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The Journal of Immunology, 2007, 178, 7531 -7539
Copyright © 2007 by The American Association of Immunologists, Inc.
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TLR Stimulation Modifies BLyS Receptor Expression in Follicular and Marginal Zone B Cells1

Laura S. Treml2,*, Gianluca Carlesso2,{dagger}, Kristen L. Hoek{dagger}, Jason E. Stadanlick*, Taku Kambayashi*, Richard J. Bram{ddagger}, Michael P. Cancro3,* and Wasif N. Khan3,{dagger}

* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; {dagger} Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; and {ddagger} Department of Pediatric and Adolescent Medicine, Mayo Clinic, Mayo Medical School, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The B lymphocyte stimulator (BLyS)4 and its receptors profoundly influence primary B cell homeostasis and selection (reviewed in Refs. 1, 2, 3). The prompt increases in B cell numbers following BLyS administration (4, 5, 6), as well as the B cell deficiencies observed in BLyS knockouts (7), BLyS receptor mutants (8, 9, 10, 11, 12), and BLyS blockade experiments (7), confirm a critical role for BLyS in maintaining naive B cells. Furthermore, BLyS has been implicated in several humoral autoimmune disorders and animal models of autoimmunity (13, 14, 15, 16), confirming its function in specificity-based selection. We and others have shown that mature B cells continuously compete for BLyS to survive (10), and that BLyS levels determine the proportion of cells that successfully complete transitional B cell differentiation (4, 17, 18, 19). Among the three known BLyS receptors, BLyS receptor 3 (BR3) interacts solely with BLyS and plays a dominant role in maintaining normal follicular (FO) and marginal zone (MZ) B cell numbers (8, 9, 11, 20, 21). The other two receptors, transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag (BCMA), can bind both BLyS and a proliferation-inducing ligand (APRIL), and affinity measurements suggest that APRIL may be their physiologically relevant ligand. Mice lacking TACI have elevated B cell numbers (22, 23), suggesting a potential negative regulatory role for this receptor, whereas BCMA appears to be involved in the control of plasmacytes, rather than mature resting B cells (24). In addition, TACI knockout mice display defective T cell-independent type II responses, further suggesting that TACI and BCMA may be involved in regulating the behavior of activated B cells.

We recently showed that BCR stimulation increases BLyS binding capacity in FO and transitional B cells, through the selective up-regulation of BR3 message (25, 26). These findings suggest that B cell activation cues can alter the levels and mix of BLyS family receptors, modulating sensitivity to BLyS or APRIL and dictating the outcomes of signals from these ligands. In this study, we have extended these analyses to B cell activation via TLRs. Although phagocytes express the greatest repertoire of TLRs, several are also expressed by B cells (27). Activation through these innate receptors differs from that achieved via BCR signaling in several ways. First, whereas BCRs are highly diverse and interact individually with a narrow range of epitopes, TLRs bind broadly expressed, conserved molecular patterns on microbial pathogens (28). Second, whereas BCR cross-linking requires concomitant T cell help to afford optimal activation, TLR ligation per se can provide the signals required for proliferation and differentiation to Ab secretion (29). In fact, BCR and TLR signals may cooperate for maximal production of pathogen-specific IgM secretion (30). Finally, T cell-dependent, BCR-driven responses lead to germinal center formation, affinity maturation, and memory cell differentiation, whereas TLR-driven responses tend toward rapid Ab-forming cell differentiation with limited affinity maturation and memory (29). Accordingly, the stimulation of murine B cells through either TLR9 or TLR4 yields a comparatively brief increase in IgM secretion and B cell proliferation (31, 32). Most TLRs signal via the adaptor protein MyD88, and initiate the classical NF-{kappa}B cascade (reviewed in Refs. 33, 34), although TLR3 and TLR4 can also signal through a MyD88-independent pathway (35).

We have used CpG DNA and LPS to stimulate B cells through TLR9 and TLR4, respectively. Our results show that stimulation with either leads to altered BLyS binding capacity by strongly up-regulating TACI and increasing BR3, although to a lesser extent. These effects are B cell intrinsic and MyD88-dependent but, surprisingly, they differ in their downstream transcriptional mediators. Finally, all FO and MZ B cells respond to CpG, whereas only MZ B cells and a subset of FO B cells respond to LPS. Furthermore, we find that APRIL, although not mitogenic, improved survival in both quiescent and BCR-stimulated B cells. These findings suggest that high TACI expression may be a hallmark of TLR activation, and that signaling through this receptor influences survival in resting and activated B cells.


