Plasmacytoid Dendritic Cells Regulate Autoreactive B

The Journal of Immunology

Plasmacytoid Dendritic Cells Regulate Autoreactive B Cell Activation via Soluble Factors and in a Cell-to-Cell Contact Manner1 Chuanlin Ding,* Yihua Cai,* Jose Marroquin,* Suzanne T. Ildstad,† and Jun Yan2*‡ Plasmacytoid dendritic cells (pDCs) are specialized type I IFN producers, which play an important role in pathogenesis of autoimmune disorders. Dysregulated autoreactive B cell activation is a hallmark in most autoimmune diseases. This study was undertaken to investigate interactions between pDCs and autoreactive B cells. After coculture of autoreactive B cells that recognize self-Ag small nuclear ribonucleoprotein particles with activated pDCs, we found that pDCs significantly enhance autoreactive B cell proliferation, autoantibody production, and survival in response to TLR and BCR stimulation. Neutralization of IFN-␣/␤ and IL-6 abrogated partially pDC-mediated enhancement of autoreactive B cell activation. Transwell studies demonstrated that pDCs could provide activation signals to autoreactive B cells via a cell-to-cell contact manner. The involvement of the ICAM-1-LFA-1 pathway was revealed as contributing to this effect. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, which showed that autoreactive B cell proliferation and activation were significantly decreased in MyD88-deficient mice compared with wild-type mice. These data suggest the dynamic interplay between pDCs and B cells is required for full activation of autoreactive B cells upon TLR or BCR stimulation. The Journal of Immunology, 2009, 183: 7140 –7149.

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lasmacytoid dendritic cells (pDCs)3 are a major source of type I IFNs in the blood, representing 0.2– 0.8% of PBMCs in both humans and mice (1). Previous studies demonstrated that pDCs play a critical role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Although absolute numbers of pDCs are decreased in the circulation of SLE patients, large numbers of activated pDCs are found in skin lesions and actively produce type I IFNs in these patients (2, 3). The pDCs appear to be activated by DNA-containing immune complexes from the patient serum in a TLR9- and CD32-dependent manner (4). U1 small nuclear ribonucleoprotein particle (snRNP) immune complexes found in SLE patients also trigger IL-6 and IFN-␣ production in pDCs via a TLR7-dependent mech-

*Tumor Immunobiology Program, James Graham Brown Cancer Center, †Institute for Cellular Therapeutic, Department of Surgery, and ‡Department of Medicine, University of Louisville, Louisville, KY 40202 Received for publication April 13, 2009. Accepted for publication September 23, 2009. 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 was supported by research funding from American College of Rheumatology and Arthritis Foundation. J.Y. is a recipient of American College of Rheumatology and Arthritis Foundation Investigator Award. This research was supported in part by National Institutes of Health Grants R01 DK52294 and HL63442 and by the University of Louisville Hospital to S.T.I. C.D. is a recipient of Arthritis Foundation Postdoctoral Fellowship Award.

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Address correspondence and reprint requests to Dr. Jun Yan, Tumor Immunobiology Program, James Graham Brown Cancer Center, Delia D. Baxter Research Building, Room 119A, University of Louisville, 580 South Preston Street, Louisville, KY 40202. E-mail address: [email protected]

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Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; SLE, systemic lupus erythematosus; snRNP, small nuclear ribonucleoprotein particle; mDC, myeloid dendritic cell; BLyS, B lymphocyte stimulating protein; BAFF, B cell-activating factor; APRIL, a proliferation-inducing ligand; Tg, transgenic; TACI, transmembrane activator and calcium modulator and cyclophilin ligand interactor; WT, wild type; FDC, follicular dendritic cell. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901175

anism (5). Even the U1 snRNP itself can directly stimulate pDCs for IFN-␣ production (6). The elevated IFN-␣ levels in serum and induction of IFN-regulated genes in PBMC in lupus patients have been shown to correlate with disease activity and severity (7–9) (10, 11). Additionally, mouse models of lupus in which the IFN receptor (IFNR) is deleted fail to develop disease manifestations (12), suggesting the crucial role of IFN-␣ in lupus pathogenesis. pDCs also regulate B cells to differentiate into plasma cells for Ab production via type I IFNs and IL-6 (13, 14). Type I IFNs stimulate B cells to generate non-Ig-secreting plasma blasts while IL-6 promotes their further differentiation into Ab-secreting plasma cells. Type I IFNs can also directly stimulate B cells for Ab production via IFNR (15), enhance Ab secretion in vivo (16), and trigger isotype switching by inducing myeloid DCs (mDCs) to up-regulate the expression of B lymphocyte stimulator (BLyS) or B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two potent B cell-activating molecules (17). Additionally, type I IFNs control B cell TLR sensitivity such as TLR7 to trigger B cell expansion and differentiation toward Ab-secreting plasma cells (18). A recent study also indicates that pDCs synergistically up-regulate costimulatory molecule CD86 expression, cytokine secretion, and plasma cell differentiation of human B cells stimulated with TLR ligand CpG-C or BCR crosslinking (14). These studies have underscored a pivotal role of pDCs in B cell activation and differentiation, mainly via soluble mediators. However, little is known about the role of pDCs in the regulation of autoreactive B cells. Dysregulated autoreactive B cells are one of the hallmarks in many autoimmune diseases. In this study, we hypothesized that pDCs regulate autoreactive B cell activation for pathogenic autoantibody production. The antisnRNP Ig H chain transgenic (Tg) mouse model was used to explore the possible interplay between pDCs and autoreactive antisnRNP B cells. Previous studies have demonstrated that antisnRNP B cells constitute the normal B cell repertoire and that the frequency of these B cells ranges from 15 to 35% in normal background mice (19). The anti-snRNP B cells of normal background

