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Dependent Manner−Inhibitory Motif Based−in an Immunoreceptor Tyrosine

Production by Plasmacytoid Dendritic Cells Ly49Q Positively Regulates Type I IFN

Noriko Toyama-Sorimachi and Andrew P. MakrigiannisHamilton-Valensky, Gerald Krystal, William G. Kerr,Marie-Line Goulet, Haggag S. Zein, Melisa Amelia Mottashed, Nicholas Ault, Chloe Corbeil,Ahmad Bakur Mahmoud, Elias Abou-Samra, Justin G. Roy, Mir Munir A. Rahim, Lee-Hwa Tai, Angela D. Troke,

http://www.jimmunol.org/content/190/8/3994doi: 10.4049/jimmunol.1200873March 2013;

2013; 190:3994-4004; Prepublished online 11J Immunol 

Referenceshttp://www.jimmunol.org/content/190/8/3994.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Ly49Q Positively Regulates Type I IFN Production byPlasmacytoid Dendritic Cells in an ImmunoreceptorTyrosine–Based Inhibitory Motif–Dependent Manner

Mir Munir A. Rahim,*,1 Lee-Hwa Tai,†,1 Angela D. Troke,* Ahmad Bakur Mahmoud,*,‡

Elias Abou-Samra,* Justin G. Roy,* Amelia Mottashed,* Nicholas Ault,*

Chloe Corbeil,* Marie-Line Goulet,* Haggag S. Zein,*,x Melisa Hamilton-Valensky,{

Gerald Krystal,{ William G. Kerr,‖,# Noriko Toyama-Sorimachi,** and

Andrew P. Makrigiannis*

Plasmacytoid dendritic cells (pDC) are the major producers of type I IFN during the initial immune response to viral infection.

Ly49Q, a C-type lectin-like receptor specific for MHC-I, possesses a cytoplasmic ITIM and is highly expressed on murine pDC.

Using Ly49Q-deficient mice, we show that, regardless of strain background, this receptor is required for maximum IFN-a

production by pDC. Furthermore, Ly49Q expression on pDC, but not myeloid dendritic cells, is necessary for optimal IL-12

secretion, MHC-II expression, activation of CD4+ T cell proliferation, and nuclear translocation of the master IFN-a regulator

IFN regulatory factor 7 in response to TLR9 agonists. In contrast, the absence of Ly49Q did not affect plasmacytoid dendritic cell–

triggering receptor expressed on myeloid cells expression or pDC viability. Genetic complementation revealed that IFN-a pro-

duction by pDC is dependent on an intact tyrosine residue in the Ly49Q cytoplasmic ITIM. However, pharmacological inhibitors

and phosphatase-deficient mice indicate that Src homology 2 domain-containing phosphatase 1 (SHP)-1, SHP-2, and SHIP

phosphatase activity is dispensable for this function. Finally, we observed that Ly49Q itself is downregulated on pDC in response

to CpG exposure in an ITIM-independent manner. In conclusion, Ly49Q enhances TLR9-mediated signaling events, leading to

IFN regulatory factor 7 nuclear translocation and expression of IFN-I genes in an ITIM-dependent manner that can proceed

without the involvement of SHP-1, SHP-2, and SHIP. The Journal of Immunology, 2013, 190: 3994–4004.

Plasmacytoid dendritic cells (pDC) are a subset of dendriticcells (DC) that are specialized in direct virus recognitionand secretion of large amounts of type I IFN (IFN-I) (1).

pDC represent ,1% of lymphoid organ cells and are primarilylocated in the T cell areas of the spleen, lymph nodes, and Peyer’spatches (1, 2). They can secrete up to 1000 times more IFN-I thanother cell types in response to microbial challenge (3). This propertyof pDC makes them an important component of the innate immuneresponse against viruses. In addition, murine pDC can secrete IL-12,IL-6, and TNF-a, and acquire the ability to directly stimulate T cellproliferation (4, 5). Ablation of pDC in mice leads to enhanced viralburden and impaired virus-specific cytotoxic T cell responses (5).pDC detect the presence of microbial nucleic acids through TLRs,which are a subset of pattern recognition receptors that recognizepathogen-associated molecular patterns. pDC selectively expressTLR7 and TLR9, which recognize ssRNA and unmethylated CpGdinucleotides of dsDNA, respectively (6).Ly49Q is a member of the large Ly49 family of type II integral

membrane receptors with extracellular C-type lectin domains (7).Genes encoding Ly49 proteins, termed Klra, are clustered togetherin the NK gene complex on mouse chromosome 6, and the geneencoding Ly49Q is part of a set of framework genes that areconserved in all known mouse Ly49 haplotypes, whereas otherLy49 genes are highly polymorphic and present in variable num-bers (8). With the exception of Ly49Q, Ly49B, and Ly49E, allother Ly49 family members are primarily expressed on NK andNKT cells (9, 10). Ly49Q is expressed on Gr1+ myeloid lineagecells in the bone marrow, spleen, and peripheral blood in all strainsof mice tested (7, 11). Virtually all pDC in blood, lymph nodes,spleen, and a subset of pDC in the bone marrow express Ly49Q(12). Furthermore, Ly49Q expression on pDC correlates with se-quential development and activation (11–13). In macrophages,

*Department of Biochemistry, Microbiology, and Immunology, University of Ottawa,Ottawa, Ontario K1H 8M5, Canada; †Centre for Innovative Cancer Research, OttawaHospital Research Institute, Ottawa, Ontario K1Y 4E9, Canada; ‡College of AppliedMedical Sciences, Taibah University, 30001 Madinah Munawwarah, Kingdom ofSaudi Arabia; xCairo University Research Park, Faculty of Agriculture, Cairo Uni-versity, Giza 12613, Egypt; {Terry Fox Laboratory, BC Cancer Agency, Vancouver,British Columbia V5Z IL3, Canada; ‖Department of Pediatrics, State University ofNew York Upstate Medical University, Syracuse, NY 13210; #Department of Micro-biology and Immunology, State University of New York Upstate Medical University,Syracuse, NY 13210; and **Department of Molecular Immunology and Inflamma-tion, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan

1M.M.A.R. and L.-H.T. contributed equally to this work.

Received for publication March 20, 2012. Accepted for publication February 7, 2013.

This work was supported by Operating Grant MOP 62841 from the Canadian In-stitutes of Health Research (to A.P.M.). A.P.M. is a Tier II Canada Research ChairHolder in Innate Pathogen Resistance.

