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International Immunology Advance Access originally published online on March 15, 2007
International Immunology 2007 19(4):465-475; doi:10.1093/intimm/dxm011
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Brian D. Hondowicz1, Shawn T. Alexander1, William J. Quinn, III2, Antonio J. Pagán1, Michele H. Metzgar1, Michael P. Cancro2 and Jan Erikson1

1 The Wistar Institute, Room 276, 3601 Spruce Street, Philadelphia, PA 19104, USA
2 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Correspondence to: J. Erikson; E-mail: jan{at}wistar.org


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

Keywords: auto-antibody, autoimmunity, cytokines


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) play central roles in B lymphocyte homeostasis and activation through binding the receptors, B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) and BLyS receptor 3 (BR3) (also known as B cell-activating factor-R) (1–4). The expression patterns of these receptors vary across B cell subsets, such that TACI and BR3 are expressed among primary B cells, while BCMA is required for the generation of long-lived, bone marrow plasma cells in mice, and the survival of plasmablasts in humans (5–7). Moreover, BLyS can interact with all three receptors, whereas APRIL binds only BCMA and TACI, but not BR3 (1–4).

Newly exported immature B cells have been classified into three transitional populations, T1 through T3 (8). Within transitional, follicular and marginal zone B cell pools, BLyS signaling via BR3 promotes survival and differentiation, as evidenced by the paucity of these cells in BR3 mutant (A/WySn) and BLyS knockout mice (9–13). In contrast, TACI-deficient mice have increased B cell numbers, suggesting that signals through this receptor play an opposing, negative regulatory function (14–16). Other data, however, point to a positive role for TACI as patients with TACI mutations have decreased serum antibodies, and signaling through TACI with APRIL augments both antibody production and isotype switching in T-independent responses (2, 17, 18). Together, these observations suggest that the actions of BLyS and APRIL may vary based on developmental and stimulatory cues which themselves may modify receptor expression patterns.

BLyS is strongly associated with humoral autoimmune syndromes, including systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren's syndrome (19–24). In addition to its role in normal homeostasis, mice that express ectopic BLyS often generate auto-antibodies (25–28). Although the mechanistic basis for this association remains unclear, these findings suggest that BLyS plays a key role in B cell tolerance. Recent work has shown that autoreactive B cells, compared with non-autoreactive B cells, have a greater requirement for BLyS (25, 28–31). In the transgenic (Tg) anti-hen egg lysozyme (HEL)/HEL B cell tolerance model, transfer of autoreactive B cells into mice replete with an endogenous B cell repertoire leads to the rapid elimination of the transferred cells (29, 30). It was postulated that this is due to an exaggerated requirement of autoreactive B cells for BLyS. In support of this model, providing excess BLyS enhances survival rates of the autoreactive anti-HEL B cells (29, 30).

While studies using B cells reactive to model antigens have provided key insights into the role of BLyS during transitional B cell selection (30), no studies have directly addressed the behavior of disease-associated B cells. Indeed, such systems have revealed additional features of autoreactive B cells, particularly their ability to become activated and secrete auto-antibodies in response to T cell help (32, 33). Accordingly, we have examined the impact of BLyS and/or its receptors on the selection and behavior of anti-chromatin B cells using VH3H9 heavy-chain Ig Tg mice before and after T cell help (33).

We selected the VH3H9 heavy chain because it had been repeatedly isolated from autoreactive B cells paired with distinct light chains (34, 35). As an Ig Tg, we have documented that the VH3H9 Tg pairs with a variety of endogenous light chains to generate both autoreactive and non-autoreactive B cells. B cells bearing one autoreactive pair, VH3H9/{lambda}1, have been studied in detail and are shown to be regulated in healthy mice, while spontaneously activated in autoimmune settings (36–38).

In non-autoimmune BALB/c mice, anti-chromatin antibodies are undetectable in the serum but VH3H9/{lambda}1 B cells are present in the bone marrow and periphery (37). In the spleen they localize to the interface between the T and B cell areas within the white pulp and consistent with their localization have reduced levels of CXCR5 and migratory capacity to CXCL13 relative to mature follicular B cells (39). In addition, the anti-chromatin B cells have a unique cell-surface phenotype that is first visible in the bone marrow as surface Ig is expressed; they appear developmentally arrested (they express low levels of B220 and CD22, high levels of HSA and are CD93+) and antigen experienced (they express lower levels of IgM, higher levels of MHC class II and CD44 and are bigger than naive B cells) (37). We have hypothesized that this phenotype is the consequence of immature anti-chromatin B cells engaging self-antigen in the absence of T cell help (40). Importantly, while these anti-chromatin B cells can be detected in the periphery, in vivo labeling experiments reveal that they have a reduced lifespan (41). The introduction of a bcl-2 Tg extended their lifespan but did not change their developmental status or localization (42).

