TLR Stimulation Modifies BLyS Expression in Follicular and Marginal Zone B Cells

This information is current as Laura S. Treml, Gianluca Carlesso, Kristen L. Hoek, Jason of October 2, 2021. E. Stadanlick, Taku Kambayashi, Richard J. Bram, Michael P. Cancro and Wasif N. Khan J Immunol 2007; 178:7531-7539; ; doi: 10.4049/jimmunol.178.12.7531 http://www.jimmunol.org/content/178/12/7531 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

TLR Stimulation Modifies BLyS Receptor Expression in Follicular and Marginal Zone B Cells1

Laura S. Treml,2* Gianluca Carlesso,2† Kristen L. Hoek,† Jason E. Stadanlick,* Taku Kambayashi,* Richard J. Bram,‡ Michael P. Cancro,3* and Wasif N. Khan3†

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in 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 expres- Downloaded from sion, 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. The Journal of Immunology, 2007, 178: 7531–7539.

he B lymphocyte stimulator (BLyS)4 and its receptors (4, 17–19). Among the three known BLyS receptors, BLyS recep- http://www.jimmunol.org/ profoundly influence primary B cell homeostasis and se- tor 3 (BR3) interacts solely with BLyS and plays a dominant role T lection (reviewed in Refs. 1–3). The prompt increases in in maintaining normal follicular (FO) and marginal zone (MZ) B B cell numbers following BLyS administration (4–6), as well as cell numbers (8, 9, 11, 20, 21). The other two receptors, trans- the B cell deficiencies observed in BLyS knockouts (7), BLyS membrane activator calcium modulator and cyclophilin ligand in- receptor mutants (8–12), and BLyS blockade experiments (7), teractor (TACI) and B cell maturation Ag (BCMA), can bind both confirm a critical role for BLyS in maintaining naive B cells. Fur- BLyS and a proliferation-inducing ligand (APRIL), and affinity thermore, BLyS has been implicated in several humoral autoim- measurements suggest that APRIL may be their physiologically mune disorders and animal models of autoimmunity (13–16), con- relevant ligand. Mice lacking TACI have elevated B cell numbers by guest on October 2, 2021 firming its function in specificity-based selection. We and others (22, 23), suggesting a potential negative regulatory role for this have shown that mature B cells continuously compete for BLyS to receptor, whereas BCMA appears to be involved in the control of survive (10), and that BLyS levels determine the proportion of plasmacytes, rather than mature resting B cells (24). In addition, cells that successfully complete transitional B cell differentiation TACI knockout mice display defective T cell-independent type II responses, further suggesting that TACI and BCMA may be in-

*Department of Pathology and Laboratory Medicine, University of Pennsylvania volved in regulating the behavior of activated B cells. School of Medicine, Philadelphia, PA 19104; †Department of Microbiology and Im- We recently showed that BCR stimulation increases BLyS bind- munology, Vanderbilt University School of Medicine, Nashville, TN 37232; and ‡De- ing capacity in FO and transitional B cells, through the selective partment of Pediatric and Adolescent Medicine, Mayo Clinic, Mayo Medical School, Rochester, MN 55905 up-regulation of BR3 message (25, 26). These findings suggest Received for publication November 27, 2006. Accepted for publication April 4, 2007. that B cell activation cues can alter the levels and mix of BLyS The costs of publication of this article were defrayed in part by the payment of page family receptors, modulating sensitivity to BLyS or APRIL and charges. This article must therefore be hereby marked advertisement in accordance dictating the outcomes of signals from these ligands. In this study, with 18 U.S.C. Section 1734 solely to indicate this fact. we have extended these analyses to B cell activation via TLRs. 1 This study is supported in part by Research Grants AI50213 and AI060729 (to Although phagocytes express the greatest repertoire of TLRs, sev- 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, eral are also expressed by B cells (27). Activation through these from the U.S. Public Health Service. innate receptors differs from that achieved via BCR signaling in 2 L.S.T. and G.C. contributed equally to this study. several ways. First, whereas BCRs are highly diverse and interact 3 Address correspondence and reprint requests to Dr. Michael P. Cancro, Department individually with a narrow range of epitopes, TLRs bind broadly of Pathology and Laboratory Medicine, 284 John Morgan Building, University of expressed, conserved molecular patterns on microbial pathogens Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104; E-mail address: [email protected] or Dr. Wasif N. Khan, Department of (28). Second, whereas BCR cross-linking requires concomitant T Microbiology and Immunology, Medical Center North A4207, Vanderbilt University cell help to afford optimal activation, TLR ligation per se can pro- School of Medicine, 1161 21st Avenue South, Nashville, TN 37232; E-mail address: [email protected] vide the signals required for proliferation and differentiation to Ab secretion (29). In fact, BCR and TLR signals may cooperate for 4 Abbreviations used in this paper: BLyS, B lymphocyte stimulator; FO, follicular; MZ, marginal zone; TACI, transmembrane activator calcium modulator and cyclo- maximal production of pathogen-specific IgM secretion (30). philin ligand interactor; APRIL, a proliferation-inducing ligand; BCMA, B cell mat- Finally, T cell-dependent, BCR-driven responses lead to ger- uration Ag; , Toll/IL-1R domain-containing adaptor-inducing IFN-␤. minal center formation, affinity maturation, and memory cell Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 differentiation, whereas TLR-driven responses tend toward www.jimmunol.org 7532 TLR REGULATION OF TACI

