Regulated Expression of BAFF-Binding Receptors during Differentiation

This information is current as Jaime R. Darce, Bonnie K. Arendt, Xiaosheng Wu and Diane of September 27, 2021. F. Jelinek J Immunol 2007; 179:7276-7286; ; doi: 10.4049/jimmunol.179.11.7276 http://www.jimmunol.org/content/179/11/7276 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation1

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

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 Downloaded from 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 induc- tion 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. The Journal of Immunology, 2007, 179: 7276–7286. http://www.jimmunol.org/ he B cell-activating factor belonging to the TNF family, that BAFF is involved in murine and human B cell differentiation BAFF3 (also known as BLyS), is required for the devel- as revealed by BAFF’s ability to enhance Ig gene diversification T opment and homeostasis of mature B cells. This was first and augment B cell differentiation into Ig-secreting cells (ISCs). demonstrated using BAFF-deficient mice, which exhibited a block However, how BAFF precisely influences these events remains in B cell maturation evidenced by a lack of transitional 2 and elusive since it has been demonstrated that in addition to BAFF-R, marginal zone B cells (1, 2), and BAFF-transgenic mice, which BAFF can also bind two other receptors, transmembrane activator developed B cell hyperplasia and accompanying autoimmunity (3, and calcium-modulating cyclophilin ligand interactor (TACI) (10– 4). The signals that drive BAFF-dependent B cell maturation in 12) and B cell maturation Ag (BCMA) (1, 13), which bind BAFF mice appear to be mediated exclusively through the BAFF receptor with intermediate and low affinity, respectively (5, 14). by guest on September 27, 2021 (BAFF-R), which binds BAFF with high affinity (2, 5). Thus, mice To date, investigation into the functions of TACI and BCMA bearing a mutated form of BAFF-R (6, 7) or BAFF-R null mice (8, during late-stage B cell biology have been largely conducted in the 9) are comparable in phenotype to BAFF-deficient mice. Still, murine system. For example, it has been suggested that TACI those murine models only provide insights into the possible role of plays an important role in negatively regulating mature B cell ho- BAFF and BAFF-R in human B cell biology. In addition, these meostasis. This was proposed after discovering that mature B cell models do not allow an assessment of the involvement of the numbers increased in TACI-deficient mice, resulting in spleno- BAFF-BAFF-R interaction during later stages of B cell megaly and in one case, a B cell lymphoma-like phenotype (15, maturation. 16). Although these data suggest that TACI may down-regulate Despite lack of a clear understanding of the effects of BAFF in mature B cell proliferation, a recent study demonstrated that when later stages of B cell differentiation, there is accumulating evidence syndecan-2 binds to TACI, B cell proliferation is augmented sug- gesting that the negative regulatory properties of TACI could be

Department of Immunology, Mayo Clinic College of Medicine, Mayo Graduate ligand specific (17). In regard to B cell differentiation, it was dis- School, Rochester MN 55905 covered that TACI also plays a pivotal role in class switch recom- Received for publication December 11, 2006. Accepted for publication September bination (CSR), since B cells in TACI-deficient mice were unable 20, 2007. to class switch even in the presence of BAFF (18). Similarly, stud- The costs of publication of this article were defrayed in part by the payment of page ies of patients suffering from common variable immunodeficiency charges. This article must therefore be hereby marked advertisement in accordance suggested that the absence in Ig H chain diversity observed is due with 18 U.S.C. Section 1734 solely to indicate this fact. to a defective form of TACI present in B cells from these indi- 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.). viduals (19, 20). Most recently, it has been proposed that TACI 2 Address correspondence and reprint requests to Dr. Diane F. Jelinek, 200 First Street expression is required for ISC differentiation, at least in the context Southwest, Rochester, MN 55905. E-mail address: [email protected] of -independent types 1 and 2 B cell responses (21, 22). 3 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the Although the requirement of TACI expression was not evaluated, TNF family; BAFF-R, BAFF receptor; ISC, Ig-secreting cell; TACI, transmembrane Castigli et al. (23) also demonstrated that signals through TACI activator and calcium-modulating cyclophilin ligand interactor; BCMA, B cell mat- uration Ag; CSR, class switch recombination; PC, plasma cell; BM, bone marrow; promoted T cell-dependent ISC differentiation, contending a pre- MB, memory B cell; GC, germinal center; BBR, BAFF-binding receptor; PB, pe- vious report claiming otherwise (24). Unfortunately, much less is ripheral blood; CD40L, CD40 ligand; AID, activation-induced cytidine deaminase; presently known about BCMA, other than it is expressed in normal MM, multiple myeloma. and malignant plasma cells (PCs) (25, 26) and that it is critical in Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 maintaining the survival of bone marrow (BM) long-lived PCs www.jimmunol.org The Journal of Immunology 7277

