Activation of Human Plasmacytoid Dendritic Cells by TLR9 Impairs Fc γRII-Mediated Uptake of Immune Complexes and Presentation by MHC Class II This information is current as of October 1, 2021. Daniel Benitez-Ribas, Paul Tacken, Cornelis J. A. Punt, I. Jolanda M. de Vries and Carl G. Figdor J Immunol 2008; 181:5219-5224; ; doi: 10.4049/jimmunol.181.8.5219

http://www.jimmunol.org/content/181/8/5219 Downloaded from

References This article cites 31 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/181/8/5219.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Human Plasmacytoid Dendritic Cells by TLR9 Impairs Fc␥RII-Mediated Uptake of Immune Complexes and Presentation by MHC Class II

Daniel Benitez-Ribas,* Paul Tacken,* Cornelis J. A. Punt,† I. Jolanda M. de Vries,1*‡ and Carl G. Figdor*

Human plasmacytoid dendritic cells (pDCs)2 exploit Ag uptake receptors like CD32a for internalization of exogenous Ags. Ac- tivation of pDC by TLR9 ligand CpG-C induces strong maturation. Surprisingly, we observed that CpG-C-stimulated pDCs showed impaired Ag-specific proliferation whereas the induction of allogeneic T cell proliferation was not affected. We demonstrated that signals from TLR9 caused a rapid down-regulation of the capacity of pDC to take-up Ab-Ag complexes without altering their CD32a expression, thus explaining the reduced Ag presentation. The recent contrasting biological responses that were observed upon TLR9 ligation in pDCs prompted us to study the effect of several TLR9 ligands. We observed that type I Downloaded from IFN-inducer CpG-A, localizing in the early endosomal compartment, did not affect CD32a function, whereas CpGs localizing in the late endosomes and inducing pDC maturation clearly inhibited CD32a-mediated Ag uptake and presentation. We conclude that TLR9 ligands not only determine the type of response, i.e., type I IFN production (innate immunity) or maturation (adaptive immunity), but also directly affect Ag presentation capacity of pDCs. We hypothesize that pDC, once activated via TLR9-ligands reaching the late endosomes, can only present initially sampled Ags and thus are protected from uptake and processing of additional potential self-Ags. The Journal of Immunology, 2008, 181: 5219–5224. http://www.jimmunol.org/

lasmacytoid pre-dendritic cells (pDCs)2 comprise one of maturation. CpG-C seems present in both types of endosomes, two major subsets of human DCs in the peripheral blood which correlates with its ability to trigger both IFN-␣ production P (1). PDCs correspond to a subset of CD11c negative cir- and pDC maturation (6). culating blood DC (2), in humans characterized by the specific Several studies have demonstrated that pDCs are able to inter- expression of C-type lectin receptors BDCA-2 and BDCA-4 (3) nalize exogenous Ags via specific surface receptors. Human pDCs and CD123 (IL-3R). Freshly isolated or nonstimulated pDCs are have been described to present Ags following internalization via

poor immune stimulators, but in response to viral and bacterial BDCA-2 (7), Fc␧RI (8), and Siglec-5 (9). Interestingly, human by guest on October 1, 2021 stimuli, mainly through TLR ligands, pDCs differentiate into a pDCs also have the capacity to efficiently cross-present HIV Ags mature type of DC capable of inducing strong immune responses upon of apoptotic debris (10), although the (pDC-derived DC). Upon viral stimulation and subsequent TLR- involved remains obscure. Furthermore, pDC exploit CD32a to mediated signaling, human pDCs produce large amounts of type I internalize immune complexes (11, 12). ␣ ␤ IFNs (IFN- / ) that stimulate T cell function by inducing Th1 Whether the Ag presenting function of pDCs, in particular that differentiation and activate NK cell cytolytic activity (4). More- of exogenous Ags, is also regulated by TLR-mediated signaling is over, type I IFNs also promote differentiation, maturation, and im- not clearly defined. Conventional (myeloid) DCs readily take up munostimulatory functions of DCs (5). soluble Ags through macropinocytosis and receptor-mediated en- For TLR9, three classes of CpG oligonucleotide ligands have docytosis. Exposure to maturation stimuli results in a short in- been described by different sequence motifs and different abilities creased capacity of Ag uptake followed by a down modulation of to stimulate IFN-␣ production and maturation of pDCs. CpG-A endocytic processes, favoring presentation of pathogen-associated localizes to early endosomes (transfering TfR-positive endosomes) Ags (13). In contrast, freshly isolated pDC have only a low ca- and mediates production, whereas CpG-B localizes in late pacity to nonspecifically take up exogenous soluble Ags and seem, endosomes (LAMP-1 positive compartments) and mediates pDC therefore, specialized in presenting endogenous viral Ag to CD4ϩ and CD8ϩ T cells, inducing specific responses (2, 14). Little is