The Journal of Immunology mice do not produce autoantibody but can be activated by stimulation such as through TLR engagement or T cell help (19, 20). Here, we demonstrate that activated pDCs trigger anti-snRNP B cells for enhanced proliferation, autoantibody production, and survival in response to TLR stimulation or BCR ligation. These effects are not only mediated by cytokines released by pDCs but are also dependent on cell-to-cell contact. Thus, pDCs provide helper signals to trigger autoreactive B cell activation that involves autoimmune pathogenesis.

Materials and Methods Mice B10.A or C57BL/6 anti-snRNP (2-12H) Tg mice were generated as previously described (19). CD40L-deficient (CD40L⫺/⫺) mice were purchased from The Jackson Laboratory. MyD88⫺/⫺ mice were obtained from Osaka University (Osaka, Japan). All mice were housed and bred in a conventional facility at the University of Louisville (Louisville, KY). Animal care and experiments were conducted in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at the University of Louisville.

Bone marrow-derived pDCs Bone marrow cells from naive B10.A mice were isolated by flushing femurs and tibiae with DMEM medium supplemented with 10% heat-inactivated FBS. The cells were then passed through a 70-␮m cell strainer. After lysis of RBCs, the cells were centrifuged and resuspended at 106 cells/ml in DMEM medium in the presence of Flt3-ligand (200 ng/ml, provided by Amgen) as described previously (21, 22). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. Every 4 days of culture, half of the medium was removed and fresh cytokine-supplemented culture medium was added back into cultures. After 7– 8 days of culture, cells were collected and labeled with anti-mouse B220 microbeads (Miltenyi Biotec) and separated using autoMACS. The purity of pDCs (CD11cint, B220⫹, mPDCA-1⫹) was ⬎90% as assessed by flow cytometry. To fix pDCs, freshly isolated or CpG (1826; Qiagen) or gardiquimod(InvivoGen) stimulated pDCs were mixed with paraformaldehyde (1% in PBS) for 4 min at 4°C and then extensively washed.

Autoreactive B cell purification and coculture of B cells and pDCs B cells were purified from 2-12H Tg mice using negative selection by magnetic bead technology. In brief, lymphocytes from splenocytes were enriched by Ficoll-Hypaque density separation. Cells were labeled with anti-mouse CD43 microbeads (Miltenyi Biotec) and separated using autoMACS. The purity of B cells was ⬎95% as assessed by flow cytometry. Coculture of B cells and pDCs were performed in RPMI 1640 medium (Caisson Laboratories), supplemented with 10% inactivated FBS, 10⫺3 M sodium pyruvate, 2 ⫻ 10⫺3 M glutamine, and 50 ␮M 2-ME. B cells (1 ⫻ 105/well, 96-well plate) were stimulated with varying amounts of CpG, gardiquimod, or anti-mouse IgM F(ab⬘)2 (Jackson ImmunoResearch Laboratories) in the presence or absence of pDCs. B cell proliferation was determined by [3H]TdR incorporation after 48 h of coculture. Cells were incubated for the last 6 h with 1 ␮Ci of [3H]TdR. Sample wells were harvested onto filters and incorporated radioactivity was counted in a BetaPlate liquid scintillation counter (Packard Bioscience). Experiments were conducted in triplicate and results were expressed as cpm ⫾ SEM. In some experiments, recombinant mouse IFN-␣ (PBL Biomedical Laboratories) or recombinant IL-6, recombinant mouse TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor)/Fc chimera fusion protein, neutralizing anti-mouse IFN-␣/␤ R2 Ab, anti-mouse IL-6 Ab (R&D Systems), anti-mouse ICAM-1 mAb (clone YNI.7.4; BioExpress), anti-mouse LFA-1 Ab (clone M17/4; eBioscience), and corresponding isotype Abs were added during the culture.

7141 plates in a total volume of 0.7 ml. B cells (5 ⫻ 105/well) were cultured in the lower compartment and pDCs (1.25 ⫻ 105) were cultured in the upper compartment of the Transwells. B cell proliferation was assayed by transferring cells in lower chambers into 96-well plates after 2-day culture and pulsing them with [3H]TdR for another 6 h of culture.

Total IgM and anti-snRNP Ab detection Total IgM levels were determined using mouse an IgM ELISA kit (Bethyl Laboratories). Detection of anti-snRNP Ab by ELISA was performed as previously described (23). Briefly, culture supernatant (5 days culture) or serial-diluted mouse sera were added to plates coated with recombinant snRNP. HRP-conjugated goat anti-mouse IgM (SouthernBiotech) was used as a secondary Ab. Assays were developed with tetramethylbenzidine conductivity substrate (BioFX Laboratories) and the OD value was measured at 450 nm. In some experiments, ABTS-1 component substrate was used and the OD at 405 nm was measured.

