Murine Plasmacytoid Dendritic Cells RIIB Prevents Antigen ...

The Journal of Immunology

Dominant Expression of the Inhibitory Fc␥RIIB Prevents Antigen Presentation by Murine Plasmacytoid Dendritic Cells1 Marcella Flores,* Dharmesh D. Desai,* Matthew Downie,* Bitao Liang,* Michael P. Reilly,† Steven E. McKenzie,† and Raphael Clynes2* Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4ⴙ and CD8ⴙ T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating Fc␥R expression (Fc␥RI, III, IV) and the dominant expression of the inhibitory receptor Fc␥RIIB. Consistent with this idea, transgenic expression of the activating human Fc␥RIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of Fc␥RIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory Fc␥Rs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity. The Journal of Immunology, 2009, 183: 7129 –7139.

T

he Fc␥Rs are expressed by hematopoietic lineage cells and function to modulate innate and adaptive immunity. On effector cells, including myeloid and NK cells, activating and inhibitory Fc␥Rs reciprocally regulate innate inflammatory responses to both soluble immune complexes (ICs)3 and Ab-opsonized cells. On APCs, including B cells, follicular dendritic cells (DCs), macrophages and DCs, Fc␥Rs mediate binding and uptake of Ag-containing ICs and together influence outcomes of responding B and T cells. On conventional dendritic cells (cDCs), Fc␥Rs provide dual functionality by modulating the DC activation state and cytokine response as well as facilitating antigenic uptake and access to the Ag-processing machinery (1–3). Foreign and self-Ag internalized by activating Fc␥Rs efficiently accesses the lysosomal degradative compartment enabling entry to the exogenous and cross-presentation pathway (1–5). Engagement of activating Fc␥Rs on cDCs leads to ITAM-mediated cellular activation, including up-regulation of costimulatory molecules and production of pro-inflammatory cytokines and chemokines thereby providing both signal 1 and signal 2 for the induction of potent CD4 and CD8 cellular re-

Received for publication April 10, 2009. Accepted for publication September 21, 2009.

sponses (1–3). The individual contributions of each of the activating Fc␥R subtypes (I, III, and IV) in modulating DC function is unknown, although their different affinities for IgG1, 2a, and 2b predict IgG subclass-dependent selectivity (6). The ITIM-containing inhibitory Fc␥RIIB is functionally distinct and prevents DC activation and alters the intracellular trafficking of internalized ICs (3, 5, 7–9). Ags endocytosed by the inhibitory Fc␥RIIB on cDCs are excluded from the lysosome and instead access a non-degradative compartment that limits the magnitude of signal 1 (5, 7) and inhibits cellular activation, with consequent reduction in signal 2 and concomitant T cell effector priming (5). The role of Fc␥Rs on plasmacytoid dendritic cells (pDCs) is less well understood. Studies of human pDCs have shown that nucleic acid-containing ICs internalized through Fc␥Rs enter an endolysosomal compartment, potentiating TLR7 (10 –13) and TLR9 (14, 15) signaling and IFN-␣ production. Furthermore, although pDCs inefficiently process and present exogenous Ags, human pDCs have been shown to be capable of Ab-enhanced Ag presentation to T cells (16), implying that Fc␥Rs on human pDCs regulate the adaptive response to self and exogenous Ags. In both situations, IC-mediated IFN-␣ induction and Ag presentation, Fc␥RIIA was found to be the dominant receptor involved (10, 16). We have investigated the expression and function of Fc␥Rs on murine pDCs and find surprisingly that only inhibitory and not activating Fc␥Rs are expressed by murine pDCs thus limiting the ability of pDCs to promote IC-mediated Ag presentation.

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.

Materials and Methods

*Department of Medicine and Microbiology, Columbia-Presbyterian Medical Center, Columbia University, New York, NY 10032; and †Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107

1

This work was supported by National Institutes of Health Grants R01 NCI 94037, T32 AI 07525, and T32 HL 072739 and by Juvenile Diabetes Research Foundation Grant 1-2006-756.

2

Address correspondence and reprint requests to Dr. Raphael Clynes, Department of Medicine and Microbiology, Columbia-Presbyterian Medical Center, Columbia University, College of Physicians and Surgeons, New York, NY 10032. E-mail address: [email protected]

3

Abbreviations used in this paper: DC, dendritic cell; IC, immune complex; cDC, conventional DC; pDC, plasmacytoid DC; BM, bone marrow; WT, wild type. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901169

Mice BALB/c, C57BL/6, C57BL/6 CD45.1⫹ (B6.SJL-Ptprca Pepcb/BoyJ), NZB/NZW, MRL/Mpj-lpr, and NOD mice were purchased from The Jackson Laboratory. Fcer1g (Fc␥R␥⫺/⫺) and Fcgr2b (Fc␥RIIB⫺/⫺) knockout mice, available on both the C57BL/6 and BALB/c background, were purchased from Taconic Farms. Fc␥RIII⫺/⫺, and Fc␥RI⫺/⫺ mice on the C57BL/6 background were provided by Jeffrey Ravetch (The Rockefeller University, New York, NY). hFc␥RIIA transgenic mice were generated as described (17). C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice (The Jackson Laboratory) and C57BL/6 OT-II mice, generously provided by