(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 ex- pressed 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 ex- pression, 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) expres- sion 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 Downloaded from 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-depen- dent mechanism (31). These investigators also demonstrated that TACI expression in response to TLR4 signals requires components

FIGURE 1. Expression of BBRs on B-lineage cells. Expression of of the NF-␬B pathway (c-Rel and p50), which are not required by http://www.jimmunol.org/ BAFF-R (A), TACI (B), and BCMA (C) on blood, tonsil, and BM B- TLR9 to augment TACI expression. The signaling pathway(s) in- lineage cells. Control Ab (filled histogram). MB were identified by expres- volved in TACI induction mediated through TLR9 was not further sion of surface CD27, which is absent on naive B cells. Top two panels, investigated and thus remains unknown. In contrast, much less is Blood and tonsil, A–C; naive B cells (CD27neg; solid-lined histogram) and known about the signals that regulate BAFF-R and BCMA. Early MB (CD27pos; dotted histogram). Bottom two panels, Tonsil and BM CD138ϩ, A–C; CD138ϩ B cells (solid-lined histogram). Data are repre- reports proposed that signals through the BCR enhanced expres- sentative of three independent experiments. sion of BAFF-R, although a specific signaling pathway(s) respon- sible for its expression remains undefined. To date, the regulation of BCMA expression has yet to be examined. by guest on September 27, 2021 (27). In addition, one report demonstrated that BCMA is induced Our study was predicated on the knowledge that there are mul- upon T cell-dependent activation of blood MB cells, which may tiple receptors that can independently bind BAFF and they are promote their survival in response to BAFF (28). likely to be variably coexpressed on B cells in a development/ In an effort to understand the specific roles of these receptors differentiation stage-specific manner. To better understand the during later stages of B cell differentiation, it is necessary to first function of each receptor in B cell biology, it is essential to first understand their expression in B-lineage cells at various stages of characterize the precise BBR profile as well as the stimuli that maturation. Expression of BAFF-R has been found on most B- regulate individual receptor expression in B cells. In this study, we lineage cells, with low levels of expression on germinal center describe our comprehensive analysis of BBR expression in normal

FIGURE 2. TACI is expressed on CD27neg B cells displaying an activated phenotype. A, Analysis of TACI expression on B cells from 10 normal in- dividuals. 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). 7278 BBR EXPRESSION IN B CELL DIFFERENTIATION

FIGURE 3. Up-regulation of TACI expression in TACIneg B cells in re- sponse to activation with various polyclonal stimuli. A, Post-sort anal- neg ysis of TACI sorted B cells. B,Ex- Downloaded from pression of surface TACI upon acti- vation of TACIneg B cells with various polyclonal activators in the presence or absence of . http://www.jimmunol.org/ by guest on September 27, 2021

human B-lineage cells. Our results demonstrating the coordinated CD138 mixture/colloid and a positive selection program on a Robosep regulation of all three receptors begin to suggest unique functions Separator. for each receptor. Specifically, our results suggest for the first time Flow cytometry that TACI is up-regulated immediately upon B cell activation and this is mediated through the ERK/MAPK pathway. Moreover, we Purified cells were stained using standard flow cytometry methodology. Briefly, cells were incubated on ice for 20 min with primary Ab before prove that up-regulation of BCMA and loss of BAFF-R occurs in washing twice with cold FACS buffer (Dulbecco’s PBS (DPBS) con- response to differentiation, suggesting that the down-regulation of taining 2 mM EDTA, 0.05% sodium azide, and 2% FCS) and subse- BAFF-R may be a requisite step in B cell differentiation into ISCs and quent incubation with various secondary reagents. After washing, cells PCs. More broadly, our results suggest the possibility that the BBR were fixed with 1% paraformaldehyde before analysis using a FACS- Calibur flow cytometer (BD Pharmingen) and FlowJo analytical soft- profile may be of use in identifying the activation history of an indi- ware (Tree Star). CD25, CD27, CD38, CD80, and CD138 Abs were vidual B cell and, as such, may also have utility in identifying the purchased from BD Pharmingen; PE conjugated mAbs against BAFF-R transformation stage of a variety of mature B cell malignancies. 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 Materials and Methods allophycocyanin-labeled streptavidin (Caltag Laboratories and Invitro- Cells gen Life Technologies) were used as secondary reagents. Cell turnover Mononuclear cells within peripheral blood (PB) from normal donors, tonsil was determined using CFSE (Molecular Probes and Invitrogen Life Technologies) intercalating dye. Cells were suspended in 0.1% FCS/ tissue from routine tonsillectomies, or BM from patients undergoing hip ϫ 6 ␮ replacement surgery were separated by Ficoll-Hypaque density gradient DPBS at a density of 20 10 cells/ml and labeled with 1.5 M CFSE centrifugation. Individuals provided written informed consent in accor- for 8 min at room temperature. Labeling was halted by adding pre- dance with the Declaration of Helsinki. The Mayo Clinic Rochester Insti- warmed FCS and then incubating at 37°C for 10 min to efflux excess tutional Review Board approved the protocol to obtain blood, tonsil, and CFSE. Cells were washed three times with 2% FCS/DPBS and cultured bone marrow tissue from volunteers. B lymphocytes were enriched to in the presence or absence of mitogen. Ͼ98% purity by magnetic cell separation using a StemCell Technology B Polyclonal activation of PB B cells cell enrichment mixture/colloid and the negative selection program on a Robosep Separator (StemCell Technologies). Similarly, BM PCs were en- PB B cells were activated with the following polyclonal stimuli: anti-Ig riched to Ͼ95% purity by magnetic cell separation using a StemCell (2 ␮g/ml agonistic anti-IgA, IgG, IgM F(abЈ)2 Abs purchased from The Journal of Immunology 7279 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

