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The Journal of Immunology, 2008, 181, 6525 -6535
Copyright © 2008 by The American Association of Immunologists, Inc.
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Human Plasmacytoid Dendritic Cells Interact with gp96 via CD91 and Regulate Inflammatory Responses1

AnnaMaria De Filippo*, Robert J. Binder{ddagger}, Chiara Camisaschi*, Valeria Beretta*, Flavio Arienti{dagger}, Antonello Villa§, Pamela Della Mina*,§, Giorgio Parmiani2,*, Licia Rivoltini* and Chiara Castelli3,*

* Unit of Immunotherapy of Human Tumours and {dagger} Unit of Immunohematology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Nazionale dei Tumori, Milano, Italy; {ddagger} Department of Immunology, University of Pittsburgh, PA 15203; and § Consorzio Microscopy and Image Analysis, Monza, Università Milano Bicocca, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Glucose-regulated stress protein gp96 is known to be involved in the host response to pathogens and to cancer. Our study explored the relationships between gp96 and human blood plasmacytoid dendritic cells (pDC) and proved that gp96 directly targets pDC by a receptor-dependent interaction. Competition studies identified CD91 as a gp96 receptor on pDC, and laser confocal imaging indicated that CD91 triggering was followed by gp96 endocytosis and trafficking into early endosomes and later into the endoplasmic reticulum compartment. Using two alternative Abs, we showed that human blood pDC reproducibly expressed CD91, although different levels of expression were detectable among the analyzed donors. Moreover, CpG-matured pDC displayed CD91 receptor up-regulation that correlated with an increased gp96 binding. Functionally, gp96-pDC interaction activated the NF-{kappa}B pathway, leading to the nuclear translocation of the NF-{kappa}B complex. gp96-treated pDC maintained an immature phenotype, while they down-modulated the release of IL-8, suggesting an anti-inflammatory role of this pathway, and they strongly up-regulated the cell surface expression of the gp96 receptor CD91. CpG-matured or gp96-treated pDC, expressing high levels of the gp96 receptor CD91, antagonized the gp96-induced activation of monocyte-derived dendritic cells in terms of cell surface phenotype and cytokine production. Altogether, these results suggest that gp96-pDC interaction might represent an active mechanism controlling the strength of the immune response to free, extracellular available gp96; this mechanism could be particularly relevant in wounds and chronic inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Upon release into the extracellular space following necrotic cell death, gp96 exerts dual immunological functions: 1) the specific transfer of its chaperoned peptides into professional APC that leads to cross-presentation to relevant T cells; and 2) APC activation, resulting in the up-regulation of costimulatory molecules and cytokine release (1). The synergism between these two roles makes gp96, purified from tumor or infected cells, an optimal vaccine eliciting specific protective immunity (2, 3, 4). gp96-APC interactions have been shown to be mediated by two membrane receptors expressed by APC, namely CD91, mainly involved in representation of heat shock protein (HSP)4-associated peptides (5, 6, 7), and TRL2, principally mediating APC activation (8). The capacity of gp96 to specifically interact with different APC including macrophages, monocytes, and myeloid-derived dendritic cells (DC) has been extensively studied in mice and humans (9, 10, 11).

However, among human APC, plasmacytoid dendritic cells (pDC) play a central role as well in molding both innate and adaptive immunity. pDC are type I IFNs producing cells mainly found in peripheral blood and secondary lymphoid organs. Through TLR7 and TLR9 engagements, pDC recognize viral RNA and bacterial DNA, respectively, and respond by secreting type I IFN. By means of this cytokine, pDC exercise their regulatory function in inflammation, activate NK cells, and initiate and sustain antiviral immune responses (12). Upon infection, pDC cross-present viral Ags to CD8+ T cells and induce Th1 polarization (13). These DC have been also found to uptake exogenous Ags through Fc{gamma}RII (14), the C-type lectin receptors blood DC antigen 2 (BDCA-2) (15) or the DC immune receptor (16), with subsequent induction of T cell activation. Moreover, a recent study showed that pDC have the ability to phagocytose infected apoptotic cells and to cross-present viral lipopeptides or HIV-1 Ags to CD8+ T cells (17).

