Human plasmacytoid dendritic cells (pDCs)2 exploit Ag uptake receptors like CD32a for internalization of exogenous Ags. Activation of pDC by TLR9 ligand CpG-C induces strong maturation. Surprisingly, we observed that CpG-C-stimulated pDCs showed impaired Ag-specific T cell 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 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.
Plasmacytoid pre-dendritic cells (pDCs)2 comprise one of two major subsets of human DCs in the peripheral blood (1). PDCs correspond to a subset of CD11c negative circulating blood DC (2), in humans characterized by the specific expression of C-type lectin receptors BDCA-2 and BDCA-4 (3) and CD123 (IL-3R). Freshly isolated or nonstimulated pDCs are poor immune stimulators, but in response to viral and bacterial stimuli, mainly through TLR ligands, pDCs differentiate into a mature type of DC capable of inducing strong immune responses (pDC-derived DC). Upon viral stimulation and subsequent TLR-mediated signaling, human pDCs produce large amounts of type I IFNs (IFN-α/β) that stimulate T cell function by inducing Th1 differentiation and activate NK cell cytolytic activity (4). Moreover, type I IFNs also promote differentiation, maturation, and immunostimulatory functions of DCs (5).
For TLR9, three classes of CpG oligonucleotide ligands have been described by different sequence motifs and different abilities to stimulate IFN-α production and maturation of pDCs. CpG-A localizes to early endosomes (transfering TfR-positive endosomes) and mediates cytokine production, whereas CpG-B localizes in late endosomes (LAMP-1 positive compartments) and mediates pDC maturation. CpG-C seems present in both types of endosomes, which correlates with its ability to trigger both IFN-α production and pDC maturation (6).
Several studies have demonstrated that pDCs are able to internalize exogenous Ags via specific surface receptors. Human pDCs have been described to present Ags following internalization via BDCA-2 (7), FcεRI (8), and Siglec-5 (9). Interestingly, human pDCs also have the capacity to efficiently cross-present HIV Ags upon phagocytosis of apoptotic debris (10), although the receptor involved remains obscure. Furthermore, pDC exploit CD32a to internalize immune complexes (11, 12).
Whether the Ag presenting function of pDCs, in particular that of exogenous Ags, is also regulated by TLR-mediated signaling is not clearly defined. Conventional (myeloid) DCs readily take up soluble Ags through macropinocytosis and receptor-mediated endocytosis. Exposure to maturation stimuli results in a short increased capacity of Ag uptake followed by a down modulation of endocytic processes, favoring presentation of pathogen-associated Ags (13). In contrast, freshly isolated pDC have only a low capacity to nonspecifically take up exogenous soluble Ags and seem, therefore, specialized in presenting endogenous viral Ag to CD4+ and CD8+ T cells, inducing specific responses (2, 14). Little is known about regulatory circuits controlling Ag uptake following pDC activation. Other phagocytic receptors such as BDCA-2 (7), FcεRI (15), and CD36 (16) are down-regulated after pDC activation, suggesting that pDCs down-modulate their capacity to take up Ags by reducing Ag receptor expression levels during their transition from pDC to pDC-derived DC. In the present study we demonstrate that the subcellular location where TLR9 agonists interact with phagocytic receptors play an important role in controlling exogenous Ags uptake. We show that TLR9-mediated signals that are associated with the late endosomes rapidly prevent internalization of immune complexes via FcR CD32a whereas TLR9-mediated 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 >95% confirmed by double staining with CD302 and CD123, data not shown) conjugated magnetic microbeads (Miltenyi Biotec) and adjusted 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 μg/ml CpG-C (M-352) for 2 h or overnight before adding keyhole limpet hemocyanin (KLH). Others CpG used were CpG-A (2216) and CpG-B (2006) (Sigma-Aldrich).
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).
pDCs (5 × 104/well) were cultured overnight with or without CpG-C and in the presence of mAbs against CD32a, BDCA-2, or isotype control(10 μg/ml). IFN-α production was analyzed with murine monoclonal capture and HRP-conjugated anti-IFN-α Abs (BenderMedsystems) using standard ELISA procedures.