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

C57BL/6J, A/J, and A/WySnJ mice were obtained from The Jackson Laboratory. Spleens from MyD88 knockout mice originally generated by Akira and colleagues (36) were obtained from Dr. M. Schmidt and Dr. K. Alugupalli (University of Massachusetts Medical School, Worcester, MA) or from Dr. S. Joyce (Vanderbilt University, Nashville, TN). c-Rel knockout mice were originally generated and their use permitted by H.-C. Liou et al. (37) and provided by M. Boothby (Vanderbilt University, Nashville, TN). TACI-deficient mice have been previously described (38). TLR4-deficient mice have been previously described (39) and were provided by Dr. S. Joyce (Vanderbilt University, Nashville, TN). Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF)-deficient spleens (40) were obtained from Dr. E. Lien (University of Massachusetts Medical School, Worcester, MA). All animal husbandry and procedures were conducted in accordance with the Animal Welfare Act.

Abs to cell surface Ag and immunofluorescent analysis

Biotinylated anti-CD23 (B3B4), anti-CD21 (7G6), anti-CD19 (1D3), and anti-IgM (ll/41) were purchased from BD Pharmingen. Biotinylated and PE-Cy7 anti-AA4.1 were purchased from eBioscience. Biotinylated BLyS was provided by Human Genome Sciences. Streptavidin Red 670 was purchased from Southern Biotechnology Associates. Anti-TACI PE (catalog no. FAB1041P) and anti-BCMA FITC (catalog no. FAB593F) were purchased from R&D Systems. Anti-BR3 (clone 7H22-E16) was purchased from Axxora Pharmaceuticals; the secondary Ab, mouse anti-rat IgG1 FITC, was purchased from Southern Biotechnology Associates.

B cell subset isolation and culture

Primary B lymphocytes were isolated using MiniMACS magnetic sorting by negative selection (CD43-depletion) to avoid inadvertent activation of B cells as previously described (41). The enrichment of B cells isolated in this manner was ~90–95% as verified by flow cytometric analysis using anti-B220 and anti-IgM Abs. All enrichments were performed at 4°C, and cells were used immediately after isolation. CD23+ B cells were prepared by MACS selection using biotin anti-CD23 and streptavidin beads as previously described (4, 25). Isolated cells were typically >92% CD23+ B cells. Additionally, FO (CD23+/CD21+) and MZ (CD23–/CD21high) B cells were FACS-sorted from MACS-enriched CD43–/AA4.1– (mature) splenic B cells using FITC-conjugated anti-CD21 and biotinylated anti-CD23 followed by allophycocyanin-streptavidin.

In vitro stimulation and proliferation assays: B cells were cultured at 1 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS (HyClone), 2 mM glutamine, 1% oxaloacetic acid (15 mg/ml), 5 mg/ml sodium pyruvate, 20 U/ml insulin, 1% nonessential amino acids, 50 µM 2-ME, and 100 U/ml gentamicin. In some experiments, CD23+ B cells were loaded with 1.25 µM CFDASE (Molecular Probes) in PBS. After a 2-min incubation, excess CFDASE, or CFSE (the deacetylated form), was quenched with an equal volume of FCS, and cells were washed once before culture. Various doses of CpG DNA (ODN 1826 5'-TCCATGACGTTCCTGACGTT) and nonstimulatory DNA (ODN 1982 5'-TCCAGGACTTCTCTCAGGTT) (Custom Primers; Invitrogen Life Technologies) and/or F(ab')2 goat anti-mouse IgM (Jackson ImmunoResearch Laboratories) or ultra-pure LPS (Axxora Life Sciences or Alexis Biochemicals) were used for stimulation, in the presence or absence of 50 ng/ml soluble recombinant BLyS (Human Genome Sciences) or APRIL (PeproTech). CFSE-labeled cells were also incubated with 1 nM TOPRO-3 (Molecular Probes) immediately before acquisition, enabling the resolution of live vs dead cells.

Flow cytometric analyses

To determine TACI induction in all B cell subpopulations, B cell subsets were identified in total CD43– B cell cultures following staining with 7-aminoactinomycin D, to exclude dead cells, and CD21-FITC, IgM-allophycocyanin, AA4.1 PE-Cy7, and CD19-allophycocyanin Cy7 for B cell subset identification. TACI induction was visualized using PE-conjugated anti-TACI. Data were collected on an LSRII cytometer and analyzed using FlowJo (Tree Star).