Address correspondence and reprint requests to Dr. Andrew P. Makrigiannis, Depart-ment of Biochemistry, Microbiology, and Immunology, University of Ottawa, RogerGuindon Hall, Room 4226, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada. E-mailaddress: amakrigi@uottawa.ca

Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; BST2, bonemarrow stromal Ag 2; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-trimethylam-monium-propane; ILT7, Ig-like transcript 7; IRF, IFN regulatory factor; KIR, killerIg-like receptor; mDC, myeloid dendritic cell; mev, viable motheaten; ODN, oligo-deoxyribonucleotide; pDC, plasmacytoid DC; PDC-TREM, PDC-triggering receptorexpressed on myeloid cells; PTP, protein tyrosine phosphatase; SH2, Src homology 2;SHP, SH2 domain-containing phosphatase 1; SSG, sodium stibogluconate; Tg, trans-genic; WT, wild-type.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

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Ly49Q expression is increased by IFN-g–induced activation, andits cross-linking induces cytoskeletal rearrangement (7). Similarly,Ly49Q regulates neutrophil polarization and migration to sites ofinflammation (14). Ly49Q also regulates osteoclast differentiationin vitro (15).Colorimetric reporter cell assay and class I MHC (MHC-I)

tetramer-binding analyses have revealed the MHC-I H-2Kb mol-ecule as a ligand for Ly49Q (16). Experiments with Ly49Q-defi-cient mice as well as pDC from wild-type (WT) mice have shownthat, despite the presence of an ITIM in the cytoplasmic domain ofLy49Q, it is stimulatory in nature and required for TLR-mediatedIFN-I production by pDC (17). Specifically, the IFN-a and IL-12produced by ex vivo pDC stimulated with CpG oligodeoxy-ribonucleotide (ODN) can be blocked with soluble mAb to Ly49Qor H-2Kb. In contrast, receptor cross-linking with plate-boundanti-Ly49Q or with recombinant H-2Kb increased cytokine pro-duction (17). In line with these observations, pDC isolated fromLy49Q-deficient mice displayed a significant defect in IFN-a andIL-12 secretion in response to CpG ODN or influenza virus com-pared with WT pDC (17). Furthermore, sera obtained from CpG-injected Ly49Q-deficient mice displayed reduced IFN-a levels.In contrast, TNF-a and IL-6 production was not compromised inLy49Q-deficient pDC (17). Thus, despite the presence of an ITIMin its cytoplasmic domain, the Ly49Q receptor is stimulatory innature and required for TLR-mediated IFN-I production by pDC.In contrast, receptors that signal through ITAM-mediated path-ways negatively regulate cytokine production by pDC. These recep-tors include Ig-like transcript 7 (ILT7), bone marrow stromal Ag 2(BST2; BDCA-2; CD317), Nkp44 on human pDC, and SiglecH onmouse pDC, all of which signal through ITAM-containing adap-tors such as DAP12 and FcεRIg (18–20). Accordingly, DAP12-deficient pDC display increased cytokine production in responseto viral infections (21). However, an exception is PDC-triggeringreceptor expressed on myeloid cells (PDC-TREM), a member ofthe triggering receptor expressed on the myeloid cell family, whichis expressed on activated pDC and augments IFN-I production ina DAP12-dependent manner (22).In a follow-up study, we demonstrated a novel mechanism by

which TLR9 signaling is controlled through the spatiotemporalregulation of membrane trafficking by the ITIM-bearing receptorLy49Q. Macrophages and pDC lacking Ly49Q showed altered dis-tribution of TLR9 and CpG ODN (23). In particular, CpG ODN-induced tubular endolysosomal extension was impaired in the ab-sence of Ly49Q. Consistent with these findings, cells lackingLy49Q showed impaired cytokine production in response to CpGODN (13).Protein tyrosine phosphatases (PTPs) play an integral role in the

regulation of immune cell activation. Immune cells express a re-markably large number of PTPs, highlighting their importance inimmune cell function (24). A number of inhibitory immune cellreceptors, including human killer Ig-like receptor (KIR) andmouse Ly49 NK receptors, possess ITIMs that, when phosphor-ylated, bind to Src homology 2 (SH2) domain-bearing PTPs (25).The majority of these PTPs inhibit activation of immune cells(24). Human KIR and mouse Ly49 ITIMs, upon phosphorylation,recruit SH2 domain-containing phosphatase 1 (SHP)-1 and SHP-2to inhibit NK cells (26–28). Some Ly49 can also recruit SHIPthrough their ITIM (29). Ly49B is expressed on a subpopulationof myeloid cells and is shown to associate with SHP-1, SHP-2, andSHIP in transfected cells (9). SHP-1 plays an important role in theregulation of cytokine production. Paradoxically, whereas SHP-1negatively regulates production of proinflammatory cytokines inlymphocytes, it has activating function in the production of IFN-I,thus contributing to immune homeostasis (30). Similarly, Ly49Q

has been shown to recruit SHP-1 and SHP-2 in an ITIM phosphor-ylation-dependent manner (7). We have shown the significance ofLy49Q phosphatase recruitment in neutrophil studies. In thesteady state, Ly49Q inhibits neutrophil adhesion by preventingfocal-complex formation. However, in the presence of inflamma-tory stimuli, Ly49Q mediates rapid neutrophil polarization andtissue infiltration in an ITIM domain–dependent manner (14).These functions appear to be mediated by distinct use of the effectorphosphatases SHP-1 and SHP-2. Ly49Q-dependent neutrophil po-larization and migration were affected by Ly49Q regulation ofmembrane raft functions (14). However, the significance of phos-phatase recruitment in pDC function is unknown.Members of the IFN regulatory factor (IRF) family of tran-

scription factors are critical for induction of the IFN-I response (31).Specifically, IRF3 and IRF7 enhance IFN-I mRNA expressionwhen expressed ectopically (32, 33). IRF7 expression is induced byIFN-a and regulates both late-phase induction as well as positivefeedback induction of the IFN-a response (34). In contrast, IRF3 isconstitutively expressed and regulates IFN-b and the early phase ofIFN-a induction (32). Hence, IRF3 and IRF7 perform distinctnonredundant functions in the induction of IFN-I genes (35). IRF7is critical for both virus-activated MyD88-independent as well asTLR-activated MyD88-dependent induction of IFN-I (36). In pDC,TLR9-mediated IFN-I production is dependent on IRF7 (36).The present study investigates the mechanism of Ly49Q aug-

mentation of the TLR-mediated IFN-I response by pDC. We dem-onstrate that the defect in IFN-a production by pDC from Ly49Q-deficient mice is restored by expression of WT Ly49Q, but not bya Ly49QYF mutant in which the tyrosine residue in the ITIM hasbeen mutated to phenylalanine. These findings confirm the criticalfunction of Ly49Q in the regulation of IFN-a production by pDC,and that this mechanism of cytokine induction is dependent on thecytoplasmic domain ITIM of Ly49Q. We also show that Ly49Qpositive regulation of IFN-a production by pDC is mediatedthrough activation and translocation of IRF7 to the nucleus. Inaddition, we investigated the role of SH2 domain–bearing phos-phatases in this process.

Materials and MethodsMice

Ly49Q-null mice were described previously (22). Ly49QWT and Ly49QYF

transgenic (Tg) mice were generated as previously described (14). Viablemotheaten (mev) mice were obtained from The Jackson Laboratory (BarHarbor, ME). SHIPflox/flox mice were described previously (29). These micewere bred with CMV-cre Tg mice (The Jackson Laboratory) to generateSHIP2/2 mice. All mice were maintained in a specific pathogen-free en-vironment. All breeding and manipulations performed on animals were inaccordance with university guidelines and approved by the University ofOttawa Animal Ethics Committee.