Here, we show that exogenous BLyS increases B cell numbers, particularly anti-chromatin B cells, while auto-antibody production and autoreactive B cell localization remain unaffected. In addition, we document that transitional anti-chromatin B cells have higher surface expression of TACI compared with transitional B cells from both Tg(–) mice and non-autoreactive Tg(+) cells. Furthermore, after receipt of T cell help, short-lived anti-chromatin antibody-secreting cells (ASCs) retain TACI but no longer express detectable levels of BR3. Together, these data point to a role for TACI in regulating B cell survival.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Male and female mice between 6 and 16 weeks of age were maintained in specific pathogen-free conditions at the Association for Assessment and Accreditation of Laboratory Animal Care accredited by Wistar Institute under the supervision of the Institutional Animal Care and Use Committee. TS1 TCR (anti-hemagglutinin) Tg BALB/c, VH3H9 BALB/c (VH3H9 Tg), Tg(–) {kappa}–/– (BALB/c) and VH3H9 BALB/c Tg {kappa}–/– (VH3H9 Tg {kappa}–/–) mice were screened by flow cytometry. The VH3H9 Tg {kappa}–/– mice were also bred to Tg mice that express the PR8 influenza hemagglutinin (HA) protein under the control of the MHC class II promoter as a source of cognate help (39). BALB/c and CB17 mice were purchased from Harlan Sprague Dawley and Charles River Laboratory, respectively.

BLyS treatment
Human recombinant BLyS was kindly provided by Thi Migone of Human Genome Sciences (Rockville, MD, USA). Two treatment protocols were used. In the first protocol, VH3H9 Tg and VH3H9 Tg {kappa}–/– mice were given daily BLyS (10 µg) or PBS intra-peritoneally for 9 days (n = 3 for all groups). In the second protocol, the same treatment was extended to 21 days using Tg(–) (n = 5), VH3H9 Tg (n = 9 without BLyS, n = 12 with BLyS) and VH3H9 Tg {kappa}–/– (n = 3 without BLyS and n = 3 with BLyS) mice.

Flow cytometry
Cells (1 x 106 to 4 x 106) were prepared and surface stained as per standard protocol (43). The following antibodies were used: anti-B220–FITC, anti-B220–PerCP-Cy5.5, anti-B220–PE-Cy7 (RA3-6B2), anti-Ig{lambda}1–biotin (R11-153), anti-{lambda}–FITC (R26-46), anti-Ig{lambda}2,3–biotin (2B6), anti-CD23–biotin (B3B4), IgMa–FITC (DS-1), anti-CXCR5–PE (2G8), rat IgG1–FITC isotype control (BD Biosciences, San Jose, USA), rat IgG2a–PE isotype control, anti-CD93–allophycocyanin (AA4.1), rat IgG1 isotype control (eBioscience, San Diego, USA), anti-TACI–PE (166010), anti-BCMA–FITC (161616) (R&D Systems, Minneapolis, USA), anti-BR3 (7H22-E16) (Axxora, San Diego, USA) and anti-IgM–PE-Cy5.5 (a generous gift from David Allman, University of Pennsylvania, USA). Secondary reagents were streptavidin–PE, strepatavidin–PE-Cy7, streptavidin–PerCP-Cy5.5, streptavidin–allophycocyanin (BD Biosciences) and anti-rat IgG1–PE (Southern Biotech, Birmingham, USA). Stained cells were run on a FACSCalibur or LSRII (Becton Dickinson, Franklin Lakes, USA) machine and analyzed using CellQuest or FlowJo software (Treestar, Ashland, USA).

Immunostaining
Spleens were frozen, sectioned and stained (37). Immunohistochemistry protocols used the following antibodies: anti-CD22–FITC (Cy34.1), anti-CD4–FITC and anti-Ig{lambda}1–biotin (R11-153) (BD Biosciences). Secondary reagents were anti-FITC–HRP and streptavidin–AP (Southern Biotech). Immunofluorescence protocols used anti-IgMa–FITC or anti-IgMa–PE, anti-IgDb–biotin (217-170), anti-CD4–FITC (GK1.5), anti-B220–FITC and streptavidin–PE (BD Biosciences).