In vivo anti-ICAM-1 mAb blocking assay and B cell proliferation assay For in vivo anti-ICAM-1 mAb blocking assay, anti-snRNP Tg mice were received blocking anti-ICAM-1 mAb 500 ␮g by i.p. injection at day ⫺3 and 250 ␮g at days ⫺1, 1, and 3, respectively. Isotype mAb was used as control. All mice were immunized i.v. with CpG (100 ␮g per mouse) on days 1 and 4. The sera were collected on day 7 for anti-snRNP Ab measurement. For in vivo B cell proliferation assay, B cells purified from splenocytes of C57BL/6 2-12H Tg mice were labeled with 5 ␮M CFSE (Molecular Probes) for 10 min at 37°C. B cells (1 ⫻ 107 per mouse) were then adoptively transferred into C57BL/6 mice or MyD88⫺/⫺ mice. Recipient mice were immunized i.v. with or without CpG (100 ␮g per mouse) on days 1 and 4. The sera were collected on day 7 for anti-snRNP Ab measurement. Splenic cells were collected and stained with mAbs for flow cytometry analysis.

Flow cytometry Cells were blocked in the presence of anti-CD16/CD32 at 4°C for 15 min before staining. All mAbs were purchased from eBioscience or BD Pharmingen. In addition to isotype controls, the following mAbs were used: anti-CD11c-FITC, anti-CD27-FITC, anti-mPDCA-1-PE, anti-CD86-PE, anti-CD40-PE, anti-CD40L-FITC, anti-CD80-PE, anti-CD83-PE, antiCD70-PE, anti-CD138-PE, anti-ICAM-PE, anti-CD19-allophycocyanin, and anti-CD45R/B220-allophycocyanin. Cells were incubated with conjugated mAbs at 4°C for 30 min and washed twice with PBS/2% FCS. Cells were analyzed by flow cytometry with a FACSCalibur cytometer and data were analyzed with FlowJo (Tree Star).

Immunofluorescence staining Fresh spleen tissues were embedded in OCT medium (Tissue-Tek OCT compound 4853; Electron Microscopy Science) by using a dry ice-cooled isopentane (Sigma-Aldrich) bath. Cryosections (7 ␮m) were fixed in icecold acetone for 15 min and air-dried at room temperature. Sections were blocked with 20% FBS in PBS for 30 min and then were stained with the following primary Abs at 4°C overnight: allophycocyanin rat-anti-mouse CD4 (1/50 dilution, clone RM4-5; BD Biosciences), biotinylated rat-antimouse PDCA-1 (1/10 dilution, clone JF05-1C2.4.1; Miltenyi Biotec). After three washes with PBS, sections were incubated with steptavidin-Alexa 594 (1/150 dilution; Molecular Probes/Invitrogen) for 45 min. Slides were mounted with the anti-fade fluorescence-mounting medium (Dako). Images were acquired by Leica TCS SP5 confocal microscope system with HC PL APO ⫻20/0.7 CS (air) objective (Leica Microsystems).

Statistical analysis Data are expressed as mean values ⫾ SEM. Statistical significance of difference was determined by the unpaired two-tailed Student’s t test. Differences were considered significant for p ⬍ 0.05. Statistical analysis was performed using Prism 4.0 (GraphPad Software).

B cell survival assay Purified B cells were stimulated with or without CpG or gardiquimod in the presence or absence of pDCs for the indicated time and stained with an Annexin V PE apoptosis detection kit (BD Biosciences). Cells were harvested and analyzed by flow cytometry (FACSCalibur; BD Biosciences).

Transwell study B cells and pDCs were cultured in the separated compartments using a Transwell system (0.4 ␮m; Costar). Cultures were performed in 24-well

Results pDCs enhance anti-snRNP B cell proliferation upon TLR stimulation or BCR engagement DCs derived from murine bone marrow cells by coculture with Flt3-ligand contain the CD11cintB220⫹mPDCA-1⫹ pDCs and conventional CD11chighB220⫺ DCs. The pDCs secreted large amounts of TNF-␣, IL-6, and IFN-␣ upon CpG stimulation (data

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FIGURE 1. pDCs enhance autoreactive B cell proliferation in response to TLR stimulation and BCR ligation. Purified anti-snRNP Tg B cells (1 ⫻ 105/well) were cultured for 48 h with the indicated concentrations of CpG (A) or gardiquimod (B) with or without pDCs (0.25 ⫻ 105/well) at a ratio of 4:1. pDCs alone with stimuli were used as controls. All cultures were then pulsed with [3H]TdR for the last 6 h. Cpm counts from pDCs alone were subtracted from those of pDC-B cell cocultures. C, B cell proliferation stimulated with varying concentrations of anti-IgM for 48 h in the presence of fixed gardiquimod or CpG-activated pDCs. Freshly isolated pDCs were used as control. Each bar represents the mean and error bars indicate SEM. Data are representative of five independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