rapid Ab-forming cell differentiation with limited affinity mat- uration and memory (29). Accordingly, the stimulation of mu- rine B cells through either TLR9 or TLR4 yields a compara- tively brief increase in IgM secretion and B cell proliferation (31, 32). Most TLRs signal via the adaptor MyD88, and initiate the classical NF-␬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 ex- tent. These effects are B cell intrinsic and MyD88-dependent but, surprisingly, they differ in their downstream transcriptional medi- ators. 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, im- proved survival in both quiescent and BCR-stimulated B cells. These findings suggest that high TACI expression may be a hall- mark of TLR activation, and that signaling through this receptor Downloaded from influences survival in resting and activated B cells.

Materials and Methods Mice

C57BL/6J, A/J, and A/WySnJ mice were obtained from The Jackson http://www.jimmunol.org/ Laboratory. 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-defi- cient mice have been previously described (39) and were provided by Dr. S. Joyce (Vanderbilt University, Nashville, TN). Toll/IL-1R domain-contain- ing adaptor-inducing IFN-␤ (TRIF)-deficient spleens (40) were obtained

from Dr. E. Lien (University of Massachusetts Medical School, Worcester, by guest on October 2, 2021

FIGURE 1. CpG stimulation preferentially up-regulates TACI expres- sion. A, CD23ϩ 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 combi- nation with 10 ␮g/ml anti-␮. Mean fluorescence intensity (MFI) of BLyS binding was determined by FACS using biotinylated BLyS and streptavi- din 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 ␮g/ml anti-␮ (open histogram), and cells stimulated with the indicated DNA sequence (filled histogram) are shown. Results are representative of three independent experiments. C, CD23ϩ splenocytes were stimulated with the indicated concentrations of ODN 1826 alone or in combination with the indicated concentrations of anti-␮. D, CD23ϩ splenocytes were isolated by MACS and incubated for 24 h in medium alone, 10 ␮g/ml anti-␮,1␮M ODN 1826, or 10 ␮g/ml anti-␮ and 1 ␮M ODN 1826 in combination. Isotype con- trols (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 rep- resentative of three independent experiments. E, Mean fluorescence intensity (MFI) of stimulated cells was divided by mean fluorescence intensity of un- p Ͻ 0.001 as determined ,ءء .stimulated cells and expressed as fold increase 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, CD23ϩ splenocytes were isolated by MACS and incubated with either 0.1 ␮M 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 (Ⅺ) and TACI expression (f) are indicated. Results are representative of three independent experiments. The Journal of Immunology 7533

