lineage cells. Our results demonstrating the coordinated regulation of all three receptors begin to suggest unique functions for each receptor. Specifically, our results suggest for the first time that TACI is up-regulated immediately upon B cell activation and this is mediated through the ERK/MAPK pathway. Moreover, we prove that up-regulation of BCMA and loss of BAFF-R occurs in response to differentiation, suggesting that the down-regulation of BAFF-R may be a requisite step in B cell differentiation into ISCs and PCs. More broadly, our results suggest the possibility that the BBR profile may be of use in identifying the activation history of an individual B cell and, as such, may also have utility in identifying the transformation stage of a variety of mature B cell malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

Mononuclear cells within peripheral blood (PB) from normal donors, tonsil tissue from routine tonsillectomies, or BM from patients undergoing hip replacement surgery were separated by Ficoll-Hypaque density gradient centrifugation. Individuals provided written informed consent in accordance with the Declaration of Helsinki. The Mayo Clinic Rochester Institutional Review Board approved the protocol to obtain blood, tonsil, and bone marrow tissue from volunteers. B lymphocytes were enriched to >98% purity by magnetic cell separation using a StemCell Technology B cell enrichment mixture/colloid and the negative selection program on a Robosep Separator (StemCell Technologies). Similarly, BM PCs were enriched to >95% purity by magnetic cell separation using a StemCell CD138 mixture/colloid and a positive selection program on a Robosep Separator.

Flow cytometry

Purified cells were stained using standard flow cytometry methodology. Briefly, cells were incubated on ice for 20 min with primary Ab before washing twice with cold FACS buffer (Dulbecco’s PBS (DPBS) containing 2 mM EDTA, 0.05% sodium azide, and 2% FCS) and subsequent incubation with various secondary reagents. After washing, cells were fixed with 1% paraformaldehyde before analysis using a FACSCalibur flow cytometer (BD Pharmingen) and FlowJo analytical software (Tree Star). CD25, CD27, CD38, CD80, and CD138 Abs were purchased from BD Pharmingen; PE conjugated mAbs against BAFF-R along with isotype control Abs were purchased from eBioscience; and biotinylated polyclonal anti-TACI and anti-BCMA Abs were purchased from R&D Systems along with goat biotinylated IgG control Ab. PE- or allophycocyanin-labeled streptavidin (Caltag Laboratories and Invitrogen Life Technologies) were used as secondary reagents. Cell turnover was determined using CFSE (Molecular Probes and Invitrogen Life Technologies) intercalating dye. Cells were suspended in 0.1% FCS/DPBS at a density of 20 x 106 cells/ml and labeled with 1.5 µM CFSE for 8 min at room temperature. Labeling was halted by adding prewarmed FCS and then incubating at 37°C for 10 min to efflux excess CFSE. Cells were washed three times with 2% FCS/DPBS and cultured in the presence or absence of mitogen.

Polyclonal activation of PB B cells

PB B cells were activated with the following polyclonal stimuli: anti-Ig (2 µg/ml agonistic anti-IgA, IgG, IgM F(ab')2 Abs purchased from Jackson ImmunoResearch Laboratories); anti-Ig/CD40 ligand (CD40L; (2 µg/ml agonistic anti-IgA, IgG, IgM F(ab')2 Abs and 0.5 µg/ml shrCD40L/TNF-related activation protein (Fitzgerald Industries International); CD40L (0.5 µg/ml shrCD40L/TRAP); CpG (oligodeoxynucleotide 2006, 5'-TCGTCGTTTTGTCGTTTTGTCGTT, synthesized by in-house core facility, 2.5 µg/ml). When indicated, B cells were also activated in the presence of various cytokine combinations. IL-2 (20 U/ml; Fitzgerald Industries International) and IL-10 (50 ng/ml; PeproTech) were used in conjunction with anti-Ig, CD40L, or anti-Ig/CD40L. IL-2 (20 U/ml) and IL-15 (10 ng/ml; PeproTech) were included with CpG. To sustain viability, B cells were often cultured in medium containing 50 ng/ml IL-4 (PeproTech). In some experiments, B cells were activated with PMA at a concentration of 10 ng/ml. To determine the effects of blocking ERK1/2 activation in long-term B cell activation experiments, B cells were pretreated for 1 h at 37°C with PD98059 (Cell Signaling Technology), U0126 (Cell Signaling Technology), or vehicle. These cells were then cultured for indicated periods of time with specified polyclonal stimuli. B cells were cultured at 37° C in a 5% CO2 incubator for the indicated lengths of time in polypropylene round-bottom tubes (BD Labware) in the presence or absence of the above stimuli at a concentration of 1 x 106 cells/ml in complete medium (RPMI 1640 with 10% FCS, L-glutamine, penicillin, and streptomycin).