not shown). To examine whether pDCs induce autoreactive B cell activation, purified anti-snRNP B cells were cocultured with pDCs in the presence of different stimuli. Autoreactive anti-snRNP Tg B cell proliferation in response to CpG stimulation was significantly enhanced in the presence of pDCs (Fig. 1A). This enhancement is proportional to the numbers of pDCs added in the culture (data not shown). Similarly, autoreactive B cell proliferation in response to TLR7 ligand gardiquimod was also significantly increased in the presence of pDCs (Fig. 1B). To explore whether BCR crosslinking-induced B cell proliferation is influenced by pDCs, anti-snRNP B cells were stimulated with anti-IgM Ab in the presence or absence of pDCs. pDCs were preactivated with TLR7 or TLR9 ligand and then fixed to eliminate any cytokine release that could impact B cell proliferation. Autoreactive B cells had a low proliferative response upon BCR ligation. However, activated pDCs significantly augment BCR-induced B cell proliferation (Fig. 1C). These results suggest that the presence of pDCs can significantly enhance anti-snRNP autoreactive B cell proliferation in response to both TLR stimulation and BCR ligation.

REGULATION OF AUTOREACTIVE B CELLS VIA pDCs

FIGURE 2. CD138 plasma cell differentiation and total IgM and antisnRNP autoAb production. A, CD138 expression on autoreactive B cells upon TLR stimulation in the presence of pDCs (n ⫽ 3). Purified antisnRNP Tg B cells were stimulated with CpG (0.1 ␮g/ml) or gardiquimod (0.5 ␮g/ml) for 72 h with or without pDCs (4:1 ratio). Cells were harvested and stained with CD19 and CD138 mAbs and the percentage of CD138⫹ cells was determined. Cells were gated on CD19⫹ B cell population. B and C, Anti-snRNP Tg B cells (5 ⫻ 105/well) were stimulated with indicated concentration of CpG or gardiquimod for 5 days with or without pDCs. The culture supernatants were assayed for total IgM (B) or anti-snRNP autoantibody production (C) by ELISA. Data are representative of three independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

pDCs induce autoreactive anti-snRNP B cells to differentiate into plasma cells and secrete anti-snRNP autoantibody Given the role of pDCs in inducing normal B cell differentiation, we examined whether pDCs would enhance autoreactive B cell differentiation. CD138 (syndecan-1) is a plasma cell marker expressed at various levels on different developmental stages of plasma cells (24). In the presence of pDCs, the CD138⫹ plasma cells were significantly increased upon CpG or gardiquimod stimulation (Fig. 2A). The total IgM levels were also augmented when B cells were cocultured with pDCs (Fig. 2B). Additionally, antisnRNP autoantibody production was enhanced by the addition of pDCs (Fig. 2C). These data indicate that pDCs indeed provide help signals for autoreactive B cell differentiation to plasma cells and subsequently for autoantibody production. pDCs promote autoreactive B cell survival in response to TLR stimulation It has been shown that macrophage or DC-mediated human B cell regulation depends on the TNF family ligand BLyS/BAFF and

The Journal of Immunology APRIL (17, 25). Engagement of BLyS (BAFF) and/or APRIL with corresponding receptors expressed on B cells leads to enhanced B cell survival via up-regulation of the antiapoptotic molecules NF-␬B and Bcl-2 (26). To determine whether autoreactive B cell survival could be enhanced by activated pDCs, anti-snRNP B cells were cocultured with activated pDCs. As shown in Fig. 3, A and B, autoreactive B cell survival was significantly enhanced from day 2 when activated pDCs were present. Further studies demonstrated that TACI/Fc fusion protein significantly decreased activated pDC-mediated enhancement of B cell survival (Fig. 3C), suggesting that BLyS and its receptor play a critical role in this effect. Enhanced autoreactive B cell proliferation and autoantibody production in the presence of pDCs are partially mediated by soluble cytokines Type I IFN and IL-6 have been reported to induce B cell differentiation and enhance Ab production. To determine the possible mediators secreted by pDCs to induce enhanced autoreactive B cell proliferation and autoantibody production, we measured autoreactive B cell activation upon TLR stimulation in the presence of rIFN-␣. Indeed, rIFN-␣ significantly increased CpG-induced autoreactive B cell proliferation and autoantibody production (Fig. 4A). However, rIL-6 did not significantly increase CpG-induced autoreactive B cell proliferation (data not shown). This could be due to the fact that CpG stimulates B cells to secrete IL-6. Thus, the addition of exogenous IL-6 cannot show the additive effect on the enhancement of CpG-induced B cell proliferation. Additionally, we used anti-IL-6 mAb or anti-type I IFN␣/␤ receptor mAb to block the IL-6 or IFN-␣/␤ activity. As shown in Fig. 4B, both neutralizing mAbs significantly, but only partially, decreased CpG-stimulated pDC supernatant-induced B cell proliferation and autoantibody production. Similar results were observed when autoreactive B cells were stimulated with TLR7 ligand (data not shown). These data suggest that cytokines IL-6 and INF-␣/␤ partially contribute to pDC-mediated enhancement of autoreactive B cell activation. Cell-to-cell contact is also required for pDC-mediated autoreactive B cell activation In addition to the soluble cytokines secreted by pDCs, it is unclear whether cell-to-cell contact is also required for pDCs to mediate these effects. To this end, pDCs and autoreactive B cells were cocultured in a transwell system. As shown in Fig. 5A, separation of pDCs and autoreactive B cells significantly reduced the costimulatory capacity of pDCs to increase autoreactive B cell proliferation. Similarly, autoAb production was also significantly decreased. Despite reduced autoreactive B cell proliferation and autoantibody production in transwell assays, B cells separated from pDCs by the transwell system still proliferated more compared with B cells stimulated with TLR ligand alone in the absence of pDCs, suggesting a contribution of soluble mediators. To further confirm that cell-to-cell contact is required for pDCmediated effect, pDCs were preactivated with TLR7 or TLR9 ligand and then fixed with paraformaldehyde to eliminate their capability for cytokine secretion (data not shown). Activated and fixed pDCs were then cocultured with autoreactive B cells. These pDCs significantly increased autoreactive B cell proliferation (Fig. 5B) and autoantibody production (data not shown) in response to TLR7 or TLR9 stimulation. This is also the case for BCR ligationinduced B cell proliferation (Fig. 1C). The enhancement is only mediated by activated pDCs, as freshly isolated pDCs did not have a similar costimulatory effect (Fig. 5C). These data suggest that activated pDCs provide costimulatory signals to autoreactive B cells via cell-to-cell contact in addition to soluble factors.