*Department of Tumor Immunology, †Department of Medical Oncology, and ‡De- known about regulatory circuits controlling Ag uptake follow- partment of Pediatric Oncology, Radboud University Nijmegen Medical Centre and ing pDC activation. Other phagocytic receptors such as Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands BDCA-2 (7), Fc␧RI (15), and CD36 (16) are down-regulated Received for publication May 20, 2008. Accepted for publication July 23, 2008. after pDC activation, suggesting that pDCs down-modulate The costs of publication of this article were defrayed in part by the payment of page their capacity to take up Ags by reducing Ag receptor expres- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sion levels during their transition from pDC to pDC-derived 1 Address correspondence and reprint requests to Dr. Jolanda De Vries, Radboud DC. In the present study we demonstrate that the subcellular University, Nijmegen, PO Box 9101, The Netherlands. E-mail address: j.devries@ location where TLR9 agonists interact with phagocytic recep- ncmls.ru.nl tors play an important role in controlling exogenous Ags up- 2 Abbreviations used in this paper: pDC, plasmacytoid ; KLH, keyhole take. We show that TLR9-mediated signals that are associated limpet hemocyanin; ODN, oligodeoxynucleotide; moDC, -derived DC. with the late endosomes rapidly prevent internalization of im- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 mune complexes via FcR CD32a whereas TLR9-mediated www.jimmunol.org 5220 TLR9 CONTROLS CD32a FUNCTION IN pDCs signals that are associated with early endosomes had no effect. Therefore, the location of TLR9 stimulation directly controls the capacity of human pDCs to respond to pathogens. Materials and Methods Cells culturing and maturation pDCs were purified from PBLs by positive isolation using anti-BDCA-4- (purity Ͼ95% confirmed by double staining with CD302 and CD123, data not shown) conjugated magnetic microbeads (Miltenyi Biotec) and ad- justed to 0.5*106 cells/ml in X-VIVO-15 (Cambrex) supplemented with 10 ng/ml IL-3 and 5% AB pooled human serum. Purified pDCs were activated (were indicated) by incubation with 5 ␮g/ml CpG-C (M-352) for2horovernight before adding keyhole limpet hemocyanin (KLH). Others CpG used were CpG-A (2216) and CpG-B (2006) (Sigma-Aldrich). Flow cytometry CD32a (clone At-10; Serotec), CD80 (clone L307.4, BD Pharmingen), CD83, and CD86 molecules were stained with mouse mAbs. Anti- BDCA-2 (clone AC144, mIgG1; Miltenyi Biotec). Secondary Abs for FACS analysis were goat-anti-mouse PE-(BD Pharmingen). Downloaded from Cytokine production pDCs (5 ϫ 104/well) were cultured overnight with or without CpG-C and in the presence of mAbs against CD32a, BDCA-2, or isotype control(10 ␮ ␣ g/ml). IFN- production was analyzed with murine monoclonal capture FIGURE 1. Proliferative T cell responses induced by pDCs. a, Allore- and HRP-conjugated anti-IFN-␣ Abs (BenderMedsystems) using standard sponse induction. CpG-C-treated pDCs induce higher T cell responses in ELISA procedures. an alloresponse compared with nontreated pDCs. Purified human pDCs http://www.jimmunol.org/ KLH internalization assays were cultured with allogeneic PBLs (Ratio pDCs:PBLs; 1:20) for 6 days, after which thymidine incorporation was measured. Left panel, One rep- Endotoxin-free protein KLH was purchased from Calbiochem. Protein Ϯ binding and internalization by pDCs was assessed by direct labeling of resentative experiment. Right panel, Data are shown as the mean SE of protein with the Alexa Fluor 488 labeling kit (Molecular Probes). pDCs three independent experiments using different donor’s pDCs and PBLs. were incubated with 10 ␮g/ml Alexa-labeled KLH in the presence of 5% Unstimulated pDCs (medium) were set as 100%. b, Ag-specific response. of human serum containing Abs against KLH. After overnight culture at CpG-C-treated pDCs elicited