Dendritic cell and mast cell culture and isolation

Dendritic cells were grown by culturing bone marrow cells of C57BL/6 mice in complete medium containing IL-4 (10 ng/ml) and GM-CSF (10 ng/ml) for 7 days as described (42). Dendritic cells were further purified from these cultures by MACS selection using CD11c beads. Mast cells were obtained by culturing C57BL/6 bone marrow cells in complete medium containing IL-3 (10 ng/ml) and stem cell factor (12.5 ng/ml) for 8 wk as described (43). Mast cells were further purified by flow cytometric sorting using anti-Fc{epsilon}RI (eBioscience) and anti-CD117 (BD Pharmingen) Abs. RNA was isolated from purified dendritic cells and mast cells using RNeasy Mini kit (Qiagen).

Quantitative real-time PCR analysis

RNA was extracted from MACS-enriched CD23+ FO B cells or FACS-sorted MZ B cells using RNeasy Mini kit (Qiagen) and used as a template to generate cDNA by reverse transcription. For real-time PCR analysis, we used TaqMan Universal Master mix (Applied Biosystems) and Stratagene Max 3000p Detection System or ABI 7300 Real-Time PCR System. Primers and FAM-labeled probes were obtained from Applied Biosystems (TaqMan Assay on Demand). The mRNA fold induction for TACI was estimated relative to 18S ribosomal RNA or GAPDH.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CpG stimulation increases BlyS binding capacity through TACI up-regulation

We first examined whether CpG, an unmethylated nucleotide sequence associated with activation through TLR9, alters BLyS receptor expression. Incubation of CD23+ B cells with stimulatory CpG DNA increased BLyS binding capacity substantially (Fig. 1, A and B), whereas nonstimulatory sequences had no effect. Moreover, this increase was greater than that observed with anti-µ alone, and combined stimulation with both anti-µ and CpG yielded additive effects up to an apparent maximum BLyS binding level (Fig. 1C).


Figure 1
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  		FIGURE 1. CpG stimulation preferentially up-regulates TACI expression. A, CD23+ splenocytes were isolated by MACS and stimulated for 24 h with the indicated concentrations of stimulatory CpG sequence (ODN 1826) or nonstimulatory GpC sequence (ODN 1982), alone or in combination with 10 µg/ml anti-µ. Mean fluorescence intensity (MFI) of BLyS binding was determined by FACS using biotinylated BLyS and streptavidin Red 670. Bars indicate means of the three replicates shown. Data are representative of three experiments. B, Histograms representative of cells analyzed in A. Unstimulated cells (gray-filled histogram), cells stimulated with 10 µg/ml anti-µ (open histogram), and cells stimulated with the indicated DNA sequence (filled histogram) are shown. Results are representative of three independent experiments. C, CD23+ splenocytes were stimulated with the indicated concentrations of ODN 1826 alone or in combination with the indicated concentrations of anti-µ. D, CD23+ splenocytes were isolated by MACS and incubated for 24 h in medium alone, 10 µg/ml anti-µ, 1 µM ODN 1826, or 10 µg/ml anti-µ and 1 µM ODN 1826 in combination. Isotype controls (shaded histogram) and stimulated cells (open histogram) are shown. Cell surface receptor expression was determined by FACS using the following stains: TACI PE, BR3/donkey-anti-rat PE, or BCMA FITC. Results are representative of three independent experiments. E, Mean fluorescence intensity (MFI) of stimulated cells was divided by mean fluorescence intensity of unstimulated cells and expressed as fold increase. **, p < 0.001 as determined by Student’s t test. F, Forward scatter and TACI plots for cells stimulated as in C. Results are representative of three independent experiments. G, CD23+ splenocytes were isolated by MACS and incubated with either 0.1 µM ODN 1826 or medium alone for the times indicated. mRNA levels were determined by real-time PCR using 18S mRNA as a control. BR3 levels ({square}) and TACI expression ({blacksquare}) are indicated. Results are representative of three independent experiments.

 
To establish which BLyS receptors contributed to these CpG-induced increases in BLyS binding capacity, we monitored surface levels of each BLyS receptor by FACS. Resting FO B cells express both TACI and BR3 on their surface, but not BCMA. Following CpG stimulation, TACI surface expression increased substantially, as evidenced by as much as a 7-fold increase in mean fluorescence intensity (Fig. 1, D and E). BR3 also increased, although to a lesser extent. The increases were independent of the cell enlargement associated with CpG-mediated mitogenesis because they did not correlate with cell size (Fig. 1F). This conclusion was further substantiated by real-time PCR for TACI and BR3 messages normalized to 18S ribosomal RNA (Fig. 1G).