7143 The ICAM-1-LFA-1 pathway is involved in the pDC-autoreactive B cell interaction Because cell-to-cell contact is required for the enhanced activation of anti-snRNP B cells mediated by pDCs, we determined the membrane molecules involved in this interaction. Many surface molecules such as CD40, CD80, CD86, CD70, and CD83 were upregulated in pDCs upon TLR ligand stimulation. CD40L was also expressed on TLR ligand-stimulated pDCs (data not shown). The CD40-CD40L pathway is crucial for B cell activation including Ab production, isotype switching, and germinal center formation (27). Signaling through CD70-CD27 can also regulate B cell activation and Ig production (28, 29). The rationale to determine the membrane molecules involved in this process is that either ligand or receptor is expressed on pDCs or B cells. Therefore, the CD40 (B cells)-CD40L (pDCs) and CD27 (B cells)-CD70 (pDCs) pathways were further investigated. However, CD40L-deficient pDCs had a similar costimulatory capacity as did wild-type (WT) pDCs to trigger anti-snRNP Tg B cell proliferation and autoantibody production (Fig. 6A and data not shown). Similarly, the CD27CD70 pathway did not have a significant role in pDC-mediated enhancement of B cell activation, as neutralizing Abs did not significantly alter the costimulatory effect of pDCs (data not shown). LFA-1 belongs to the integrin family of cell adhesion molecules and is expressed on leukocytes, including B cells (30). Its main ligand, ICAM-1 (CD54), is expressed widely on DCs, leukocytes, and endothelial cells. LFA-1/ICAM-1 has been implicated in the physical interaction between B cells and conventional DCs and B cells and follicular DCs (FDCs) (31, 32). The interaction of ICAM-1-LFA-1 reduces the B cell activation threshold via facilitating B cell adhesion and synapse formation (33). ICAM-1 expression levels on pDCs were significantly increased upon TLR ligand stimulation (Fig. 6B). To determine whether the ICAM-1LFA-1 pathway involves pDC-B cell interaction, neutralizing antiLFA or anti-ICAM-1 mAbs were used. As shown in Fig. 6C, antiICAM-1- or anti-LFA-1-neutralizing mAbs, but not isotype control mAbs, significantly abrogated activated, fixed pDC-mediated autoreactive B cell proliferation in response to both TLR and BCR stimulation. Similarly, autoantibody production was also significantly reduced (data not shown). To further examine whether the ICAM-1-LFA-1 pathway plays a critical role in autoreactive B cell activation in vivo, we treated anti-snRNP Tg mice with anti-ICAM-1 blocking mAb and then immunized mice with CpG. As shown in Fig. 6D, anti-snRNP autoantibody production was significantly lower in anti-ICAM-1 mAb-treated mice compared with that in isotype mAb-treated mice. Decreased B cell proliferation, costimulatory activation, and autoantibody production in vivo in the absence of the help of pDCs To explore whether pDCs also provide helper signals for autoreactive B cell in vivo activation, anti-snRNP B cells were purified, fluorescent labeled, and then adoptively transferred into WT or MyD88⫺/⫺ mice. Recipient mice were then immunized with or without TLR7/9 ligand and B cell proliferation and activation were examined. In this system, TLR7/9 ligand activates both antisnRNP B cells and pDCs in WT recipient mice. However, only anti-snRNP B cells are responsive to TLR stimulation in MyD88deficient recipient mice. As shown in Fig. 7A, anti-snRNP B cells proliferated vigorously in WT recipient mice immunized with TLR9 ligand. In contrast, autoreactive B cells did not proliferate as much in MyD88⫺/⫺ recipient mice. As controls, anti-snRNP B cells had minimal proliferation in WT or MyD88⫺/⫺ mice without

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FIGURE 3. Enhanced B cell survival in the presence of pDCs. Purified anti-snRNP Tg B cells were cultured in the presence or absence of CpG (0.1 ␮g/ml) or gardiquimod (0.5 ␮g/ml) with or without pDCs. B cell survival was evaluated by annexin V and 7-aminoactinomycin D staining. Representative dot plots (A) and the percentage of live cells are shown (n ⫽ 4; B). C, Anti-snRNP B cells were incubated with TACI/Fc fusion protein or control protein (100 ng/ml) for 20 min and then stimulated with CpG-activated pDC supernatants for 3 days. B cell survival was assessed by annexin V and 7-aminoactinomycin D staining (n ⫽ 3). ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.