a reduced Ag-specific T cell response as 37°C, cells were washed and analyzed by flow cytometry. Internalization compared with unstimulated pDCs. pDCs isolated from moDC vaccinated of Alexa-labeled KLH was confirmed by confocal laser scanning micros- melanoma patients were incubated in the presence of 5% human serum copy. Cells were fixed on poly-L-lysine-coated glass slides, followed by containing IgGs directed against KLH (postvaccination serum). pDCs by guest on October 1, 2021 staining with MHC class II mAb (clone Q5/13) or IgG2a isotype control as were pretreated for 2 or 12 h with 5 ␮g/ml CpG-C or medium. Subse- a secondary mAb goat-anti-mouse Alexa 568 was used. Cells were imaged quently, 1 ␮g/ml KLH was added. After O/N incubation, pDCs were with a Bio-Rad MRC 1024 confocal system operating on a Nikon Optiphot microscope and a Nikon ϫ60 planApo 1.4 oil immersion lens. Pictures washed and cocultured with autologous PBLs for 96 h, after which were analyzed with Bio-Rad Lasersharp 2000 and Adobe Photoshop 7.0 thymidine incorporation was measured. Left panel shows one represen- (Adobe Systems) software. tative experiment. Right panel data (stimulation index ϭ SI) are shown as the mean Ϯ SE of three independent experiments using different Cellular responses to KLH donor’s pDCs and PBLs. Significant differences from control according Ͻ ء pDCs were incubated overnight with 5% postvaccination serum in the pres- to Student’s t test: , p 0.05. ence of 10 ␮g/ml KLH. KLH-loaded pDCs were washed and cocultured with KLH-responsive PBLs derived from monocyte-derived DC (moDC) vaccinated patients. After 4 days of coculture, a tritiated thymidine incor- tivated pDCs were compared with nonstimulated pDCs (21 and poration assay was performed. Tritiated thymidine (1 ␮Ci/well; MP Bio- 59% reduction after 2 and 12 h pretreatment, respectively, n ϭ 3; medicals) was added to the cell cultures and incorporation was measured after 16 h. Fig. 1b). This reduced capacity to generate Ag-specific T cell re- sponse suggested a functional interaction between TLR9 signaling Statistical analyses and CD32a-mediated Ag presentation. Statistical differences were determined using independent-samples t test procedure. Significance was accepted at the p Յ 0.05 level. CD32a-mediated signaling does not modulate TLR9-induced CD80/CD86 expression nor type I IFN secretion by pDC Results One potential pathway of CD32a-mediated inhibition of immune Preactivation of human pDCs by ligation of TLR9 enhances stimulatory capacity of pDCs might be the prevention of up-reg- allo-Ag responses but inhibits presentation of exogenous Ags in ulation of costimulatory molecules. We determined whether MHC class II CD32a-mediated signals after receptor cross-linking affected pDC When highly purified human pDCs were activated with TLR9 ago- maturation in the presence or absence of TLR9 stimulation. As we nists, distinct effects on T cell proliferation were observed. As show in Fig. 2a, upon CD32a cross-linking using specific mAb, depicted in Fig. 1a and in agreement with previous findings (17), neither up- nor down-regulation of CD80, CD83, and CD86 was oligodeoxynucleotides (ODNs) CpG-C-treated pDCs showed en- observed when pDCs were cultured with rhIL-3 or TLR9 was trig- hanced allogeneic T cell proliferation when compared with IL-3- gered, respectively. cultured pDCs. Surprisingly and in contrast to the enhanced al- Next, we investigated the possibility that CD32a-mediated sig- loresponsiveness (Fig. 1a), we observed that the induction of nals down-regulate specific T cell responses because of modula- KLH-specific T cell response to pDCs loaded with KLH-immune tion of IFN-␣ production. This hypothesis was supported by sev- complexes was significantly decreased when TLR9 agonist preac- eral reports indicating that IFN-␣ negatively influences T cell The Journal of Immunology 5221