Flow cytometry

The following Abs were obtained from commercial sources: allophy-cocyanin–mPDCA-1 (anti–BST2; Miltenyi Biotec), FITC-440c (anti-SiglecH), biotin-MHCII, FITC-CD11c, allophycocyanin-CD45R (anti-B220) (eBioscience), FITC-2E6 (anti-Ly49Q) (MBL International),PE-PDC-TREM (BioLegend), and PE-mouse IL-12 (BD Pharmingen).PE-streptavidin and 7-aminoactinomycin D (7-AAD) were purchasedfrom eBioscience. Biotin-NS34 (anti-Ly49Q) was generated as previouslydescribed (7). Splenocytes were prepared by injecting the spleens withcollagenase-D (Roche). The injected spleens were minced and incubated in5 ml collagenase-D (0.5 mg/ml) for 20 min at 37˚C. EDTA (Life Tech-nologies) was added at 1 mM concentration and incubated for additional 5min to dislodge cells. Spleen fragments were crushed and passed througha 70-mm cell strainer (Fisher Scientific) to remove large debris. RBCs werelysed with ACK lysis buffer for 5 min at 4˚C. Cells were washed and kept incold PBS. For FACS analysis, cells were washed with FACS buffer (PBS,0.5% BSA, 0.02% NaN3) and incubated with fluorochrome-conjugated Abs

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for 20 min at 4˚C. For secondary staining, cells were washed in FACS bufferand further incubated with the appropriate Abs for 20 min at 4˚C. For in-tracellular staining, cells were fixed with Cytofix/Cytoperm reagent (BDBiosciences) and stained following manufacturer’s instructions. To staindead/apoptotic cells, 7-AAD was added at 0.25 mg/106 cells and incubatedfor 5 min at room temperature. Cells were finally washed in FACS bufferand analyzed with a CyAN flow cytometer and Kaluza software (BeckmanCoulter).

In vivo pDC stimulation

Mice were injected i.v. through the tail vein with 10 mg CpG-B (ODN 2006)or CpG-A DNA (ODN 2216) (Hycult Biotech) and 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP; Roche) preparation (170 ml CpGODN diluted in sterile PBS plus 30 ml DOTAP). Serum was prepared fromblood obtained through the cheek vein 6 h after CpG ODN injection.Serum cytokine levels were analyzed by ELISA, as previously described(17), or FlowCytomix bead assay (eBioscience) following the manu-facturer’s instructions.

In vitro pDC and DC2.4 stimulation

pDC were isolated from spleens using an AutoMACS-pro instrument(Miltenyi Biotec) and pDC isolation kit-II (Miltenyi Biotec), according tothe manufacturer’s protocol. Isolated pDC were seeded at 105 cells/well ina flat-bottom 96-well plate in 200 ml pDC medium (RPMI 1640, 10% FBS,100 U/ml penicillin, 100 mg/ml streptomycin, 2.05 mM L-glutamine, and55 mM 2-ME) for 16 h at 37˚C and 5% CO2. CpG ODN, at the indicatedconcentrations, and sodium stibogluconate (SSG; 10 or 100 mg/ml; Cal-biochem) were added in the corresponding wells. Culture supernatant wascollected and stored at 280˚C for analysis with ELISA or FlowCytomixbead assay. For surface Ly49Q analysis, isolated pDC were treated withCpG-A, CpG-B ODN (10 mg/ml), or LPS (1 mg/ml), as above, for 4 h. TheDC2.4 cell line was transfected with Ly49Q (B6 allele) or ITIM-mutatedLy49QYF using lipofectamine transfection reagent (Invitrogen), and stableclones were selected. Cells were treated with CpG ODN (10 mg/ml), LPS(1 mg/ml), poly(I:C) (15 mg/ml), or imiquimod (3 mg/ml) in vitro for 4 h.Ly49Q and MHC-I expression was analyzed by flow cytometry.

Immunofluorescent staining and confocal microscopy

In vivo stimulated pDC were isolated with an AutoMACS-pro instrument,as described above. pDC were resuspended in PBS and cytospun onto glassslides (Fisher Scientific) at 800 rpm for 3 min. Slides were allowed to dryovernight and fixed in methanol for 10 min at 220˚C. Following threewashes with PBS, cells were permeabilized with 0.2% Triton X-100 inPBS for 10 min at room temperature. Slides were washed three times withPBS and blocked with 5% BSA in PBS containing 0.2% Tween 20 (PBS-T) for 1 h at room temperature. Slides were then incubated with a 1:250dilution of rabbit polyclonal Ab against mouse IRF7 (Abcam) in blockingbuffer for 2 h at room temperature. After three washes with PBS-T, slideswere incubated with a 1:500 dilution of Alexa-488–conjugated anti-rabbitIgG Ab (Invitrogen) in PBS-T with 3% BSA for 45 min at room tem-perature. Slides were then washed with PBS-T and incubated with DAPI(Invitrogen) at 1 mg/ml in PBS for 10 min at room temperature. Finally,slides were washed three times in PBS and mounted with a coverslip usingMowiol 4-88 mounting medium (Calbiochem) following the manu-facturer’s instructions. After setting for 24 h at 4˚C, confocal images wereacquired with a laser-scanning microscope LSM 510 META (Zeiss) usingthe 340 oil-emersion objective.

IFN-a measurement

Mouse IFN-a concentration in culture supernatant and serum was deter-mined by sandwich ELISA, as described previously (17), or by Flow-Cytomix bead assay (eBioscience). For ELISA, samples were analyzed at1:10 dilution and OD was read at 450 nm with a PowerWave XS2microtitre plate reader (BioTek). FlowCytomix bead assay was performedas per the manufacturer’s instructions and analyzed with a CyAN flowcytometer (Beckman Coulter) and FlowCytomix Pro 2.2 software (eBio-science).

Western blot analysis

To increase pDC numbers, mice were injected s.c. with B16-Flt3L cells (agenerous gift of S. Vidal, McGill University, Montreal, Montreal, QC, Canada),as described elsewhere (37). pDC were isolated from spleens with anAutoMACS-pro instrument, as described above. Proteins were extracted fromisolated pDC using radioimmunoprecipitation assay buffer that contained 50mM Tris (pH 8), 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40 detergent, and 0.1% SDS in presence of proteinase inhibitor mixture

(Calbiochem). The amount of protein in the lysate was determined by Bio-Rad protein assay reagent (Bio-Rad), as per the manufacturer’s instruc-tions. Protein samples were resolved on SDS-PAGE and transferred toa membrane. The membranes were blotted with Abs against IRF7 (Abcam),GAPDH (Abcam), SHP-1, SHP-2, and SHIP (generous gifts of A. Veil-lette, Institut de Recherches Clinique de Montreal, Montreal, QC, Canada).Signals were detected with HRP-conjugated secondary Ab and ECL PlusWestern blot detection system (Amersham).