Anti-nuclear antibodies
Serum was collected from mice before BLyS injection and at day 9 or day 21 after treatment. The presence of anti-nuclear antibodies (ANAs) in serum was detected using permeabilized HEp-2 cells as the substrate as previously described (38). Serum was considered ANA+ if homogeneous nuclear staining was observed (36).

Real-time quantitative PCR
Splenocytes were stained with anti-B220–FITC, anti-{lambda}1–biotin, anti-CD93–allophycocyanin and streptavidin–PE. The populations that were sorted are indicated in Fig. 4(E). All sorted populations were >90% pure except VH3H9 Tg {lambda}1+ CD93– B cells that were only 60% pure in two experiments and >90% in one experiment. RNA was collected from 0.5x 106 to 2.0 x 106 sorted splenocytes. Cells were washed twice with PBS before RNA was isolated using the Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). The cDNA was made using SuperScript First-Strand Synthesis (Invitrogen, Carlsbad, USA). TACI and BR3 cDNA was amplified according to Applied Biosystems.


Figure 4
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  		Fig. 4. BR3 and TACI expression on VH3H9 B cells. (A) Representative histograms of BR3 and TACI from transitional B cells in Tg(–) mice. Shaded areas indicate staining from an isotype control antibody (n = 3). (B) BR3 and TACI staining of transitional and mature B cells from Tg(–), VH3H9 Tg and VH3H9 Tg {kappa}–/– mice. In each histogram, the shaded areas represent staining from an isotype control antibody, the solid line CD93+ cells and the dashed line CD93– cells (n = 3). All staining in (A) and (B) was done using a pan-{lambda} antibody because the {lambda}1 is the only reagent that reacted with the anti-rat IgG1 used for BR3 staining. The {lambda}1 B cells account for >95% of all {lambda}-expressing B cells in VH3H9 Tg and VH3H9 Tg {kappa}–/– mice. (C) Representative histograms of TACI staining for {lambda}2,3 B cells from Tg(–), VH3H9 Tg and VH3H9 Tg {kappa}–/– mice (n = 2 for Tg(–) mice and n = 3 for VH3H9 Tg and VH3H9 Tg {kappa}–/– mice). (D) The average ± standard deviation of the geometric means of the indicated B cell population. Asterisk indicates significantly lower (P < 0.05) levels of TACI expression compared with Tg(–) IgM+ CD93– B cells and VH3H9 Tg IgM+ {lambda}+ cells (n = 3). The right panel shows the ratio of the BR3 geometric mean to TACI geometric mean. (E) Real-time PCR was used to measure BR3 (left) and TACI (middle) mRNA levels in the indicated B cell populations. The right panel shows the ratio of BR3 to TACI levels. For each group, n = 3, except VH3H9 Tg CD93+ where n = 4. Asterisk denotes significantly (P < 0.05) different than all other groups.

 
CB17 transfer
In all, 12x 106 to 52 x 106 CB17 [a congenic strain carrying the Ig heavy-chain allele (Igh-1b) from a C57BL/Ka on a BALB/c background] spleen cells were transferred intravenously (i.v.) into the tail vein of BALB/c (Iga) Tg(–), Tg(–) {kappa}–/– or VH3H9 Tg {kappa}–/– mice. Spleens were harvested 16–20 h later. Tissue sections were stained for IgMa, IgDb and CD4.

Th, influenza and anti-chromatin B cell injections
Axillary, brachial, popliteal, cervical and inguinal lymph nodes were harvested and dispersed using sterile glass slides. Lymph node cells from TS1 TCR Tg mice were stained with anti-CD25–FITC (7D4) and anti-CD4–allophycocyanin (RM4.5) (BD Biosciences) and sorted using a Cytomation MoFlo. Purity was always >97% for CD4+ CD25– cells. A total of 2 x 106 CD4+ CD25– TS1 T cells were suspended in sterile PBS with 1000 hemagglutinating units of purified PR8 influenza virus and injected i.v. into CB17 recipient mice (allotype Igb). Splenocytes from VH3H9 Tg HA+ {kappa}–/– mice were depleted of RBCs and an aliquot was stained by flow cytometry to determine the frequency of anti-chromatin B cells (B220+ Ig{lambda}1+). CB17 recipient mice were injected with spleen preparations containing 5 x 106 anti-chromatin B cells (allotype Iga) (44).