KLH internalization assays
Endotoxin-free protein KLH was purchased from Calbiochem. Protein binding and internalization by pDCs was assessed by direct labeling of protein with the Alexa Fluor 488 labeling kit (Molecular Probes). pDCs were incubated with 10 μg/ml Alexa-labeled KLH in the presence of 5% of human serum containing Abs against KLH. After overnight culture at 37°C, cells were washed and analyzed by flow cytometry. Internalization of Alexa-labeled KLH was confirmed by confocal laser scanning microscopy. Cells were fixed on poly-l-lysine-coated glass slides, followed by staining with MHC class II mAb (clone Q5/13) or IgG2a isotype control as a secondary mAb goat-anti-mouse Alexa 568 was used. Cells were imaged with a Bio-Rad MRC 1024 confocal system operating on a Nikon Optiphot microscope and a Nikon ×60 planApo 1.4 oil immersion lens. Pictures were analyzed with Bio-Rad Lasersharp 2000 and Adobe Photoshop 7.0 (Adobe Systems) software.
Cellular responses to KLH
pDCs were incubated overnight with 5% postvaccination serum in the presence of 10 μg/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 incorporation assay was performed. Tritiated thymidine (1 μCi/well; MP Biomedicals) was added to the cell cultures and incorporation was measured after 16 h.
Statistical differences were determined using independent-samples t test procedure. Significance was accepted at the p ≤ 0.05 level.
Preactivation of human pDCs by ligation of TLR9 enhances allo-Ag responses but inhibits presentation of exogenous Ags in MHC class II
When highly purified human pDCs were activated with TLR9 agonists, distinct effects on T cell proliferation were observed. As depicted in Fig. 1,a and in agreement with previous findings (17), oligodeoxynucleotides (ODNs) CpG-C-treated pDCs showed enhanced allogeneic T cell proliferation when compared with IL-3-cultured pDCs. Surprisingly and in contrast to the enhanced alloresponsiveness (Fig. 1,a), we observed that the induction of KLH-specific T cell response to pDCs loaded with KLH-immune complexes was significantly decreased when TLR9 agonist preactivated pDCs were compared with nonstimulated pDCs (21 and 59% reduction after 2 and 12 h pretreatment, respectively, n = 3; Fig. 1 b). This reduced capacity to generate Ag-specific T cell response suggested a functional interaction between TLR9 signaling and CD32a-mediated Ag presentation.
CD32a-mediated signaling does not modulate TLR9-induced CD80/CD86 expression nor type I IFN secretion by pDC
One potential pathway of CD32a-mediated inhibition of immune stimulatory capacity of pDCs might be the prevention of up-regulation of costimulatory molecules. We determined whether CD32a-mediated signals after receptor cross-linking affected pDC maturation in the presence or absence of TLR9 stimulation. As we show in Fig. 2 a, upon CD32a cross-linking using specific mAb, neither up- nor down-regulation of CD80, CD83, and CD86 was observed when pDCs were cultured with rhIL-3 or TLR9 was triggered, respectively.
Next, we investigated the possibility that CD32a-mediated signals down-regulate specific T cell responses because of modulation of IFN-α production. This hypothesis was supported by several reports indicating that IFN-α negatively influences T cell proliferation (18, 19) and that cross-linking of pDC surface receptors can directly affect IFN-α secretion. As we show in Fig. 2 b, the CD32a cross-linking upon pDC activation did not affect IFN-α secretion, in contrast to BDCA-2, C-type lectin known to profoundly inhibit IFN-α secretion (7). In addition, engagement of CD32a by immune complexes did not affect costimulatory molecule expression levels or IFN-α secretion (data not shown). Collectively, these results show that phenotype and cytokine secretion of fresh or stimulated pDCs was not affected upon CD32a cross-linking.
Impaired uptake of immune complexes after preactivation of pDC through TLR9 is not caused by down-regulation of CD32a
Given that other phagocytic receptors such as BDCA-2 (7), FcεRI (15), and CD36 (16) are down-regulated after pDC activation, we investigated whether CD32a expression on pDCs was modified after activation. Preactivation of pDCs with CpG-C clearly leads to maturation as evidenced by IFNα production and up-regulation of costimulatory molecules, but this was not accompanied by decreased CD32a expression levels (Fig. 3). Next we determined whether the uptake of immune complexes by pDCs was reduced in the presence of CpG-C. As early as 2 h after exposure, substantially reduced amounts of internalized protein were observed, which became more apparent after overnight incubation (Fig. 4). This direct effect of CpG-C on CD32a-mediated uptake of immune complexes is further substantiated by inspection of individual cells by confocal microscopy (Fig. 4 a, right). We observed that pDCs were either positive for labeled-CpG-C (in blue) or contained KLH-alexa-488 immune complexes (in green) indicating that once CpG-C is present inside pDCs these cells are not capable anymore to endocytose protein via CD32a.