Analysis of Ig secretion

Ig secretion was measured using a standard Ig H chain-specific ELISA. Briefly, 96-well microtiter plates (Nalge Nunc International) were independently coated with anti-IgA, anti-IgG, and anti-IgM Abs (BioSource International). Plates were then blocked with 1x casein (BioFX Laboratories). After several washes, culture supernatants were added to coated plates and incubated for 2 h. Igs were detected colorimetrically using anti-IgA, anti-IgG, and anti-IgM HRP-labeled Abs (BioSource International) and a Molecular Devices microplate reader. Standard curves were generated to quantitate ELISA results using known amounts of purified human IgA, IgG, and IgM Abs (Jackson ImmunoResearch Laboratories). The detection limit of the assays was 1 µg/ml to 0.1 ng/ml for IgG and IgM and 2 µg/ml to 0.1 ng/ml for IgA. o-Phenylenediamine dihydrochloride ELISA substrate for HRP along with stable peroxide substrate buffer were purchased from Pierce.

Immunoblot analysis

We incubated purified PB B cells at a density of 1 x 106 cells/ml for 1 h at 37°C with either 20 µM PD98059 or U0126, or an equivalent volume of DMSO carrier (final DMSO concentration was 0.001%). After treatment with inhibitors or vehicle, cells were stimulated for the indicated time points. After stimulation, cells were collected and incubated on ice with lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 5 mM Na2EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM EGTA, 10 mM sodium fluoride, 1 mM PMSF, and 1 mM tetrasodium pyrophosphate) supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 2 mM Na3VO4, 100 µM β-glycerophosphate, and 1 mM PMSF). Lysates were cleared of insoluble material by centrifugation for 10 min at 15,000 x g. Lysates (10–20 µg/lane) were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore) for immunoblotting. Membranes were blocked for 1 h at 37°C in 5% Blotto (Santa Cruz Biotechnology) supplemented with 0.2% Tween 20 and then blotted overnight with anti-phospho-ERK or anti-ERK Abs (Cell Signal Technology) following the manufacturer’s protocol. Immunoreactive proteins were detected using an ECL detection system (Super Signal; Pierce) and autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Baseline expression of BBRs on human B-lineage cells

We were first interested in determining the expression of BBRs on B cells at varying stages of maturation, as well as from various tissues. We found that BAFF-R is uniformly expressed on naive B cells, as well as MB cells and tonsillar PCs (Fig. 1A). However, bone marrow PCs, unlike tonsillar PCs, did not express BAFF-R on their surface (Fig. 1A). Furthermore, BCMA was expressed by tonsillar MB cells in addition to PCs from both tonsil and BM tissues (Fig. 1B). Bone marrow PCs, however, expressed lower levels of BCMA compared with tonsillar PCs (Fig. 1B). In addition, expression of BCMA was absent in naive B cells as well as PB MB cells (Fig. 1B). As for TACI expression, we discovered that it is predominantly expressed by MB cells and tonsillar and BM PCs, although to a lesser degree in the latter population (Fig. 1C). Of interest, we found that a small subset of CD27neg naive B cells also expressed low levels of TACI and this was observed in both blood and tonsil tissues (Fig. 1C). Analysis of blood B cells from 10 additional healthy individuals further supported our finding that TACI is expressed by a subset of CD27neg naive B cells (Fig. 2A). The CD27neg TACI-positive subset also expressed activation markers such as CD25 and CD80 (Fig. 2, A and B), suggesting that TACI expression is associated with B cell activation.


Figure 1
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  		FIGURE 1. Expression of BBRs on B-lineage cells. Expression of BAFF-R (A), TACI (B), and BCMA (C) on blood, tonsil, and BM B-lineage cells. Control Ab (filled histogram). MB were identified by expression of surface CD27, which is absent on naive B cells. Top two panels, Blood and tonsil, A–C; naive B cells (CD27neg; solid-lined histogram) and MB (CD27pos; dotted histogram). Bottom two panels, Tonsil and BM CD138+, A–C; CD138+ B cells (solid-lined histogram). Data are representative of three independent experiments.