CpG-mediated BLyS receptor regulation is MyD88-dependent and B cell-intrinsic

TLR9 stimulation proceeds through MyD88, which targets NF-{kappa}B for nuclear translocation to activate gene expression (33, 34). Thus, to substantiate MyD88-NF-{kappa}B as the major pathway involved in CpG-mediated TACI up-regulation, we assessed B cells isolated from MyD88 and c-Rel/NF-{kappa}B knockout mice. The results show that MyD88-deficient B cells fail to increase BLyS binding or TACI expression in response to stimulatory CpG DNA (Fig. 2, A and B), whereas c-Rel is dispensable for this response (Fig. 2, C and D and data not shown). Consistent with these results, MyD88 is required for up-regulation of TACI transcription in response to CpG, but c-Rel is not (Fig. 2E). MyD88 is also required for the proliferative and prosurvival effects of CpG DNA stimulation (Fig. 2F). Although the c-Rel-deficient cells are generally less viable, c-Rel deficiency abrogates neither proliferation nor survival in response to CpG DNA, even in the presence of BLyS (Fig. 2G and data not shown). Thus, CpG-mediated increases in survival and TACI expression occur through a MyD88-dependent but c-Rel-independent mechanism.


Figure 2
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  		FIGURE 2. CpG-induced BLyS receptor up-regulation is MyD88-dependent and c-Rel-independent. A, Wild-type or MyD88 knockout cells were stimulated as indicated for 24 h, then BLyS binding was determined by FACS. MyD88-(B) and c-Rel-deficient (C) B cells were stimulated with either 1 µM CpG or 10 µg/ml anti-µ for 24 h, then TACI expression was determined by FACS. Isotype control (dotted line histogram), unstimulated cells (gray-filled histogram), and stimulated cells (thick line histogram) are represented. Results are representative of three independent experiments. D, B cells isolated by either CD23-positive selection (top) or CD43-negative selection (bottom) were stimulated with either CpG or anti-µ and TACI expression was determined by FACS as in B. The response of wild-type (wt) cells ({square}) and of c-Rel-deficient (crel–/–) cells (Figure 2) are shown. *, p < 0.05 as determined by Student’s t test. E, CD23+ splenocytes from the indicated mice were stimulated with either CpG or anti-µ for 4 h, and quantitative real-time PCR was performed to determine the fold change in the steady-state TACI mRNA compared with 18S mRNA. F, CD23+ splenocytes from MyD88 knockout mice or wild-type mice were loaded with CFSE and incubated either with 10 µg/ml anti-µ or 1 µM ODN 1826. After 72 h, the cells were harvested, stained with TOPRO-3, and analyzed by flow cytometry. Results are representative of three independent experiments. Numbers indicate percentage of total cells in each gate. G, Wild-type ({blacksquare}) or c-Rel-deficient ({square}) cells were incubated in medium or 1 µM ODN 1826. Cultures included 60 ng/ml BLyS or 200 ng/ml APRIL as indicated. *, p < 0.05 as determined by Student’s t test. H, CD23+ splenocytes from MyD88 knockout mice and congenic (Ly5.2) mice were mixed 50/50 and incubated for 24 h either with 10 µg/ml anti-µ or 1 µM ODN 1826. The congenic marker and TACI expression were then determined by FACS. Isotype control (dotted line histogram) is shown for each cell type, and unstimulated cells (gray-shaded histogram) and TACI expression (open histogram) on stimulated cells are also represented shown. Results are representative of three independent experiments.

 
B cells are most likely stimulated by CpG DNA via TLR9, directly increasing expression of BLyS receptors. Alternatively, the increase in BLyS receptors could be indirectly mediated by cytokines released from other cells in response to CpG DNA. MyD88 dependence and the availability of gene-targeted mice afforded the opportunity to distinguish these possibilities. MyD88 knockout and congenic wild-type B cells were cocultured with stimulatory CpG DNA for 24 h. The cells were then harvested and stained for BLyS receptor and congenic marker expression. Only wild-type B cells increased TACI expression (Fig. 2H), indicating that the BLyS receptor increase following CpG stimulation requires TLR9-MyD88 signaling in the B cells themselves, demonstrating direct B cell intrinsic mechanisms.