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 Sciences. Streptavidin Red 670 was pur- chased from Southern Biotechnology Associates. Anti-TACI PE (catalog no. FAB1041P) and anti-BCMA FITC (catalog no. FAB593F) were pur- chased 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 ϳ90–95% 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. CD23ϩ B cells were prepared by MACS selection using biotin anti-CD23 and streptavidin beads as previ- Downloaded from ously described (4, 25). Isolated cells were typically Ͼ92% CD23ϩ B cells. Additionally, FO (CD23ϩ/CD21ϩ) and MZ (CD23Ϫ/CD21high) B cells were FACS-sorted from MACS-enriched CD43Ϫ/AA4.1Ϫ (mature) splenic B cells using FITC-conjugated anti-CD21 and biotinylated anti-CD23 fol- lowed by allophycocyanin-streptavidin. In vitro stimulation and proliferation assays: B cells were cultured at 1 ϫ 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS

(HyClone), 2 mM glutamine, 1% oxaloacetic acid (15 mg/ml), 5 mg/ml http://www.jimmunol.org/ sodium pyruvate, 20 U/ml insulin, 1% nonessential amino acids, 50 ␮M 2-ME, and 100 U/ml gentamicin. In some experiments, CD23ϩ B cells were loaded with 1.25 ␮M 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 5Ј-TCCATGACGTTC CTGACGTT) and nonstimulatory DNA (ODN 1982 5Ј-TCCAGGACT TCTCTCAGGTT) (Custom Primers; Invitrogen Life Technologies) and/or Ј 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 by guest on October 2, 2021 BLyS (Human Genome Sciences) or APRIL (PeproTech). CFSE-labeled cells were also incubated with 1 nM TOPRO-3 (Molecular Probes) imme- diately before acquisition, enabling the resolution of live vs dead cells. Flow cytometric analyses FIGURE 2. CpG-induced BLyS receptor up-regulation is MyD88-depen- dent and c-Rel-independent. A, Wild-type or MyD88 knockout cells were To determine TACI induction in all B cell subpopulations, B cell subsets stimulated as indicated for 24 h, then BLyS binding was determined by FACS. Ϫ were identified in total CD43 B cell cultures following staining with MyD88-(B) and c-Rel-deficient (C) B cells were stimulated with either 1 ␮M 7-aminoactinomycin D, to exclude dead cells, and CD21-FITC, IgM-allo- CpG or 10 ␮g/ml anti-␮ for 24 h, then TACI expression was determined by phycocyanin, AA4.1 PE-Cy7, and CD19-allophycocyanin Cy7 for B cell FACS. Isotype control (dotted line histogram), unstimulated cells (gray-filled subset identification. TACI induction was visualized using PE-conjugated anti-TACI. Data were collected on an LSRII cytometer and analyzed using histogram), and stimulated cells (thick line histogram) are represented. Results FlowJo (Tree Star). are representative of three independent experiments. D, B cells isolated by either CD23-positive selection (top) or CD43-negative selection (bottom) were and mast cell culture and isolation stimulated with either CpG or anti-␮ and TACI expression was determined by FACS as in B. The response of wild-type (wt) cells (Ⅺ) and of c-Rel-deficient Dendritic cells were grown by culturing cells of C57BL/6 Ϫ Ϫ .p Ͻ 0.05 as determined by Student’s t test ,ء .crel / ) cells (u) are shown) mice in complete medium containing IL-4 (10 ng/ml) and GM-CSF (10 ϩ ng/ml) for 7 days as described (42). Dendritic cells were further purified E, CD23 splenocytes from the indicated mice were stimulated with either from these cultures by MACS selection using CD11c beads. Mast cells CpG or anti-␮ for 4 h, and quantitative real-time PCR was performed to de- were obtained by culturing C57BL/6 bone marrow cells in complete me- termine the fold change in the steady-state TACI mRNA compared with 18S dium containing IL-3 (10 ng/ml) and stem cell factor (12.5 ng/ml) for 8 wk mRNA. F, CD23ϩ splenocytes from MyD88 knockout mice or wild-type mice as described (43). Mast cells were further purified by flow cytometric sort- were loaded with CFSE and incubated either with 10 ␮g/ml anti-␮ or 1 ␮M ing using anti-Fc␧RI (eBioscience) and anti-CD117 (BD Pharmingen) Abs. ODN 1826. After 72 h, the cells were harvested, stained with TOPRO-3, and RNA was isolated from purified dendritic cells and mast cells using analyzed by flow cytometry. Results are representative of three independent RNeasy Mini (Qiagen). experiments. Numbers indicate percentage of total cells in each gate. G, Wild- f Ⅺ ␮ Quantitative real-time PCR analysis type ( ) or c-Rel-deficient ( ) cells were incubated in medium or 1 M ODN 1826. Cultures included 60 ng/ml BLyS or 200 ng/ml APRIL as indicated. ϩ p Ͻ 0.05 as determined by Student’s t test. H, CD23ϩ splenocytes from MyD88 ,ء -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 knockout mice and congenic (Ly5.2) mice were mixed 50/50 and incubated for to generate cDNA by reverse transcription. For real-time PCR analysis, we 24 h either with 10 ␮g/ml anti-␮ or 1 ␮M ODN 1826. The congenic marker and used TaqMan Universal Master mix (Applied Biosystems) and Stratagene TACI expression were then determined by FACS. Isotype control (dotted line Max 3000p Detection System or ABI 7300 Real-Time PCR System. Prim- ers and FAM-labeled probes were obtained from Applied Biosystems histogram) is shown for each cell type, and unstimulated cells (gray-shaded his- (TaqMan Assay on Demand). The mRNA fold induction for TACI was togram) and TACI expression (open histogram) on stimulated cells are also rep- estimated relative to 18S ribosomal RNA or GAPDH. resented shown. Results are representative of three independent experiments. 7534 TLR REGULATION OF TACI Downloaded from http://www.jimmunol.org/ 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) CD23ϩ splenocytes were stimulated with either 10 ␮g/ml anti-␮ or 1 ␮M ODN 1826 and either 60 ng/ml BLyS or 200 ng/ml APRIL. After 72 h, the ;p Ͻ 0.05 ,ء .cells were harvested, stained with TOPRO-3, and analyzed by flow cytometry. Results are representative of three independent experiments .p Ͻ 0.0001 as determined by Student’s t test ,ءءء ;p Ͻ 0.001 ,ءء