Interestingly, in addition to this positive stimulatory activity on the immune system, pDC are also endowed with tolerogenic functions. They have been shown to induce anergy in human CD4+ T cells (18) and to prime IL-10-producing regulatory T cells (19, 20). Alloantigen-presenting pDC mediate Ag-specific regulatory T cell development and allograft tolerance (21), while their infiltration in tumor lesions such as in breast carcinoma is associated with poor prognosis, suggesting a potential involvement of this cell subset in tumor progression and/or in cancer-related immune suppressive pathways (22). As pDC can also be found in inflamed tissues (23, 24, 25, 26) and in tumors (27, 28, 29), their encounter with gp96 released by necrotic tissue or dying cells is likely to occur in vivo (30).

In the present study, we explore the relations between gp96 and pDC and demonstrate that gp96 specifically interacts with this subset of DC in a receptor-mediated fashion and that CD91 is involved in this contact. On the basis of the data described herein, we propose that, through CD91 triggering, pDC may be a part of a control mechanism limiting the strength of the immune response induced by free extracellular available gp96.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of pDC and monocytes

Buffy coats were obtained from healthy blood donors from the blood donor association at the Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Nazionale Tumori (Milan, Italy). PBMC were isolated by standard density-gradient centrifugation over Ficoll-Paque Plus (Amersham Biosciences). pDC were isolated as untouched cells by immunomagnetic beads using a human pDC isolation kit (Miltenyi Biotec). Purity of isolated pDC was assessed with a PE- or allophycocyanin-conjugated BDCA-2 mAb, and 96–98% purity was always detected (Miltenyi Biotec).

Peripheral blood monocytes were negatively isolated from PBMCs by immunomagnetic beads (monocyte isolation kit II, Miltenyi Biotec), according to the manufacturer’s instructions (≥90% purity).

gp96 purification

gp96 from human placenta was purified as previously described (31) and kindly provided by Antigenics. Electrophoresis of the purified protein was conducted by 10% SDS-PAGE, and Western blotting was performed using polyvinylidene difluoride membranes (Amersham Pharmacia). The membranes were blocked using 5% skim milk solution, probed with the rat monoclonal anti-gp96 Ab (clone 9G10.F8.2) (NeoMarkers), and visualized using ECL detection reagent (Amersham Biosciences). Alternatively, SDS-PAGE gel was stained using Coomassie blue (Bio-Rad Laboratories).

Protein conjugation to fluorochromes

gp96 and BSA (Sigma-Aldrich) were conjugated to FITC using the FluoroTag FITC conjugation kit (Sigma-Aldrich) according to the manufacturer’s instructions for the small-scale conjugation. Alternatively, gp96 and BSA were labeled using the Alexa Fluor (AF)488 mAb labeling kit (Molecular Probes) according to the manufacturer’s instructions. Final labeled protein concentrations and fluorescein/protein molar ratios were determined by the absorbance reading of the conjugate samples (dilution 1/10 in PBS) at 280 and 495 nm for FITC and at 280 and 494 nm for AF488 according to the manufacturer’s instructions.

Electrophoresis of the gp96/BSA-FITC complexes was conducted by 10% SDS-PAGE, and Western blotting was performed using polyvinylidene difluoride membranes (Amersham Pharmacia). The membranes were blocked using 5% skim milk solution, probed with the rabbit F(ab')2 polyclonal anti-FITC/HRP (1/1000 dilution) (Dako) and visualized using ECL detection reagent (Amersham Biosciences). Quality of gp96/BSA-AF488 complexes was analyzed by FACS and laser confocal microscopy analysis.

gp96 binding to monocytes and pDC

PBMC (1 x 106cells/sample), freshly purified monocytes (5 x 105cells/sample), and pDC (1.5 x 105cells/sample) were washed with FACS buffer (PBS + 0.5% FCS) and then incubated for 30 min at 4°C with gp96-FITC or BSA-FITC at the indicated dose. Anti-CD14 Ab (BD Biosciences) or anti-BDCA-2 and anti-CD123 Abs were used to gate monocytes and pDC in PBMC, respectively. After being washed, cells were resuspended in 2% formalin solution before FACS analysis at the FACSCalibur flow cytometer station (BD Biosciences). BSA-FITC or BSA-AF488 (50 µg/ml) was used as negative control.