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.

Jackson ImmunoResearch Laboratories); anti-Ig/CD40 ligand (CD40L; CO2 incubator for the indicated lengths of time in polypropylene round- (2 ␮g/ml agonistic anti-IgA, IgG, IgM F(abЈ)2 Abs and 0.5 ␮g/ml bottom tubes (BD Labware) in the presence or absence of the above shrCD40L/TNF-related activation (Fitzgerald Industries Inter- stimuli at a concentration of 1 ϫ 106 cells/ml in complete medium national); CD40L (0.5 ␮g/ml shrCD40L/TRAP); CpG (oligode- (RPMI 1640 with 10% FCS, L-glutamine, penicillin, and streptomycin). oxynucleotide 2006, 5Ј-TCGTCGTTTTGTCGTTTTGTCGTT, synthe- sized by in-house core facility, 2.5 ␮g/ml). When indicated, B cells Analysis of Ig secretion were also activated in the presence of various combinations. IL-2 (20 U/ml; Fitzgerald Industries International) and IL-10 (50 ng/ml; Ig secretion was measured using a standard Ig H chain-specific ELISA. PeproTech) were used in conjunction with anti-Ig, CD40L, or anti-Ig/ Briefly, 96-well microtiter plates (Nalge Nunc International) were in- CD40L. IL-2 (20 U/ml) and IL-15 (10 ng/ml; PeproTech) were included dependently coated with anti-IgA, anti-IgG, and anti-IgM Abs (Bio- with CpG. To sustain viability, B cells were often cultured in medium Source International). Plates were then blocked with 1ϫ casein (BioFX containing 50 ng/ml IL-4 (PeproTech). In some experiments, B cells Laboratories). After several washes, culture supernatants were added to were activated with PMA at a concentration of 10 ng/ml. To determine coated plates and incubated for 2 h. Igs were detected colorimetrically the effects of blocking ERK1/2 activation in long-term B cell activation using anti-IgA, anti-IgG, and anti-IgM HRP-labeled Abs (BioSource experiments, B cells were pretreated for1hat37°C with PD98059 International) and a Molecular Devices microplate reader. Standard (Cell Signaling Technology), U0126 (Cell Signaling Technology), or curves were generated to quantitate ELISA results using known vehicle. These cells were then cultured for indicated periods of time amounts of purified human IgA, IgG, and IgM Abs (Jackson Immu- with specified polyclonal stimuli. B cells were cultured at 37° C in a 5% noResearch Laboratories). The detection limit of the assays was 1 7280 BBR EXPRESSION IN B CELL DIFFERENTIATION

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.

␮g/ml to 0.1 ng/ml for IgG and IgM and 2 ␮g/ml to 0.1 ng/ml for IgA. Results o-Phenylenediamine dihydrochloride ELISA substrate for HRP along Baseline expression of BBRs on human B-lineage cells Downloaded from with stable peroxide substrate buffer were purchased from Pierce. We were first interested in determining the expression of BBRs Immunoblot analysis on B cells at varying stages of maturation, as well as from We incubated purified PB B cells at a density of 1 ϫ 106 cells/ml for various tissues. We found that BAFF-R is uniformly expressed ␮ 1 h at 37°C with either 20 M PD98059 or U0126, or an equivalent on naive B cells, as well as MB cells and tonsillar PCs (Fig. volume of DMSO carrier (final DMSO concentration was 0.001%). Af-