 

Figure 2
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  		FIGURE 2. TACI is expressed on CD27neg B cells displaying an activated phenotype. A, Analysis of TACI expression on B cells from 10 normal individuals. B, B cell populations gated on expression of surface CD27 and TACI. C, Expression of CD25 and CD80 on the gated populations. Control Ab (filled histogram), MB (thin-lined histogram), TACIneg B cells (dotted histogram), and TACIposCD27neg B cells (thick-lined histogram).

 
TACI expression is induced upon activation of TACI-negative B cells

Because we initially found TACI expressed on the surface of MB cells and a subpopulation of CD27neg B cells, both of which display an activated phenotype, we next wanted to determine whether TACI expression could be induced in response to in vitro activation and, if so, what type of stimulus was required.

For these studies, we isolated TACI-negative B cells from PB B cell preparations and then treated these cells with various polyclonal stimuli in the presence or absence of cytokines (Fig. 3). We found that activation of naive B cells through their BCR led to a marginal expression of TACI, which was slightly increased when CD40L was added to the culture (Fig. 3B). In contrast, activation of naive B cells with CD40L alone did not yield TACI expression (Fig. 3B). However, when these cells were activated with either anti-Ig or CD40L in the added presence of IL-2 and IL-10, we observed a significant increase in TACI expression. TACI induction was even greater when cells were stimulated with the combination of anti-Ig, CD40L, IL-2, and IL-10 (Fig. 3B). Of note, TACI expression was not induced when cells were cultured with IL-2 and IL-10 alone, suggesting that initial activation may be required (Fig. 3B). In addition, we activated naive B cells using CpG oligodeoxynucleotides, which engage and signal through TLR 9. We found that CpG treatment alone significantly induced expression of TACI in these cells and that expression levels did not further increase upon addition of IL-2 and IL-15, cytokines which are known to enhance B cell responsiveness to CpG. Culture of B cells with IL-2 and IL-15 alone was without effect in inducing TACI expression (Fig. 3B). In addition, we observed that TACI is transcriptionally regulated. Thus, freshly isolated naive B cells did not express TACI mRNA, but following activation, TACI mRNA was detectable using RT-PCR (data not shown).


Figure 3
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  		FIGURE 3. Up-regulation of TACI expression in TACIneg B cells in response to activation with various polyclonal stimuli. A, Post-sort analysis of TACIneg sorted B cells. B, Expression of surface TACI upon activation of TACIneg B cells with various polyclonal activators in the presence or absence of cytokines.

 
Up-regulation of TACI occurs early in B cell activation and it is independent of cell division

We next wished to study the kinetics of TACI induction upon CpG/IL-2,15 and anti-Ig/CD40L/IL-2,10 activation, since these two stimuli resulted in a robust expression of TACI. We assessed surface TACI levels on resting or activated PB B cells daily for a period of 5 days. Our data revealed that unstimulated B cells cultured with IL-4 to preserve viability maintained a differential expression of TACI even after 5 days in culture (Fig. 4A). Although IL-4 maintained B cell viability (32) (data not shown), it did not affect the levels of surface TACI expression when compared with B cells cultured in medium alone (Figs. 3B and 4A and data not shown). However, as shown in Fig. 4, B and C, we noticed that TACI is rapidly induced, whereby maximum levels of expression were achieved 2–3 days following activation with either stimulus. Moreover, within 24 h of activation, we found that TACI was up-regulated in a fraction of B cells. To determine whether cell division was required before up-regulation of TACI expression, we monitored TACI levels and cell division concurrently using CFSE, which is a reagent commonly used to detect dividing cells. As shown in Fig. 5A, TACI expression is found in B cells that have high CFSE staining intensity, which represent B cells that have not yet divided in response to CpG activation at 72 h. These data suggest that TACI expression occurs independent of cell division. Still, our data above clearly demonstrate that TACI is quickly induced upon activation of B cells, thereby implying it is an activation marker. We next examined the expression levels of TACI and CD25 (a well-known activation marker induced early in B cell activation). Following 24 h of CpG/IL-2,15 activation, virtually all of the B cells acquired CD25 expression, including TACI-negative B cells (Fig. 5B). These results suggest that CD25 expression precedes TACI expression.


Figure 4
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  		FIGURE 4. Kinetics of TACI expression upon PB B cell activation. Analysis of TACI expression in B cells cultured with IL-4 (A), CpG/IL-2,15 (B), or anti-Ig/CD40L/IL-2,10 (C) at various time points. Forward (FSC) vs side scatter (SSC) dot plots of each time point are presented to demonstrate the gated B cells included in this analysis.