Binding competition

Competitors were used in molar ratios (gp96-FITC/competitor) as follow: unlabeled gp96 (1:1, 1:10, 1:50, and 1:100), {alpha}2-macroglobulin (1:1, 1:10, and 1:50), anti-CD91 polyclonal Ab from Antigenics (1:1, 1:10, 1:50, and 1:100), anti-CD91 mAb from BD Pharmingen (1:1, 1:10, and 1:50), albumin (1:1, 1:10, 1:50, and 1:100), and Ab-matched isotype control (1:1, 1:10, and 1:50). PBMC (1 x 106cells/sample) were stained for PE-conjugated Ab to BDCA-2 and biotin-conjugated CD123 for 30 min at 4°C, then washed using FACS buffer (PBS + 0.5% FCS), stained with anti-biotin streptavidin-PerCP for an additional 30 min, and washed and fixed in 2% paraformaldehyde (PFA) for 1 min on ice. Following extensive wash in FACS buffer, cells were resuspended in 100 µl of PBS and added with competitor and gp96-FITC (10 µg/ml) simultaneously. Cells were incubated for 15 min on ice, washed, and resuspended in PBS. FACS analysis was performed on the FACSCalibur station.

gp96 intracellular pathway in pDC

Freshly purified pDC were incubated with gp96-AF488 (50 µg/ml) alone or together with Alexa Fluor 594-conjugated human transferrin (50 µg/ml, Invitrogen) for 30 min on ice to prevent endocytosis. Cells were washed using PBS + 2% BSA and either fixed in 4% PFA for 15 min on ice or, before being fixed, brought to 37°C for 5 or 10 min to allow endocytosis. Following an extensive wash, cells were resuspended in 100 µl of PBS, transferred on gelatin-coated slides, and allowed to dry out overnight at room temperature (RT). After rehydration, cells were treated briefly with 0.1 M glycine in PBS (pH 7.4) followed by 0.3% Triton X-100 buffer. Cells were later stained with rabbit anti-calnexin polyclonal Ab (1/200 dilution, StressGen Biotechnologies) or mouse anti-LAMP-2 (lysosome-associated membrane protein 2) mAb (1/250 dilution, Cell Signaling Technology, provided by Invitrogen) overnight at 4°C. Subsequently, cells were stained with rhodamine-conjugated goat anti-rabbit IgG (H+L) (1/100 dilution, Rockland) or rhodamine-conjugated goat anti-mouse IgG (H+L) secondary Ab, respectively, for 1 h at RT. Slides were mounted in 95% glycerol mounting medium and analyzed by a confocal microscope (Radiance 2100, Bio-Rad Laboratories) equipped with a krypton/argon laser.

NF-{kappa}B pathway activation

Purified pDC (2 x 105cells/sample) kept in complete medium (RPMI 1640 + 10% heat-inactivated FCS, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine) (BioWhittaker) were stimulated with type A+B CpG (3 µg/ml each), gp96 (50 µg/ml), or left untreated for 3 h at 37°C. Cells were washed extensively with PBS + 2% BSA and resuspended in 4% PFA fixing solution for 15 min at RT. Following extensive washing, cells were permeabilized with 100% ice-cold methanol for 10 min at –20°C, washed again, and then blocked for 30 min at RT using PBS + 2% BSA. Cells were later stained with mouse anti-NF-{kappa}B p65 mAb (BD Biosciences) and diluted 1/300 in PBS + 2% BSA for 2 h at 4°C. After extensive washing, cells were stained with AF488 goat anti-mouse (1/500 dilution, Invitrogen) for 1 h at RT. Cells were finally resuspended in 100 µl of PBS, transferred on gelatin-coated slides, and allowed to dry out overnight at RT. For DNA staining, slides were treated with RNase A (1 mg/ml) and TOTO-3 iodide (642/660, Molecular Probes/Invitrogen) at 1/400 dilution in PBS buffer 0.12 M for 10 min at RT. Following a final wash with PBS, glass slides were mounted with 95% glycerol in PBS and analyzed by a confocal microscope (Radiance 2100) equipped with a krypton/argon laser.

For the flow cytometry analysis, freshly purified pDC (1.5 x 105cells/sample) were stimulated for 3 h with gp96 (50 µg/ml) or CpG (type A+B, 3 µg/ml each) with or without 0.5 or 1 µM of NF-{kappa}B inhibitor (I{kappa}B kinase 2 (IKK2) inhibitor IV, Merck Chemicals). Cells were then washed, fixed in 4% PFA for 15 min at RT, washed again, and permeabilized with Perm Buffer III (BD Biosciences) for 30 min on ice. Washed cells were stained with Alexa Fluor 647-conjugated anti-phosphorylated NF-{kappa}B p65 mAb (BD Biosciences) for 30 min at 4°C. Cells were washed, resuspended in PBS, and analyzed on a FACSCalibur station.