ter treatment with inhibitors or vehicle, cells were stimulated for the 1A). However, bone marrow PCs, unlike tonsillar PCs, did not http://www.jimmunol.org/ indicated time points. After stimulation, cells were collected and incu- express BAFF-R on their surface (Fig. 1A). Furthermore, bated on ice with lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 5 BCMA was expressed by tonsillar MB cells in addition to PCs mM Na EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM 2 from both tonsil and BM tissues (Fig. 1B). Bone marrow PCs, EGTA, 10 mM sodium fluoride, 1 mM PMSF, and 1 mM tetrasodium pyrophosphate) supplemented with protease inhibitors (10 ␮g/ml leu- however, expressed lower levels of BCMA compared with ton- ␮ ␮ ␮ peptin, 10 g/ml aprotinin, 10 g/ml pepstatin, 2 mM Na3VO4, 100 M sillar PCs (Fig. 1B). In addition, expression of BCMA was ab- ␤-glycerophosphate, and 1 mM PMSF). Lysates were cleared of insol- sent in naive B cells as well as PB MB cells (Fig. 1B). As for ϫ uble material by centrifugation for 10 min at 15,000 g. Lysates TACI expression, we discovered that it is predominantly ex- (10–20 ␮g/lane) were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore) for immunoblotting. Membranes pressed by MB cells and tonsillar and BM PCs, although to a were blocked for1hat37°C in 5% Blotto (Santa Cruz Biotechnology) lesser degree in the latter population (Fig. 1C). Of interest, we by guest on September 27, 2021 supplemented with 0.2% Tween 20 and then blotted overnight with found that a small subset of CD27neg naive B cells also ex- anti-phospho-ERK or anti-ERK Abs (Cell Signal Technology) follow- pressed low levels of TACI and this was observed in both blood ing the manufacturer’s protocol. Immunoreactive were de- tected using an ECL detection system (Super Signal; Pierce) and and tonsil tissues (Fig. 1C). Analysis of blood B cells from 10 autoradiography. additional healthy individuals further supported our finding that

FIGURE 6. PMA-induced up-regulation of TACI expression is blocked by PD98059. A, Phos- phorylation of ERK1/2 in freshly isolated B cell ly- sates (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 con- trols. 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-spe- cific Ab. The Journal of Immunology 7281

FIGURE 7. U0126 blocks CpG-induced up-reg- ulation 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, Phos- phorylation 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. Downloaded from http://www.jimmunol.org/

TACI is expressed by a subset of CD27neg naive B cells (Fig. Up-regulation of TACI occurs early in B cell activation and 2A). The CD27neg TACI-positive subset also expressed activa- it is independent of cell division tion markers such as CD25 and CD80 (Fig. 2, A and B), sug- We next wished to study the kinetics of TACI induction upon gesting that TACI expression is associated with B cell CpG/IL-2,15 and anti-Ig/CD40L/IL-2,10 activation, since these activation. two stimuli resulted in a robust expression of TACI. We assessed TACI expression is induced upon activation of TACI-negative surface TACI levels on resting or activated PB B cells daily for a period of 5 days. Our data revealed that unstimulated B cells cul- B cells by guest on September 27, 2021 tured with IL-4 to preserve viability maintained a differential ex- Because we initially found TACI expressed on the surface of MB pression of TACI even after 5 days in culture (Fig. 4A). Although cells and a subpopulation of CD27neg B cells, both of which dis- IL-4 maintained B cell viability (32) (data not shown), it did not play an activated phenotype, we next wanted to determine whether affect the levels of surface TACI expression when compared with TACI expression could be induced in response to in vitro activa- B cells cultured in medium alone (Figs. 3B and 4A and data not tion and, if so, what type of stimulus was required. shown). However, as shown in Fig. 4, B and C, we noticed that For these studies, we isolated TACI-negative B cells from PB B TACI is rapidly induced, whereby maximum levels of expression cell preparations and then treated these cells with various poly- were achieved 2–3 days following activation with either stimulus. clonal stimuli in the presence or absence of cytokines (Fig. 3). We Moreover, within 24 h of activation, we found that TACI was found that activation of naive B cells through their BCR led to a up-regulated in a fraction of B cells. To determine whether cell marginal expression of TACI, which was slightly increased when division was required before up-regulation of TACI expression, we CD40L was added to the culture (Fig. 3B). In contrast, activation monitored TACI levels and cell division concurrently using CFSE, of naive B cells with CD40L alone did not yield TACI expression which is a reagent commonly used to detect dividing cells. As (Fig. 3B). However, when these cells were activated with either A anti-Ig or CD40L in the added presence of IL-2 and IL-10, we shown in Fig. 5 , TACI expression is found in B cells that have observed a significant increase in TACI expression. TACI induc- high CFSE staining intensity, which represent B cells that have not tion was even greater when cells were stimulated with the combi- yet divided in response to CpG activation at 72 h. These data nation of anti-Ig, CD40L, IL-2, and IL-10 (Fig. 3B). Of note, TACI suggest that TACI expression occurs independent of cell division. expression was not induced when cells were cultured with IL-2 Still, our data above clearly demonstrate that TACI is quickly in- and IL-10 alone, suggesting that initial activation may be required duced upon activation of B cells, thereby implying it is an activa- (Fig. 3B). In addition, we activated naive B cells using CpG oli- tion marker. We next examined the expression levels of TACI and godeoxynucleotides, which engage and signal through TLR 9. We CD25 (a well-known activation marker induced early in B cell found that CpG treatment alone significantly induced expression of activation). Following 24 h of CpG/IL-2,15 activation, virtually all TACI in these cells and that expression levels did not further in- of the B cells acquired CD25 expression, including TACI-negative crease upon addition of IL-2 and IL-15, cytokines which are B cells (Fig. 5B). These results suggest that CD25 expression pre- known to enhance B cell responsiveness to CpG. Culture of B cells cedes TACI expression. with IL-2 and IL-15 alone was without effect in inducing TACI expression (Fig. 3B). In addition, we observed that TACI is tran- scriptionally regulated. Thus, freshly isolated naive B cells did not TACI expression requires the ERK1/2 signaling pathway express TACI mRNA, but following activation, TACI mRNA was So far, our data demonstrate that TACI is quickly up-regulated in detectable using RT-PCR (data not shown). B cells in response to activation by various polyclonal stimuli. We 7282 BBR EXPRESSION IN B CELL DIFFERENTIATION