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FIGURE 4. Type I IFNs and IL-6 partially contribute to the enhancement of autoreactive B cell activation via pDCs. A, Purified anti-snRNP Tg B cells (1 ⫻ 105/well) were stimulated by CpG with or without recombinant mouse IFN-␣ (500 U/ml). B cell proliferation and anti-snRNP autoantibody production were determined. B, pDCs were stimulated with CpG (0.3 ␮g/ml) for 24 h. The culture supernatants were harvested and mixed with purified anti-snRNP Tg B cells (1 ⫻ 105/well) for 48 h (proliferation assay) or 120 h (autoantibody assay) in the presence of anti-IFN-␣/␤ R2 mAb (1 ␮g/ml) and/or anti-IL-6 mAb (1 ␮g/ml) or isotype control mAbs. Data are representative of three independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

TLR9 ligand immunization. Additionally, costimulatory molecule CD86 expression levels on autoreactive B cells were also significantly lower in MyD88⫺/⫺ mice compared with those in WT mice (Fig. 7B). Similarly, autoreactive B cells in MyD88⫺/⫺ recipient mice produced significantly fewer autoantibodies with respect to those in WT mice (Fig. 7C). These data suggest that activation of autoreactive B cells in vivo requires helper signals from pDCs. To determine in vivo interaction between pDCs and autoreactive B cells, autoreactive B cells were CFSE labeled and then adoptively transferred into WT or MyD88⫺/⫺ mice. Mice were immunized with or without TLR9 ligand and spleen sections from different groups were stained with pDCs and CD4 mAbs. In the spleens of PBS immunized WT or MyD88⫺/⫺ mice, pDCs were localized in T zone and marginal zone with lower numbers (Fig. 7D). Autoreactive B cells resided in the follicle. MyD88⫺/⫺ mice with CpG immunization displayed similar pattern as did PBS immunized mice. However, pDCs were significantly increased and localized in both T zone and marginal zone in WT mice immunized with CpG. Autoreactive B cells were predominantly located in the T-B cell interface and marginal zone with pDCs, suggesting pDC-autoreactive B cell interaction.

Discussion In this study, we found that activated pDCs interact with autoreactive B cells for augmented proliferation and autoantibody production in response to TLR and BCR stimulation. TLR7/9 ligands can directly activate autoreactive B cells for proliferation and autoantibody production despite their anergic phenotype. However, their full activation appears to be dependent on activated pDCs. Activated pDCs not only provide soluble mediators including type I IFN, IL-6, and BLyS/BAFF to stimulate autoreactive B cell differentiation to Ab-secreting plasma cells and promote B cell sur-

FIGURE 5. Cell-to-cell contact is required for pDC-mediated enhanced B cell proliferation. A, Purified anti-snRNP Tg B cells (5 ⫻ 105/well) and pDCs (1.25 ⫻ 105/well) were cocultured in separated compartments using a transwell system in the presence of CpG (0.1 ␮g/ml) or gardiquimod (0.5 ␮g/ml) for 48 h. The data suggest that enhanced B cell proliferation in the presence of pDCs is significantly reduced in transwell assays. B, Purified anti-snRNP Tg B cells (1 ⫻ 105/well) were cultured in the presence of CpG (0.1 ␮g/ml) or gardiquimod (0.1 ␮g/ml) with or without fixed, preactivated pDCs (1.25 ⫻ 105/well). C, Freshly isolated or CpG-activated pDCs were fixed and cocultured with purified anti-snRNP Tg B cells (1 ⫻ 105/well) in the presence of CpG (0.1 ␮g/ml). The percentage of enhancement was calculated using the formula: cpm(B cells ⫹ pDCs)/cpm(B cell alone) ⫻ 100% (n ⫽ 3). ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

vival but also to directly interact with autoreactive B cells via the ICAM-1-LFA-1 costimulatory pathway. There is accumulating evidence to suggest that autoreactive B cells can be activated to secrete pathogenic autoantibodies via a T cell-independent pathway. For example, activation of anti-snRNP B cells and AM14 rheumatoid factor B cells via TLR ligands does not absolutely require T cells but depends on the TLR pathway (34, 35). However, it is unknown whether autoreactive B cell activation in this manner requires interaction with other cell types. Previous studies have shown that anti-chromatin-CpG complex or