FIGURE 2. Effect of CD32 cross-linking on pDC phenotype and type I IFN secretion. Freshly isolated pDCs were stimulated with 5 ␮g/ml CpG-C or medium in the presence of murine anti-CD32 or matching iso- type control Abs. Cells and supernatants were harvested after 24 h and stained with anti-CD80, CD83, and CD86 Abs (a) and cytokine detection (b), respectively. a, CpG-C induced mauration of pDCs and this was not affected by CD32 cross-linking. b, Abs directed against CD32 did not influence type 1 IFN production whereas Downloaded from BDCA-2 induced a strong inhibition of cytokine pro- duction as expected (7). Absolute cytokine levels; me- dium (25 Ϯ 15 ng/ml), AT-10 (27 Ϯ 19 ng/ml), and BDCA-2 (0.1 Ϯ 0.04 ng/ml). One representative of three independent experiments. http://www.jimmunol.org/ by guest on October 1, 2021

proliferation (18, 19) and that cross-linking of pDC surface recep- focal microscopy (Fig. 4a, right). We observed that pDCs were tors can directly affect IFN-␣ secretion. As we show in Fig. 2b, the either positive for labeled-CpG-C (in blue) or contained KLH- CD32a cross-linking upon pDC activation did not affect IFN-␣ alexa-488 immune complexes (in green) indicating that once secretion, in contrast to BDCA-2, C-type lectin known to pro- CpG-C is present inside pDCs these cells are not capable any- foundly inhibit IFN-␣ secretion (7). In addition, engagement of more to endocytose protein via CD32a. CD32a by immune complexes did not affect costimulatory mole- cule expression levels or IFN-␣ secretion (data not shown). Col- Endosomal maturation is a prerequisite for CpG-C-induced lectively, these results show that phenotype and cytokine secretion inhibition of immune complex internalization of fresh or stimulated pDCs was not affected upon CD32a Endosomal maturation has been described to be essential for cross-linking. CpG-C signals to induce cytokine and chemokine production via TLR9 (20). Chloroquine prevents endosomal maturation Impaired uptake of immune complexes after preactivation of primarily through inhibition of vesicular acidification (21) (22). pDC through TLR9 is not caused by down-regulation of CD32a To prove that endosomal maturation is also important to control Given that other phagocytic receptors such as BDCA-2 (7), CD32a function through CpG-C signaling, pDCs were preacti- Fc␧RI (15), and CD36 (16) are down-regulated after pDC ac- vated for 4 h with CpG-C in the presence or absence of chlo- tivation, we investigated whether CD32a expression on pDCs roquine. Subsequently, the capacity of pretreated pDCs to in- was modified after activation. Preactivation of pDCs with ternalize KLH immune complexes was tested. Intriguingly, the CpG-C clearly leads to maturation as evidenced by IFN␣ pro- results in Fig. 5a show that in the presence of chloroquine, the duction and up-regulation of costimulatory molecules, but this internalization of immune complexes is fully restored to control was not accompanied by decreased CD32a expression levels levels. Chloroquine hardly altered protein uptake by nonacti- (Fig. 3). Next we determined whether the uptake of immune vated pDCs, excluding that the treatment was toxic. As ex- complexes by pDCs was reduced in the presence of CpG-C. As pected, treatment with chloroquine also completely inhibited early as 2 h after exposure, substantially reduced amounts of the IFN-␣ secretion by activated pDCs as previously reported internalized protein were observed, which became more appar- (data not shown) (20). Taken together, these data reveal that ent after overnight incubation (Fig. 4). This direct effect of endosomal acidification, and therefore TLR9-mediating signal- CpG-C on CD32a-mediated uptake of immune complexes is ing, is required to inhibit CD32a-mediated uptake of immune further substantiated by inspection of individual cells by con- complexes by CpG-C. 5222 TLR9 CONTROLS CD32a FUNCTION IN pDCs Downloaded from http://www.jimmunol.org/

FIGURE 3. CD32 expression following CpG-C-mediated pDC stimu- lation. a, Flow cytometric analysis of CD32 surface expression (black line: CD32; dotted line: isotype control) on cultured pDCs in the presence of FIGURE 4. KLH-specific uptake by pDCs. Alexa Fluor 488-labeled CpG-C or IL-3 containing medium. CD80 up-regulation is included as a KLH internalization by human isolated pDCs was analyzed in the presence maturation marker for pDCs. a, One representative experiment. b, Mean of of serum from donors displaying Ab responses to KLH. pDCs were cul- different experiments (n ϭ 4), CD32 positive expressing pDCs (IL-3 cul- ϭ Ϯ ϭ Ϯ tured with 5% of serum and pretreated 2 or 12 h with or without CpG-C tured pDCs mean 64% 15; CpG-C treated pDCs mean 71% 20), ␮