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 activa- tion is mediated through protein kinase C, which subsequently triggers downstream ERK and JNK signaling pathways. To un- cover 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 acti- vation, 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 Downloaded from 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. These studies prompted us to investigate whether TACI up-reg-

ulation, mediated by CpG activation, also required the ERK1/2 http://www.jimmunol.org/ pathway. However, before treating cells with ERK1/2 inhibitors, we first determined whether CpG activation also resulted in phos- phorylation 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 phosphoryla- FIGURE 8. Surface BAFF-R and BCMA expression following B cell tion in response to CpG stimulation of human PB B cells, we activation. Expression of BAFF-R (A) or BCMA (B) on B cells cultured by guest on September 27, 2021 with IL-4 or activated with CD40L/IL-2,10 or CpG/IL-2,15 for 5 days. observed phosphorylation of ERK as early as 15 min after activa- Data are representative of five independent experiments. tion (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 next wished to determine the signals that may be involved in reg- U0126, we were able to completely block CpG-induced phosphor- ulating TACI up-regulation. Because the polyclonal activators ylation of ERK1/2 (Fig. 7B). In addition, phosphorylation of used thus far signal through multiple pathways, in experiments not ERK1/2 at later time points (60 and 120 min) was also blocked by shown, we used chemical agonists of specific pathways, i.e., PMA U0126 (data not shown). We next evaluated the expression of and calcium ionophore, to determine whether activation of protein TACI in B cells activated with CpG in the presence or absence of

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, Ex- pression 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. The Journal of Immunology 7283

FIGURE 10. Inverse expression of BAFF-R and BCMA is associated with B cell differentiation into ISCs. Secreted Ig levels in response to A) Downloaded from CD40L/IL-2,10 or B) CpG/IL-2,15 in the presence or absence of anti-Ig Abs.Openbars(activationwithoutanti- Ig antibodies) filled bars (activation in the presence of anti-Ig antibodies). C) BAFF-R and BCMA levels in B cells http://www.jimmunol.org/ 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. by guest on September 27, 2021