Phenotype and maturation of pDC

Freshly purified pDC (5 x 105cells/sample) were matured for 24 or 48 h at 37°C in complete medium (described above) supplemented with IL-3 (10 ng/ml), IL-3 + gp96 (50 µg/ml) (Antigenics), or IL-3 + CpG10103 (1.5 µg/ml) (TCG TCG TTT CGT CGT TTT GTC, type B oligodeoxynucleotide sequence, Coley Pharmaceutical Group). Nonmatured or matured pDC were then stained with PE-conjugated mAb to CD80, biotin-conjugated/streptavidin PerCP Ab to BDCA-2, and allophycocyanin-conjugated mAb to CD83. FACS analysis was performed on a FACSCalibur station. Expression of CD91 was assessed on purified pDC by indirect immunofluorescence using mouse monoclonal anti-CD91 Ab (BD Pharmingen) or rabbit polyclonal anti-CD91 Ab (Antigenics) followed by secondary anti-rabbit or anti-mouse FITC- or PE-labeled Ab.

Alternatively, total PBMC (2.5 x 106cells/sample) were matured for 48 h at 37°C in regular growing medium supplemented with gp96 (50 µg/ml) or with CpG10103 (1.5 µg/ml) as control. pDC were identified within the total PBMC using the following Ab: FITC-conjugated Ab to lineage, allophycocyanin-conjugated Ab to CD11c, PE-Cy5-conjugated Ab to CD123, PE-Cy7-conjugated Ab to HLA-DR, and PE-conjugated mAb to CD80, CD83, or CD91 (all purchased from BD Biosciences). The five-color analysis was performed on a FACS CyAn (Dako) at the Goccia Laboratory (San Raffaele Scientific Institute, Milan).

Functional maturation of pDC

Freshly purified pDC (1.25 x 105cells/well) were stimulated in a 96-well plate for 24 h at 37°C in complete medium (described above) supplemented with IL-3 (10 ng/ml), IL-3 + gp96 (50 µg/ml) (Antigenics), IL-3 + CpG10103 (1.5 µg/ml) (type B), IL-3 + CpG2336 (1.5 µg/ml) (GGG GAC GAC GTC GTG GGG GGG, type A oligodeoxynucleotides sequence, Coley Pharmaceutical Group), IL-3 + gp96 + CpG10103 or IL-3 + gp96 + CpG2336. Supernatant was collected and stored at –20°C before cytometric bead array (CBA) analysis.

Cytometric bead assays (BD Biosciences) were conducted according to the manufacturer’s instructions. Three different kits with specific capture bead mixtures were exploited: CBA human inflammation kit, for the detection of IL-8, IL-1β, IL-6, IL-10, TNF-{alpha}, and IL-12p70; CBA human Th1/Th2 kit, which quantitatively measures the release of IL-2, IL-4, IL-6, IL-10, TNF, and IFN-{gamma}; and the CBA human chemokine kit, for the detection of CXCL8 (IL-8), CCL5 (RANTES), CXCL9 (MIG), CCL2 (MCP-1), and CXCL10 (IP-10). Samples were analyzed using a FACSCan flow cytometer station (BD Biosciences). Data were acquired and analyzed using the BD CBA software. Data were displayed as two-color dot plots (FL-2 vs FL-3). Standard curves were plotted using a four-parameter logistic curve-fitting model, and cytokine/chemokine concentrations were calculated on its basis. Human instant ELISA system (Bender MedSystems) was performed to quantitatively detect soluble IFN-{alpha} released in the supernatant of pDC cell cultures following stimulations. The assay and calculation of results were performed according to the manufacturer’s instructions.

Maturation of monocyte-derived DCs (MoDC) in the presence of pDC

Monocytes, negatively purified from PBMC by immunomagnetic beads (monocyte isolation kit II, Miltenyi Biotec), were resuspended and cultured for 6 days in RPMI 1640, 10% FCS, 2 mM L-glutamine (Cambrex) with 50 ng/ml GM-CSF (Myelogen, Schering-Plough), and 20 ng/ml IL-4 (PeproTech). MoDC maturation was induced by overnight culture with 50 µg/ml of gp96 or 2 µg/ml LPS 0111:B4 (Sigma-Aldrich).