Fc␥RIIB IS THE SOLE Fc␥R ON MURINE pDC

7130 Alan Frey (New York University, New York, NY), were both crossed to CD45.1⫹ mice.

DC isolation from spleen and from expanded bone marrow (BM) cultures BM cells (2 ⫻ 106 cells/ml) were cultured for 8 –9 days in RPMI 1640/10% FCS supplemented with 100 –200 ␮g/ml human Flt3 ligand (hFLT3L, generously provided by Amgen) or 20 ␮g/ml murine Flt3 ligand as indicated (mFLT3L; R&D Systems) and typically consisted of 70% cDCs/30% pDCs and 40% cDCs/60% pDCs, respectively. cDCs were also prepared from GM-CSF cultures using supernatant from a J558L cells transfected with the mouse gm-csf gene (provided by A. Houghton, Memorial SloanKettering Cancer Center, New York, NY). GM-CSF cultures typically generate ⬎90% CD11b⫹CD11c⫹ cDCs on day 7. FACS analysis of splenic DC populations was performed on gated splenic cells after RBC lysis, either with or without prior depletion of CD19⫹ B cells. For preparations of protein or RNA extracts, four-color flow sorting was used to obtain high purity CD11c⫹B220⫹ CD11b⫺ BST2⫹ splenic pDC and CD11c⫹ B220⫺ CD11b⫹ splenic cDC populations (with mAbs HL3, RA3– 6B2, M1/70 mAbs obtained from BD Pharmingen; 120G8 was a gift from Georgio Trinchieri, Schering-Plough, Palo Alto, CA).

In vitro and in vivo Ag presentation assays of BM-derived DC subsets cDCs were enriched from day 8 FLT3L expanded BM cultures with CD11b microbeads (Miltenyi Biotec). pDCs were enriched from the CD11b negative fractions by subsequent positive selection with B220 microbeads. GM-CSF cDCs were harvested at day 6 or 7 and used without enrichment. For in vitro studies both cDCs and pDCs were incubated for 24 h in 96-well plates with the indicated Ags together with either 2 ⫻ 105 CD8 OTZ-I or CD4 OTZ-II T hybridoma cells, expressing the NFAT-responsive ␤-galactosidase gene, and ␤-galactosidase activity assayed as described (7). Where indicated, FLT3L expanded BM cDC and pDC subsets were bead enriched as before, and then pulsed/washed with 10 ␮g/ml OVA-ICs before incubation (at a 1:10 ratio) with OT-I or OT-II splenocytes previously depleted of APCs with biotinylated CD11c⫹, CD19⫹, CD11b⫹, DX5⫹, GR-1⫹, B220⫹, and Ter119⫹ mAbs and streptavidin microbeads. For in vivo studies 1 ⫻ 106 bead-enriched, Ag-pulsed and washed FLT-3 BMderived pDCs or cDCs were injected via tail vein into recipient mice that had received 4 ⫻ 106 CFSE-labeled CD45.1⫹ CD8⫹ OT-I cells, and CD4⫹ OT-II cells, the previous day. T proliferative responses were assessed after 3 days by CFSE dilution in gated, live CD45.1⫹CD4⫹ and CD45.1⫹CD8⫹ splenic populations.

Ex vivo Ag presentation of splenic DCs C57BL/6 mice were injected via tail vein with 10 mg of OVA or OVA-ICs (100 ␮g of OVA/250 ␮g of rabbit anti-OVA IgG). Spleens were harvested 2 h later and sorted to obtain CD11c⫹ B220⫹ CD11b⫺ and BST2⫹ pDCs and CD11c⫹ B220⫺ BST2⫺ cDCs, and cocultured at a ratio of either 1:4 or 1:20 (DC:T) for 3– 4 days with CFSE-labeled CD8⫹ OT-I or CD4⫹ OT-II splenocytes previously depleted of CD11c⫹, CD19⫹, CD11b⫹, DX5⫹, GR-1⫹, and Ter119⫹ cells.

IC binding and internalization Day 8 FLT3L expanded BMDCs were incubated on ice (binding assay) or at 37oC (internalization/accumulation assay) with fluorescent ICs (10 ␮g/ml Alexa Fluor 488 OVA (Invitrogen):50 ␮g/ml polyclonal rabbit antiOVA IgG) and then washed and stained for flow cytometric analysis with CD11c, CD11b, and B220 mAbs to identify pDC and cDC populations.

Cellular IC processing DQ-OVA-ICs were made by mixing DQ-OVA (Invitrogen) and polyclonal rabbit anti-OVA IgG at a 1:5 ratio (by weight) at 37°C for 30 min. Day 8 or 9 FLT3L BMDCs were incubated with free DQ-OVA or DQ-OVA-ICs at 37°C and harvested at indicated times and then stained for CD11c, CD11b, and CD45R positive populations. For in vivo studies mice were injected i.v. with DQ OVA-ICs (25 ␮g of DQ-OVA and 100 ␮g of rabbit anti-OVA IgG) or with DQ-OVA alone (25 or 250 ␮g). Spleens were harvested 4 h postinjection and processing of pDCs or cDCs identified flow cytometrically by anti-BST2, CD11c, and CD11b staining. Oxyburst or FcOxyburst ICs (Invitrogen) were used at a final concentration of 150 ␮g/ml and 400 ␮g/ml, respectively, and incubated with RBC-lysed splenocytes for 45 min at 37°C. pDCs and cDCs were identified and assayed by FACS.