Chromatin ELISAs
ELISA plates (ThermoLabSystems, Waltham, MA, USA) were coated with 2 µg ml–1 of chromatin (a generous gift of M. Monestier, Temple University, PA, USA) overnight at 4°C. The remaining steps were conducted at room temperature. All washes were conducted at least eight times in 1x PBS/0.05% Tween 20. Following the coating step, plates were washed, blocked with 1% BSA/PBS/azide for at least 1 h and washed again. Sera were then added at increasing dilutions, as indicated in figure, and incubated for a minimum of 1 h. Plates were washed and incubated with developing antibody (anti-IgMa–biotin, BD Biosciences) for at least 1 h. Finally, plates were washed, incubated with streptavidin–AP (Southern Biotech) for at least 1 h, washed and developed for 14–18 h. The plates were developed with Immunopure p-nitrophenyl phosphate (Pierce, Rockford, IL, USA) as the substrate. Absorbances were read at dual wavelength, 405/650 nm using a microplate reader.

Statistical analysis
Statistical significance was determined via the Student's t-test provided by Microsoft Excel software unless otherwise noted. Significance was ascribed when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The effect of BLyS treatment on anti-chromatin B cell numbers
As BLyS receptor expression and responsiveness are developmentally regulated, we first determined the maturation status of anti-chromatin B cells in two settings prior to BLyS treatment. In VH3H9 Tg mice, the {lambda}1+ anti-chromatin B cells are a minority of the B cells (5–10%) (Fig. 1A). While the majority of splenic B cells in Tg(–) (BALB/c) mice are mature (CD93–), as are the {lambda}1(–) B cells in VH3H9 Tg mice (Fig. 1A and C), the VH3H9 {lambda}1 anti-chromatin B cells are mostly transitional (CD93+, range = 75.6–86.3%, average = 81.5 ± 3.7%, n = 8) (Fig. 1C) (45). To increase the frequency of anti-chromatin B cells, VH3H9 Tg mice were mated to mice deficient in kappa light chains (VH3H9 Tg {kappa}–/–). Of the available light chains, only {lambda}1 generates an autoreacitve receptor when paired with VH3H9; both {lambda}2 and {lambda}x do not and {lambda}3 has not been detected (46, 47). The VH3H9/{lambda}1 anti-chromatin B cells in the VH3H9 Tg {kappa}–/– mice dominate the B cell repertoire (70–80%) (Fig. 1A, top row). Here, the percentage of transitional VH3H9/{lambda}1+ anti-chromatin B cells is lower and more variable (CD93+, range = 24.6–82.8%, average = 58.9 ± 15.1%, n = 14) (Fig. 1A and B). Additionally, Tg(–) {kappa}–/– and VH3H9 Tg(–) {kappa}–/– mice have fewer total B cells in the spleen (Fig. 1C). Thus, in the absence of competition for BLyS and/or with a reduction in total B cell numbers, anti-chromatin B cells develop a more mature phenotype.


Figure 1
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  		Fig. 1. CD93 expression on Tg(–) and anti-chromatin B cells. (A) The top row shows representative B220 and {lambda}1 staining of spleen cells from Tg(–) (BALB/c) (n = 4), VH3H9 Tg (n = 8) and VH3H9 Tg {kappa}–/– (n = 14) mice. The percentage shown in the flow cytometry plots are the average percentage of {lambda}1+ B220+ cells from total B220+ cells. The bottom rows are the representative histograms of the CD93 levels of B220+ {lambda}1+ (bold line) or {lambda}1(–) (dashed line) B cells. (B) Graph of CD93 expression of indicated B cells populations from Tg(–) (n = 4), VH3H9 Tg (n = 8) and VH3H9 Tg {kappa}–/– (n = 14). Asterisk indicates significantly different (P < 0.05) compared with other CD93+ or CD93–. (C) Graph of total number of B220+ cells in the spleen from Tg(–) (n = 12), VH3H9 Tg (n = 10), Tg(–) {kappa}–/– (n = 4) and VH3H9 Tg {kappa}–/– (n = 11). The total numbers of B220+ cells were determined by multiplying the percentage of B220+ cells by the total number of viable cells as determined by trypan blue exclusion. Asterisk indicates significantly different (P < 0.05) compared with kappa-sufficient mice.

 
To determine if BLyS regulates anti-chromatin B cell maturation, exogenous BLyS was administered. We hypothesized that BLyS may be limiting in VH3H9 Tg mice, where anti-chromatin B cells have to compete with other B cells, but not when the competition is reduced in VH3H9 Tg {kappa}–/– mice. After 9 days of BLyS treatment, the percentage of anti-chromatin B cells increased after BLyS treatment in VH3H9 Tg and VH3H9 Tg {kappa}–/– mice (Fig. 2A). The fold increase in mature and transitional cell number from VH3H9 Tg {kappa}–/– mice was 2.3 and 1.9, respectively (Fig. 2B, right panel). The mature and transitional anti-chromatin B cells from VH3H9 Tg mice increased in cell number 15- and 5-fold, respectively. This more dramatic increase is consistent with the hypothesis that BLyS is a limiting resource for anti-chromatin B cells when they are part of a more diverse B cell repertoire.