T cell proliferation assay

Sorted pDC and myeloid dendritic cells (mDC) were seeded at 5 3 104

cells/well of a 96-well round-bottom plate. Sorted BALB/c CD4+ T cellswere seeded at 1.5 3 105 cells/well. CpG-B ODN (3 mg/ml) was added tocorresponding wells. Cultures were maintained for 3 d at 37˚C, and 1 mCi[3H]thymidine (Beckman Coulter) was added for the last 16 h of incubation.Cultures were harvested on filter plates (Perkin-Elmer) using Unifilter-96Harvester (Perkin-Elmer), and [3H]thymidine incorporation was measuredwith Packard TopCount NKT scintillation counter (Perkin-Elmer).

Statistical analysis

Unless otherwise indicated, all p values were calculated with a two-tailedStudent t test.

ResultsDefective TLR9-mediated IFN-a production byLy49Q-deficient pDC on the B6 genetic background

We have previously shown a profound defect in TLR-mediated IFN-a production by pDC lacking Ly49Q (17). For the purpose of thecurrent study, the Ly49Q-deficient mice, which were originallygenerated on the 129S1 genetic background, were bred onto theC57BL/6 (B6) genetic background for 12 generations. These miceare essentially congenic for the region of mouse chromosome 6,which contains the Klra (Ly49) gene cluster and the disrupted Ly49q1gene from 129-strain mice (data not shown). B6.Ly49129 congenicmice, which have the 129S1-derived Ly49 gene cluster on a B6genetic background, were used as WT controls (38). Mouse strain-specific differences in secretion of IFN-a by pDC are well docu-mented. In particular, 129-strain mice produce higher levels of IFN-athan B6 mice when challenged with CpG ODN (2). The productionof IFN-a after systemic stimulation by CpG ODN is due solely topDC, as shown by IFN-a enhanced GFP knock-in reporter mice (39).To determine whether the strain-specific differences in IFN-a

production are due to Ly49 haplotype variation, splenic pDC wereisolated from B6, 129S1, and B6.Ly49129 congenic mice bynegative selection and stimulated overnight with CpG-B ODN.IFN-a levels were analyzed in the culture supernatant by ELISA.As expected, B6 pDC produced significantly lower levels of IFN-aas compared with 129S1 pDC (Fig. 1A). pDC from B6.Ly49129S1

congenic mice produced IFN-a levels similar to that of B6 mice(Fig. 1A), suggesting that the background of the NK gene complexgenes, including Ly49, does not affect pDC IFN-a production. Todetermine IFN-a production capacity in Ly49Q-deficient mice ona B6 background, B6.Ly49129S1 congenic (WT) and Ly49Q-defi-cient mice were injected with CpG ODN i.v., and serum IFN-alevels were evaluated after 6 h. As expected, CpG-AODN inducedhigher serum IFN-a levels than CpG-B ODN treatment in WTmice. Serum IFN-a levels were significantly lower in Ly49Q-deficient compared with the WT mice when treated with CpG-Aas well as CpG-B ODNs (Fig. 1B). We also compared IFN-aproduction capacity of pDC isolated from Ly49Q-deficient miceon B6 background when stimulated overnight with various con-centrations of CpG ODN in vitro. Ly49Q-deficient pDC consis-tently produced significantly reduced levels of IFN-a as comparedwith the pDC from WT control mice at various concentrations ofCpG-B ODN (Fig. 1C). A similar defect in IFN-a production byLy49Q-deficient pDC was observed when stimulated with CpG-A

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ODN at low concentrations (Fig. 1D). At higher concentration (10mg/ml), CpG-A ODN was able to induce significant levels of IFN-a production by Ly49Q-deficient pDC in vitro, almost approach-ing the levels produced by WT pDC (Fig. 1D). These data togetherconfirm our previous finding that Ly49Q positively regulatesTLR9-mediated IFN-a production by pDC. Furthermore, thisfunction is conserved in mice of different genetic backgrounds andwith different CpG ODN types. Because CpG-B ODN consistentlyinduce good levels of IFN-a production by WT and not Ly49Q-deficient pDC, in vitro as well as in vivo, it was used for the re-mainder of this study.

Defective IL-12 production and T cell stimulation byLy49Q-deficient pDC, but not mDC

In addition to IFN-a, TLR9 stimulation also induces IL-12 pro-duction by murine pDC (4, 5). We previously reported lower serumIL-12 levels in Ly49Q-deficient compared with WT mice on the129S1 background (17). To test IL-12 production by Ly49Q-deficient mice on B6 background, WT and Ly49Q-deficient micewere treated with CpG-B ODN in vivo for 6 h and flow cytometryanalysis was performed to detect intracellular IL-12 in pDC andmDC ex vivo. Although CpG-B ODN treatment induced IL-12production by WT pDC, it failed to do so in pDC from Ly49Q-deficient mice (Fig. 2A). In contrast, equal numbers of mDC fromWTand Ly49Q-deficient mice stained positively for intracellular IL-12 (Fig. 2B). These data demonstrate a requirement for Ly49Q inTLR9-induced IL-12 production specifically in pDC and not mDC.

In vitro treatment with CpG-B ODN has been shown to inducepDC maturation and acquisition of the ability to stimulate T cellproliferation (40, 41). We measured class II MHC (MHC-II) levelson pDC from WT and Ly49Q-deficient mice treated with CpG-BODN in vitro. MHC-II upregulation was almost 50% lower onLy49Q-deficient pDC compared with WT pDC (Fig. 2C). To de-termine whether this corresponded to a lower T cell stimulationcapacity in Ly49Q-deficient pDC, a mixed lymphocyte reactionwas performed in which pDC or mDC from WT and Ly49Q-de-ficient mice were cultured with sorted BALB/c CD4+ T cells in thepresence of CpG-B ODN, and T cell proliferation was measuredby radioactive thymidine incorporation. In agreement with pre-vious reports (42), the ability of pDC to stimulate T cells is muchlower than mDC. Whereas mDC from both WT and Ly49Q-deficient mice induced equally high levels of CD4+ T cell pro-liferation, Ly49Q-deficient pDC were significantly less efficient ininducing CD4+ T cell proliferation compared with WT pDC whentreated with CpG-B ODN in vitro (Fig. 2D). CpG-B ODN treat-ment alone did not induce proliferation on mDC, pDC, or CD4+

T cells in this assay. These data suggest that CpG-B ODN-inducedmaturation and acquisition of T cell proliferation capacity aredisrupted in Ly49Q-deficient pDC.