Confocal Microscopy OVA-containing ICs were made as above. Flu ICs were made by mixing heat-killed A/WSN/33 influenza (gift of Peter Palese, Mt. Sinai School of Medicine, New York, NY) and immune sera from C57BL/6 mice previously infected with live A/WSN/33 influenza. ICs were incubated with day 8 FLT3L BM cultures adherent to microwell slides. Cells were stained with anti-BST2 Alexa-488 or CD11b-Alexa-647, together with either antimouse IgG-PE, anti-rabbit IgG-PE, or anti-rabbit IgG-488. Images were acquired with either a Zeiss LSM 510 META scanning confocal microscrope and a Plan-Apochromat 100⫻ objective with an NA of 1.4, or a Plan-Neofluor with an NA of 1.3 or with a Leica TCS SP5X Supercontinuum Confocal and a 100⫻ objective and analyzed by deconvolution algorithims.

Flow cytometric analysis of Fc␥R expression FLT3L expanded BM cultures and splenic DC populations were analyzed for murine Fc␥R expression using the following Abs: anti-mouse Fc␥RI (X54 –5/7.1; BD Pharmingen), anti-Fc␥RII/III (2.4G2; BD Pharmingen), anti-Fc␥RIV (9G8.1; gift from Jeffrey Ravetch, Rockefeller University, NewYork, NY).

RT-PCR analysis of Fc␥R expression RNA was extracted from cells by using TRIzol (Invitrogen). cDNA was synthesized using Superscript III First-Strand cDNA synthesis kit (Invitrogen). The following primers were used to assess Fc␥R expression: Fc␥RI: 5⬘-CAATGCCAAGTGACCCTGTGC3-⬘ and 5⬘-ACTGCTGTCCTCCGT GGCTACC3-⬘; Fc␥RIIB isoforms: 5⬘-GCCTGTCACCATCACTGTCCA AGGGCCCAA-3⬘ and 5⬘-AATGTGGTTCTGGTAATCATGCTCTGTT TCTTC-3⬘; Fc␥RIII: 5⬘-TCCGAAGGCTGTGGTGAAACTG-3⬘ and 5⬘CGTAGAAATAAAGGCCCGTGTCC-3⬘; Fc␥RIV: 5⬘-CTAGGCGATCC AGGGTCTCCAT-3⬘ and 5⬘-GCGTGCGCATTGCTGTATCA-3⬘; HGPRT primers: 5⬘-CCACAGGACTAGAACACCTGCTAA-3⬘ and 5⬘-AGCTACT GTAATGATCAGTCAACG-3⬘.

Statistical Analysis Differences between groups were evaluated by ANOVA for multiple group analysis, with p values provided as indicated using one-tailed paired t tests performed between pairs of groups. Difference between slopes of dose response measurements were tested by linear regression.

Results Murine pDCs do not present ICs via the exogenous or cross-presentation pathways ICs facilitate T cell priming by cDCs through induction of DC maturation and enhancement of antigenic uptake/processing (1–3). To determine whether pDCs share this functional attribute with cDC, bead-enriched CD11c⫹ B220⫹ FLT3L pDCs were compared with bead-enriched CD11c⫹ CD11b⫹ FLT3L cDCs and GMcDCs for their ability to present OVA-ICs by the exogenous and cross-presentation pathways using NFAT-responsive LacZ⫹ CD8⫹ MHC I-OVA peptide-restricted OT-I hybridomas (OTZ-I) and LacZ⫹ CD4⫹ MHC II-OVA peptide-specific OT-II hybridomas (OTZ-II) (Fig. 1). As expected, cDCs from GM-CSF and FLT3L expanded BM cultures triggered activation of OTZ-I and OTZ-II hybrids in the presence of OVA-ICs. IC-mediated Ag presentation capacity to both OTZ-I and OTZ-II cells was abolished in FcR␥⫺/⫺ cDCs and was enhanced in Fc␥RIIB⫺/⫺ cDCs, consistent with the notion that for cDCs activating Fc␥Rs are responsible for IC-mediated Ag presentation whereas the inhibitory Fc␥RIIB reciprocally regulates this process. Wild-type (WT) GMCSF cDCs were superior to WT hFLT3L cDCs for the activation of both OTZ-I and OTZ-II hybrids, likely due at least in part, to the higher ratio of expression of activating:inhibitory Fc␥Rs exhibited by GM-CSF cDCs (data not shown). This is most apparent for cross-presentation of ICs to OTZ-I hybrids, where FLT3L cDCs failed to activate T cells unless genetically deficient in Fc␥RIIB. In contrast, pDCs were unable to activate OTZ-I or OTZ-II cells, regardless of the Fc␥R genotype, pDC cell number (Fig. 1A) or OVA-IC concentration (Fig. 1B). This was not due to a lack of


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FIGURE 1. Murine BM pDCs do not present OVA-ICs in vitro. A, OTZ-I CD8⫹ or OTZ-II CD4⫹ T cell hybridoma activation (LacZ OD) after 36 h of incubation with increasing numbers of either MACS bead-purified pDCs and cDCs loaded with either OVA peptides (unfilled symbols, 10 nM OT-I peptide or 10 ␮M OT-II peptide) or OVA-ICs (filled symbols: 50 ␮g/ml rabbit anti-OVA IgG and 10 ␮g/ml OVA). BM cDCs were either expanded in hFLT3L (solid line) or GM-CSF (dotted lines). DC genotypes are WT (circles), Fc␥RIIB⫺/⫺ (triangles) and Fc␥R ␥⫺/⫺ (squares). p ⱕ 0.001 (ⴱⴱ) for OTZ-I ⫹ WT (GM-CSF cDC vs hFLT3L pDC); p ⱕ 0.001 (ⴱⴱ) for OTZ-I ⫹ hFLT3L Fc␥RII⫺/⫺ (pDC vs cDC); p ⱕ 0.001 (ⴱⴱ) for OTZ-II ⫹ WT (GM-CSF cDC vs hFLT3L pDC); p ⱕ 0.001 (ⴱⴱ) for OTZ-II ⫹ WT FLT3L (pDC vs cDC). B, LacZ OD hybridoma activation of OTZ-I CD8⫹ or OTZ-II CD4⫹ T cells incubated for 36 h with increasing concentrations of OVA-ICs (filled symbols) or peptides (unfilled symbols) and a fixed number (105 cells/96-well) MACS bead purified hFLT3L pDCs and cDCs. DC genotypes are WT (circles), Fc␥RIIB⫺/⫺ (triangles) and Fc␥R ␥⫺/⫺ (squares). The data are representative of seven experiments total. p ⫽ 0.032 (ⴱ) for OTZ-I ⫹ hFLT3L Fc␥RIIB⫺/⫺ (cDCs vs pDCs); p ⫽ 0.01 (ⴱ) for OTZ-II ⫹ hFLT3L WT (cDCs vs pDCs); p ⫽ 0.041 (ⴱ) for OTZ-I ⫹ hFLT3L Fc␥RIIB⫺/⫺ (cDCs vs pDCs).