 

Figure 5
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  		FIGURE 5. TACI is induced as a result of B cell activation, but is independent of proliferation. A, Analysis of TACI expression levels along with cell division monitored by CFSE dilution assay at 72 h. B, Expression analysis of TACI and CD25 at 24 h of CpG/IL-2,15 activation.

 
TACI expression requires the ERK1/2 signaling pathway

So far, our data demonstrate that TACI is quickly up-regulated in B cells in response to activation by various polyclonal stimuli. We next wished to determine the signals that may be involved in regulating TACI up-regulation. Because the polyclonal activators used thus far signal through multiple pathways, in experiments not shown, we used chemical agonists of specific pathways, i.e., PMA and calcium ionophore, to determine whether activation of protein kinase C or induction of intracellular calcium release could trigger TACI induction. These studies demonstrated that TACI expression was induced in B cells following stimulation with PMA, but not by the calcium ionophore ionomycin (data not shown). PMA activation is mediated through protein kinase C, which subsequently triggers downstream ERK and JNK signaling pathways. To uncover the downstream signals that regulate TACI expression in PMA-activated B cells, we chose to use known pharmacological inhibitors of these downstream pathways. To block ERK1/2 activation, we used ERK-specific inhibitors PD98059 and U0126. As shown in Fig. 6A, phosphorylation of ERK1/2, resulting from PMA activation, is attenuated in the presence of PD98059 and U0126 (data not shown). We then activated B cells for 120 h with PMA in the presence or absence of PD98059 or DMSO vehicle and monitored TACI expression at said time. Our data reveal that TACI up-regulation induced by PMA activation is blocked when B cells are treated with the ERK1/2-specific inhibitor PD98059 (Fig. 6B). Because it is possible that the inhibitor simply blocked B cell activation, we also evaluated the activation status of B cells treated with PD98059 by monitoring induction of CD25 expression. As shown in Fig. 6B, CD25 was induced in B cells treated with PMA in the presence or absence of PD98059. These data suggest that B cell activation was not affected by PD98059 treatment.


Figure 6
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  		FIGURE 6. PMA-induced up-regulation of TACI expression is blocked by PD98059. A, Phosphorylation of ERK1/2 in freshly isolated B cell lysates (C, control) and in B cells activated with PMA for 3 min in the presence or absence of PD98059. ERK1/2 protein levels were used as loading controls. B, Expression of TACI and CD25 in response to PMA treatment with or without PD98059. Expression measured at 120 h of activation. Filled histograms, Ig control; open histograms, TACI-specific Ab.

 
These studies prompted us to investigate whether TACI up-regulation, mediated by CpG activation, also required the ERK1/2 pathway. However, before treating cells with ERK1/2 inhibitors, we first determined whether CpG activation also resulted in phosphorylation of ERK1/2. Despite an early report suggesting that activation of the WEHI-231 B cell line with CpG alone does not trigger activation of ERK1/2 (33), two recent reports revealed that CpG activation does result in the phosphorylation of ERK1/2 in murine B cells (34, 35). In our analysis of ERK1/2 phosphorylation in response to CpG stimulation of human PB B cells, we observed phosphorylation of ERK as early as 15 min after activation (Fig. 7A), with maximum responsiveness observed at 30 min. ERK1/2 phosphorylation was significantly diminished at the 60- and 120-min time points (Fig. 7A). Using the ERK inhibitor U0126, we were able to completely block CpG-induced phosphorylation of ERK1/2 (Fig. 7B). In addition, phosphorylation of ERK1/2 at later time points (60 and 120 min) was also blocked by U0126 (data not shown). We next evaluated the expression of TACI in B cells activated with CpG in the presence or absence of U0126. As we previously observed in our PMA studies, U0126 treatment blocked the up-regulation of TACI induced by CpG stimulation at 72 h (Fig. 7C). Similarly, we found that U0126 treatment also blocked TACI expression at 120 h (data not shown). Despite blocking TACI expression, up-regulation of CD25 was not affected by U0126 treatment (Fig. 7C). Thus, these data imply that the ERK pathway is significantly involved in regulating the induction of TACI in response to B cell activation.


Figure 7
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  		FIGURE 7. U0126 blocks CpG-induced up-regulation of TACI. A, ERK1/2 phosphorylation in freshly isolated B cells (C, control) and in B cells activated with CpG at various time points. B, Phosphorylation of ERK1/2 after treatment with CpG (30 min) in the presence or absence of U0126. C, Effects of U0126 on TACI and CD25 expression at 72 h of activation with CpG only (without IL-2 and IL-15). Filled histograms, Ig control; open histograms, TACI-specific Ab.