before adding 10 g/ml alexa-labeled KLH. a, Representative experiment by guest on October 1, 2021 and mean fluorescence intensity (IL-3 cultured pDCs mean ϭ 23% Ϯ 8; showing decreased uptake of pDCs pretreated with CpG-C for 2 and 12 h. CpG-C-treated pDCs mean ϭ 20% Ϯ 8). Cells were analyzed by flow cytometry (left) and confocal microscopy (right). b, Results of three independent experiments using pDCs of different donors. Data represent mean percentages Ϯ SE of KLH uptake relative to Regulation of CD32a function by different TLR9 ligands uptake by control pDC (set to 100%). Since different endosomal compartments associate with the differ- ent signaling pathways regulating biological responses to TLR9 activation in pDCs, we tested the respective TLR9 ligands on (data not shown), CD80, CD83, and CD86 and type I IFN secre- CD32a function. From our results (Fig. 5b), we conclude that, in tion. In contrast, the presentation of exogenous immune com- particular, signaling related to the late endosomes is important in plexed Ags to specific T cells was remarkably inhibited. CD32a mediated since CpG-B and CpG-C had the We previously demonstrated that pDCs present immune com- strongest effect on the binding and uptake of immune complexes. plexed exogenous proteins after uptake via CD32a, exploiting Due to this strong CpG-B- and -C-mediated effect and the finding KLH as a model Ag (12, 23). Given the fact that CpG-C-activated that CpG-A did not have any effect, CD32a function it is not likely pDCs induce strong alloresponses, it seems unlikely that their re- related to signaling resulting in or from IFN-␣ production (IRF-7) duced capacity to stimulate Ag-specific immune responses was (Fig. 5d). This notion is supported by our findings that addition of related to secretion of suppressive , like IL-10, or expres- exogenous IFN-␣ to pDCs did not inhibit the KLH-immune com- sion of inhibitory molecules induced by TLR agonist stimulation. plex uptake (data not shown). Moreover, while in CpG-C pre- The interaction between immune complexes and CD32a might in- treated pDCs Ag-specific T cell specific proliferation is reduced, duce negative signals that modulate TLR9-mediated pDCs matu- we did not observe reduced T cell proliferation when the pDCs ration and thus explain the observed inhibition of Ag presentation were pretreated with CpG-A (Fig. 5c). in MHC class II. Although, indeed, different subtypes of CD32 have been described–i.e., activating CD32a (Fc␥RIIa) and CD32c Discussion (Fc␥RIIc) and inhibitory CD32b (Fc␥RIIb)–human pDCs only ex- In the present study, we demonstrate that the capacity of human press the activating CD32a subtype (24) (data not shown). Since pDCs to present exogenous Ags in the context of MHC class II is CD32a has been reported as a potent immune activating receptor, tightly regulated upon activation via TLR9 agonists. In agreement and can initiate the release of inflammatory cytokines (25), its with previous findings (17), we observed that class C-type CpG engagement might also affect pDC function. However, CD32a is ODNs (CpG-C)-treated pDCs showed enhanced allogeneic T cell differently regulated depending on cell type, nature of the stimuli proliferation when compared with IL-3-cultured pDCs. This cor- as well as coexpression of other Fc␥Rs containing ITIM motifs responded to increased expression levels of MHC classes I and II (26). Receptor triggering might dramatically affect pDC function, The Journal of Immunology 5223

FIGURE 5. The effect of chloroquine and different TLR9 ligands on the capacity of pDCs to take up immune complexes. a, pDCs were incubated 4 h with medium or CpG-C in the presence or absence of chloroquine (0.05 mM). The concentration used in this experiment did not cause cell death. Subsequently, KLH-alexa 488 was added to pDCs in the presence of serum containing Abs directed against KLH and the amount of internalized protein was analyzed. Data are mean percentages Ϯ SD of internalized KLH (medium control was set to 100%, n ϭ 2). In the presence of chloroquine, the inhibitory effect of CpG-C was abrogated, and the values were restored to basal lev- els. b, Alexa Fluor 488-labeled KLH internalization by hu- man isolated pDCs in the presence of serum from donors displaying Ab responses to KLH. pDCs were cultured in the presence of 5% of serum, and pretreated for 2 h with CpG-A, -B, -C, or medium before adding labeled-KLH. Experiments were performed three times with pDCs iso- lated from different donors. A representative experiment showing decreased uptake of pDCs pretreated with CpG-B

and CpG-C (both localizing in the late endosomes). Lo- Downloaded from calization of a TLR9 ligand in the early endosomes (CpG-A) does not effect the uptake of immune complexes. c, The capacity of pDCs to present Ag to specific T cells was not affected when pDCs were pretreated with CpG-A in contrast to CpG-C pretreatment. pDCs isolated from moDC-vaccinated melanoma patients were incubated in