Figure 2
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  		Fig. 2. CD93 expression and anti-chromatin B cell numbers after BLyS treatment. (A) After 9 days of PBS or BLyS treatment, the percentage of CD93+ cells is shown from splenic B220+ {lambda}1+ cells from VH3H9 Tg and VH3H9 Tg {kappa}–/– mice (n = 3). (B) The number of B cells recovered after PBS or BLyS treatment (left panel). The total number of B cells from VH3H9 Tg mice PBS treated 46 x 106 ± 9 x 106, BLyS treated 127 x 106 ± 31 x 106, VH3H9 Tg {kappa}–/– PBS treated 41 x 106 ± 3 x 106 and BLyS treated 78 x 106 ± 21 x 106. The fold increase in B cell numbers after BLyS treatment (right panel) was determined by dividing the average number of B cells from BLyS-treated mice by the average number of B cells from PBS-treated mice.

 
The role of exogenous BLyS on anti-chromatin B cell localization and CXCR5 expression
Previous studies with the anti-HEL model have given mixed results regarding the location of autoreactive HEL B cells after BLyS treatment or using Tg BLyS mice (29, 30). To determine the splenic localization of anti-chromatin B cells before and after BLyS treatment, immunohistochemistry was performed. While {lambda}1+ B cells from Tg(–) mice were observed in the B cell follicle (Fig. 3A), the majority of the anti-chromatin B cells in VH3H9 Tg mice were excluded from the B cell follicle (Fig. 3A) (37). After BLyS treatment, anti-chromatin B cells were present in greater numbers, but many of the cells were still located at the T/B interface or PALS (Fig. 3A).


Figure 3
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  		Fig. 3. Splenic localization and CXCR5 expression of anti-chromatin B cells from BLyS-treated VH3H9 Tg(–) mice. (A) Representative immunohistochemistry sections of spleens from Tg(–) (n = 3), VH3H9 Tg (n = 14), BLyS-treated (21 days) VH3H9 Tg (n = 14), VH3H9 Tg {kappa}–/– (n = 3) and BLyS-treated (21 days) VH3H9 Tg {kappa}–/– mice. The sections are stained for CD22, CD4 and Ig{lambda}1+ cells. (B) CB17 (Igb) spleen cells were transferred into Tg(–) and VH3H9 Tg {kappa}–/– (Iga) mice and 16–20 h later spleens were stained for CD4, IgMa and IgDb. The sections are representative of two experiments. (C) A representative histogram of CXCR5 staining from splenocytes of the indicated mice. Gates for B cells were set as shown in Fig. 1(A). (D) CXCR5 staining of CD93+ or CD93– {lambda}1+ B cells in a VH3H9 Tg {kappa}–/– spleen.

 
Anti-chromatin B cells in VH3H9 Tg {kappa}–/– mice are located peripherally to the T cell area, but their position relative to the few non-autoreactive B cells is hard to discern (Fig. 3A). To clarify this, we adoptively transferred non-Tg spleen cells with the anticipation that they would delineate the B cell follicle (48). Specifically, spleen cells from Ig allotype-mismatched CB17 mice were transferred into VH3H9 Tg {kappa}–/–, Tg(–) {kappa}–/– or Tg(–) mice and immunofluorescence was performed 16–20 h later. The transferred CB17 B cells localized with the endogenous B cells in Tg(–) mice (Fig. 3B) and Tg(–) {kappa}–/– mice (data not shown). When transferred into VH3H9 Tg {kappa}–/– mice, however, the majority of the transferred cells were segregated from the endogenous B cells forming a ring on the outside of the VH3H9 Tg {kappa}–/– B cells. Thus, the localization of the VH3H9 Tg {kappa}–/– anti-chromatin B cells appears similar to that of anti-chromatin B cells from VH3H9 Tg mice where they are positioned at the T/B interface (Fig. 3B) (37).