intrinsic Ag-presenting capacity as pDCs were able to present exogenously loaded OT-I or -II peptides, albeit at reduced levels as compared with cDCs. To address whether the inability of FLT3L BM-expanded pDCs to participate in IC-mediated T cell priming was limited to in vitro settings, OVA or OVA-IC-pulsed BM-derived pDC and cDCs were transferred i.v. into recipient mice previously injected with CD45.1⫹ CFSE-labeled CD8⫹ OT-I cells and CD4⫹ OT-II transgenic T cells. When loaded in vitro with high doses of OVA alone (1 mg/ml) both FLT3L expanded pDCs and cDCs primed CD4⫹ and CD8⫹ T cell responses (Fig. 2A) demonstrating that pDCs were capable of OVA processing and presentation. Similar to the situation in vitro, pDCs did not exhibit any Fc␥R-dependent ICmediated T cell proliferation, with comparable responses seen for WT and Fc␥R null pDCs. (Fig. 2, B and C). As expected, WT GM-CSF or FLT3L expanded cDC induced robust IC-mediated OT-I and OT-II T cell proliferation as compared with their Fc␥R null counterparts (Fig. 2, B and C). As was seen for T hybrid activation (Fig. 1), GM-CSF cultured BMDCs more potently activated OT-I and OT-II cells than their FLT3L cDC counterparts (Fig. 2C).

We next examined whether primary splenic pDCs were capable of priming T cells through antigenic engagement of Fc␥Rs. Splenic DCs were loaded in vivo by tail vein injection with either 100 ␮g of OVA-ICs (Fig. 3, A and B) or 10 mg of OVA (Fig. 3C) and then tested the Ag presentation capacity of isolated splenic pDC and cDC subsets ex vivo. Using this approach, splenic pDCs presented soluble OVA to OT-I and OT-II cells only after TLR stimulation, as described (18) (Fig. 3C and data not shown). However, splenic pDCs were again incapable of presenting Ab-opsonized OVA even with TLR activation (Fig. 3A) or at 3-fold higher DC:T cell ratios (Fig. 3B). As expected, splenic cDCs were able to prime OT-I and OT-II cells after loading in vivo with either OVA or OVA-ICs. Murine pDCs express Fc␥RIIB but not activating Fc␥Rs The expression of activating (Fc␥RI, III, and IV) and inhibitory Fc␥Rs (Fc␥RIIB) was examined on both hFLT3L BM pDCs and splenic pDCs from C57BL/6 and BALB/c mice (data not shown). As expected, FLT3L cDCs expressed all Fc␥Rs, although the expression of Fc␥RIV was low and variable (Fig. 4A). Strikingly, FLT3L BM pDCs lacked surface expression of all activating

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FIGURE 2. Murine BM pDCs do not present OVA-ICs in vivo. WT (CD45.2⫹) recipients of 1 ⫻ 106 CFSE labeled CD45.1⫹ OT-I T cells (black) and OT-II T cells (white) were immunized i.v. with Ag-loaded WT or Fc␥R null FLT3L BM pDCs or cDCs. T cell proliferative responses (percent-divided gated CD45.1⫹ splenocytes) are shown 3 days after immunization. A, Immunizing mFLT3L BM DCs were loaded with 1 mg/ml OVA. B, Immunizing hFLT3L pDCs or GM-CSF cDCs were loaded with either OVA-ICs (50 ␮g/ml rabbit anti-OVA IgG/10 ␮g/ml OVA) or OVA peptides (10 nM OT-I peptide or 10 ␮M OT-II peptide). One representative experiment of three is shown in A (four mice per group) and B (one mouse per group). C, Cumulative data of three independent experiments done as shown in B, showing Fc␥R-mediated proliferation, calculated as: (percentage of gated T cells divided in WT DC conditions) ⫺ (percentage of gated T cells divided in Fc␥R null DC conditions). Each dot represents the Fc␥R-mediated proliferation (WT DC-Fc␥R null DC) between single pairs of recipients. ⴱ, p ⬍ 0.05 for pDC vs hFLT3L cDC); ⴱⴱ, p ⬍ 0.01 for GM-CSF cDC vs pDC.