Normal in vivo pDC activation and survival in Ly49Q-deficientmice

Reduced IFN-a production by Ly49Q-deficient pDC could be dueto insufficient activation or survival of pDC lacking Ly49Q. pDC

FIGURE 1. Ly49Q deficiency results in defective CpG ODN-induced IFN-a production by pDC irrespective of genetic background. Splenic pDC were

isolated and treated with CpG ODN in vitro at the indicated concentrations. In vivo stimulation for 6 h was performed with CpG ODN + DOTAP via tail

vein injection. IFN-a was measured in culture supernatant and serum by ELISA or bead assay. (A) IFN-a production by isolated pDC from B6, 129S1, and

B6.Ly49129 congenic mice after CpG-B ODN stimulation in vitro. (B) Blood IFN-a levels from Ly49Q+/+ and Ly49Q2/2 B6-background mice 6 h after

in vivo CpG-A and CpG-B ODN + DOTAP stimulation. (C) IFN-a production by isolated pDC from Ly49Q+/+ (B6.Ly49129) and Ly49Q2/2 (B6 back-

ground) mice after CpG-B ODN stimulation in vitro. (D) IFN-a production by isolated pDC from Ly49Q+/+ and Ly49Q2/2 mice after CpG-A ODN

stimulation in vitro. Each symbol represents the data from an individual mouse. The mean is indicated by a small horizontal bar. All p values are indicated.

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activation was assessed by flow cytometric analysis of PDC-TREMexpression after in vivo CpG-B ODN challenge. PDC-TREM isexpressed on pDC activated by TLR7 and TLR9 ligands, andpositively regulates IFN-a production (22). Ly49Q-deficient andWT mice were injected i.v. with CpG-B ODN, and PDC-TREM ex-pression on pDC was analyzed 6 h later by flow cytometry. CpG-BODN challenge induced expression of PDC-TREM on pDC.However, no difference in the percentage of pDC expressing PDC-TREM after CpG-B ODN challenge was observed between WTand Ly49Q-deficient mice (Fig. 3A), in agreement with priorstudies of CD86 expression (17). These data suggest that the effectof Ly49Q on IFN-a production by pDC is independent of expres-sion of activation markers on pDC.pDC survival after in vivo CpG-B ODN challenge was assessed

by 7-AAD staining and flow cytometry. Ly49Q-deficient and WTmice were injected i.v. with CpG-B ODN, and pDC were stainedwith 7-AAD after 6 h. CpG-B ODN challenge induced pDC cell

death to the same level in WT and Ly49Q-deficient mice (Fig. 3B).This led to a similar decrease in splenic pDC numbers in WT andLy49Q-deficient mice after CpG-B ODN challenge (Fig. 3C). Asrecently reported (43), pDC activation via TLR9 resulted in in-creased numbers of dying pDC. No significant differences in thefrequency of apoptotic/dead pDC were observed prior to or afterCpG-B ODN challenge between WT and Ly49Q-deficient mice.Together these results suggest normal activation and survival ofpDC in the absence of Ly49Q expression.

Essential role of the Ly49Q ITIM in TLR9-mediated IFN-aproduction by pDC

Ly49Q possesses a cytoplasmic ITIM that is able to recruitphosphatases when phosphorylated (7). To determine the necessityof the Ly49Q ITIM in augmentation of TLR9-mediated IFN-aproduction by pDC, Ly49Q-deficient mice were bred with mice Tgfor WT (Ly49QWT) or ITIM-mutated (Ly49QYF) Ly49Q in which

FIGURE 2. Ly49Q deficiency results in defective IL-

12 production and CD4+ T cell proliferation by pDC,

but not mDC. Mice were injected (i.v.) with CpG-B

ODN + DOTAP for 6 h. IL-12 was detected by intra-

cellular flow cytometric analysis on pDC (Siglec-H+

BST2+) and mDC (CDllc+B2202). (A) Intracellular

detection of IL-12 in pDC from Ly49Q+/+ and Ly49Q2/2

mice after in vivo CpG-B ODN stimulation. (B) Intra-

cellular detection of IL-12 in mDC from Ly49Q+/+ and

Ly49Q2/2 mice after CpG-B ODN stimulation. (C) Mean

fluorescence intensity (MFI) of class II MHC (MHC-II),

measured by flow cytometric analysis, on isolated pDC

from Ly49Q+/+ (black bars) and Ly49Q2/2 mice (white

bars) after CpG-B ODN stimulation in vitro. (D) Pro-

liferation of BALB/c splenic CD4+ T cells incubated

with isolated pDC or mDC in the presence of CpG-B

ODN. T cell proliferation was measured by [3H]thymidine

incorporation. Controls include mDC alone, pDC alone,

or T cells alone. Black bars represent WT; white bars

represent Ly49Q-deficient mice.

FIGURE 3. pDC activation and survival are not affected by Ly49Q deficiency. Ly49Q+/+ and Ly49Q2/2 mice were injected (i.v.) with CpG-B ODN +

DOTAP for 6 h. Mice not injected with CpG-B + DOTAP were used as unstimulated (Unstim.) controls. (A) Surface expression of PDC-TREM on splenic

pDC (Siglec-H+BST2+) as measured by flow cytometry to assess pDC activation. (B) The 7-AAD staining of splenic pDC (Siglec-H+BST2+) as measured

by flow cytometry to assess apoptotic/dead cells. (C) pDC (Siglec-H+BST2+) numbers in the spleen. Data represent the mean value 6 SD of four to eight

samples from individual mice.

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the tyrosine residue in the ITIM is substituted with phenylalanineto generate Ly49Q2/2.QWT Tg and Ly49Q2/2.QYF Tg mice, respec-tively. Ly49Q expression on pDC was assessed ex vivo by flowcytometry and was found to be restored to normal levels on Ly49Q-deficient pDC carrying the Ly49QWT or Ly49QYF Tg (Fig. 4A).To determine whether Ly49Q Tg expression restored IFN-a

production in Ly49Q-deficient pDC, WT, Ly49Q-deficient,Ly49Q2/2.QWT Tg, and Ly49Q2/2.QYF Tg mice on a B6 back-ground were injected with CpG-B ODN i.v. and serum IFN-alevels were evaluated after 6 h. Expression of the Ly49QWT Tgalmost completely restored IFN-a production by Ly49Q-deficientpDC to the same level as the WT pDC (Fig. 4B). In contrast, theITIM-mutated Ly49QYF Tg expression failed to restore IFN-aproduction by Ly49Q-deficient pDC (Fig. 4B). Similar resultswere obtained with pDC isolated from WT, Ly49Q-deficient,Ly49Q2/2.QWT Tg, and Ly49Q2/2.QYF Tg mice treated withCpG-B ODN in vitro (Fig. 4C). These data confirm the necessaryrole of Ly49Q in the regulation of IFN-a production by pDC.Furthermore, the cytoplasmic ITIM of Ly49Q is shown to becritical for CpG-mediated IFN-a production by pDC.