U0126. As we previously observed in our PMA studies, U0126 with IL-4 for 5 days (Fig. 8). However, in response to activation with treatment blocked the up-regulation of TACI induced by CpG either CD40L/IL-2,10 or CpG/IL-2,15, a subpopulation of B cells stimulation at 72 h (Fig. 7C). Similarly, we found that U0126 exhibited a loss of BAFF-R (Fig. 8). Similarly, we noticed that both treatment also blocked TACI expression at 120 h (data not shown). of these activation schemes induced the expression of BCMA, but Despite blocking TACI expression, up-regulation of CD25 was not only in a small fraction of B cells (Fig. 8). We next wanted to address affected by U0126 treatment (Fig. 7C). Thus, these data imply that whether: 1) BAFF-R and BCMA were inversely expressed in the the ERK pathway is significantly involved in regulating the induc- same B cell population and 2) which B cells were losing or gaining tion of TACI in response to B cell activation. BAFF-R and BCMA, respectively. Because CpG/IL-2,15 activation was more effective at promoting the loss of BAFF-R and up-regula- Activated memory B cells lose BAFF-R and gain tion of BCMA in a larger fraction of B cells in comparison to CD40L/ BCMA expression IL-2,10 activation (Fig. 8), this mode of stimulation was used in sub- In addition to understanding the expression of TACI, we also inves- sequent studies. To determine whether loss of BAFF-R was tigated the expression profile of BAFF-R and BCMA as a result of B coincident with acquisition of BCMA, we activated B cells with CpG/ cell activation. As described above, PB B cells uniformly express IL-2,15 and analyzed BAFF-R and BCMA expression levels daily BAFF-R and lack BCMA upon isolation (Fig. 1). We discovered that over a period of 5 days. As shown in Fig. 9A, the same population of this BAFF-R and BCMA expression profile does not change with B cells that lost expression of BAFF-R gained BCMA expression on time in B cells cultured in medium alone (data not shown) or cultured their surface, suggesting that expression of BCMA may require the 7284 BBR EXPRESSION IN B CELL DIFFERENTIATION loss of BAFF-R. Moreover, unlike the relatively rapid kinetics of BBRs during B cell activation and differentiation. Because of the TACI up-regulation, the inverse expression levels of BAFF-R and highly regulated and coordinated expression of all three receptors, BCMA were acquired later in activation, beginning at 72 h and be- we propose that the BBR profile may be a useful biological foot- coming more evident at 120 h (Fig. 9A). Because we were stimulating print that marks the developmental stage of a human B cell. 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 BAFF-R expression and up-regulating BCMA. Therefore, we costained CpG/IL-2,15-ac- Although it is clear that formation of a mature B cell compartment tivated B cells with anti-CD27 Abs to distinguish between naive and requires signals from BAFF through the BAFF-R (2, 5), the precise memory B cells, along with Abs against BAFF-R or BCMA. We stage at which developing B cells acquire BAFF-R expression re- analyzed BAFF-R and BCMA expression on CD27neg B cells and mains unclear. Nevertheless, it has been demonstrated that transitional two populations of CD27pos B cells that varied in absolute levels of B cells exit the BM already expressing BAFF-R (40). However, their CD27 (CD27low and CD27high; Fig. 9B). Upon activation, CD27neg B BAFF-R expression levels are lower than mature B cells (40), sug- cells retained the expression of BAFF-R and did not up-regulate gesting that further B cell maturation yields an increase in BAFF-R BCMA (Fig. 9C). In contrast, CD27pos B cells lost substantial levels expression. Of note, the functional significance of higher levels of of surface BAFF-R and gained expression of BCMA (Fig. 9C). More- BAFF-R expression remains unknown. Our analysis of BAFF-R on over, we found that the majority of the cells that lost BAFF-R ex- CD19ϩ PB and tonsillar B cells is consistent with other studies (28, pression and expressed higher levels of BCMA were within the 29); however, we extend the literature by demonstrating that naive CD27high population (Fig. 9C). and memory PB B cells express comparable levels of BAFF-R. In