Fc␥Rs and expressed Fc␥RIIB as their dominant Fc␥R. To address Fc␥R expression in vivo, splenic DCs were enriched through negative selection by a CD19 magnetic bead column and DC subsets (CD11c⫹ CD11b⫹ cDCs and CD11c⫹B220⫹BST2⫹ pDCs) were compared for Fc␥R expression by flow cytometry. Confirming the expression profile with BM-derived pDCs, murine splenic pDCs express high surface levels of the inhibitory Fc␥RIIB but minimal or no surface activating Fc␥Rs, including Fc␥RI, Fc␥RIII, and IV. Activating Fc␥Rs are transcriptionally silent in pDCs The failure of pDCs to express all activating Fc␥R on the cell surface might have been explained by a lack of expression of Fc␥R ␥, since expression of this adapter is required for membrane expression of Fc␥R ␣-subunits (19). However, both CD11b⫹ cDC and B220⫹ pDC populations in FLT3L cultured BMDCs expressed the FcR ␥-subunit by Western analysis (data not shown), indicating that the lack of surface expression of activating Fc␥Rs is not a consequence of the lack of expression of the Fc␥R ␥-subunit. Expression of Fc␥R ␣-subunits was directly examined at the RNA level from FACS-sorted CD11c⫹ splenic populations (Fig. 4B). Using Fc␥R-subtype-specific primers for RT-PCR analysis, transcripts for all Fc␥R isoforms were detectable in CD11b⫹ CD11c⫹ DC populations, including the ␣-subunits for Fc␥RI, IIB1, IIB2, III, and IV. However, only Fc␥RIIB1 and B2 transcripts were found in sorted pDC populations and transcription of all three activating Fc␥R ␣-subunits was lacking. Thus, the unique surface expression of inhibitory Fc␥Rs on pDCs is the consequence of lineage-specific transcriptional silence of activating Fc␥Rs.

Activating Fc␥R expression on pDCs is not up-regulated by inflammatory cytokines or TLR ligands The expression of activating Fc␥Rs, in particular Fc␥RI, is known to be up-regulated by STAT1-mediated cytokine stimulation, including IFN-␥ (20). Thus we examined surface expression of activating Fc␥Rs on hFLT3L BM pDCs after stimulation with a variety of cytokines and TLR ligands known to activate DCs (Fig. 4, C and D). Although Fc␥RI was up-regulated in response to IFN-␣, IFN-␥, and LPS in CD11b⫹ cDC, neither Fc␥RI (Fig. 4C) nor Fc␥RIII (data not shown) were up-regulated in stimulated pDCs. Similarly, neither TLR9 or TLR7 ligands induced the activating Fc␥Rs, Fc␥RI (Fig. 4D) or Fc␥RIII (data not shown) on pDCs. All ligands induced strong CD86 up-regulation from both DC subsets (data not shown). Thus, dominant expression of the inhibitory Fc␥RIIb on murine pDCs is not due to cellular quiescence and persists even in the presence of cytokine or TLR-mediated cellular activation. pDCs bind and internalize ICs in an Fc␥RIIB-dependent manner To assess whether murine pDCs bind and internalize ICs through Fc␥RIIB, day 8 FLT3L BM-derived pDCs and cDCs were compared for binding of Alexa-488-conjugated OVA/rabbit anti-OVA-ICs by flow cytometry (Fig. 5A). IC binding was exhibited by both WT and Fc␥RIIB-deficient cDCs; however, binding by Fc␥RIIB⫺/⫺ pDCs was completely abolished and was identical to the Fc␥R null control. These findings were supported by confocal microscopy (Fig. 5B, left) and confirms


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FIGURE 3. Splenic pDCs fail to present ICs to T cells even in the presence of CpG. Sorted splenic DCs obtained 4 h after i.v. injection of either OVA (10 mg) or OVA-ICs (100 ␮g of anti-OVA/20 ␮g of OVA) were cultured at either 10,000 cells/well (A) or 30,000 cells/well (B) and for 4 days (C) with APC-depleted 2 ⫻ 105 CFSE-labeled CD4⫹ OT-II or CD8⫹ OT-I splenocytes in the presence or absence of 1 ␮M CpG-B. Data are representative of at least two experiments each.

as expected that OVA-IC binding to pDCs is completely dependent upon Fc␥RIIB expression. Similarly, binding of murine polyclonal influenza viral ICs again demonstrated a complete dependence on Fc␥RIIB (Fig. 5B, right). Fc␥RIIB exists in two major isoforms, B1 and B2. Both isoforms are expressed by GM-CSF cDCs (7) and by splenic pDCs (Fig. 4). Fc␥RIIB2 is endocytic whereas Fc␥RIIB1, the larger isoform, contains an endocytosis inhibitory motif preventing antigenic uptake (21),

although some studies using transfected B cell lines have suggested that this inhibition is not absolute (22). Thus, it was important to determine whether pDCs were capable of internalizing ICs. Consistent with the Fc␥R expression data (Fig. 4), accumulation of ICs by pDCs was completely dependent on Fc␥RIIB and indicated that the endocytic competent Fc␥RIIB2 isoform was functionally present on murine pDCs (Fig. 5C). By confocal microscopy, internalized ICs were apparent within 30 min of incubation at 37°C (Fig. 5D).