Endosomal maturation is a prerequisite for CpG-C-induced inhibition of immune complex internalization
Endosomal maturation has been described to be essential for CpG-C signals to induce cytokine and chemokine production via TLR9 (20). Chloroquine prevents endosomal maturation primarily through inhibition of vesicular acidification (21) (22). To prove that endosomal maturation is also important to control CD32a function through CpG-C signaling, pDCs were preactivated for 4 h with CpG-C in the presence or absence of chloroquine. Subsequently, the capacity of pretreated pDCs to internalize KLH immune complexes was tested. Intriguingly, the results in Fig. 5 a show that in the presence of chloroquine, the internalization of immune complexes is fully restored to control levels. Chloroquine hardly altered protein uptake by nonactivated pDCs, excluding that the treatment was toxic. As expected, treatment with chloroquine also completely inhibited the IFN-α secretion by activated pDCs as previously reported (data not shown) (20). Taken together, these data reveal that endosomal acidification, and therefore TLR9-mediating signaling, is required to inhibit CD32a-mediated uptake of immune complexes by CpG-C.
Regulation of CD32a function by different TLR9 ligands
Since different endosomal compartments associate with the different signaling pathways regulating biological responses to TLR9 activation in pDCs, we tested the respective TLR9 ligands on CD32a function. From our results (Fig. 5,b), we conclude that, in particular, signaling related to the late endosomes is important in CD32a mediated endocytosis since CpG-B and CpG-C had the strongest effect on the binding and uptake of immune complexes. Due to this strong CpG-B- and -C-mediated effect and the finding that CpG-A did not have any effect, CD32a function it is not likely related to signaling resulting in or from IFN-α production (IRF-7) (Fig. 5,d). This notion is supported by our findings that addition of exogenous IFN-α to pDCs did not inhibit the KLH-immune complex uptake (data not shown). Moreover, while in CpG-C pretreated pDCs Ag-specific T cell specific proliferation is reduced, we did not observe reduced T cell proliferation when the pDCs were pretreated with CpG-A (Fig. 5 c).
In the present study, we demonstrate that the capacity of human pDCs to present exogenous Ags in the context of MHC class II is tightly regulated upon activation via TLR9 agonists. In agreement with previous findings (17), we observed that class C-type CpG ODNs (CpG-C)-treated pDCs showed enhanced allogeneic T cell proliferation when compared with IL-3-cultured pDCs. This corresponded to increased expression levels of MHC classes I and II (data not shown), CD80, CD83, and CD86 and type I IFN secretion. In contrast, the presentation of exogenous immune complexed Ags to specific T cells was remarkably inhibited.
We previously demonstrated that pDCs present immune complexed exogenous proteins after uptake via CD32a, exploiting KLH as a model Ag (12, 23). Given the fact that CpG-C-activated pDCs induce strong alloresponses, it seems unlikely that their reduced capacity to stimulate Ag-specific immune responses was related to secretion of suppressive cytokines, like IL-10, or expression of inhibitory molecules induced by TLR agonist stimulation. The interaction between immune complexes and CD32a might induce negative signals that modulate TLR9-mediated pDCs maturation and thus explain the observed inhibition of Ag presentation in MHC class II. Although, indeed, different subtypes of CD32 have been described–i.e., activating CD32a (FcγRIIa) and CD32c (FcγRIIc) and inhibitory CD32b (FcγRIIb)–human pDCs only express the activating CD32a subtype (24) (data not shown). Since CD32a has been reported as a potent immune activating receptor, and can initiate the release of inflammatory cytokines (25), its engagement might also affect pDC function. However, CD32a is differently regulated depending on cell type, nature of the stimuli as well as coexpression of other FcγRs containing ITIM motifs (26). Receptor triggering might dramatically affect pDC function, as was show for the C-type lectin BDCA-2 and DCIR. Both receptors dramatically inhibit IFN-α secretion induced upon engagement with various ligands (7, 27). By contrast, our findings clearly demonstrate that cross-linking of CD32a does not inhibit the IFN-α production by TLR9-stimulated pDCs providing further evidence that inhibition of T cell proliferation is not affected by CD32a-mediated signaling. This notion is further supported by the finding that IFN-α production was not altered upon cross-linking of CD32a on freshly isolated pDC (data not shown). Together, these results indicate that, distinct from C-type lectin-like receptors BDCA-2 or DCIR, CD32a in itself, after immune complex induced cross-linking on pDCs, does not act as an immune activating or inhibiting receptor and, therefore, does not likely interfere with TLR9-derived signals.