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FIGURE 6. The LFA-1-ICAM-1 pathway, but not the CD40-CD40L pathway, contributes to the pDC-autoreactive B cell interaction. A, CpG-activated pDCs from WT or CD40L⫺/⫺ mice were fixed and then cocultured with purified anti-snRNP Tg B cells (1 ⫻ 105/well) in the presence of CpG (0.1 ␮g/ml). B, ICAM-1 expression levels on pDCs stimulated with CpG (0.1 ␮g/ml) or gardiquimod (0.1 ␮g/ml) for 48 h were analyzed by flow cytometry and the mean fluorescence intensity (MFI) was shown. C, Purified B cells (1 ⫻ 105/well) were cultured with fixed, preactivated pDCs (0.25 ⫻ 105/well) in the presence of CpG (0.1 ␮g/ml), gardiquimod (0.1 ␮g/ml), or anti-IgM (10 ␮g/ml) with or without blocking anti-ICAM-1 mAb (1 ␮g/ml) or anti-LFA-1 mAb (10 ␮g/ml) and corresponding isotype control mAbs. D, Anti-snRNP Tg mice (n ⫽ 6 or 7) were injected with anti-ICAM-1 blocking mAb or isotype mAb at days ⫺3, ⫺1, 1, and 3. Mice were immunized with CpG at days 1 and 4. Sera were collected at day 7 to detect anti-snRNP Ab level (1/100 dilution). Data are representative of two or more independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

U1 snRNP immune complex can activate pDCs to produce type I IFN (4, 5). These immune complexes also activate autoreactive B cells simultaneously. Thus, activated pDCs could provide further costimulatory activity to autoreactive B cells, leading to augmented pathogenic autoantibody secretion. It may also stimulate autoreactive B cells to be potent APCs for autoreactive T cell activation. Interestingly, blockade of TLR7 and TLR9 with immunoregulatory sequence 954 in lupus-prone mice resulted in significant reduction of autoantibody production and amelioration of disease progression (36). Note that enhanced autoreactive B cell activation mediated by activated pDCs occurs when autoreactive B cells were stimulated with lower concentration of TLR or BCR stimulation. The pDC-mediated enhancement effect was less significant when autoreactive B cells were stimulated with higher concentrations of TLR ligands or BCR ligation (data not shown). This phenomenon may reflect the physiological situation in which trace amounts of endogenous TLR ligands induce autoreactive B cell activation to generate canonical autoantibodies through a pDC-mediated activation amplification loop. Indeed, SLE patients

have elevated levels of circulating plasma DNA that is enriched in hypomethylated CpGs (37). Genomic DNA is also hypomethylated in SLE patients. These danger triggers could serve as initial stimuli to induce autoreactive B cell activation through interaction with pDCs in a T cell-independent manner. This effect could be also possibly extended to other B cells with different Ag specificity. Indeed, anti-nitrophenyl B cell activation was also augmented in the presence of activated pDCs (data not shown). Another possible mediator is pDC-secreted exosome. Previous studies have shown that activated DCs secrete bona fide exosomes (38). These exosomes express MHC class II and costimulatory molecules such as ICAM-1 and play a critical role in T cell activation (38, 39). To address whether pDC exosomes have a stimulatory effect on autoreactive B cell activation, supernatants from CpG or gardiquimod-activated pDCs were harvested and ultracentrifuged to deprive exosomes. Autoreactive B cell proliferation was comparable between noncentrifuged and centrifuged gardiquimod-activated pDC supernatants (data not shown). In contrast, the stimulatory effect of CpG-activated pDC supernatants on


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FIGURE 7. Autoreactive B cell proliferation and activation in vivo. Purified autoreactive B cells were labeled with CFSE and adoptively transferred into C57BL/6 mice or MyD88⫺/⫺ mice. Recipient mice were immunized i.v. with CpG at 100 ␮g/injection on days 1 and 4 and sacrificed at day 7. B cell division and CD86 expression were examined by flow cytometry. Anti-snRNP IgM response was measured by ELISA. A, Proliferation of transferred B cells and the percentage of proliferated B cells gated on CFSE⫹ population. B, CD86 expression on autoreactive B cells. Mean fluorescence intensity (MFI) of CD86 expression on the CFSE⫹ cells is shown. C, Anti-snRNP IgM response in recipient mice of a 1/100 dilution of serum. The OD value of the preimmune sera subtracted from the postimmune sera is shown. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01. D, Location of pDCs and autoreactive B cells. Anti-snRNP Tg B cells were purified and labeled with CFSE (green) and then adoptively transferred into WT or MyD88⫺/⫺ mice. Recipient mice were then injected i.v. with 100 ␮g CpG or PBS for 24 h. Sections of spleens were stained with anti-CD4 (T cells; purple) and anti-mPDCA-1 (pDCs; red) mAbs. Bar, 100 ␮m. E, Schematic model of pDC-autoreactive B cell interaction.