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FIGURE 4. Murine pDCs selectively express Fc␥RIIB. A, Flow cytometry for Fc␥R expression of hFLT3L BMDCs or splenic DCs. Fc␥RI and Fc␥RIV expression was assessed directly with specific anti-murine Fc␥RI and IV mAbs. Filled gray histograms are staining of Fc␥R null DCs and black histograms are WT DCs. Fc␥RIII expression was assessed indirectly with the anti-FcRII/III mAb 2.4G2, by comparing 2.4G2 binding between Fc␥R null (filled gray) and Fc␥RIIB⫺/⫺ (black) DCs. Conversely, Fc␥RIIB expression was examined by comparing mAb 2.4G2 staining between Fc␥R null (filled gray) and Fc␥R ␥⫺/⫺ (black) cells, the latter devoid in expression of Fc␥RIII. Data are representative of at least five experiments and were reproduced using mFLT3L and hFLT3L cultures with similar results. B, RT-PCR of FACS isolated splenic DCs from WT C57BL/6 mice. Representative data of three experiments are shown. C, Flow cytometry for Fc␥RI expression from hFLT3L WT BMDCs 24 h after exposure to indicated treatments (black line) vs control Fc␥R null DCs (shaded gray). D, Flow cytometry of Fc␥RI expression of hFLT3L WT BMDCs after overnight exposure to indicated treatments. Shaded gray histograms are untreated (no tx) Fc␥R null cells; black and red histograms are untreated and TLR-treated WT DCs, respectively. Data representative of at least two experiments are shown.

ICs internalized by murine pDCs fail to reach a degradative compartment To assess antigenic delivery to the proteolytic degradative compartment, the self-quenching fluorescent reagent, DQ-OVA was employed, which fluoresces only upon hydrolysis. When FLT3L cDCs were cultured with DQ-OVA-ICs, fluorescence increased over time, the result of proteolytic processing of the internalized IC cargo (Fig. 6, A and B). As expected the rate of processing by cDCs was increased with elimination of Fc␥RIIB, consistent with previous findings in cDCs that internalization via this inhibitory receptor delivers Ag to a non-degradative cellular compartment (7). Unlike cDCs, BM FLT3L expanded pDCs, however, did not degrade the internalized DQ-OVA-ICs, and the fluorescence of DQ-OVA-ICs was not improved in the absence of Fc␥RIIB (Fig. 6, A and B). The inability of pDCs to degrade IC cargo was not an inherent deficiency in endosomal processing as both cDC and pDCs were able to degrade free DQ-OVA (at 10-fold higher concentrations) at the same rate and regardless of specific Fc␥R expression. To determine whether splenic pDCs were equally deficient as their BM expanded counterparts in processing ICs, 25 ␮g of DQOVA-ICs or DQ-OVA alone was injected i.v. into WT C57BL/6

recipients and spleens were harvested 4 – 6 h post injection. Processing of DQ-OVA-ICs was enhanced for splenic cDCs, as compared with cDCs from mice injected with equal amounts of injected free DQ-OVA alone, consistent with activating Fc␥R-mediated delivery to the degradative pathway (Fig. 6C). In contrast, splenic pDCs, like their BM expanded counterparts, did not display an enhancement in IC processing and yet were capable of processing DQ-OVA reagent when injected at 10-fold higher doses of DQ-OVA (250 ␮g) (Fig. 6D). pDCs from autoimmune-prone mouse strains do not degrade ICs Splenic pDCs from a variety of mouse strains were examined to insure that the lack of activating Fc␥R function on murine pDCs was not simply restricted to C57BL/6 and BALB/c mice. To accomplish this, we used a similar reagent to DQ-OVA, FcOxyburst which consists of BSA-ICs coupled to a fluorochrome active only in an oxidative environment. Fluorescence of this reagent requires internalization via activating Fc␥Rs (5) and provides a measure of activating Fc␥R function. In contrast to cDCs, WT splenic pDCs from all strains examined, including three autoimmune prone strains, failed to process FcOxyburst (Fig. 7A). Splenic pDCs from


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FIGURE 5. Murine pDCs bind and accumulate ICs in an Fc␥RIIB dependent manner. IC binding: A, Flow cytometry for OVA-Alexa 488 binding to hFLT3L BMDC cultures incubated with OVA-Alexa 488 ICs on ice for 30 min. WT DCs (black), Fc␥RIIB⫺/⫺ DCs (red) are compared with Fc␥R null DCs (gray). Data are representative of at least four experiments. B, Confocal microscopic images (⫻100) of hFLT3L BM pDCs surface-stained with BST2 mAbs (green) incubated with either OVA-ICs (detected with anti-rabbit Ig PE; red) or A/WSN/33 Flu-ICs (detected with anti-mouse IgG Alexa 555; red) for 30 min on ice. IC internalization: C, Change in MFI of hFLT3L BMDC cultures incubated with OVA-Alexa 488 ICs at 37°C and harvested at indicated times. WT DCs (black) are compared with Fc␥R null DCs (gray). Results are cumulative of at least three experiments. ⴱ, p ⫽ 0.024 for WT pDCs vs Fc␥RIIB⫺/⫺ pDCs. ⴱ, p ⫽ 0.031 for WT cDCs vs Fc␥R null cDCs. D, Confocal fluorescent microscopy and DIC images of mFLT3L BM pDCs incubated with OVA-Alexa 488 ICs (green) and DAPI (blue) showing internalized ICs after 30 min at 37°C.