MoDC and pDC cocultures were performed in 24-well plates with transwell inserts to prevent cell-to-cell contact (Nunc). Immature MoDC (4 x 105cells/sample) were placed in the lower chamber in medium conditioned with 50 µg/ml gp96. The upper chamber contained equal numbers of pDC immature or matured as described above and then fixed with 4% PFA. Total volume in each well was 0.6 ml. After 24 h of incubation, conditioned medium and MoDC were collected. MoDC were analyzed for the cell surface expression of CD80 and CD86 using PE or FITC-conjugated mAb (BD Biosciences) while conditioned media were evaluated for the cytokine content using the CBA assay (BD Biosciences) following the manufacturer’s instructions. Data were acquired using a FACScan flow cytometer station and analyzed using the BD CBA software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human gp96 binds pDC

To gain an insight into the possibility of a specific interaction of gp96 with human pDC and to characterize its functional outcomes, we investigated the capacity of blood pDC to bind human gp96. gp96 having a purity >95% (Fig. 1A) was labeled with FITC. Western blot analysis with anti-FITC mouse Ab confirms the purity and the optimal quality of FITC-labeled gp96 as well as FITC-labeled BSA, which was used as negative control protein (Fig. 1B). Purified and labeled gp96 was also assessed for its capacity to specifically bind isolated human monocytes. Monocytes were incubated for 30 min at 4°C with titrated doses of gp96-FITC and then analyzed by flow cytometry to quantify protein binding. Fig. 1C (top panel) shows that at 50 µg/ml, gp96-FITC efficiently binds to monocytes and that increasing protein concentration (100 µg/ml) produces higher binding in a dose-response fashion (Fig. 1C, bottom panel). BSA-FITC staining indicates levels of unspecific binding. These data show a specific interaction of human gp96 with monocytes and confirm similar findings previously reported for murine gp96 (11).


Figure 1
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  		FIGURE 1. Characterization of the gp96 preparation. A, gp96 (1 µg) was analyzed by 10% SDS-PAGE. B, FITC-conjugated gp96 and control BSA (1 µg of each) were assessed by immunoblotting with anti-FITC/HRP Ab. C, gp96-FITC binding to monocytes is specific and saturable. Titrated amounts of gp96-FITC and BSA-FITC complexes were incubated with freshly isolated human monocytes (5 x 105 cells/sample) for 30 min and analyzed by flow cytometry. Black filled histograms show gp96-FITC saturable binding, while gray filled histograms show BSA-FITC unspecific binding. Open histograms show autofluorescence. The percentages of positive cells stained with each complex as well as the protein concentrations used are indicated.

 
We then proceeded to test the binding of gp96-FITC to pDC. pDC were identified within PBMC as CD123high and BDCA-2+ cells (Fig. 2A) or they were isolated from PBMC (0.3% of the total PBMC population) by purification with the immunomagnetic cell sorting system (MACS) (purity yield ≥96%) (Fig. 2B). Freshly isolated PBMC or purified pDC were incubated at 4°C with 50 µg/ml FITC-labeled gp96 or BSA. FACS analysis showed that pDC were able to strongly and specifically bind FITC-labeled gp96, as FITC-labeled BSA resulted only in background level of staining (Fig. 2, A and B, right panels). Saturability of gp96-pDC interaction was tested and a constant number of PBMC were stained with increasing quantity of FITC-labeled gp96. At each protein concentration, the mean fluorescent intensity of pDC, gated as CD123highBDCA-2+ cells, was measured by FACS analysis. As shown in Fig. 2C (left panel), the gp96 binding was dependent on protein concentration, and saturable binding was reached at ~50 µg/ml of protein. Binding of gp96 to MoDC (Fig. 2C, right panel) was also evaluated, resulting in a similar binding saturation, although with a higher affinity as indicated by the Kd values of the Scatchard analysis.


Figure 2
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  		FIGURE 2. Human blood pDC specifically bind gp96. A, pDC were gated inside freshly isolated healthy donors’ PBMC (1 x 106 cells/sample) as CD123+BDCA-2+ cells (middle panel). gp96-FITC (black line) or BSA-FITC staining (filled histogram), obtained with 50 µg/ml of each complex, was analyzed on the gated cells (0.3% of the total PBMC). More than five independent experiments on different healthy donors were performed. B, Freshly purified pDC from a healthy donor’s PBMC (1.5 x 105 cells/sample) were stained first with anti-CD123 and anti-BDCA-2 Ab to assess purity (middle panel), and subsequently with 50 µg/ml of gp96-FITC (black line) or BSA-FITC (filled histogram). More than five independent experiments on different healthy donors were performed. C, gp96-FITC binding saturation on pDC. Freshly isolated PBMC (1 x 106 cells/sample) were stained for BDCA-2 and CD123 to identify pDC and then incubated for 30 min at 4°C with increasing amount of gp96-FITC complex, as indicated in the figure (left panel). To compare the binding saturation, 6-day-cultured MoDC were stained for CD1a and then incubated with gp96-FITC complex at the same increasing concentrations (right panel). Scatchard plot analysis and the values of Kd (expressed in nM) are indicated in the figure.