B cells was significantly decreased after ultracentrifugation (data not shown). However, we found that the ultracentrifugation also removed the remaining CpG in supernatants since control medium with CpG lost its stimulatory effect after ultracentrifugation. Thus, it is unlikely that exosomes secreted by activated pDCs contribute to pDC-mediated enhancement of autoreactive B cell activation. This notion is supported by a recent study showing that activated DC exosomes did not induce B cell proliferation (40). We found that pDCs regulate autoreactive B cell activation via soluble mediators as well as through cell-to-cell contact. It is not

surprising that soluble cytokines including type I IFN and IL-6 are involved in autoreactive B cell differentiation into autoantibodysecreting plasma cells as reported previously in human B cell studies (13, 14). The intriguing finding is that cell-to-cell contact between pDCs and autoreactive B cells is also necessary for augmented autoreactive B cell activation. This notion is supported by both transwell assays and by using fixed, preactivated pDCs to eliminate cytokine secretion capability. Our initial attempts to discover possible costimulatory molecule pathways involved in this interaction were focused mainly on the CD40-CD40L and CD27-CD70

7148 pathways, given their importance in regulating B cell activation (27, 28). To our surprise, neither of these pathways showed significant roles in the pDC-autoreactive B cell interaction. The ICAM-1-LFA-1 pathway has been shown to be critical in regulating B cell activation threshold (33). Therefore, we investigated this pathway and found that blockade of the ICAM-1-LFA-1 interaction significantly attenuates pDC-induced enhancement of autoreactive B cell activation in vitro. Moreover, in vivo blockade of ICAM-1-LFA-1 interaction using blocking mAb significantly decreased autoantibody production. CpG-DNA derived from lupus serum has been shown to induce ICAM-1 up-regulation (41). Notably, ICAM-1 is also expressed on B cells, and its expression levels were significantly increased upon CpG or gardiquimod stimulation. However, CpG-, gardiquimod-, or anti-IgM-induced B cell proliferation was not inhibited by blocking anti-ICAM-1 mAb (data not shown), suggesting that decreased B cell proliferation is not due to inhibition of B cell itself. Although ICAM-1 is constitutively expressed on pDCs, freshly isolated pDCs failed to induce enhanced B cell activation. This may be related to the conformational change of LFA-1-ICAM-1 after TLR7/9 ligand stimulation. LFA-1 (␣L␤2) belongs to the ␤2-integrin family and mediates cellto-cell and cell-to-extracellular matrix adhesions essential for immune and inflammatory responses (42). One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding ␣L-I domain from low affinity to high affinity (43). The inserted (I) domain of LFA-1 contains the ligand-binding epitope, and a conformational change in this region during activation increases ligand affinity (44). Thus, up-regulated ICAM-1 on pDCs may bind to the high affinity of LFA-1 to induce “outside-in” signals to autoreactive B cells. Interestingly, loss of LFA-1 or ICAM-1 significantly reduces anti-dsDNA autoantibody production and decreases glomerulonephritis (45, 46), suggesting critical roles of the ICAM-1-LFA-1 pathway for autoreactive B cell regulation. We further demonstrated that autoreactive B cell activation in vivo also requires pDC help. It is clear that autoreactive B cell proliferation, CD86 costimulatory molecule expression levels, and autoantibody production were significantly reduced in MyD88-deficient recipient mice as compared with those in WT recipient mice. Autoreactive anti-snRNP B cells are of an immature phenotype and have decreased CD86 expression to induce autoreactive T cell tolerance (20, 47). Enhanced CD86 expression may suggest possible autoreactive T cell activation. In mice, pDCs and conventional mDCs both express TLR7 and TLR9 (48). We initially used a pDC-depleting mAb to address this issue. However, pDCs were not effectively depleted in our systems. Therefore, the reduced autoreactive B cell activation in vivo could be contributed by both DC types. Indeed, mDCs have been shown to stimulate B cell proliferation and differentiation through the secretion of soluble mediators including IL-6 and IL-12 as well as membrane molecules such as BAFF/APRIL (49, 50). Moreover, activated DCs from lupus-prone mice directly increase B cell effector functions (40). A recent study demonstrated that pDCs and mDCs may differentially regulate B cell responses (51). pDCs, but not mDCs, predominantly enhance B cell activation upon TLR7/8 ligand stimulation and, to a lesser extent, TLR9 ligand stimulation. Nevertheless, in vivo trafficking studies show that pDCs are significantly expanded upon TLR stimulation and trafficked into both T zone and marginal zone to interact with autoreactive B cells. These data suggest that interactions between pDCs and autoreactive B cells take place in vivo to induce augmented B cell activation. On the basis of our results, we postulate a schematic model to explain how pDCs interact with autoreactive B cells for pathogenic autoantibody production (Fig. 7E). Endogenous plasma DNA/ RNA or DNA/RNA-containing immune complexes activate pDCs

REGULATION OF AUTOREACTIVE B CELLS VIA pDCs to secrete type I IFNs, IL-6, and BLyS/BAFF. Activated pDCs up-regulate ICAM-1 expression and engage LFA-1 on autoreactive B cells. Simultaneously, autoreactive B cells are activated by plasma DNA/RNA or DNA/RNA-containing immune complexes or are engaged by self-Ag stimulation via BCR. Secreted type I IFNs and IL-6 enhance autoreactive B cell differentiation, and BLyS/BAFF promotes autoreactive B cell survival. Additionally, up-regulated ICAM-1 on activated pDCs binds high-affinity LFA-1 on B cells to lower activation threshold for autoreactive B cells. Taken together, these events lead to augmented autoreactive B cell expansion, plasma cell differentiation, and autoantibody production. Further understanding of the cellular and molecular events of the pDC-B cell interaction will help us to comprehend autoreactive B cell activation, differentiation, or autoimmunity.

Disclosures The authors have no financial conflicts of interest.

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