the presence of 5% human serum containing IgGs directed http://www.jimmunol.org/ against KLH (postvaccination serum). pDCs were either pretreated for2horCpG(5␮g/ml) was added together with 1 ␮g/ml KLH. After O/N incubation, pDCs were washed and cocultured with autologous PBLs for 96 h, after which thymidine incorporation was measured. Data are mean proliferation indices relative to medium control for experiments performed in triplicate. d, Model of Ag uptake inhibition. Upon CpG-C activation, pDCs initiate their transition from pre to plasmacytoid-derived DCs. En- docytotic molecules like BDCA-2 are down-regulated re- by guest on October 1, 2021 sulting in a decreased Ag uptake capacity. In our study, we show that activation of pDCs does not lead to CD32a down-regulation but the receptor is inactivated. This inac- tivation impairs further exogenous Ag uptake and presen- tation in MHC class II. The inhibitory effect using CpG-B and CpG-C can be linked to the NF-␬B pathway associ- ated with the late endosome signaling, suggesting a highly regulated Ag uptake capacity of human pDCs (6). as was show for the C-type lectin BDCA-2 and DCIR. Both re- CD36 (16), and DCIR (27). In contrast, the C-type lectin DEC205 ceptors dramatically inhibit IFN-␣ secretion induced upon engage- (CD205) appears to lose its endocytic capacity in monocyte-de- ment with various ligands (7, 27). By contrast, our findings clearly rived DC upon TLR triggering rather than being down-regulated at demonstrate that cross-linking of CD32a does not inhibit the the surface expression level upon maturation stimuli (30). The ex- IFN-␣ production by TLR9-stimulated pDCs providing further ev- pression of CD32a remained unaltered after pDC activation using idence that inhibition of T cell proliferation is not affected by ODN-CpG. These observations demonstrate that TLR9-mediated CD32a-mediated signaling. This notion is further supported by the signals directly control CD32a at the functional level and not at the finding that IFN-␣ production was not altered upon cross-linking expression level. This is supported by recent data on human neu- of CD32a on freshly isolated pDC (data not shown). Together, trophils showing that phorbol ester PMA inhibits the ligand bind- these results indicate that, distinct from C-type lectin-like receptors ing capacity of CD32a (31). BDCA-2 or DCIR, CD32a in itself, after immune complex induced Three classes of CpG ODN ligands for TLR9 have been de- cross-linking on pDCs, does not act as an immune activating or scribed by different sequence motifs and different abilities to stim- inhibiting receptor and, therefore, does not likely interfere with ulate IFN-␣ production and maturation of pDCs (6). CpG-A lo- TLR9-derived signals. calizes to early endosomes (Transferrin TfR-positive endosomes) APCs generally lose their Ag uptake capacity upon maturation and mediates cytokine production, whereas CpG-B localizes in late either by reducing membrane receptor expression or by affecting endosomes (LAMP-1 positive compartments) and mediates pDC its internalization. This is described for many Ag uptake receptors maturation. CpG-C is present in both types of endosomes, which including DC-SIGN and the mannose receptor (28) (29). Also on correlates with the ability to trigger both IFN-␣ production and pDCs, maturation stimuli indeed down-regulate expression of sev- pDC maturation. Interestingly, we can distinguish two different eral putative Ag receptors including: Fc␧RI (15), BDCA-2 (7), effects of CpGs on the CD32a inactivation, whereas CpG-B and 5224 TLR9 CONTROLS CD32a FUNCTION IN pDCs

CpG-C have a strong inhibitory effect, CpG-A hardly affects the 8. Foster, B., D. D. Metcalfe, and C. Prussin. 2003. Human dendritic cell 1 and ␧ immune complexes uptake. dendritic cell 2 subsets express Fc RI: correlation with serum IgE and allergic asthma. J. Allergy Clin. Immunol. 112: 1132–1138. It will be important to unravel the signaling pathway that results 9. Lock, K., J. Zhang, J. Lu, S. H. Lee, and P. R. Crocker. 2004. Expression of in CD32a inactivation upon TLR9 stimulation. We hypothesize CD33-related siglecs on human mononuclear , monocyte-derived den- dritic cells, and plasmacytoid dendritic cells. Immunobiology 209: 199–207. that the function of CD32a is regulated via pathways similar to 10. Hoeffel, G., A. C. Ripoche, D. Matheoud, M. Nascimbeni, N. Escriou, P. Lebon, those regulating costimulatory molecule expression (NF-␬B sig- F. Heshmati, J. G. Guillet, M. Gannage, S. Caillat-Zucman, et al. 2007. naling resulting in inhibition of uptake) and cytokine expression crosspresentation by human plasmacytoid dendritic cells. Immunity 27: 481–492. 11. Balmelli, C., I. E. Vincent, H. Rau, L. Guzylack-Piriou, K. McCullough, and (IRF-7 signaling with no effect on uptake) (Fig. 5d). Unfortu- A. Summerfield. 2005. Fc␥RII-dependent sensitisation of natural -pro- nately, unraveling signaling pathways controlling CD32a function ducing cells for viral infection and interferon-␣ responses. Eur. J. Immunol. 35: of pDCs are hampered by the fact that human pDCs are rare blood 2406–2415. 12. Benitez-Ribas, D., G. J. Adema, G. Winkels, I. S. Klasen, C. J. Punt, cells and cannot be grown in vitro in sufficient numbers for ex- C. G. Figdor, and I. J. de Vries. 2006. Plasmacytoid dendritic cells of melanoma tensive biochemical analysis. patients present exogenous proteins to CD4ϩ T cells after Fc␥RII-mediated up- In conclusion, we have demonstrated that, in human pDCs, take. J. Exp. Med. 203: 1629–1635. 13. West, M. A., R. P. Wallin, S. P. Matthews, H. G. Svensson, R. Zaru, TLR9-mediated signals associated with the late endosomes are re- H. G. Ljunggren, A. R. Prescott, and C. Watts. 2004. Enhanced dendritic cell sponsible for the diminished uptake of immune complexed Ags via antigen capture via toll-like receptor-induced remodeling. Science 305: 1153–1157. CD32a. This represents a novel Ag uptake control mechanism lim- 14. Krug, A., R. Veeraswamy, A. Pekosz, O. Kanagawa, E. R. Unanue, M. Colonna, iting presentation of Ags encountered after pDC activation and and M. Cella. 2003. Interferon-producing cells fail to induce proliferation of maturation. Moreover, we show that CpG-B and CpG-C, both lo- naive T cells but can promote expansion and T helper 1 differentiation of antigen- experienced unpolarized T cells. J. Exp. Med. 197: 899–906. calizing to the late endosomes, but not CpG-A, reduce uptake of