 
Activated memory B cells lose BAFF-R and gain BCMA expression

In addition to understanding the expression of TACI, we also investigated the expression profile of BAFF-R and BCMA as a result of B cell activation. As described above, PB B cells uniformly express BAFF-R and lack BCMA upon isolation (Fig. 1). We discovered that this BAFF-R and BCMA expression profile does not change with time in B cells cultured in medium alone (data not shown) or cultured with IL-4 for 5 days (Fig. 8). However, in response to activation with either CD40L/IL-2,10 or CpG/IL-2,15, a subpopulation of B cells exhibited a loss of BAFF-R (Fig. 8). Similarly, we noticed that both of these activation schemes induced the expression of BCMA, but only in a small fraction of B cells (Fig. 8). We next wanted to address whether: 1) BAFF-R and BCMA were inversely expressed in the same B cell population and 2) which B cells were losing or gaining BAFF-R and BCMA, respectively. Because CpG/IL-2,15 activation was more effective at promoting the loss of BAFF-R and up-regulation of BCMA in a larger fraction of B cells in comparison to CD40L/IL-2,10 activation (Fig. 8), this mode of stimulation was used in subsequent studies. To determine whether loss of BAFF-R was coincident with acquisition of BCMA, we activated B cells with CpG/IL-2,15 and analyzed BAFF-R and BCMA expression levels daily over a period of 5 days. As shown in Fig. 9A, the same population of B cells that lost expression of BAFF-R gained BCMA expression on their surface, suggesting that expression of BCMA may require the loss of BAFF-R. Moreover, unlike the relatively rapid kinetics of TACI up-regulation, the inverse expression levels of BAFF-R and BCMA were acquired later in activation, beginning at 72 h and becoming more evident at 120 h (Fig. 9A). Because we were stimulating total PB B cells, which encompass both naive and memory B cells, it was not clear which B cell population was down-regulating BAFF-R and up-regulating BCMA. Therefore, we costained CpG/IL-2,15-activated B cells with anti-CD27 Abs to distinguish between naive and memory B cells, along with Abs against BAFF-R or BCMA. We analyzed BAFF-R and BCMA expression on CD27neg B cells and two populations of CD27pos B cells that varied in absolute levels of CD27 (CD27low and CD27high; Fig. 9B). Upon activation, CD27neg B cells retained the expression of BAFF-R and did not up-regulate BCMA (Fig. 9C). In contrast, CD27pos B cells lost substantial levels of surface BAFF-R and gained expression of BCMA (Fig. 9C). Moreover, we found that the majority of the cells that lost BAFF-R expression and expressed higher levels of BCMA were within the CD27high population (Fig. 9C).


Figure 8
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  		FIGURE 8. Surface BAFF-R and BCMA expression following B cell activation. Expression of BAFF-R (A) or BCMA (B) on B cells cultured with IL-4 or activated with CD40L/IL-2,10 or CpG/IL-2,15 for 5 days. Data are representative of five independent experiments.

 

Figure 9
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  		FIGURE 9. BAFF-R and BCMA are differentially expressed on CpG/IL-2,15- activated memory B cells. A, Expression of BAFF-R and BCMA at different time points in B cells that were activated with CpG/IL-2,15. B, CD27 expression on CpG/c-activated B cells. C, Expression of BAFF-R and BCMA on CD27neg and two populations of CD27pos B cells, CD27high and CD27low. Control Ab (filled histogram), CD27neg B cells (thick-lined histogram), CD27low (dotted histogram), and CD27high (thin-lined histogram). Data are representative of three different experiments.

 
Inverse expression of BAFF-R and BCMA occurs as a result of B cell differentiation into ISCs