all strains were capable of oxidizing OVA, when pulsed with 10fold higher concentrations of unopsonized Oxyburst (data not shown). pDCs are not matured by IC stimulation In addition to potentiating signal 1, TCR engagement, Fc␥R-mediated uptake by conventional DCs also triggers a potent signal 2 for T cells through the up-regulation of costimulatory molecules. To examine whether murine pDCs were also deficient in up-regulating signal 2 following IC exposure, BM pDCs were incubated with OVA-ICs or CpG-B and maturation immunophenotypic markers assessed 24 h later (Fig. 8). As expected, cDCs responded to OVA-ICs by increased expression of CD86, MHC II and partial up-regulation of CD80 and CD40. The IC-triggered increase in

costimulatory markers by cDCs was ITAM dependent, since FcR␥⫺/⫺ cDCs failed to mature following IC treatment (data not shown). In contrast, WT BM pDCs (nor Fc␥RIIB⫺/⫺ pDCs, data not shown) were not matured by ICs. Thus, murine pDCs are limited in their ability to activate T cells with ICs by lacking both ITAM-mediated antigenic processing and maturation. Transgenic expression of hFc␥RIIA rescues the ability of murine pDCs to present OVA-ICs The activating Fc␥RIIA, a gene not present in the mouse, is the dominant Fc␥R expressed by pDCs in human blood. Transgenic expression of human Fc␥RIIA (17) driven by its endogenous promoter (including 45 kb of 5⬘-flanking region containing and 7 kb of 3⬘-flanking region) led to its expression on the surface

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FIGURE 6. ICs do not access a degradative compartment in murine pDCs. A, Change in MFI from baseline (fluorescence at 4°C) of hFLT3L BMDC cultures incubated with DQ-OVA-ICs (100 ␮g/ml anti-OVA IgG/20 ␮g/ml OVA) or with DQ-OVA alone (200 ␮g/ml) at 37°C for 90 min and harvested at indicated times. Cumulative data are shown from three separate experiments. ⴱ, p ⫽ 0.013 WT (pDC vs cDC); ⴱⴱ, p ⫽ 0.003 Fc␥RIIB⫺/⫺ (pDC vs cDC). B, Confocal microscopy of WT hFLT3L BMDC cultures incubated with DQ-OVA-ICs for 60 min and stained (in blue) with either anti-CD11b or anti-BST2. C, Left panel: Flow cytometry of splenic pDCs or cDCs from mice injected i.v. 4 h before harvest with equal doses of either DQ-OVA-ICs or with DQ-OVA alone (25 ␮g/mouse OVA component). Representative data of two experiments shown and quantified in the right panel as “immune complex enhancement” calculated as the IC-mediated increase in MFI of splenic DCs (OVA-IC minus OVA alone). Cumulative data are shown from two experiments each with three mice per group. The value of p (0.03) of pDC vs cDC is shown. D, Flow cytometry of splenic pDCs from mice injected or not, 4 h before harvest, with a high dose (3 mg) of DQ-OVA alone; contour plots and overlay histogram. Filled gray histogram-no treatment (No Tx); black histogram-DQ-OVA. Results are representative of three experiments, each done with three mice.

of mouse BM expanded pDCs (Fig. 9A). IC binding by splenic hFc␥RIIA transgenic pDCs (Fig. 9B) led to robust antigenic processing of DQ-OVA-ICs (Fig. 9C), in a manner completely inhibitable by the Fc␥RIIA-specific blocking mAb IV.3. The Fc␥RIIA-dependent processing also led to potent T cell priming capacity (Fig. 9D). Thus, murine pDCs are capable of processing and presenting endocytosed ICs if internalized by activating Fc␥Rs.

Discussion Both activating and inhibitory Fc␥Rs are expressed by murine myeloid lineage cells although their relative expression depends on cell-type and activation status. Murine CD8⫹ CD11c⫹ and CD8⫺ CD11c⫹ DCs express both activating and inhibitory Fc␥Rs (23) that reciprocally regulate the adaptive T cell response through ITAM and ITIM modulation of both antigenic processing and DC maturation. The dominant expression of Fc␥RIIB in the mouse, exhibited by both splenic pDCs and FLT3L pDCs likely explains their inability to activate T cells despite Fc␥R-mediated internalization of OVA-containing ICs. Due to the capacity of Fc␥RIIB to retain intact Ag (7), it remains possible, however, that cell-surface ICs on pDCs may directly promote humoral if not cellular immunity. pDCs express the ITAM-adapter Fc␥R ␥-chain eliminating the possibility that the lack of surface expression of activating Fc␥Rs

is a consequence of retention of Fc␥R ␣-subunits in the endoplasmic reticulum (19). Instead, the lack of activating Fc␥R surface expression on murine pDCs is due to the absence of transcriptional expression of all three activating Fc␥Rs ␣-subunits. A recent transcriptional profile of murine splenic pDCs published by an independent group confirms this result (24). The lack of functional activating Fc␥Rs on pDCs in the mouse appears to be generally true across different murine strains, including MRL/Mpj-lpr and (NZB/NZW)F1 strains genetically susceptible to the development of IC-associated autoimmunity. Although it remains possible that activating Fc␥R expression on murine pDCs is up-regulated at sites of inflammation, activation dependent up-regulation was not seen after stimulation by several relevant pro-inflammatory cytokines and TLR7 and TLR9 ligands. Current understanding of development of DC subsets remains incomplete, but the finding that pDCs and B lymphocytes express Fc␥RIIB as their dominant Fc␥R adds to the shared markers of these two cell types (expression of B220, SpiB, BCAP, and BCR genetic rearrangements) suggestive of potential lineage relationships and/or shared transcriptional regulatory programs between pDCs and B lymphocytes. Our results conflict with those of Bjorck et al. (25), in which OVA-IC-loaded pDCs were shown to be able to induce T cell proliferative responses ex vivo. However, these studies did not examine the role of Fc␥Rs genetically and could not exclude


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FIGURE 7. pDCs from autoimmune prone strains do not degrade ICs. Splenocytes from the indicated strains of mice were incubated at 37°C (black histogram) or at 4°C (shaded gray) with 150 ␮g of FcOxyburst for 45 min and analyzed by FACS for fluorescence by pDCs (BST2⫹CD11cmidCD11b⫺) and cDCs (CD11chighBST2⫺CD11b⫹). In each histogram, the difference in MFI (MFI37°C-MFI4°C) is provided. Data are representative of three experiments.