15. Schroeder, J. T., A. P. Bieneman, H. Xiao, K. L. Chichester, K. Vasagar, S. Saini, Downloaded from immune complexes by pDCs, thus confirming and extending the and M. C. Liu. 2005. TLR9- and Fc␧RI-mediated responses oppose one another hypothesis that major differences in TLR9 ligand localization and in plasmacytoid dendritic cells by down-regulating receptor expression. J. Im- munol. 175: 5724–5731. signaling can dramatically affect pDC cell function. In addition, 16. Cella, M., F. Facchetti, A. Lanzavecchia, and M. Colonna. 2000. Plasmacytoid our findings demonstrate that TLR9-mediated signaling differently dendritic cells activated by influenza virus and CD40L drive a potent TH1 po- regulates FcR-mediated Ag uptake (i.e., function of receptor) when larization. Nat. Immunol. 1: 305–310. 17. Rothenfusser, S., E. Tuma, S. Endres, and G. Hartmann. 2002. Plasmacytoid compared with lectin like receptor-mediated Ag uptake (i.e., dendritic cells: the key to CpG. Hum. Immunol. 63: 1111–1119. down-regulation of the receptor). 18. Erickson, S., O. Sangfelt, J. Castro, M. Heyman, S. Einhorn, and D. Grander. http://www.jimmunol.org/ 1999. Interferon-␣ inhibits proliferation in human T by abrogation It is tempting to speculate that the endosomal localization of of interleukin 2-induced changes in cell cycle-regulatory proteins. Cell Growth viral or bacterial-derived products determines the biological re- Differ. 10: 575–582. sponse to TLR9 activation in pDCs. Upon stimulation in the late 19. Aman, M., T. Tretter, I. Eisenbeis, G. Bug, T. Decker, W. Aulitzky, H. Tilg, C. Huber, and C. Peschel. 1996. Interferon-␣ stimulates production of interleu- endosomes, but not in the early endosomes, endocytic activity is kin-10 in activated CD4ϩ T cells and . Blood 87: 4731–4736. reduced to facilitate presentation of Ags that are associated with 20. Osawa, Y., S. Iho, R. Takauji, H. Takatsuka, S. Yamamoto, T. Takahashi, the TLR9 ligands rather than presentation of nonrelated Ags that S. Horiguchi, Y. Urasaki, T. Matsuki, and S. Fujieda. 2006. Collaborative action of NF-␬B and p38 MAPK is involved in CpG DNA-induced IFN-␣ and chemo- are acquired subsequent to TLR9 stimulation. This might prevent kine production in human plasmacytoid dendritic cells. J. Immunol. 177: pDC-induced as uptake and presentation of im- 4841–4852. by guest on October 1, 2021 mune-complexed self Ags is hampered in matured pDCs. 21. Hacker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K. Heeg, G. B. Lipford, and H. Wagner. 1998. CpG-DNA-specific activation of antigen-presenting cells requires stress activity and is preceded by non- Acknowledgments specific endocytosis and endosomal maturation. EMBO J. 17: 6230–6240. We thank Nicole Scharenborg, Marieke Kerkhoff, Annemiek de Boer, and 22. Lande, R., J. Gregorio, V. Facchinetti, B. Chatterjee, Y. H. Wang, B. Homey, W. Cao, Y. H. Wang, B. Su, F. O. Nestle, et al. 2007. Plasmacytoid dendritic cells Mandy van de Rakt for technical assistance. Drs. Gosse Adema and Fried- sense self-DNA coupled with antimicrobial peptide. Nature 449: 564–569. erike Meyer-Wentrup are acknowledged for discussions. 23. De Vries, I. J., D. J. Krooshoop, N. M. Scharenborg, W. J. Lesterhuis, J. H. Diepstra, G. N. Van Muijen, S. P. Strijk, T. J. Ruers, O. C. Boerman, Disclosures W. J. Oyen, et al. 2003. Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state. Can- The authors have no financial conflict of interest. cer Res. 63: 12–17. 24. Bave, U., M. Magnusson, M. L. Eloranta, A. Perers, G. V. Alm, and References L. Ronnblom. 