TACI has been reported as a negative regulator for both B cell survival and proliferation (22, 23, 38), yet it is increased on the surface of CpG-treated B cells. We therefore examined whether APRIL or BLyS could alter survival or proliferation (Fig. 3, A and B). Neither BLyS nor APRIL influenced the extent of proliferation in any treatment group, indicating that these cytokines lack direct mitogenic activity. As expected, BLyS improved survival in all treatment groups, in agreement with previous findings that BLyS increases survival of FO and transitional B cells (4, 44, 45). APRIL also increased survival in the unstimulated and BCR-stimulated groups, although somewhat less potently than BLyS in unstimulated cells (Fig. 3B). In contrast to BLyS, however, APRIL had little survival-promoting effect in the CpG-stimulated cells.


Figure 3
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  		FIGURE 3. APRIL supports survival in unstimulated and BCR-stimulated cells. Wild-type (A and B), A/WySnJ (C and D), or TACI knockout (E and F) CD23+ splenocytes were stimulated with either 10 µg/ml anti-µ or 1 µM ODN 1826 and either 60 ng/ml BLyS or 200 ng/ml APRIL. After 72 h, the cells were harvested, stained with TOPRO-3, and analyzed by flow cytometry. Results are representative of three independent experiments. *, p < 0.05; **, p < 0.001; ***, p < 0.0001 as determined by Student’s t test.

 
To further examine the role of BR3 and TACI in the improving survival in response to CpG DNA, we repeated this experiment in the BR3 mutant, A/WySnJ, and in TACI-deficient cells (Fig. 3, C–F). In the A/WySnJ, both APRIL and BLyS had modest prosurvival effects on all cells, regardless of concomitant stimulation. Both cytokines can bind TACI, which may explain the slight improvement seen. However, the levels of survival with CpG stimulation were equivalent between the A/WySnJ cells and the A/J controls (data not shown), demonstrating that this effect is not mediated through BR3.

The TACI-deficient cells respond similarly to wild-type controls in all treatments except in anti-µ-stimulated cells incubated with APRIL. In this instance, survival was equivalent to anti-µ-stimulated cells that received no cytokine. Three conclusions follow from these data: first, survival of unstimulated cells incubated with APRIL is improved through a TACI-independent mechanism; second, APRIL increases survival of cells stimulated through their BCR by signaling through TACI; and third, TACI is not necessary for the prosurvival effects of CpG DNA.

To examine possible autocrine production of BLyS or APRIL, we performed quantitative real-time PCR on FACS-sorted FO cells (B220+, AA4.1–, IgMint, and CD21/35int) incubated with either anti-µ or CpG DNA (data not shown). Neither stimulation induced APRIL expression above unstimulated levels, eliminating this mechanism as a possibility for increased survival. Both anti-µ and CpG DNA increased BLyS expression 5- to 6-fold over unstimulated cells; however, cell survival in these two groups is not equivalent (Fig. 3A), particularly in A/WySnJ cells. Thus, although autocrine BLyS production may contribute to the prosurvival effects of CpG DNA, it cannot solely account for this effect.

LPS induced TACI expression is MyD88- and c-Rel-dependent

To investigate whether TACI up-regulation is a general characteristic of TLR-induced B cell activation, we stimulated total B cells with the TLR4 agonist, LPS. LPS increased BLyS binding and TACI expression in B cells (Fig. 4, A and B). This induction was TLR4-mediated, as TLR4-deficient B cells failed to produce this response (Fig. 4B). Together with the observation that TLR9 induces TACI expression (Fig. 1), these results suggest a general role for TLRs in the regulation of TACI expression.


Figure 4
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  		FIGURE 4. LPS-induced up-regulation of TACI requires MyD88. A, Total splenic B cells were stimulated for 24 h with 10 µg/ml anti-µ or 5.0 µg/ml LPS. BLyS binding was determined by flow cytometric analysis using biotinylated BLyS and streptavidin-PE. B, Total splenic B cells were isolated from TLR4-deficient (tlr4–/–) or wild-type (wt) mice stimulated as in A, and cell surface TACI levels were determined using PE-labeled rat anti-mouse TACI. Total (C) or CD23+ (D) B cells from wild-type (wt) or MyD88 knockout (myD88–/–) mice were stimulated with 10 µg/ml anti-µ, 5.0 µg/ml LPS, 3.3 µg/ml anti-µ, and 5.0 µg/ml LPS in combination or with medium alone for 18 h and TACI levels determined as in B. E, CD23+ B cells from MyD88 knockout and wild-type littermate control mice were stimulated for 4 h with LPS (1 µg/ml) or anti-mouse-CD40 (10 µg/ml) as indicated, and quantitative real-time PCR was performed to determine the fold change in the steady-state levels of TACI mRNA compared with 18S mRNA. **, p < 0.001 as determined by Student’s t test. F, Total B cells from TRIF-deficient (trif–/–) or wild-type (wt) spleens were stimulated with 10 µg/ml LPS. Cells were harvested at 24 h, and TACI levels were determined by FACS. Results are isotype control (dotted line histogram), unstimulated cells (gray-shaded histogram), and stimulated cells (open histogram) for experiments shown.