 
Specific gp96 binding to pDC involves CD91

Consistent with results in both the murine and human systems (8, 9, 10, 11), our data suggest that gp96-pDC interaction involves receptor-mediated binding. Since CD91 has been described to function as a gp96 receptor for other murine or human APC (5, 6, 32), we speculated that the same molecule could be involved in gp96 binding to pDC. Therefore, human pDC gated as CD123highBDCA-2+ cells in PBMC of healthy donors were assessed for the expression of CD91 using two sovereign Abs raised independently against CD91, namely Ab1, which is a polyclonal Ab kindly provided by Antigenics, and Ab2, a mAb commercially available from BD Pharmingen. By FACS analysis, both anti-CD91 Abs revealed a consistent expression of CD91 on the surface of pDC (Fig. 3A), although at variable levels among the healthy donors examined. More precisely, while pDC from all donors were shown to express CD91, the levels of expression varied and donors exhibiting low (0.5–15% positive gated cells), intermediate (16–60%), or high (61–99%) percentages of CD91+ cells were identified (Fig. 3B and Table I).


Figure 3
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  		FIGURE 3. HSP receptor CD91 is expressed on human blood pDC and its expression is variable among donors. A, pDC (3 x 105 cells/sample) gated on freshly isolated donors’ PBMC were stained with two different anti-CD91 Abs (open histograms): Ab1 is a polyclonal Ab from Antigenics; Ab2 is a mAb from BD Pharmingen. Filled histograms represent isotype control staining. Percentages of CD91+ cells are indicated. More than five independent experiments on different healthy donors were performed. B, CD91 receptor expression is variable among donors. pDC (3 x 105 cells/sample) gated on healthy donors’ PBMC were stained with anti-CD91 Ab1. The three levels of CD91 expression (low, intermediate, and high) are represented by the open histograms of donors 3, 11, and 17. The percentage values of positive cells are indicated. Filled histograms are the isotype control staining. To standardize the values of percentage reported, instrument detector setup was kept constant throughout the experiments by maintaining the voltage of FL2 channel in the same intensity range (i.e., 641–667 log units).

 

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  		Table I. CD91 surface expression on healthy donors’ pDCa

 
gp96 binding to CD91 expressed by pDC was confirmed by competition studies. Titrated molar excesses of unlabeled gp96 or {alpha}2-macroglobulin (a known CD91 ligand) (5) were able to inhibit the binding of gp96-FITC to CD91 (Fig. 4A). In contrast, control protein albumin, which does not interact with CD91, was unable to compete with gp96-FITC binding, supporting the specificity of the observed competition. Furthermore, Ab1 or Ab2 Abs to CD91 inhibited the binding of gp96-FITC to cells, although with different efficiency (Fig. 4B). The corresponding control Ab isotypes (rabbit for Ab1 and mouse IgG for Ab2) did not inhibit gp96-FITC staining. The ability of ligands for and Abs to CD91 to compete with gp96 binding strongly suggests that CD91 is acting as a receptor for gp96 on pDC.


Figure 4
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  		FIGURE 4. gp96, {alpha}2-microglobulin (alpha 2-M), and anti-CD91 Ab1 and Ab2 compete with gp96-FITC for binding. A and B, Total PBMC (1 x 106 cells/sample) were stained for CD123 and BDCA-2 and subsequently fixed. Following washing, cells were incubated with gp96-FITC/competitor mixtures, with competitor proteins used at increasing molar amounts as indicated. gp96-FITC was used at a final concentration of 10 µg/ml. Cells were extensively washed to remove excess protein, and FITC fluorescence was analyzed by flow cytometry on CD123high and BDCA-2+. Percentages of gp96-FITC binding inhibition are plotted vs increasing molar amounts of competitor proteins.