addition, our analysis of primary B-lineage cells at various stages of Downloaded from Inverse expression of BAFF-R and BCMA occurs as a result of maturation reveals that BAFF-R expression decreases and eventually B cell differentiation into ISCs is lost, as B cells differentiate and eventually become PCs. With re- A previous report by Avery et al. (36) revealed that as MB cells spect to reduced BAFF-R expression, Ng et al. (29) and Chiu et al. commit to the PC lineage they increase their expression of CD27. (26) previously described low BAFF-R levels in GC B cells and ton- Since we demonstrate above that CD27high B cells have, for the most sillar PCs; however, we demonstrate for the first time the complete part, lost BAFF-R and gained BCMA, we hypothesized that this ex- loss of BAFF-R expression in normal human BM PCs. Our observa- http://www.jimmunol.org/ pression profile is a consequence of differentiation. Our hypothesis tions that T cell-dependent or T cell-independent in vitro activation of was further substantiated by our initial ex vivo analysis of BM PCs, PB MB cells results in decreased BAFF-R levels as these cells dif- which lack BAFF-R yet express TACI and BCMA (Fig. 1). To block ferentiate into ISCs corroborates findings by Zhang et al. (30) and B cell differentiation into ISCs, we implemented the use of anti-Ig extends those studies by showing that the differentiation process itself receptor Abs (anti-Ig), since it has been previously demonstrated that specifically decreases BAFF-R expression. Thus, we demonstrate that constant signals through the BCR block ISC differentiation induced CD40L/IL-2,10- or CpG/IL-2,15-stimulated extinction of BAFF-R by CD40L/IL-2,10 and CpG/IL-2,15 activation (34, 37–39). In Fig. expression is significantly compromised by sustained BCR signaling, 10, we show that activation with CD40L/IL-2,10 (Fig. 10A) or CpG/ an action known to inhibit B cell differentiation into ISCs (Fig. 10, A IL-2,15 (Fig. 10B) drives differentiation of B cells, measured by se- and B, and Refs. 34 and 37–39). Furthermore, we show for the first by guest on September 27, 2021 cretion of Ig (IgA, IgG, and IgM). However, when anti-Ig Abs are time differences in expression of BAFF-R on CD138pos tonsillar vs added, secretion of IgA, IgG, and IgM was blocked (Fig. 10, A and B). BM PCs. Taken together with our in vitro studies tracking BAFF-R In addition to these data, we also found no evidence of cytoplasmic Ig levels during B cell differentiation, we speculate that PC BAFF-R staining in B cells activated with either stimulus in the presence of expression status may provide a tool to discriminate between short- cross-linking anti-BCR Abs (data not shown). These data established lived and long-lived PCs. Implicit in this speculation is the possibility a method of blocking differentiation, thus we next evaluated the ex- that BAFF-R continues to play a role in the short-lived PCs. Given pression of BAFF-R and BCMA in B cells treated in the presence or that chemokine receptors have been shown to play a key role in PC absence of anti-Ig Abs. Unlike CpG/IL-2,15 or CD40L/IL-2,10 acti- homing to the BM (41, 42), it is possible that signals delivered vation, we found no decrease in BAFF-R nor up-regulation of BCMA through BAFF-R attenuate expression of receptors required for PCs to in B cells activated in the presence of anti-Ig Abs (Fig. 10, C and D). make this transition. Of note, although the inverse expression of BAFF-R and BCMA was abrogated by anti-Ig treatment, addition of anti-Ig to either CD40L/ TACI expression IL-2,10 or CpG/IL-2,15 activation did not negatively affect TACI ex- Several reports in the literature describe TACI expression on hu- pression (Fig. 4C and data not shown). These data suggest that up- man B-lineage cells; however, these analyses have largely been regulation of BCMA along with loss of BAFF-R expression is not a restricted to tonsillar B cells, which are enriched in numbers of consequence of B cell activation but instead differentiation into ISCs. activated cells (26, 29, 30). Our studies considerably expand on the published reports by demonstrating that TACI is uniformly ex- Discussion pressed by PB and tonsillar MB cells as well as PCs from tonsil The precise role(s) of BBRs during B cell differentiation into ef- and BM tissues. In addition, we describe for the first time a sub- fector MB cells or PCs has not been yet defined, in part because a population of CD27neg PB B cells that coexpress TACI. This sub- consensus view of BBR expression on mature B-lineage cells be- population was routinely detectable in normal healthy adults and fore and after activation has not been reached. Thus, although there was shown to coexpress cell activation markers. Moreover, we are numerous reports characterizing BBR expression levels on B found that TACI expression is quickly induced upon in vitro ac- cells, there remain various inconsistencies in reported BBR pro- tivation and this is potentially mediated specifically through the files. The goal of our study was 2-fold: 1) comprehensively char- activation of the ERK pathway, since blocking with ERK-specific acterize the BBR expression profile on several human B cell sub- inhibitors significantly attenuates activation-induced TACI expres- sets ranging from resting naive B cells to BM PCs and 2) sion. Currently, there are no reports identifying the transcription determine the specific effects of a range of stimuli on BBR ex- factors that may be involved in regulating TACI expression. How- pression on PB naive and MB cells. Our data demonstrate how ever, since we found that TACI expression depends on ERK1/2 different antigenic stimuli unequivocally mediate expression of activation, which regulates the activity of the transcription factor The Journal of Immunology 7285