 
In contrast to TLR9, which interacts with DNA intracellularly and signals exclusively via MyD88, TLR4 is displayed on the cell surface and uses both MyD88-dependent and -independent signaling mechanisms (46). To determine whether LPS-induced up-regulation of TACI involves MyD88 or c-Rel/NF-{kappa}B, the expression levels of TACI were compared in CD23+ and total MyD88- and c-Rel-deficient and wild-type B cells following LPS stimulation. Similar to the results obtained with CpG, LPS up-regulated cell surface and mRNA expression of TACI in wild-type B cells but not in MyD88-deficient B cells (Fig. 4, C–E). Likewise, TRIF-deficient B cells also failed to induce TACI in response to LPS, as measured by both FACS and mRNA levels (Fig. 4F and data not shown), indicating that TLR4 up-regulation of TACI proceeds via both MyD88-dependent and -independent mechanisms. This finding is consistent with Hirotani et al. (47), who identified a cluster of genes that require both the MyD88- and TRIF-dependent pathways. Furthermore, in contrast to CpG, LPS-induced expression of TACI was impaired in B cells deficient for c-Rel (Fig. 5). Together these findings suggest that TLR4-dependent up-regulation of BLyS receptors proceeds via MyD88 and TRIF. In addition, unlike TLR9, TLR4-induced TACI expression is c-Rel-dependent.


Figure 5
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  		FIGURE 5. LPS induced TACI up-regulation is c-Rel-dependent. Total (A) or CD23+ (B) B cells isolated from wild-type (wt) or c-Rel-deficient (crel–/–) mice were stimulated as indicated for 24 h, followed by quantification of TACI expression levels by FACS. **, p < 0.001 as determined by Student’s t test. C, CD23+ B cells purified from c-Rel-deficient (crel–/–) or wild-type (wt) mice were stimulated for 4 h as indicated, and quantitative real-time PCR was performed to determine the fold change in the steady-state levels of TACI mRNA compared with 18S mRNA. *, p < 0.05 as determined by Student’s t test.

 
TLR4 stimulation differentially increases TACI expression in MZ vs FO B cells

We observed that unlike either CpG or anti-µ (Fig. 2), LPS-induced TACI expression was not uniform among either CD23+ or total B cells (Fig. 4, C and D). Analysis of splenic B cell subsets revealed that LPS-induced TACI expression occurred predominantly in MZ B cells (Fig. 6B). This observation was confirmed with analysis of FACS-sorted FO and MZ B cells at the level of cell surface protein as well as mRNA expression (Fig. 6, C and D). Together, these results show that in contrast to CpG, which induces TACI in all B cell populations analyzed, LPS up-regulates TACI predominantly in MZ B cells. Likewise, CD69 is also increased most on MZ B cells (Fig. 6E), indicating preferential activation of this subset. The observation that only a subset of FO B cells responds to LPS was further investigated in a kinetic experiment in which purified FO B cells were stimulated with LPS for 3 days (Fig. 6F). The FO subset responsive to LPS proliferates over this time period, as evidenced in the CFSE profiles comparing cells expressing high levels of TACI with those that lack TACI up-regulation. This expansion leads to a proportional enlargement of the TACIhigh subpopulation, and a corresponding reduction in the proportion of TACIlow cells.