Role of SH2 domain–bearing phosphatases in IFN-aproduction by pDC

The Ly49Q ITIM recruits SH2 domain–bearing phosphataseSHP-1 and SHP-2 when phosphorylated (7). Therefore, we inves-

tigated the possible role of these phosphatases in the regulation of

IFN-a production by pDC. First, the expression of phosphatases in

pDC was determined by Western blot analysis. To obtain enough

pDC for this assay, B6.Ly49129 congenic mice were injected s.c.

with B16-Flt3L cells, which have been shown to increase pDC

numbers in mice (37). Western blot analysis of splenic pDC

protein extracts showed the presence of SHP-1, SHP-2, and SHIP

phosphatases in these cells (Fig. 5).We next evaluated the role of these phosphatases in pDC IFN-a

production with the use of SHP-1–mutant meV mice (44), as well

as SSG, a chemical inhibitor of SHP-1 and SHP-2 (45). Splenic

pDC were isolated from meV mice and put in culture overnight

with various concentrations of CpG-B ODN. meV pDC produced

similar levels of IFN-a as compared with WT pDC at all CpG-B

ODN concentrations tested (Fig. 6A). This suggests that SHP-1

FIGURE 4. Ly49Q ITIM-dependent res-

cue of defective IFN-a production by Ly49Q-

deficient pDC. Ly49Q2/2 mice were bred

with Ly49QWT Tg (Ly49Q2/2.QWT Tg) and

Ly49QYF Tg (Ly49Q2/2.QYF Tg) mice to

restore Ly49Q expression on pDC. (A)

Scatter plots represent flow cytometric anal-

ysis of Ly49Q surface expression on splenic

pDC from the indicated mouse strains. Mean

fluorescence intensity (MFI) is indicated. (B)

Mice of the indicated genotypes were injec-

ted (i.v.) with CpG-B ODN + DOTAP. After

6 h, serum IFN-a levels were measured by

ELISA. (C) IFN-a production by isolated

pDC from mice of the indicated genotypes

after CpG-B ODN stimulation in vitro. IFN-a

levels were measured in culture supernatant

by bead assay. The horizontal bar represents

mean values. Each symbol represents the

data from a single mouse. All p values are

indicated.

FIGURE 5. Expression of SH2-bearing phospha-

tases in murine pDC. Western blot analysis of pDC

protein extracts. Protein samples were resolved by

SDS-PAGE and transferred to a membrane. The mem-

brane was blotted with antisera against SHP-1 and SHIP

(A), or SHP-2 (B) and detected by chemiluminescence.

The membrane was stripped and blotted with Ab against

GAPDH. Protein samples from cell lines (NIH3T3, 293T,

and EL4) were used as controls.

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deficiency alone does not disrupt TLR9-mediated IFN-a produc-tion by pDC. Similarly, when WT pDC were stimulated with CpG-BODN in the presence of SSG at concentrations that inhibitedSHP-1 specifically (10 mg/ml) or at concentrations that inhibit bothSHP-1 and SHP-2 (100 mg/ml), no reduction in IFN-a productionwas observed (Fig. 6B). To evaluate the role of SHIP in pDC IFN-aproduction, splenic pDC were isolated fromWT, ship+/2, and ship2/2

mice and put in culture overnight with CpG-B ODN. SHIP-deficientpDC produced similar levels of IFN-a as compared with WT pDC(Fig. 6C). These experiments demonstrate that SHP-1, SHP-2, andSHIP are individually dispensable for pDC IFN-a production.

Ly49Q-induced augmentation of pDC IFN-a production ismediated through IRF7 transcription factor activation

Transcription factor IRF7 is critical for TLR9-mediated IFN-Iproduction by pDC (36), and is constitutively expressed at highlevels in human pDC (46). We confirmed that IRF7 is also con-stitutively expressed in mouse pDC at high levels by Western blotanalysis of pDC protein extracts (Fig. 7A). To determine whetherIRF7 is involved in Ly49Q-induced augmentation of IFN-a pro-duction by pDC after CpG-B ODN challenge, we assessed IRF7activation and nuclear translocation by immunofluorescent stain-ing and confocal microscopy. WT and Ly49Q-deficient mice wereinjected with CpG-B ODN i.v., and splenic pDC were isolated 6 hlater. Immunofluorescent staining of IRF7 revealed nucleartranslocation of IRF7 in CpG-B ODN-treated WT pDC (Fig. 7B).In contrast, fewer Ly49Q-deficient pDC showed translocation ofIRF7 to the nucleus after CpG-B ODN challenge (Fig. 7B). Thesedata suggest that the signaling events downstream of Ly49Q thatregulate TLR9-mediated IFN-a production by pDC culminate inthe activation of IRF7 and its translocation to the nucleus to in-duce IFN-a expression.

Evidence for a possible role of Ly49Q in CpG ODN trafficking

Our previous study has shown that TLR9 signaling is controlledthrough the spatiotemporal regulation of membrane trafficking by

FIGURE 6. Role of SH2-bearing

phosphatases in CpG-B ODN-in-

duced IFN-a production by pDC.

(A) IFN-a production by SHP-1–

deficient (meV) pDC after in vitro

CpG-B ODN treatment. Splenic pDC

from WT and meV mice were treated

with different concentrations of CpG-

B ODN. IFN-a was measured in

culture supernatant by ELISA. Each

symbol represents the data from an

individual mouse. (B) IFN-a pro-

duction by isolated pDC in the pres-

ence of SSG. Isolated splenic pDC

from WT mice were treated with

different concentrations of CpG-B

ODN and SSG. IFN-a was measured

in culture supernatant by ELISA. (C)

IFN-a production by SHIP-deficient

(ship2/2) pDC after in vitro CpG-B

ODN treatment. Splenic pDC from

WT, ship+/2, and ship2/2 mice were

treated with CpG-B ODN. IFN-a was

measured in culture supernatant by

bead assay. The horizontal bars rep-

resent mean values. Each symbol

represents the data from pDC isolated

from a single mouse. A representative

experiment from three similar exper-

iments is shown.

FIGURE 7. CpG-B ODN-induced nuclear translocation of IRF7 is de-

fective in Ly49Q-deficient pDC. (A) Western blot analysis of pDC protein

extracts. Protein samples were resolved by SDS-PAGE and transferred to

a membrane. The membrane used in Fig. 5Awas stripped and blotted with

Ab against IRF7 and detected by chemiluminescence. The membrane

subsequently was stripped and blotted with Ab against GAPDH. Protein

samples from cell lines (NIH3T3, 293T, and EL4) were used as controls.

(B) Confocal microscopy imaging of IRF7 nuclear translocation after pDC

stimulation. Ly49Q+/+ and Ly49Q2/2 mice were injected (i.v.) with CpG-B

ODN + DOTAP. After 6 h, splenic pDC were isolated and cytospun on

glass slides. pDC were stained with anti-IRF7 Ab and a fluorescently la-

beled secondary Ab. Nuclei were visualized with DAPI staining. Confocal

images were acquired with a 340 oil immersion objective. The images are

representative of three independent experiments.