Results sults show that MyD88-deficient B cells fail to increase BLyS CpG stimulation increases BlyS binding capacity through TACI binding or TACI expression in response to stimulatory CpG DNA up-regulation (Fig. 2, A and B), whereas c-Rel is dispensable for this response by guest on October 2, 2021 (Fig. 2, C and D and data not shown). Consistent with these results, We first examined whether CpG, an unmethylated nucleotide se- MyD88 is required for up-regulation of TACI transcription in re- quence associated with activation through TLR9, alters BLyS re- ϩ sponse to CpG, but c-Rel is not (Fig. 2E). MyD88 is also required ceptor expression. Incubation of CD23 B cells with stimulatory for the proliferative and prosurvival effects of CpG DNA stimu- CpG DNA increased BLyS binding capacity substantially (Fig. 1, F A and B), whereas nonstimulatory sequences had no effect. More- lation (Fig. 2 ). Although the c-Rel-deficient cells are generally over, this increase was greater than that observed with anti-␮ less viable, c-Rel deficiency abrogates neither proliferation nor alone, and combined stimulation with both anti-␮ and CpG yielded survival in response to CpG DNA, even in the presence of BLyS additive effects up to an apparent maximum BLyS binding level (Fig. 2G and data not shown). Thus, CpG-mediated increases in (Fig. 1C). survival and TACI expression occur through a MyD88-dependent To establish which BLyS receptors contributed to these CpG- but c-Rel-independent mechanism. induced increases in BLyS binding capacity, we monitored surface B cells are most likely stimulated by CpG DNA via TLR9, levels of each BLyS receptor by FACS. Resting FO B cells express directly increasing expression of BLyS receptors. Alternatively, both TACI and BR3 on their surface, but not BCMA. Following the increase in BLyS receptors could be indirectly mediated by CpG stimulation, TACI surface expression increased substantially, released from other cells in response to CpG DNA. as evidenced by as much as a 7-fold increase in mean fluorescence MyD88 dependence and the availability of -targeted mice af- intensity (Fig. 1, D and E). BR3 also increased, although to a lesser forded the opportunity to distinguish these possibilities. MyD88 extent. The increases were independent of the cell enlargement knockout and congenic wild-type B cells were cocultured with associated with CpG-mediated mitogenesis because they did not stimulatory CpG DNA for 24 h. The cells were then harvested and correlate with cell size (Fig. 1F). This conclusion was further sub- stained for BLyS receptor and congenic marker expression. Only stantiated by real-time PCR for TACI and BR3 messages normal- wild-type B cells increased TACI expression (Fig. 2H), indicating ized to 18S ribosomal RNA (Fig. 1G). that the BLyS receptor increase following CpG stimulation re- quires TLR9-MyD88 signaling in the B cells themselves, demon- CpG-mediated BLyS receptor regulation is MyD88-dependent strating direct B cell intrinsic mechanisms. and B cell-intrinsic TACI has been reported as a negative regulator for both B cell TLR9 stimulation proceeds through MyD88, which targets NF-␬B survival and proliferation (22, 23, 38), yet it is increased on the for nuclear translocation to activate gene expression (33, 34). surface of CpG-treated B cells. We therefore examined whether Thus, to substantiate MyD88-NF-␬B as the major pathway in- APRIL or BLyS could alter survival or proliferation (Fig. 3, A and volved in CpG-mediated TACI up-regulation, we assessed B cells B). Neither BLyS nor APRIL influenced the extent of proliferation isolated from MyD88 and c-Rel/NF-␬B knockout mice. The re- in any treatment group, indicating that these cytokines lack direct The Journal of Immunology 7535