A previous report by Avery et al. (36) revealed that as MB cells commit to the PC lineage they increase their expression of CD27. Since we demonstrate above that CD27high B cells have, for the most part, lost BAFF-R and gained BCMA, we hypothesized that this expression profile is a consequence of differentiation. Our hypothesis was further substantiated by our initial ex vivo analysis of BM PCs, which lack BAFF-R yet express TACI and BCMA (Fig. 1). To block B cell differentiation into ISCs, we implemented the use of anti-Ig receptor Abs (anti-Ig), since it has been previously demonstrated that constant signals through the BCR block ISC differentiation induced by CD40L/IL-2,10 and CpG/IL-2,15 activation (34, 37, 38, 39). In Fig. 10, we show that activation with CD40L/IL-2,10 (Fig. 10A) or CpG/IL-2,15 (Fig. 10B) drives differentiation of B cells, measured by secretion of Ig (IgA, IgG, and IgM). However, when anti-Ig Abs are added, secretion of IgA, IgG, and IgM was blocked (Fig. 10, A and B). In addition to these data, we also found no evidence of cytoplasmic Ig staining in B cells activated with either stimulus in the presence of cross-linking anti-BCR Abs (data not shown). These data established a method of blocking differentiation, thus we next evaluated the expression of BAFF-R and BCMA in B cells treated in the presence or absence of anti-Ig Abs. Unlike CpG/IL-2,15 or CD40L/IL-2,10 activation, we found no decrease in BAFF-R nor up-regulation of BCMA in B cells activated in the presence of anti-Ig Abs (Fig. 10, C and D). Of note, although the inverse expression of BAFF-R and BCMA was abrogated by anti-Ig treatment, addition of anti-Ig to either CD40L/IL-2,10 or CpG/IL-2,15 activation did not negatively affect TACI expression (Fig. 4C and data not shown). These data suggest that up-regulation of BCMA along with loss of BAFF-R expression is not a consequence of B cell activation but instead differentiation into ISCs.


Figure 10
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  		FIGURE 10. Inverse expression of BAFF-R and BCMA is associated with B cell differentiation into ISCs. Secreted Ig levels in response to A) CD40L/IL-2,10 or B) CpG/IL-2,15 in the presence or absence of anti-Ig Abs. Open bars (activation without anti-Ig antibodies) filled bars (activation in the presence of anti-Ig antibodies). C) BAFF-R and BCMA levels in B cells activated with CD40L/IL-2,10 or CpG/IL-2,15 for 120 h. D) BAFF-R and BCMA levels in B cells activated with anti-Ig/CD40L/IL-2,10 or anti-Ig/CpG/IL-2,15 for 120 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The precise role(s) of BBRs during B cell differentiation into effector MB cells or PCs has not been yet defined, in part because a consensus view of BBR expression on mature B-lineage cells before and after activation has not been reached. Thus, although there are numerous reports characterizing BBR expression levels on B cells, there remain various inconsistencies in reported BBR profiles. The goal of our study was 2-fold: 1) comprehensively characterize the BBR expression profile on several human B cell subsets ranging from resting naive B cells to BM PCs and 2) determine the specific effects of a range of stimuli on BBR expression on PB naive and MB cells. Our data demonstrate how different antigenic stimuli unequivocally mediate expression of BBRs during B cell activation and differentiation. Because of the highly regulated and coordinated expression of all three receptors, we propose that the BBR profile may be a useful biological footprint that marks the developmental stage of a human B cell.

BAFF-R expression

Although it is clear that formation of a mature B cell compartment requires signals from BAFF through the BAFF-R (2, 5), the precise stage at which developing B cells acquire BAFF-R expression remains unclear. Nevertheless, it has been demonstrated that transitional B cells exit the BM already expressing BAFF-R (40). However, their BAFF-R expression levels are lower than mature B cells (40), suggesting that further B cell maturation yields an increase in BAFF-R expression. Of note, the functional significance of higher levels of BAFF-R expression remains unknown. Our analysis of BAFF-R on CD19+ PB and tonsillar B cells is consistent with other studies (28, 29); however, we extend the literature by demonstrating that naive and memory PB B cells express comparable levels of BAFF-R. In addition, our analysis of primary B-lineage cells at various stages of maturation reveals that BAFF-R expression decreases and eventually is lost, as B cells differentiate and eventually become PCs. With respect to reduced BAFF-R expression, Ng et al. (29) and Chiu et al. (26) previously described low BAFF-R levels in GC B cells and tonsillar PCs; however, we demonstrate for the first time the complete loss of BAFF-R expression in normal human BM PCs. Our observations that T cell-dependent or T cell-independent in vitro activation of PB MB cells results in decreased BAFF-R levels as these cells differentiate into ISCs corroborates findings by Zhang et al. (30) and extends those studies by showing that the differentiation process itself specifically decreases BAFF-R expression. Thus, we demonstrate that CD40L/IL-2,10- or CpG/IL-2,15-stimulated extinction of BAFF-R expression is significantly compromised by sustained BCR signaling, an action known to inhibit B cell differentiation into ISCs (Fig. 10, A and B, and Refs. 34 and 37, 38, 39). Furthermore, we show for the first time differences in expression of BAFF-R on CD138pos tonsillar vs BM PCs. Taken together with our in vitro studies tracking BAFF-R levels during B cell differentiation, we speculate that PC BAFF-R expression status may provide a tool to discriminate between short-lived and long-lived PCs. Implicit in this speculation is the possibility that BAFF-R continues to play a role in the short-lived PCs. Given that chemokine receptors have been shown to play a key role in PC homing to the BM (41, 42), it is possible that signals delivered through BAFF-R attenuate expression of receptors required for PCs to make this transition.