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the ITIM-bearing receptor Ly49Q. In macrophages and pDC, TLR9and CpG distribution is altered in the absence of Ly49Q (23). Arecent study proposed a role for KIR3DL2 in uptake and traf-ficking of CpG ODN in NK cells (47). KIRs are the functionalhomologs of mouse Ly49 receptors on NK cells. KIR3DL2 wasdownmodulated from the NK cell surface and colocalized withCpG ODN in early endosomes where TLR9 is encountered. Adirect binding of KIR3DL2 and CpG ODN was also demonstrated(47). To explore the role of Ly49Q in CpG ODN uptake andtrafficking, we used DC2.4 cell line ectopically expressingLy49QWT or ITIM-mutated Ly49QYF on the cell surface. We ob-served a dramatic downmodulation of Ly49Q from cell surface inpresence of CpG-A and CpG-B ODN (Fig. 8A, 8C). This down-modulation was seen as early as 2 h after CpG ODN treatment(data not shown). Similar downmodulation of ITIM-mutatedLy49QYF was also observed in the presence of CpG ODN, indi-cating an ITIM-independent mechanism (Fig. 8A, 8C). Ly49Qwas not downmodulated in presence of other TLR ligands such asLPS, poly(I:C), and imiquimod (Fig. 8B). In contrast to Ly49Qdownmodulation, MHC-I surface expression on the same cellswas unaffected in the presence of any of the TLR ligands tested

(Fig. 8A, 8B). When isolated pDC from the BALB/c mouse strainwere treated similarly, a slight but significant downmodulation ofLy49Q surface expression was observed with CpG-A and CpG-BODN treatments, but not with LPS (Fig. 8C). These data suggesta possible role for Ly49Q in recognition and internalization ofCpG ODN from the cell surface, which is independent of theITIM. Further experiments are required to determine the nature ofbinding (direct versus indirect) and the role of Ly49Q and its ITIMin the trafficking of CpG ODN to early endosomes.

DiscussionWe have previously shown that Ly49Q–MHC-I interaction posi-tively regulates TLR-mediated cytokine production by pDC (17).Furthermore, we have reported that Ly49Q colocalizes with CpGODN in the endosome/lysosome compartment of peritoneal macro-phages and the murine macrophage cell line RAW264 (23). Thiscolocalization appears to be crucial for intracellular trafficking ofCpG ODN and TLR9 and cytokine production in response to CpGODN (23). The present study confirms and extends these findingsby demonstrating the critical function of the Ly49Q cytoplasmicdomain ITIM in the regulation of IFN-a production by pDC

FIGURE 8. Modulation of Ly49Q surface expression in response to TLR9 agonists. DC2.4 cell line expressing Ly49QWT or ITIM-mutated Ly49QYF was

treated with (A) CpG-A and CpG-B ODN, and (B) LPS, poly(I:C), and imiquimod for 4 h. Surface expression of Ly49Q and MHC-I was analyzed by flow

cytometry and presented as histograms. (C) Relative Ly49Q mean fluorescence intensity (MFI) analyzed by flow cytometry on DC2.4 cell line expressing

Ly49QWT or ITIM-mutated Ly49QYF treated with CpG-A, CpG-B ODN, LPS, poly(I:C), and imiquimod for 4 h. (D) Isolated pDC from the BALB/c mouse

strain were treated with CpG-A, CpG-B ODN, or LPS for 4 h. Surface expression of Ly49Q was analyzed by flow cytometry and is presented as histograms

(left panel). Relative Ly49QMFI is presented as bar graph (right panel). One representative experiment of three independent experiments is shown. p values

are indicated.

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through Tg complementation. Specifically, we observed a failureof Ly49QYF mutant receptor to restore IFN-a production inLy49Q-deficient mice when stimulated with CpG-B ODN (Fig.4B, 4C). WT Ly49Q transgene expression, in contrast, fully re-stored this function in Ly49Q-deficient mice. We also demonstratethat Ly49Q augmentation of the TLR-mediated IFN-I response bypDC is mediated through activation and translocation of IRF7transcription factor to the nucleus (Fig. 7B). In addition, Ly49-deficient pDC fail to produce IL-12 (Fig. 2A) and failed to acquirethe capacity to stimulate CD4+ T cells in response to CpG-B ODNstimulation (Fig. 2D). However, pDC activation as measured byupregulation of PDC-TREM when stimulated with CpG-B ODNin vivo appears to be independent of IFN-a production and isunaffected by Ly49Q deficiency (Fig. 3A). Thus, unlike ITIM-bearing Ly49 receptors that impart inhibitory signals in NK cells,Ly49Q positively regulates TLR signaling in pDC. In line withthis observation, receptors such as ILT7, BST2, Nkp44, andSiglecH, which signal through ITAM-containing DAP12 andFcεRIg adaptor molecules and are expected to be stimulatory,negatively regulate cytokine production by pDC (19–21).Ly49Q forms a framework gene that is conserved in all known

mouse Ly49 haplotypes (8). Several alleles of Ly49Q have beenreported, some of which contain polymorphisms in the ligand-binding domain, resulting in different binding affinities to MHC-I (11, 16). Our genetic studies with B6.Ly49129 congenic mice,which have the 129S1-derived Ly49 gene cluster on B6 geneticbackground, demonstrate that the difference in the level of IFN-aproduction by different mouse strains is independent of strainorigin of Ly49Q and the rest of Ly49 gene cluster. B6.Ly49129

congenic pDC produce IFN-a to the same level as B6 pDC uponCpG-B ODN stimulation despite having 129S1-derived Ly49 genecluster (Fig. 1A). Finally, we observed that Ly49Q positivelyregulates TLR9-mediated IFN-a production by pDC in two dif-ferent genetic backgrounds, 129S1 and C57BL/6 (Fig. 1B–D),suggesting that this regulation mechanism is not a strain-specificphenomenon. Furthermore, this was evident with both CpG-A andCpG-B ODN stimulations. However, significant levels of IFN-aproduction were induced in Ly49Q-deficient pDC by higherconcentrations of CpG-A ODN when stimulation was performedin vitro only (Fig. 1D). Guiducci et al. (41) have shown that thehigher order structure (multimeric CpG-A, monomeric CpG-B)and endosomal location of CpG ODNs influence pDC responses.We show evidence that Ly49Q may act as sensor for CpG ODNsand aid in their uptake and delivery to endosomal compartments(discussed below). The multimeric structure of CpG-A ODN mayallow its nonspecific uptake by pDC at high concentrationsin vitro. Alternatively, other low-affinity DNA sensors may aid inuptake and shuttling of CpG-A ODN to endosomes in the absenceof Ly49Q when high concentrations of CpG-A ODN are available.This is not seen with CpG-B ODN possibly due to its monomericstructure not being suitable for spontaneous uptake.Ly49Q has been shown to recruit SHP-1 and SHP-2 in an ITIM

phosphorylation-dependent manner (7), and SHP-1 deficiency resultsin reduced TLR-induced IFN-I production in total splenocytes (30).Furthermore, tyrosine phosphorylation plays an important role inmediating signals downstream of Ly49 receptor molecules. In-hibitory human KIR and mouse Ly49 NK receptors possess ITIMsthat, when phosphorylated, bind to SH2 domain–bearing PTPs(25). Recruitment of SH2 domain–bearing PTPs SHP-1 and SHP-2by KIR and Ly49 ITIMs results in inhibition of NK cell functions(26–28). Our studies with SHP-1–mutant meV mice and SHP-1/SHP-2 chemical inhibitor (SSG) did not provide conclusive evi-dence for the involvement of these two phosphatases in Ly49Q-mediated enhancement of IFN-a production in pDC. pDC from