FIGURE 4. LPS-induced up-regulation of TACI re- quires MyD88. A, Total splenic B cells were stimulated for 24 h with 10 ␮g/ml anti-␮ or 5.0 ␮g/ml LPS. BLyS binding was determined by flow cytometric analysis us- ing biotinylated BLyS and streptavidin-PE. B, Total splenic B cells were isolated from TLR4-deficient (tlr4Ϫ/Ϫ) or wild-type (wt) mice stimulated as in A, and cell surface TACI levels were determined using PE-la- beled rat anti-mouse TACI. Total (C) or CD23ϩ (D)B cells from wild-type (wt) or MyD88 knockout (myD88Ϫ/Ϫ) mice were stimulated with 10 ␮g/ml anti- ␮, 5.0 ␮g/ml LPS, 3.3 ␮g/ml anti-␮, and 5.0 ␮g/ml LPS in combination or with medium alone for 18 h and TACI levels determined as in B. E, CD23ϩ B cells from

MyD88 knockout and wild-type littermate control mice Downloaded from were stimulated for 4 h with LPS (1 ␮g/ml) or anti- mouse-CD40 (10 ␮g/ml) as indicated, and quantitative real-time PCR was performed to determine the fold change in the steady-state levels of TACI mRNA com- p Ͻ 0.001 as determined by ,ءء .pared with 18S mRNA Student’s t test. F, Total B cells from TRIF-deficient (trifϪ/Ϫ) or wild-type (wt) spleens were stimulated with http://www.jimmunol.org/ 10 ␮g/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 his- togram) for experiments shown. by guest on October 2, 2021