TACI expression

Several reports in the literature describe TACI expression on human B-lineage cells; however, these analyses have largely been restricted to tonsillar B cells, which are enriched in numbers of activated cells (26, 29, 30). Our studies considerably expand on the published reports by demonstrating that TACI is uniformly expressed by PB and tonsillar MB cells as well as PCs from tonsil and BM tissues. In addition, we describe for the first time a subpopulation of CD27neg PB B cells that coexpress TACI. This subpopulation was routinely detectable in normal healthy adults and was shown to coexpress cell activation markers. Moreover, we found that TACI expression is quickly induced upon in vitro activation and this is potentially mediated specifically through the activation of the ERK pathway, since blocking with ERK-specific inhibitors significantly attenuates activation-induced TACI expression. Currently, there are no reports identifying the transcription factors that may be involved in regulating TACI expression. However, since we found that TACI expression depends on ERK1/2 activation, which regulates the activity of the transcription factor TCF/Elk-1, it is interesting to speculate that TCF/Elk-1 may play a role in regulating TACI levels. Indeed, we have analyzed the sequence upstream of the TACI gene and determined that there are two conserved TCF/Elk-1 binding sites at –58 and –48, in both human and mouse DNA (data not shown). A regulatory role for TCF/Elk-1 in activation-induced TACI up-regulation is currently under investigation. Finally, our demonstration that TACI is rapidly induced upon activation as well as expressed in preactivated B cells and PCs suggest possible roles for TACI during early B cell responses as well as later stages of B cell differentiation.

With respect to B cell differentiation, it has been recently demonstrated that TACI is necessary for CSR in mice and humans (18, 19, 20). However, to date, there exits no biochemical or molecular evidence describing the TACI-mediated signals involved in regulating CSR events. Still, there is some evidence that TACI could regulate the expression of activation-induced cytidine deaminase (AID), which is the enzyme responsible for both CSR and somatic hypermutation (43). In light of our observations that a subpopulation of CD27neg B cells expresses TACI, we are currently investigating AID expression levels in TACI-positive CD27neg B cells, as well as determining whether signals through TACI can regulate AID expression and function in these cells. Of interest, it was recently demonstrated that AID is found in a subpopulation of naive B cells possessing an activated phenotype (44). In regard to requiring TACI for differentiation into ISCs, we found that blocking TACI up-regulation (via ERK inhibitors) blocked differentiation of B cells into ISCs. This was assessed by measuring Ig secretion and BCMA expression, which was not induced in these cells (data not shown).

BCMA expression

Both BAFF-R and TACI have been extensively studied and implicated in B cell homeostasis and development. Conversely, knowledge of BCMA is limited and thought to only affect the homeostasis of long-lived PCs. However, we describe that BCMA is expressed not only by tonsillar and BM PCs, but also by MB cells found within tonsil tissues, suggesting that this receptor may also be involved in MB cell biology. Furthermore, we also demonstrate that unlike TACI, BCMA is not rapidly up-regulated in response to activation. Instead, its expression is induced as a result of MB cell differentiation into ISCs. We found increased BCMA expression in cells that expressed higher levels of CD27, which has been correlated with their commitment to differentiate into PCs (36). Moreover, we demonstrate for the first time that a form of T cell-independent B cell activation (via CpG/IL-2,15) can induce BCMA expression. This implies that BCMA expression can potentially be acquired outside GC reactions. Although the specific signals that drive BCMA induction are presently unknown, we discovered that its expression is coupled with the loss of BAFF-R. Furthermore, constant activation through the BCR blocks its expression as well as the down-regulation of BAFF-R.