CXCR5 expression is required for B and T cell follicular entry (49, 50). Transitional B cells have lower levels of CXCR5 relative to follicular B cells and accumulate at the T/B border (51). Mature follicular B cells can also be observed at the T/B boundary upon contact with antigen, but in this case, the movement is associated with an increase in CCR7 and not a change in CXCR5 expression (52, 53). Before BLyS treatment, anti-chromatin B cells from VH3H9 Tg mice expressed reduced levels of CXCR5 relative to follicular B cells, consistent with their transitional status and exclusion from the B cell follicle (Fig. 3A and C). After BLyS treatment, when there were more mature anti-chromatin B cells, the CXCR5 expression was higher and more diffuse but still lower than Tg(–) follicular B cells (Figs. 2A, B and 3C). In the VH3H9 Tg {kappa}–/– mice, the mature anti-chromatin B cells accounted for the higher CXCR5 expression levels while both immature and mature anti-chromatin B cells appear follicularly excluded (Fig. 3B and D).

Auto-antibody production and BLyS treatment
Since BLyS Tg C57BL/6 mice have elevated levels of auto-antibodies (27), we determined if daily BLyS treatment for 9 days resulted in anti-chromatin antibody production. VH3H9 Tg or VH3H9 Tg {kappa}–/– mice before or after BLyS treatment were negative for {lambda}1+ ANAs (n = 3, data not shown). Furthermore, no ASCs (CD138+ {lambda}1+) were visible in tissue sections from the spleen (data not shown). Since 9 days may not have been a sufficient time to accumulate serum auto-antibodies, mice were treated for 21 days, but here again, BLyS treatment did not result in serum ANAs (n = 5, Tg(–); n = 12, VH3H9 Tg; n = 3, VH3H9 Tg {kappa}–/–, data not shown).

Homeostatic BLyS receptor expression
BLyS receptor expression is affected by the maturation status of the B cell and may be modulated with antigen encounter (5, 54). Therefore, we investigated the status of the BLyS receptors on distinct B cell populations. We determined BR3, TACI and BCMA protein expression by flow cytometry, and BR3 and TACI mRNA by real-time PCR. BCMA protein expression on all B cell populations was very low and similar to staining with an isotype control antibody (data not shown). In Tg(–) mice, BR3 was lowest on the immature T1 fraction and similarly higher on the immature T2/T3 fraction and mature B cells (Fig. 4A and B). Regardless of maturation status or B cell repertoire complexity, the expression of BR3 on anti-chromatin B cells was uniform and most similar to T2/T3/mature levels on Tg(–) B cells (Fig. 4B and C). TACI protein expression on Tg(–) B cells increased as the cells proceeded from the T1 to T3 fraction and was highest on mature B cells (Fig. 4A and B). These data correlate with TACI mRNA levels previously published (5). In contrast, Ng et al. (55) reported that T2 B cells have higher TACI levels than follicular B cells. While our study and that of Ng et al. used different anti-TACI reagents, a more likely possibility for this discrepancy is that the markers used to define T2 B cells in the Ng et al. study (IgM, CD21 and CD23) will include marginal zone precursors (56). While TACI levels on the marginal zone precursors have not been measured, marginal zone B cells themselves have high levels of TACI (55).

Anti-chromatin B cells phenotypically most closely resemble transitional B cells in that they are CD93+. BR3 mRNA and protein expression are similar to levels seen on Tg(–) T2/T3/mature B cells (Fig. 4B, D and E). Strikingly, however, TACI surface and mRNA expression from anti-chromatin B cells was significantly higher than Tg(–) transitional B cell populations and was most like that of mature B cells from Tg(–) mice (Fig. 4B, D and E). Non-autoreactive B cells from VH3H9 Tg mice displayed a TACI profile identical to their Tg(–) counterparts with CD93+ cells being TACIlow and CD93– cells TACIhigh (Fig. 4B and C). Therefore, the ratio of BR3 to TACI expression is much lower on transitional anti-chromatin B cells compared with other transitional B cells (Fig. 4D and E), which may have important consequences for anti-chromatin B cell survival. It has been proposed that it is the relative level of BR3 to TACI that is critical for B cell survival in different developmental niches (57).

TACI, BR3 and BCMA expression levels on anti-chromatin B cells in the presence of T cell help
Because the status of different BLyS receptors during an immune response is relatively unknown, we determined TACI, BR3 and BCMA expression levels on anti-chromatin B cells after receiving T cell help (Fig. 5A) (58). Using an adoptive transfer model (Fig. 5A), we have shown that anti-chromatin B cells survive and differentiate into short-lived ASCs when provided with T cell help, while those transferred without Th cells do not survive to day 8 (Fig. 5B and C) (39, 58).