mitogenic activity. As expected, BLyS improved survival in all To examine possible autocrine production of BLyS or APRIL, treatment groups, in agreement with previous findings that BLyS we performed quantitative real-time PCR on FACS-sorted FO increases survival of FO and transitional B cells (4, 44, 45). APRIL cells (B220ϩ, AA4.1Ϫ, IgMint, and CD21/35int) incubated with also increased survival in the unstimulated and BCR-stimulated either anti-␮ or CpG DNA (data not shown). Neither stimulation groups, although somewhat less potently than BLyS in unstimu- induced APRIL expression above unstimulated levels, eliminating lated cells (Fig. 3B). In contrast to BLyS, however, APRIL had this mechanism as a possibility for increased survival. Both anti-␮ little survival-promoting effect in the CpG-stimulated cells. and CpG DNA increased BLyS expression 5- to 6-fold over un- To further examine the role of BR3 and TACI in the improving stimulated cells; however, cell survival in these two groups is not survival in response to CpG DNA, we repeated this experiment in equivalent (Fig. 3A), particularly in A/WySnJ cells. Thus, although the BR3 mutant, A/WySnJ, and in TACI-deficient cells (Fig. 3, autocrine BLyS production may contribute to the prosurvival ef- C–F). In the A/WySnJ, both APRIL and BLyS had modest pro- fects of CpG DNA, it cannot solely account for this effect. survival effects on all cells, regardless of concomitant stimulation. Both cytokines can bind TACI, which may explain the slight im- LPS induced TACI expression is MyD88- and c-Rel-dependent provement seen. However, the levels of survival with CpG stim- To investigate whether TACI up-regulation is a general character- ulation were equivalent between the A/WySnJ cells and the A/J istic of TLR-induced B cell activation, we stimulated total B cells controls (data not shown), demonstrating that this effect is not with the TLR4 agonist, LPS. LPS increased BLyS binding and mediated through BR3. TACI expression in B cells (Fig. 4, A and B). This induction was The TACI-deficient cells respond similarly to wild-type controls TLR4-mediated, as TLR4-deficient B cells failed to produce this in all treatments except in anti-␮-stimulated cells incubated response (Fig. 4B). Together with the observation that TLR9 in- with APRIL. In this instance, survival was equivalent to anti- duces TACI expression (Fig. 1), these results suggest a general ␮-stimulated cells that received no . Three conclusions role for TLRs in the regulation of TACI expression. follow from these data: first, survival of unstimulated cells incu- In contrast to TLR9, which interacts with DNA intracellularly bated with APRIL is improved through a TACI-independent and signals exclusively via MyD88, TLR4 is displayed on the cell mechanism; second, APRIL increases survival of cells stimulated surface and uses both MyD88-dependent and -independent signal- through their BCR by signaling through TACI; and third, TACI is ing mechanisms (46). To determine whether LPS-induced up-reg- not necessary for the prosurvival effects of CpG DNA. ulation of TACI involves MyD88 or c-Rel/NF-␬B, the expression 7536 TLR REGULATION OF TACI

nantly 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 in- duces TACI in all B cell populations analyzed, LPS up-regulates TACI predominantly in MZ B cells. Likewise, CD69 is also in- creased most on MZ B cells (Fig. 6E), indicating preferential ac- tivation of this subset. The observation that only a subset of FO B cells responds to LPS was further investigated in a kinetic exper- iment 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. To verify that MZ and FO B cell expression of TLRs, quanti- tative 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 Downloaded from 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). http://www.jimmunol.org/