Figure 6
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  		FIGURE 6. MZ B cells respond more uniformly and more rapidly than FO B cells to LPS. A, Fresh splenocytes were stained as indicated to reveal B cell subsets. B, Total splenic B cells were incubated either in medium alone or with 5 µg/ml LPS for 24 h, then stained to identify TACI expression on B cell subsets gated as indicated in A. Dotted lines, isotype controls. C, FACS-sorted MZ and FO B cells were stimulated as indicated, and TACI expression was determined by flow cytometry. D, FACS-purified MZ and FO B cells were stimulated for 4 h with anti-µ (10 µg/ml), LPS (5 µg/ml), a combination of anti-µ and LPS (3.3 and 5 µg/ml, respectively), or medium alone and analyzed for steady-state levels of TACI mRNA compared with 18S mRNA by quantitative real-time PCR. *, p < 0.05, **; p < 0.001 as determined by Student’s t test. E, Freshly isolated splenic B cells were stimulated as indicated for 12 h, followed by quantification of CD69 expression by FACS. Identification of each B cell subsets was performed on gated CD19+ cells, based on the expression of CD21/IgMhigh CD23– (MZ), IgMhigh CD21lowCD23– (T1), IgM/CD21high CD23+ (T2), and IgMlow, CD21/CD23+ (FoB) cells. *, p < 0.05 as determined by Student’s t test. F, CD23+ splenic B cells were labeled with CFSE and stimulated with either 1 µM CpG or 10 µg/ml LPS for 3 days. Cells were harvested at 24, 48, and 72 h and stained for TACI. Cells were analyzed by flow cytometry. Live cells as determined by TOPRO-3 exclusion are shown. Results are representative of three independent experiments. G, Freshly isolated splenocytes were FACS-sorted into B220+ AA4– IgMint CD21/35int (FO) or B220+ AA4– IgMhigh CD21/35high (MZ) subsets and analyzed by quantitative real-time PCR for expression of TLR2, TLR4, and TLR9, and GAPDH. Dendritic cell (DC) and mast cell mRNA results are included as controls. Results are representative of three independent experiments. H, CD23+ splenocytes were incubated with either 60 ng/ml BLyS or 200 ng/ml APRIL for the indicated times. Cells were harvested and analyzed for expression of TLR4 and TLR9, and 18S mRNA by quantitative real-time PCR. Results are representative of three independent experiments.

 
To verify that MZ and FO B cell expression of TLRs, quantitative real-time PCR was performed on FACS-sorted splenocytes, using mast cells and dendritic cells as controls. As shown in Fig. 6G, MZ cells transcribe more TLR2, TLR4, and TLR9 than FO cells. Although TLR2 mRNA levels were much higher in dendritic cells than in any other cell type, TLR4 levels were equivalent between MZ and dendritic cells, and TLR9 levels were highest in MZ cells. Mast cells expressed slightly higher levels of TLR4 mRNA than any of the other cell types assayed. Neither TLR4 nor TLR9 increased after incubation with BLyS or APRIL (Fig. 6H).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies examine BLyS receptor expression in B cells undergoing TLR-driven activation. The results indicate that both TLR9 and TLR4 ligation lead to increased BLyS binding capacity through the up-regulation of TACI and, to a lesser extent, BR3. This finding contrasts with the Ng et al. (48), who showed that increases in TACI were comparable in BCR and CpG stimulations of B2 cells. We found these shifts to reflect B cell intrinsic, MyD88-dependent mechanisms. Although all FO and MZ B cells are TLR9 responsive, TLR4 stimulation activates MZ B cells and a subset of FO B cells. Overall, TLR stimulation shifts BLyS receptor profiles from one biased toward BR3 on naive cells to one dominated by TACI on TLR-stimulated cells. In conjunction with previous work, these observations suggest a mechanism whereby the mode of B cell activation dictates relative sensitivity and downstream outcomes of APRIL and BLyS signaling.

Our results are consistent with studies that show a role for BLyS and APRIL in T-independent immune responses involving TACI-dependent mechanisms. For example, TACI knockout mice display defective T cell-independent type II responses (23, 38), and Castigli et al. (49) established TACI as important in isotype switching. Furthermore, studies by He et al. (50) suggest that BAFF cooperates with CpG DNA in this process. Our observation that APRIL augments quiescent B cell survival is in accord with this view as well. These viability-promoting effects are most likely delivered through TACI because naive B cells express neither BCMA nor syndecan-1, and APRIL cannot bind BR3. It is unclear why TLR9-stimulated cells remain sensitive to the survival promoting effect of BLyS, whereas APRIL had little effect following CpG stimulation despite elevated TACI levels. Cycling and quiescent cells may differ in their sensitivities to the two cytokines, but we found that once a cell enters cell cycle, TACI levels remain uniformly high (data not shown). Alternatively, TLR9 and TACI signaling may converge on a common and redundant downstream prosurvival signaling pathway. We favor this explanation for several reasons. First, MyD88-dependent signaling proceeds at least in part via NF-{kappa}B1 mediators, and recent data indicate that TACI signals primarily through the NF-{kappa}B1 pathway (M. Gupta and M. P. Cancro, manuscript submitted). Furthermore, this explanation would account for continued sensitivity to BLyS among CpG-stimulated cells because BR3 ligation also activates the nonclassical NF-{kappa}B2 pathway (51, 52), thus providing an additive survival effect. Regardless of the exact mechanisms through which TACI signaling enhances survival, these findings are most consistent with TACI having a role in sustaining both resting and activated B cells.