 
Interestingly, CD91 receptor expression on pDC was up-regulated by pDC maturation. Indeed, purified pDC incubated for 24 h with IL-3, which is necessary for in vitro survival (12), enhanced their CD91 expression from 20% to 77% positivity (Fig. 5A, top and middle panels). Moreover, full maturation of pDC achieved by treatment with IL-3 and type B CpG caused an even greater increase of CD91 expression, with 93% of cells becoming positive for the receptor (Fig. 5A, bottom panel). CD91 up-regulation occurred after maturation regardless of initial receptor expression. pDC displaying a low level of CD91 expression, as shown for donor 1, increased CD91 positivity from 0.5% to 36% upon maturation (Fig. 5B, top panel). The increased expression of CD91 in IL-3 and CpG-B matured pDC tightly correlated to an enhanced ability of these cells to bind gp96. The binding of gp96 by matured pDC strongly increased with respect to immature pDC (Fig. 5B, middle panel), and this enhanced binding capacity was specific for gp96 since it could not be detected with BSA (Fig. 5B, bottom panel). These results show that pDC express variable levels of CD91 between donors, but this expression is inducible by maturating stimuli such as IL-3 and CpG. Inducibility of CD91 expression parallels a greater capacity of these cells to specifically interact with gp96.


Figure 5
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  		FIGURE 5. CD91 receptor expression on pDC is up-regulated following cell maturation, parallel to an increase in gp96 binding. A, Up-regulation of CD91 following cell maturation. Freshly purified pDC (1.5 x 105 cells/sample) from a healthy donor were matured for 24 h with IL-3 only (10 ng/ml) or IL-3 + CpG-B (1.5 µg/ml), and then stained with anti-CD91 Ab2 (filled histograms). Open histograms are the isotype control staining. Percentage of CD91+ cells and range values are indicated in the figure. Three independent experiments on different healthy donors were performed. B, Up-regulation of CD91 receptor expression corresponds to increased gp96-FITC binding. pDC (1 x 106 cells) purified from donor 1 (low expression of CD91) were matured for 24 h with IL-3 + CpG-B. CD91 expression, using Ab1 (upper panel), and gp96-FITC binding (middle panel) were analyzed before and after maturation. As the percentage values in the figure indicate, the CD91 up-regulation corresponds to an increase in gp96-FITC binding. BSA-FITC binding was used as a control for unspecific protein binding (lower panel). Isotype control staining is represented by the dashed lines. Several experiments were performed on different healthy donors, and the range of values are indicated.

 
Trafficking of internalized gp96 to the endoplasmic reticulum (ER) of pDC

After showing that gp96 does interact in a receptor-mediated fashion with human pDC, we analyzed the fate of gp96 after receptor engagement by laser confocal microscopy. Binding of gp96-AF488 to human pDC cell surfaces revealed a patched pattern of staining localized at the cell surface. Further incubation of gp96-labeled cells at 37°C for 5 (Fig. 6a–c) or 10 min (Fig. 6d–i) led to efficient endocytosis of gp96-AF488 complexes. To analyze the subcellular trafficking itinerary of internalized gp96, pDC were also coincubated with Alexa Fluor 549-conjugated transferrin as a marker for early endosomes (Fig. 6b). Staining for calnexin or LAMP-2 as markers for ER and lysosomes, respectively (Fig. 6, e and h), were then performed. Confocal analysis demonstrated that, after only 5 min of internalization at 37°C, gp96-AF488 colocalized with transferrin-Alexa Fluor 549 (Fig. 6c), indicating a rapid receptor-mediated endocytosis of the HSP, as previously reported (9), as well as subsequent localization of internalized gp96 into pDC early endosomes. After 10 min of incubation, cytoplasmic staining of gp96 was observed (Fig. 6d) that matched with anti-calnexin Ab staining (Fig. 6, e and f), demonstrating gp96 localization into the ER. In line with previous data in murine APC, such as macrophages and bone-marrow derived DC (10), no costaining with anti-LAMP-2 Ab was detected, suggesting gp96 exclusion from lysosome compartment (Fig. 6, h and i) even at later times of observation (data not shown).