Figure 5
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  		Fig. 5. TACI, BR3 and BCMA expression on anti-chromatin B cells after T cell help. (A) Outline of protocol for administering T cell help to anti-chromatin B cells. (B) Immunohistochemistry staining of spleens of recipient CB17 (Igb) mice that received Th cells and anti-chromatin B cells (IgMa) (left) or anti-chromatin B cells alone (right). Spleen sections were stained with anti-CD22 (brown) and anti-IgMa (blue). Pictures are representative of atleast three experiments per condition. (C) Anti-chromatin ELISA with or without T cells help. Data are from one experiment and are representative of more than three experiments. (D) Eight days after T cell help, spleen cells were stained for anti-chromatin B cells with IgMa and {lambda}1. The IgMa+ {lambda}1+ cells were gated on and TACI and BR3 expression was determined. Shaded histograms represent staining from an isotype control antibody and the unshaded plots are stained with anti-TACI or BR3 antibodies (n = 3) or BCMA (n = 2) as indicated. (E) Eight days after T cell help, TACI, BR3 and BCMA expression was determined from the spleen. Cells were gated on CD138+ B220– IgMa+ and then TACI, BR3 and BCMA expression was determined with an isotype control antibody shown in gray (n = 2–3).

 
Of the surviving anti-chromatin B cells post-T cell help, 11.4 ± 4.5% (n = 5) have acquired an ASC phenotype (CD138+, intracellular IgMa+, B220low) and are visible at the bridging channels to the red pulp (Fig. 5B). Likewise, auto-antibodies are detected in the serum (Fig. 5C) (44, 58). To determine expression of the BLyS receptors on anti-chromatin ASCs, we gated on CD138+ and intracellular IgMa+ cells in the spleen and bone marrow (Fig. 5E and data not shown). Consistent with their short-lived status (59), there was no detectable IgMa+ ASCs in the bone marrow (data not shown). The anti-chromatin ASCs in the spleen expressed only TACI, with BCMA and BR3 staining being negligible (Fig. 5E), whereas the non-ASCs present at day 8 expressed both TACI and BR3 (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
A prominent characteristic of SLE is the presence of anti-chromatin antibodies (60). In addition, many lupus patients have elevated BLyS levels and Tg BLyS over-expression can result in SLE-associated auto-antibodies (22, 61–64). These observations prompted us to examine the role of BLyS on anti-chromatin B cell regulation.

In order to investigate the effect of BLyS on anti-chromatin B cells, we used two VH3H9 Tg mouse models that differ in the complexity of their B cell repertoire and number of B cells. Anti-chromatin B cells are part of a diverse B cell repertoire in VH3H9 Tg mice and are phenotypically most similar to transitional cells in that they are CD93+. In the VH3H9 Tg {kappa}–/– mice where there are fewer B cells and less interclonal competition for BLyS, the anti-chromatin B cells are more mature. This is consistent with data from the anti-HEL B cell tolerance model and supports the hypothesis that autoreactive B cells compete poorly with non-autoreactive B cells for entry into the mature B cell pool (29, 30).

BLyS increases B cell numbers with the most exaggerated increase coming from anti-chromatin B cells in VH3H9 Tg mice where the greatest level of competition for BLyS would be expected to occur. Previous studies have reported conflicting results regarding the ability of BLyS to act as a maturation factor for transitional B cells (65, 66). In the study by Rolink et al. (66), sorted immature cells (CD93+) were placed in culture with BLyS and 3 days later most of the B cells were mature (CD93–). However, Batten et al. (65) demonstrated that T2 B cells cultured in the presence of BLyS underwent maturation only with the addition of anti-µ. Our study does not allow us to distinguish whether BLyS is driving the differentiation of anti-chromatin B cells versus their increased survival, which in turn permits their maturation. Nevertheless, our data were consistent with that of others and supports the hypothesis that autoreactive B cells have a greater requirement for BLyS than non-autoreactive B cells for their survival (29, 30).

Neither BLyS treatment nor the prolonging of lifespan through the introduction of a bcl-2 Tg resulted in anti-chromatin B cells entering the follicle (42). These data were analogous to those reported for autoreactive HEL B cells when transferred into soluble HEL-expressing mice treated with exogenous BLyS (29). They differ from other studies using the HEL B cell tolerance model in the context of a BLyS Tg (30). There are many experimental variables that differ within the HEL model studies as well as between the anti-chromatin model described here that could account for these distinct outcomes. One important difference is the genetic backgrounds used. Both the HEL model and BLyS Tg are on the C57BL/6 background, whereas the anti-chromatin model is on the BALB/c background. It has been previously shown that C57BL/6 mice are more autoimmune prone than BALB/c mice (67–69).