Although signaling through TLR4 and TLR9 strongly up-regulates TACI expression, the underlying mechanisms differ, as does the breadth of the B cells affected. Both TLRs require MyD88 to up-regulate TACI expression. However, whereas TLR4-mediated TACI up-regulation requires c-Rel and p50 (Fig. 5 and data not shown), TLR9-induced TACI expression is largely c-Rel- and p50-independent (Fig. 2 and data not shown). These observations suggest that the increases in TACI transcription in each case rely on different NF-{kappa}B dimers or proceed via different mediators. Because the major known target of TLR signaling is the NF-{kappa}B1 pathway, it is possible that the other transactivation subunit of the NF-{kappa}B1 pathway, RelA, serves as the mediator of NF-{kappa}B-dependent gene expression in response to TLR9 stimulation. Recent studies have suggested that specific {kappa}B site sequences in the promoters of target genes determine the dimer specificity and coactivator requirements (53, 54). Thus, use of distinct NF-{kappa}B transactivation subunits by different TLRs may target distinct "pathogen-specific" NF-{kappa}B-responsive genes and may play an important role in modulating immune responses to the various pathogens that are recognized by distinct members of the TLR family.

All MZ B cells respond to either TLR9 or TLR4 stimulation. However, only a subset CD23+ B cells is LPS responsive. At optimal doses of LPS, these cells comprise ~20% of CD23+ splenic B cells; in cell sorting experiments, we have established that this subset exists in both the AA4.1+ and AA4.1– fractions (data not shown). Whether these are B cells poised for selection into the MZ from the transitional and FO pools, or instead represent a stable TLR4-bearing subset within developing and mature FO cells needs to be examined. Regardless of the exact origin of this subset, our results suggest that immune responses to various pathogens may be regulated both by the choice of TLR and in turn the subset of B cells recruited following challenge. Determining TLR expression profiles within peripheral B cell subsets should reveal details of this potential immunoregulatory mechanism.

Although these issues necessitate resolution, the overarching effect of TLR stimulation remains a preferential increase in TACI expression. This response contrasts with BCR- and CD40-mediated shifts in BLyS receptor family expression, which favor BR3, as well as the prevalence of BCMA among long-lived memory plasma cells (data not shown) (24). Recent work has shown that mutations rendering TACI nonfunctional correlate with combined variable immunodeficiency disease and IgA deficiency in humans (55, 56). The frequent association of these syndromes with impaired bacterial polysaccharide responses, together with the defective T cell-independent type II responses in TACI-deficient mice, suggest a role for TACI in these types of responses (38, 57). In this regard, it is tempting to speculate that TACI-centric signaling outcomes are a hallmark of rapid but short-lived Ab-forming cell responses. This possibility encourages caution in using TLR ligands as vaccine adjuvants, inasmuch as they may favor short-lived Ab-forming cell responses and thwart the formation of memory B cells.


   Acknowledgment
 
We thank Dr. Egil Lien for critical reading of the manuscript.


   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 study is supported in part by Research Grants AI50213 and AI060729 (to W.N.K.) and Grant AI54488 (to M.P.C.) from the U.S. Public Health Service. K.L.H. and L.S.T. are supported by Training Grants AI069770 and RR07063, respectively, from the U.S. Public Health Service. Back

2 L.S.T. and G.C. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Michael P. Cancro, Department of Pathology and Laboratory Medicine, 284 John Morgan Building, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104; E-mail address: cancro{at}mail.med.upenn.edu or Dr. Wasif N. Khan, Department of Microbiology and Immunology, Medical Center North A4207, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232; E-mail address: wasif.khan{at}vanderbilt.edu Back

4 Abbreviations used in this paper: BLyS, B lymphocyte stimulator; FO, follicular; MZ, marginal zone; TACI, transmembrane activator calcium modulator and cyclophilin ligand interactor; APRIL, a proliferation-inducing ligand; BCMA, B cell maturation Ag; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-beta. Back

Received for publication November 27, 2006. Accepted for publication April 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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