Discussion These studies examine BLyS receptor expression in B cells un- dergoing TLR-driven activation. The results indicate that both TLR9 and TLR4 ligation lead to increased BLyS binding capacity FIGURE 5. LPS induced TACI up-regulation is c-Rel-dependent. Total through the up-regulation of TACI and, to a lesser extent, BR3. ϩ (A) or CD23 (B) B cells isolated from wild-type (wt) or c-Rel-deficient This finding contrasts with the Ng et al. (48), who showed that Ϫ/Ϫ (crel ) mice were stimulated as indicated for 24 h, followed by quanti- increases in TACI were comparable in BCR and CpG stimulations Ͻ by guest on October 2, 2021 ءء fication of TACI expression levels by FACS. , p 0.001 as determined of B2 cells. We found these shifts to reflect B cell intrinsic, by Student’s t test. C, CD23ϩ B cells purified from c-Rel-deficient Ϫ Ϫ MyD88-dependent mechanisms. Although all FO and MZ B cells (crel / ) or wild-type (wt) mice were stimulated for4hasindicated, and quantitative real-time PCR was performed to determine the fold change in are TLR9 responsive, TLR4 stimulation activates MZ B cells and -p Ͻ a subset of FO B cells. Overall, TLR stimulation shifts BLyS re ,ء .the steady-state levels of TACI mRNA compared with 18S mRNA 0.05 as determined by Student’s t test. ceptor 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 ϩ levels of TACI were compared in CD23 and total MyD88- and the mode of B cell activation dictates relative sensitivity and c-Rel-deficient and wild-type B cells following LPS stimulation. downstream outcomes of APRIL and BLyS signaling. Similar to the results obtained with CpG, LPS up-regulated cell Our results are consistent with studies that show a role for BLyS surface and mRNA expression of TACI in wild-type B cells but and APRIL in T-independent immune responses involving TACI- not in MyD88-deficient B cells (Fig. 4, C–E). Likewise, TRIF- dependent mechanisms. For example, TACI knockout mice dis- deficient B cells also failed to induce TACI in response to LPS, as play defective T cell-independent type II responses (23, 38), and measured by both FACS and mRNA levels (Fig. 4F and data not Castigli et al. (49) established TACI as important in isotype shown), indicating that TLR4 up-regulation of TACI proceeds via switching. Furthermore, studies by He et al. (50) suggest that both MyD88-dependent and -independent mechanisms. This find- BAFF cooperates with CpG DNA in this process. Our observation ing is consistent with Hirotani et al. (47), who identified a cluster that APRIL augments quiescent B cell survival is in accord with of that require both the MyD88- and TRIF-dependent path- this view as well. These viability-promoting effects are most likely ways. Furthermore, in contrast to CpG, LPS-induced expression of delivered through TACI because naive B cells express neither TACI was impaired in B cells deficient for c-Rel (Fig. 5). Together BCMA nor syndecan-1, and APRIL cannot bind BR3. It is unclear these findings suggest that TLR4-dependent up-regulation of why TLR9-stimulated cells remain sensitive to the survival pro- BLyS receptors proceeds via MyD88 and TRIF. In addition, unlike moting effect of BLyS, whereas APRIL had little effect following TLR9, TLR4-induced TACI expression is c-Rel-dependent. CpG stimulation despite elevated TACI levels. Cycling and qui- escent cells may differ in their sensitivities to the two cytokines, TLR4 stimulation differentially increases TACI expression in MZ but we found that once a cell enters cell cycle, TACI levels remain vs FO B cells uniformly high (data not shown). Alternatively, TLR9 and TACI We observed that unlike either CpG or anti-␮ (Fig. 2), LPS-in- signaling may converge on a common and redundant downstream duced TACI expression was not uniform among either CD23ϩ or prosurvival signaling pathway. We favor this explanation for sev- total B cells (Fig. 4, C and D). Analysis of splenic B cell subsets eral reasons. First, MyD88-dependent signaling proceeds at least revealed that LPS-induced TACI expression occurred predomi- in part via NF-␬B1 mediators, and recent data indicate that TACI The Journal of Immunology 7537 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

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 ␮g/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-␮ (10 ␮g/ml), LPS (5 ␮g/ml), a combination of anti-␮ and LPS (3.3 ,ء .and 5 ␮g/ml, respectively), or medium alone and analyzed for steady-state levels of TACI mRNA compared with 18S mRNA by quantitative real-time PCR p Ͻ 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 ;ءء ,p Ͻ 0.05 of CD69 expression by FACS. Identification of each B cell subsets was performed on gated CD19ϩ cells, based on the expression of CD21/IgMhigh CD23Ϫ (MZ), p Ͻ 0.05 as determined by Student’s t test. F, CD23ϩ splenic ,ء .IgMhigh CD21lowCD23Ϫ (T1), IgM/CD21high CD23ϩ (T2), and IgMlow, CD21/CD23ϩ (FoB) cells B cells were labeled with CFSE and stimulated with either 1 ␮M CpG or 10 ␮g/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 B220ϩ AA4Ϫ IgMint CD21/35int (FO) or B220ϩ AA4Ϫ 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, CD23ϩ 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. 7538 TLR REGULATION OF TACI signals primarily through the NF-␬B1 pathway (M. Gupta and Disclosures M. P. Cancro, manuscript submitted). Furthermore, this explana- The authors have no financial conflict of interest. tion would account for continued sensitivity to BLyS among CpG- stimulated cells because BR3 ligation also activates the nonclas- References sical NF-␬B2 pathway (51, 52), thus providing an additive survival 1. Harless Smith, S. M., and M. P. Cancro. 2003. BLyS: the pivotal determinant of effect. Regardless of the exact mechanisms through which TACI peripheral B cell selection and lifespan. Curr. Pharm. Des. 9: 1833–1847. 2. Mackay, F., P. Schneider, P. Rennert, and J. Browning. 2003. 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