APCs generally lose their Ag uptake capacity upon maturation either by reducing membrane receptor expression or by affecting its internalization. This is described for many Ag uptake receptors including DC-SIGN and the mannose receptor (28) (29). Also on pDCs, maturation stimuli indeed down-regulate expression of several putative Ag receptors including: FcεRI (15), BDCA-2 (7), CD36 (16), and DCIR (27). In contrast, the C-type lectin DEC205 (CD205) appears to lose its endocytic capacity in monocyte-derived DC upon TLR triggering rather than being down-regulated at the surface expression level upon maturation stimuli (30). The expression of CD32a remained unaltered after pDC activation using ODN-CpG. These observations demonstrate that TLR9-mediated signals directly control CD32a at the functional level and not at the expression level. This is supported by recent data on human neutrophils showing that phorbol ester PMA inhibits the ligand binding capacity of CD32a (31).
Three classes of CpG ODN ligands for TLR9 have been described by different sequence motifs and different abilities to stimulate IFN-α production and maturation of pDCs (6). CpG-A localizes to early endosomes (Transferrin TfR-positive endosomes) and mediates cytokine production, whereas CpG-B localizes in late endosomes (LAMP-1 positive compartments) and mediates pDC maturation. CpG-C is present in both types of endosomes, which correlates with the ability to trigger both IFN-α production and pDC maturation. Interestingly, we can distinguish two different effects of CpGs on the CD32a inactivation, whereas CpG-B and CpG-C have a strong inhibitory effect, CpG-A hardly affects the immune complexes uptake.
It will be important to unravel the signaling pathway that results in CD32a inactivation upon TLR9 stimulation. We hypothesize that the function of CD32a is regulated via pathways similar to those regulating costimulatory molecule expression (NF-κB signaling resulting in inhibition of uptake) and cytokine expression (IRF-7 signaling with no effect on uptake) (Fig. 5 d). Unfortunately, unraveling signaling pathways controlling CD32a function of pDCs are hampered by the fact that human pDCs are rare blood cells and cannot be grown in vitro in sufficient numbers for extensive biochemical analysis.
In conclusion, we have demonstrated that, in human pDCs, TLR9-mediated signals associated with the late endosomes are responsible for the diminished uptake of immune complexed Ags via CD32a. This represents a novel Ag uptake control mechanism limiting presentation of Ags encountered after pDC activation and maturation. Moreover, we show that CpG-B and CpG-C, both localizing to the late endosomes, but not CpG-A, reduce uptake of immune complexes by pDCs, thus confirming and extending the hypothesis that major differences in TLR9 ligand localization and signaling can dramatically affect pDC cell function. In addition, our findings demonstrate that TLR9-mediated signaling differently regulates FcR-mediated Ag uptake (i.e., function of receptor) when compared with lectin like receptor-mediated Ag uptake (i.e., down-regulation of the receptor).
It is tempting to speculate that the endosomal localization of viral or bacterial-derived products determines the biological response to TLR9 activation in pDCs. Upon stimulation in the late endosomes, but not in the early endosomes, endocytic activity is reduced to facilitate presentation of Ags that are associated with the TLR9 ligands rather than presentation of nonrelated Ags that are acquired subsequent to TLR9 stimulation. This might prevent pDC-induced autoimmunity as uptake and presentation of immune-complexed self Ags is hampered in matured pDCs.
We thank Nicole Scharenborg, Marieke Kerkhoff, Annemiek de Boer, and Mandy van de Rakt for technical assistance. Drs. Gosse Adema and Friederike Meyer-Wentrup are acknowledged for discussions.
The authors have no financial conflict of interest.
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.
Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; KLH, keyhole limpet hemocyanin; ODN, oligodeoxynucleotide; moDC, monocyte-derived DC.