While anti-chromatin B cell numbers increased after 9 and 21 days of BLyS treatment, there were no detectable ANAs in the serum or ASCs by immunohistochemistry. Likewise, there was no evidence presented that BLyS promotes anti-HEL auto-antibodies in the HEL tolerance model (29, 30). These data appear in contrast to BLyS Tg mice that express elevated auto-antibodies after being exposed to ectopic levels of BLyS for up to 6 months (25, 27, 28). However, the levels of auto-antibodies produced from the different BLyS Tg mice are variable and are always associated with hypergammaglobulinemia (25, 27).

One hypothesis to account for the presence of auto-antibodies in BLyS Tg mice and lupus patients is that increasing the length of time anti-chromatin B cells persist, increases the probability that they become targets for secondary stimulation. Given that the defects in SLE patients are multigenic, we suggest that an alteration in pro-survival factors, for example, BLyS and Bcl-2 family members (42, 70–72), increases the susceptibility of anti-chromatin B cells to activation via T cell help. Indeed anti-chromatin B cells are induced to secrete auto-antibodies in response to T cell help (39, 44, 58). Interestingly, their response to T-independent stimuli, such as LPS and CpG, like anergic anti-HEL B cells, is defective (40, 73, 74).

BR3 is a survival factor for B cells while TACI has been implicated in playing a negative role (75). Therefore, one might predict that the ratio of BR3 to TACI would have important consequences for B cell survival (57). Indeed, transitional anti-chromatin B cells with a low ratio of BR3 to TACI have a short half-life in vivo (37). If TACI is solely a negative regulator, then BLyS treatment would result in decreased survival of the TACIhigh anti-chromatin B cells. However, this was not the case; exogenous BLyS increased anti-chromatin B cell numbers at a higher proportion than non-VH3H9/{lambda}1 B cells. One possibility to account for this is that BLyS treatment changes the ratio of the two TACI-binding ligands, BLyS and APRIL, and higher levels of BLyS relative to APRIL may yield a pro-survival signal while lower BLyS to APRIL levels may promote death. Experiments are currently under way to test this hypothesis.

To determine the BR3, TACI and BCMA expression levels on B cells during an in vivo immune response, we evaluated the levels of BR3, TACI and BCMA on anti-chromatin B cells after receiving T cell help using an adoptive transfer model (39, 59). When anti-chromatin B cells are transferred into recipient mice without T cell help, they are not detectable by day 8, consistent with their short half-life in vivo (37), whereas in the presence of T cell help they survive (44, 58). Interestingly, while the BLyS receptor expression levels were unchanged on the majority of the transferred anti-chromatin B cells, those that had differentiated to ASCs lost BR3 expression. Since the induced anti-chromatin ASCs are only short lived (59), these data suggest that TACI expression may directly account for the short-lived nature of the ASCs. Likewise, recent data from NP immunized C57BL/6 mice have also shown that short-term ASCs express TACI with reduced amounts of BR3 (76). Together, these data indicate that TACI expression is a characteristic of short-lived ASCs, and not a unique feature of autoreactive plasma cells. Overall, these data indicate that TACI may play distinct roles depending on the activation or differentiation status of the B cell. Clearly, more studies are needed on how the signals emanating from the BLyS receptors are integrated and how that might be modulated with B cell receptor engagement and maturation.


   Acknowledgements
 
The authors thank Gina N. Wharton and Simone Willms for their excellent technical assistance and L. Ng for critical review of the manuscript. Additionally, we would like to thank Matt Farabaugh and J. S. Faust from the Wistar Flow Cytometry Facility and Richard D. Schretzenmair from the University of Pennsylvania Flow Cytometry and Cell Sorting Facility. J.E. is supported by the National Institutes of Health (AI32137 and AR47913) and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health, M.P.C. is supported by AI054488 [GenBank] and B.D.H. by 2T32AI007518.


   Abbreviations
 
ANA, anti-nuclear antibody
APRIL, a proliferation-inducing ligand
ASC, antibody-secreting cell
BCMA, B cell maturation antigen
BLyS, B lymphocyte stimulator
BR3, BLyS receptor 3
HA, hemagglutinin
HEL, hen egg lysozyme
i.v., intravenously
SLE, systemic lupus erythematosus
TACI, transmembrane activator and CAML interactor
Tg, transgenic

   Notes
 
Transmitting editor: C. Goodnow

Received 21 August 2006, accepted 19 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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BAFF and MyD88 signals promote a lupuslike disease independent of T cells
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