2003. Fc␥RIIa is expressed on natural IFN-␣-producing cells 1. Soumelis, V., and Y. J. Liu. 2006. From plasmacytoid to dendritic cell: morpho- (plasmacytoid dendritic cells) and is required for the IFN-␣ production induced logical and functional switches during plasmacytoid pre-dendritic cell differen- by apoptotic cells combined with lupus IgG. J. Immunol. 171: 3296–3302. tiation. Eur. J. Immunol. 36: 2286–2292. 25. Boruchov, A. M., G. Heller, M. C. Veri, E. Bonvini, J. V. Ravetch, and 2. Grouard, G., M. C. Rissoan, L. Filgueira, I. Durand, J. Banchereau, and Y. J. Liu. J. W. Young. 2005. Activating and inhibitory IgG Fc receptors on human DCs 1997. The enigmatic plasmacytoid T cells develop into dendritic cells with in- mediate opposing functions. J. Clin. Invest. 115: 2914–2923. terleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185: 1101–1111. 26. Kalergis, A. M., and J. V. Ravetch. 2002. Inducing tumor immunity through the 3. Dzionek, A., A. Fuchs, P. Schmidt, S. Cremer, M. Zysk, S. Miltenyi, D. W. Buck, selective engagement of activating Fc␥ receptors on dendritic cells. J. Exp. Med. and J. Schmitz. 2000. BDCA-2, BDCA-3, and BDCA-4: three markers for dis- 195: 1653–1659. tinct subsets of dendritic cells in human peripheral blood. J. Immunol. 165: 27. Meyer-Wentrup, F., D. Benitez-Ribas, P. J. Tacken, C. J. Punt, C. G. Figdor, 6037–6046. I. J. de Vries, and G. J. Adema. 2008. Targeting DCIR on human plasmacytoid 4. Romagnani, C., M. Della Chiesa, S. Kohler, B. Moewes, A. Radbruch, dendritic cells results in and inhibits IFN-␣ production. L. Moretta, A. Moretta, and A. Thiel. 2005. Activation of human NK cells by Blood 111: 4245–4253. plasmacytoid dendritic cells and its modulation by CD4ϩ T helper cells and 28. Relloso, M., A. Puig-Kroger, O. M. Pello, J. L. Rodriguez-Fernandez, CD4ϩ CD25hi T regulatory cells. Eur. J. Immunol. 35: 2452–2458. G. de la Rosa, N. Longo, J. Navarro, M. A. Munoz-Fernandez, 5. Yoneyama, H., K. Matsuno, E. Toda, T. Nishiwaki, N. Matsuo, A. Nakano, P. Sanchez-Mateos, and A. L. Corbi. 2002. DC-SIGN (CD209) expression is IL-4 S. Narumi, B. Lu, C. Gerard, S. Ishikawa, and K. Matsushima. 2005. Plasmacy- dependent and is negatively regulated by IFN, TGF-␤, and anti-inflammatory toid DCs help lymph node DCs to induce anti-HSV CTLs. J. Exp. Med. 202: agents. J. Immunol. 168: 2634–2643. 425–435. 29. Geijtenbeek, T. B. H., R. Torensma, S. J. van Vliet, G. C. F. van Duijnhoven, 6. Guiducci, C., G. Ott, J. H. Chan, E. Damon, C. Calacsan, T. Matray, K. D. Lee, G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, R. L. Coffman, and F. J. Barrat. 2006. Properties regulating the nature of the a novel dendritic cell-specific ICAM-3 receptor that supports primary immune plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J. Exp. responses. Cell 100: 575–585. Med. 203: 1999–2008. 30. Kato, M., K. J. McDonald, S. Khan, I. L. Ross, S. Vuckovic, K. Chen, 7. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, D. Munster, K. P. Macdonald, and D. Hart. 2006. Expression of human DEC-205 G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al. 2001. BDCA-2, a (CD205) multilectin receptor on leukocytes. Int. Immunol. 18: 857–869. novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen 31. Nagarajan, S., N. H. Fifadara, and P. Selvaraj. 2005. Signal-specific activation capture and is a potent inhibitor of interferon ␣/␤ induction. J. Exp. Med. 194: and regulation of human neutrophil Fc␥ receptors. J. Immunol. 174: 1823–1834. 5423–5432.