meV mice and WT pDC stimulated with CpG-B ODN in thepresence of inhibitor SSG did not show any significant defect inIFN-a production (Fig. 6A, 6B). Other SH2 domain–bearingphosphatases, such as SHIP, have been shown to be recruited bythe phosphorylated ITIM of other Ly49 (29). IFN-a production bySHIP-deficient pDC is also normal in response to CpG-B ODNstimulation (Fig. 6C). In contrast to our results, An et al. (30) haveshown that SHP-1 increases TLR-mediated production of IFN-Iby inhibiting activation of kinase IRAK1. However, these experi-ments were carried out with total splenocytes, conventional DC,and bone marrow–derived DC. They also measured cytokine pro-duction by these cells in response to poly(I:C) and LPS, which areligands for TLR3 and 4, respectively. PTPs regulate many aspectsof immune cell functions and may have varied roles in different celltypes (24). The discrepancy between the current study and the oneby An et al. (30) can be attributed to the differences in the exper-imental designs. Similarly, SHP-2 has been shown to negativelyregulate TLR3-mediated but not TLR2-, TLR7-, and TLR9-mediated cytokine production by peritoneal macrophages (48). Itis also possible that signaling events downstream of Ly49Q reg-ulate the TLR9 signaling cascade independent of the recruitmentof phosphatases. This is evident from experiments showing in-creased tyrosine phosphorylation of proteins after mAb-mediatedcross-linking of Ly49Q on macrophages (7).Positive regulation of the TLR9 signaling cascade by Ly49Q

upon stimulation with CpG-B ODN culminates in activation andtranslocation of IRF7 transcription factor to the nucleus. We showin this study that IRF7 translocation to the nucleus is impaired inLy49Q-deficient pDC (Fig. 7B). Both IRF3 and IRF7 positivelyregulate IFN-I gene transcription (32, 33). Although IRF3 isconstitutively expressed (32), IRF7 expression is induced by IFN-a and is required for the late-phase induction of IFN-a genes andfeedback regulation of IFN-I production (34). However, theseexperiments were conducted in mouse embryonic fibroblast cellsand may not be a true representation of IRF7 function and ex-pression in pDC. pDC are unique in that they can secrete up to1000 times more IFN-I than other cell type in response to mi-crobial challenge (3). Furthermore, in pDC, TLR9-mediated IFN-Iproduction is completely dependent on IRF7 (36). IRF7 is con-stitutively expressed at high levels in human pDC (46). Similarly,IRF7 protein can be detected at high levels in naive mouse pDC(Fig. 7A). Impaired translocation of IRF7 to the nucleus ofLy49Q-deficient pDC most likely has a significant impact on theexpression of IFN-I genes and may explain the inability of thesecells to produce IFN-a when stimulated with CpG-B ODN.Recent work by Swiecki et al. (43) has demonstrated that IFN-a

produced after viral infection of mice induces pDC death andsignificantly reduces their numbers. We recapitulate this phe-nomenon in the current study with CpG-B ODN challenge in bothWTand Ly49Q-deficient mice (Fig. 3B). Because Ly49Q-deficientmice are impaired in the production of IFN-a through TLR9 ac-tivation by CpG-B ODN, one would expect lesser pDC death inthese mice as compared with the WT mice. However, we observedsimilar levels of pDC death, measured by 7-AAD staining, in WTand Ly49Q-deficient mice receiving CpG-B ODN injections. IFN-a–induced pDC death, shown by Swiecki et al. (43), may requiresmall levels of IFN-a. Although impaired, IFN-a production isnot completely absent in Ly49Q-deficient mice. This small levelof IFN-a upon CpG-B ODN challenge may be sufficient to inducepDC death in Ly49Q-deficient mice. Alternatively, Ly49Q-defi-cient pDC may be more susceptible to IFN-a–induced death orupon activation through mechanisms independent of IFN-a.Our previous studies have demonstrated that Ly49Q plays an

important role in membrane trafficking to regulate TLR9 signaling.

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CpG-induced tubular endolysosomal extension is impaired in theabsence of Ly49Q, resulting in altered distribution of TLR9 andCpG ODN in macrophages and pDC lacking Ly49Q (23). Con-sequently, cells lacking Ly49Q showed impaired cytokine pro-duction in response to CpG ODN (13). The role of Ly49Q and itsITIM as a sensor of microbial products has not been explored. Studieswith human NK cells have implicated inhibitory KIR3DL2 indirect binding and trafficking of CpG ODN to early endosomeswhere TLR9 is encountered (47). Our preliminary experimentssuggest a similar function of Ly49Q in CpG ODN uptake, whichappears to be independent of Ly49Q ITIM (Fig. 8). Further ex-periments are required to address the role of Ly49Q and its ITIMin trafficking of CpG ODN to endosomal compartment and thenature of interaction with CpG ODN.To take advantage of the discovery of MHC-I regulation of pDC

function in mice, the existence of an analogous receptor performingthe same function in human pDC needs to be confirmed. At present,the human functional analog of Ly49Q is unknown, although there areseveral promising candidates. CD300a/c are two related members ofthe Ig superfamily expressed on leukocytes, including NK cells andpDC. CD300a contains three ITIMs, whereas CD300c contains noobvious signaling motifs and most likely signals through an un-identified ITAM-containing adaptor. Ab cross-linking (cross-reactivitywith both CD300a/c) results in enhanced IFN-a, but suppressesTNF-a and IL-6 production by pDC in response to CpG-ODN (49).The endogenous ligands are unknown, and it is not yet clearwhether these effects are due to ITIM or ITAM signaling. Inter-estingly, human pDC preferentially express ILT7 (LILRA4),which couples with a signaling adapter to activate an ITAM-mediated signaling pathway. ILT7 protein directly binds to BST2(CD317) protein, the expression of which is found on cells pre-exposed to IFN-a or on the surface of human cancer cells.Counterintuitively, the interaction between ILT7 and BST2 resultsin the reduction of IFN-a by pDC in response to TLR agonists(50). Finally, the C-type lectin-related dendritic cell immuno-receptor is expressed on pDC, contains a cytoplasmic ITIM, butinhibits IFN-I production upon Ab-mediated cross-linking (51).The physiological ligand of dendritic cell immunoreceptor is un-known. Although none of these human pDC receptors contain allthe functional features of mouse Ly49Q (cytoplasmic ITIM,MHC-I as endogenous ligand, positively modulates pDC duringTLR-induced cytokine responses), the existence of these humanpDC receptors, sometimes with paradoxical signaling outcomes,suggests our pDC studies in mice warrant further research. If thehuman functional analog to Ly49Q is discovered, it will open thedoor to the possibility of targeting specific molecules via mAb orsmall molecular inhibitors or other technology to try and manip-ulate pDC function during autoimmune responses.

AcknowledgmentsWe are grateful to Dr. Silvia Vidal (McGill University, Montreal, QC, Can-

ada) for providing the B16Flt3 cells and to Dr. Andre Veillette (Institut de

Recherches Clinique de Montreal, Montreal, QC, Canada) for providing

SHP-1, SHP-2, and SHIP Abs.

DisclosuresThe authors have no financial conflicts of interest.

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