Because BCMA is not coexpressed with BAFF-R in BM PCs, it suggests that these receptors are not compatible. Furthermore, we believe that BAFF-R plays a pivotal role in deciding the fate of MB cells engaged in T cell-independent responses. We recently demonstrated that BAFF negatively affects the CpG-induced differentiation of MB cells into ISCs (32). In unpublished observations (J. R. Darce et al.), we find that this negative effect is regulated through BAFF-R and not TACI, since we have evidence showing that APRIL does not affect ISC differentiation. Therefore, it is quite possible that once B cells are committed to becoming a PC (CD27high), they lose BAFF-R to prevent a BAFF-mediated block in differentiation. Studies are currently in progress to confirm this hypothesis. Furthermore, we propose that the BAFF-R/BCMA balance may be involved in a B cell’s decision to either differentiate into a MB cell or PC.

Utility of BBR expression profiles

Collectively, our data support the conclusion that a given B cell’s BBR expression profile is useful because of its ability to add a level of phenotypic characterization that is not currently possible using markers such as CD27 and CD138. As we described above, we found two populations of CD27neg B cells, which can be further divided by their expression of TACI. Similarly, CD138pos cells can be further divided into two populations using BAFF-R expression, since it is expressed in tonsillar but not BM CD138pos PCs. Characterization of the BBR profile on neoplastic B cells may similarly be of use in better understanding their activation history and differentiation status. For example, identification of the normal B cell counterpart of the leukemic B cell clone in patients with B cell chronic lymphocytic leukemia has yet to be determined. Indeed, preliminary studies reveal interesting differences in BAFF-R, TACI, and BCMA expression levels that appear to correlate with the level of Ig H chain variable region somatic hypermutation status (D. F. Jelinek et al., unpublished observations). Because Ig mutation status is a valuable prognostic factor in this disease (45, 46), BBR expression profiling may be of additional use in further subcategorizing patient disease outcome. Precise BBR expression profiling data are also likely to be of use in better understanding the biology of other human B cell malignancies. We have previously demonstrated that multiple myeloma (MM) cells express BBRs, which may be important in regulating disease pathology (25). Moreover, we reported that in contrast to normal BM PCs (this study), BAFF-R was expressed by myeloma cells obtained from some patients along with TACI and BCMA. This phenotype is remarkably similar to the BBR profile of CD138pos tonsillar PCs described in this report and it is reasonable to speculate that the BBR expression profile in MM may have clinical significance. Thus, it is conceivable that there will be differences in disease outcome from patients with a BM PC transformation vs transformation of PCs from secondary lymphoid structures. In this regard, there is one report in the literature suggesting a link between TACI expression and MM disease outcome (47). However, it should be noted this conclusion was reached on the basis of gene expression profiling data and did not include an analysis of TACI expression at the protein level.

In summary, B cell differentiation is accompanied by the coordinated regulation of expression of all three BBRs. This coordinated regulation strongly suggests that each receptor plays a distinct role during B cell development and differentiation and that the absolute expression level of each receptor is a key factor. Although identification of the precise role that each receptor plays during human B cell differentiation was beyond the scope of these studies, our data do provide a framework for future studies focusing on unique activating and inhibitory signals that may be delivered through each receptor. Lastly, our data also describe for the first time the utility of profiling BBR expression on normal human B cells as a means to assess the activation history of B-lineage cells. We anticipate that the BBR profile will also be of use in better understanding the biological heterogeneity intrinsic to a number of human B cell malignancies.


   Acknowledgments
 
We thank Sook Kyung Chang and Renee Tschumper for experimental advice and helpful discussions and members of the FACS core facility for their technical support with sample analysis and cell sorting. We also thank Steven Mihalcik for his analysis of the TACI sequence using the rVista and MATCH software programs and Roberta DeGoey and Steve Zincke for help in processing BM samples and isolation of PCs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants CA105258 and CA062242 (to D.F.J.) and a predoctoral fellowship AI061838 (to J.R.D.). Back

2 Address correspondence and reprint requests to Dr. Diane F. Jelinek, 200 First Street Southwest, Rochester, MN 55905. E-mail address: Jelinek.Diane{at}mayo.edu Back

3 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; BAFF-R, BAFF receptor; ISC, Ig-secreting cell; TACI, transmembrane activator and calcium-modulating cyclophilin ligand interactor; BCMA, B cell maturation Ag; CSR, class switch recombination; PC, plasma cell; BM, bone marrow; MB, memory B cell; GC, germinal center; BBR, BAFF-binding receptor; PB, peripheral blood; CD40L, CD40 ligand; AID, activation-induced cytidine deaminase; MM, multiple myeloma. Back

Received for publication December 11, 2006. Accepted for publication September 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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