Fc␥R-independent uptake and presentation of OVA by pDCs, which might have been seen had Fc␥R-deficient DCs been used (Figs. 2B and 6A). Moreover, in these studies, Ag presentation capacity to T cells attributed to pDCs may have instead been provided by CD4⫹ or CD8⫹ APCs within these positively selected OT-I or OT-II cell populations. Indeed, unless spleen APCs are actively removed as performed in this current work, bead-selected splenic T cells proliferate vigorously when incubated with OVA-containing ICs even without added APCs (data not shown). These data add to our understanding of the APC capacity, and lack thereof, of murine pDCs and their functional discrimination from cDCs. Ag presentation capacity of pDCs is alternatively dependent on the anatomic location of the pDC (26), the activation state of the pDC (18, 27, 28), and whether the responding T cells are naive or memory (29). Initial studies of FLT3L derived cultured murine pDCs showed that pDCs were

poor stimulators of allogeneic and Ag-specific T cell responses when compared with their cDC counterparts. In particular, exogenous Ags are particularly poorly presented (29, 30) due to inefficient endocytosis (31), a lack of antigenic exogenous processing machinery and/or altered regulation of MHC II turnover (32). However, this poor Ag presentation capacity has been contentious (33) and other reports have demonstrated that pDCs can prime T cells. Targeted antigenic uptake through several endocytic receptors on murine pDCs (Siglec (34), mPDCA-1 (26)), or human pDCs (Fc␥RIIA (16), DCIR (35)) enables Agspecific T cell activation. Microbial Ags can be presented by pDCs through the endogenous and exogenous pathways (27, 36) although again when directly compared with cDCs, viral Ags were presented much more poorly by pDCs (28). Some (18), but not all (37–39) studies have noted that exogenous Ag presentation is inducible in splenic pDCs after TLR activation or may be a specialized attribute of pDCs restricted to specific

FIGURE 8. Fc␥R-mediated maturation is not induced in murine pDCs. Flow cytometry of costimulatory molecules on hFLT3L DCs after overnight incubation with either nothing (filled gray), OVA-ICs (gray, 50 ␮g/ml anti-OVA IgG/10 ␮g/ml OVA) or CpG-B (black, 1 ␮M). Data are representative of at least three experiments.


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FIGURE 9. Transgenic expression of hFc␥RIIA rescues the ability of murine pDCs to present OVA Ics. A, hFc␥RIIA expression of mFLT3L BM pDCs from hFc␥RIIA transgenic mice. pDCs are stained with mouse mAb anti-hFc␥RIIA mAb IV.3 (black line) compared with anti-mouse IgG secondary alone (shaded gray) and analyzed by FACS. B, OVA-IC binding of splenic Fc␥RIIA transgenic pDCs incubated on ice with Alexa 488-OVA-ICs with (gray) or without (black) blocking mAb IV.3 compared with no IC control (gray filled) and analyzed by FACs. C, DQ-OVA fluorescence of splenic pDCs incubated at 37°C with DQ-OVA-ICs with (gray) or without (black) blocking mAb IV.3 compared with 4°C control (shaded gray). D, CFSE dilutional profile of CD8⫹ OT-I T cells incubated with mFLT3L BM pDCs pulsed with OVA-IC (left panel) or SIINFEKL (right panel). pDCs from WT mice (gray line) and Fc␥RIIA transgenic mice (black line) are compared. Results are representative of three experiments and were reproduced using mFLT3L and hFLT3L cultures with similar results.

anatomic locations, e.g., peripheral lymph nodes (26). In our studies, CpG failed to induce IC-mediated Ag presentation by splenic pDCs after i.v. IC delivery but technical difficulties limited similar assessment of LN pDCs following s.c. injection of Ag. In contrast to the mouse, human pDCs obtained from the blood express the activating Fc␥RIIA and not the inhibitory Fc␥RIIB, although Fc␥RIIB expression may be subject to regulation by cytokines (8, 12, 40). Consequently, human pDCs present ICs to T cells in vitro (16, 41) through the exogenous pathway, in a manner inhibitable by blocking Abs to Fc␥RIIA. Using the human pDC cell line CAL-1 we have shown that antigenic processing of DQ-OVA-ICs is enhanced in the presence of Fc␥RIIB-specific blocking Abs (data not shown) consistent with competitive antigenic degradative and retention pathways regulated by activating/inhibitory human Fc␥Rs. Transgenic expression of hFc␥RIIA, a gene not present in the mouse genome, recapitulates the human situation and imparts IC processing/presentation capacity to murine pDCs. The aberrant pDC expression of Fc␥RIIA in transgenic mice may contribute to the systemic spontaneous autoimmunity that is seen in these mice (17). The divergent expression of activating and inhibitory Fc␥Rs by pDCs in the mouse and human as demonstrated here, suggest distinct, species-specific roles for pDCs in linking the humoral and cellular adaptive immunity. Transcriptional repression of activating Fc␥Rs in murine pDCs, suggest species-specific and lineage-specific transcriptional regulatory factors in DC subsets. Current efforts are aimed at determining whether Fc␥RIIA or IIB internalization of TLR-containing ICs also differentially modulates the innate pDC cytokine response

as a consequence of cooperative ITAM activation (42– 47), recruitment of the phosphatase SHIP (48) and/or endosomal trafficking/signaling persistence (49).

Disclosures The authors have no financial conflict of interest.

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