TCF/Elk-1, it is interesting to speculate that TCF/Elk-1 may play BCMA balance may be involved in a B cell’s decision to either a role in regulating TACI levels. Indeed, we have analyzed the differentiate into a MB cell or PC. sequence upstream of the TACI gene and determined that there are Ϫ Ϫ two conserved TCF/Elk-1 binding sites at 58 and 48, in both Utility of BBR expression profiles human and mouse DNA (data not shown). A regulatory role for TCF/Elk-1 in activation-induced TACI up-regulation is currently Collectively, our data support the conclusion that a given B cell’s under investigation. Finally, our demonstration that TACI is rap- BBR expression profile is useful because of its ability to add a level idly induced upon activation as well as expressed in preactivated B of phenotypic characterization that is not currently possible using cells and PCs suggest possible roles for TACI during early B cell markers such as CD27 and CD138. As we described above, we found neg responses as well as later stages of B cell differentiation. two populations of CD27 B cells, which can be further divided by pos With respect to B cell differentiation, it has been recently dem- their expression of TACI. Similarly, CD138 cells can be further onstrated that TACI is necessary for CSR in mice and divided into two populations using BAFF-R expression, since it is pos (18–20). However, to date, there exits no biochemical or molec- expressed in tonsillar but not BM CD138 PCs. Characterization of ular evidence describing the TACI-mediated signals involved in the BBR profile on neoplastic B cells may similarly be of use in better regulating CSR events. Still, there is some evidence that TACI understanding their activation history and differentiation status. For could regulate the expression of activation-induced cytidine example, identification of the normal B cell counterpart of the leuke- deaminase (AID), which is the enzyme responsible for both CSR mic B cell clone in patients with B cell chronic lymphocytic leukemia and somatic hypermutation (43). In of our observations that has yet to be determined. Indeed, preliminary studies reveal interest- a subpopulation of CD27neg B cells expresses TACI, we are cur- ing differences in BAFF-R, TACI, and BCMA expression levels that Downloaded from rently investigating AID expression levels in TACI-positive appear to correlate with the level of Ig H chain variable region somatic CD27neg B cells, as well as determining whether signals through hypermutation status (D. F. Jelinek et al., unpublished observations). TACI can regulate AID expression and function in these cells. Of Because Ig mutation status is a valuable prognostic factor in this dis- interest, it was recently demonstrated that AID is found in a sub- ease (45, 46), BBR expression profiling may be of additional use in population of naive B cells possessing an activated phenotype (44). further subcategorizing patient disease outcome. Precise BBR expres- sion profiling data are also likely to be of use in better understanding In regard to requiring TACI for differentiation into ISCs, we found http://www.jimmunol.org/ that blocking TACI up-regulation (via ERK inhibitors) blocked the biology of other human B cell malignancies. We have previously differentiation of B cells into ISCs. This was assessed by measur- demonstrated that multiple myeloma (MM) cells express BBRs, ing Ig secretion and BCMA expression, which was not induced in which may be important in regulating disease pathology (25). More- these cells (data not shown). over, we reported that in contrast to normal BM PCs (this study), BAFF-R was expressed by myeloma cells obtained from some pa- BCMA expression tients along with TACI and BCMA. This phenotype is remarkably pos Both BAFF-R and TACI have been extensively studied and im- similar to the BBR profile of CD138 tonsillar PCs described in this plicated in B cell homeostasis and development. Conversely, report and it is reasonable to speculate that the BBR expression profile knowledge of BCMA is limited and thought to only affect the in MM may have clinical significance. Thus, it is conceivable that by guest on September 27, 2021 homeostasis of long-lived PCs. However, we describe that BCMA there will be differences in disease outcome from patients with a BM is expressed not only by tonsillar and BM PCs, but also by MB PC transformation vs transformation of PCs from secondary lym- cells found within tonsil tissues, suggesting that this receptor may phoid structures. In this regard, there is one report in the literature also be involved in MB cell biology. Furthermore, we also dem- suggesting a link between TACI expression and MM disease outcome onstrate that unlike TACI, BCMA is not rapidly up-regulated in (47). However, it should be noted this conclusion was reached on the response to activation. Instead, its expression is induced as a result basis of gene expression profiling data and did not include an analysis of MB cell differentiation into ISCs. We found increased BCMA of TACI expression at the protein level. expression in cells that expressed higher levels of CD27, which has In summary, B cell differentiation is accompanied by the coor- been correlated with their commitment to differentiate into PCs dinated regulation of expression of all three BBRs. This coordi- (36). Moreover, we demonstrate for the first time that a form of T nated regulation strongly suggests that each receptor plays a dis- cell-independent B cell activation (via CpG/IL-2,15) can induce tinct role during B cell development and differentiation and that the BCMA expression. This implies that BCMA expression can po- absolute expression level of each receptor is a key factor. Although tentially be acquired outside GC reactions. Although the specific identification of the precise role that each receptor plays during signals that drive BCMA induction are presently unknown, we human B cell differentiation was beyond the scope of these studies, discovered that its expression is coupled with the loss of BAFF-R. our data do provide a framework for future studies focusing on Furthermore, constant activation through the BCR blocks its ex- unique activating and inhibitory signals that may be delivered pression as well as the down-regulation of BAFF-R. through each receptor. Lastly, our data also describe for the first Because BCMA is not coexpressed with BAFF-R in BM PCs, it time the utility of profiling BBR expression on normal human B suggests that these receptors are not compatible. Furthermore, we cells as a means to assess the activation history of B-lineage cells. believe that BAFF-R plays a pivotal role in deciding the fate of We anticipate that the BBR profile will also be of use in better MB cells engaged in T cell-independent responses. We recently understanding the biological heterogeneity intrinsic to a number of demonstrated that BAFF negatively affects the CpG-induced dif- human B cell malignancies. ferentiation of MB cells into ISCs (32). In unpublished observa- tions (J. R. Darce et al.), we find that this negative effect is regu- Acknowledgments lated through BAFF-R and not TACI, since we have evidence We thank Sook Kyung Chang and Renee Tschumper for experimental showing that APRIL does not affect ISC differentiation. Therefore, advice and helpful discussions and members of the FACS core facility for it is quite possible that once B cells are committed to becoming a their technical support with sample analysis and cell sorting. We also thank high PC (CD27 ), they lose BAFF-R to prevent a BAFF-mediated Steven Mihalcik for his analysis of the TACI sequence using the rVista and block in differentiation. Studies are currently in progress to con- MATCH software programs and Roberta DeGoey and Steve Zincke for firm this hypothesis. Furthermore, we propose that the BAFF-R/ help in processing BM samples and isolation of PCs. 7286 BBR EXPRESSION IN B CELL DIFFERENTIATION

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