Figure 6
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  		FIGURE 6. gp96 trafficking and subcellular ER localization in pDC. Freshly purified pDC were incubated with gp96-AF488 (50 µg/ml) and transferrin (50 µg/ml) for 30 min on ice and then transferred for 5 min at 37°C (a–c); cells incubated with gp96-AF488 (50 µg/ml) for 30 min on ice, transferred for 10 min at 37°C, and stained with anti-calnexin (d–f) or with anti-LAMP-2 Abs (g–i). Analyses were performed by laser confocal microscope at x60 magnification.

 
gp96 induces the nuclear translocation of NF-{kappa}B

One of the outcomes of gp96 interaction with APC is cell activation via intracellular signaling (30). Therefore, to determine the effect of gp96 interaction and internalization in human pDC, we investigated the nuclear translocation of NF-{kappa}B after gp96-treatment (Fig. 7). While in untreated pDC, NF-{kappa}B was mainly located within the cytoplasm, as expected for cells in steady-state condition (Fig. 7A, top panels), treatment of freshly purified pDC with gp96 or CpG, used as positive control, elicited translocation of NF-{kappa}B into the nucleus, as evident in the merged images (Fig. 7A, right panels).


Figure 7
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  		FIGURE 7. gp96 treatment of pDC elicits NF-{kappa}B nuclear translocation. A, Freshly purified pDC (2 x 105 cells/sample) untreated or stimulated 3 h with gp96 (50 µg/ml) or CpG (type A+B, 3 µg/ml each) were stained with anti-NF-{kappa}B p65 mAb. For DNA staining, slides are treated with RNase A (1 mg/ml) and TOTO-3 iodide (1/400) for 10 min at RT. Cells were analyzed by a laser confocal microscope at x60 magnification. B, Freshly purified pDC (1.5 x 105 cells/sample) were stimulated for 3 h with gp96 (50 µg/ml, upper panels) or CpG (A+B, 3 µg/ml each) (bottom panels) with or without 0.5 or 1 µM of NF-{kappa}B inhibitor. Cells were then stained with anti-phosphorylated NF-{kappa}B p65 mAb. Percentages of positive cells with respect to the isotype staining are indicated. Average values (±SEM) of three independent experiments are expressed in the graphs. *, p < 0.05.

 
A more quantitative analysis of the NF-{kappa}B activation was performed by flow cytometry using an anti-phosphorylated NF-{kappa}B p65 mAb and a NF-{kappa}B specific inhibitor (Fig. 7B). Freshly purified pDC were stimulated for 3 h with gp96 (Fig. 7B, top panels) or CpG (Fig. 7B, bottom panels) with or without titrated amounts of IKK2 inhibitor. Although a constitutive activation of NF-{kappa}B is present in purified pDC, its level of phosphorylation is increased following gp96 stimulation. The number of cells positive for NF-{kappa}B phosphorylation returned closer to and below the baseline level (no treatment staining) in the presence of IKK2 inhibitor. Similar results were obtained in CpG-stimulated pDC.

Consistently with data in murine APC, these results demonstrate that the binding of gp96 to pDC triggers intracellular activating signaling resulting in the nuclear translocation of NF-{kappa}B.

Phenotypic characterization of pDC treated with gp96

We then tested whether NF-{kappa}B nuclear translocation observed in gp96-stimulated pDC translated into external phenotypic and functional changes. pDC were thus purified, maintained in the obligatory IL-3-containing medium for cell survival, and then incubated with gp96 for subsequent analysis of costimulatory molecule expression. In this experimental setting, no up-regulation of CD83 was observed upon treatment with gp96, while the expression of CD80, already very high in the presence of IL-3 alone and further increased by CpG, showed no change after incubation with gp96 (Fig. 8A). In line with these results, the ability of pDC to release TNF, IL-6, IL-8, or IFN-{alpha}, efficiently triggered by CpG stimulation, was instead unaffected by gp96 (Fig. 8B). On the other hand, gp96 was able to significantly inhibit the constitutive secretion of IL-8 by unstimulated pDC (see arrow in Fig. 8B) but did not interfere with the release of this cytokine in CpG-A- or CpG-B-stimulated pDC.


Figure 8
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  		FIGURE 8. gp96 does not induce direct maturation of pDC but it up-regulates CD91 receptor expression. A, Freshly purified pDC (5 x 105 cells/sample) were matured 24 h at 37°C in complete medium (described earlier) supplemented with IL-3 only (10 ng/ml), IL-3 + gp96 (50 µg/ml), or IL-3 + CpG-B (1.5 µg/ml). Cells were then stained with BDCA-2-, CD80-, and CD83-specific Ab. B, Freshly purified pDC (1.25 x 105 cells/well) were matured as in A and in complete medium supplemented with IL-3 + CpG-A (1.5 µg/ml), IL-3 + gp96 + CpG-A, or IL-3 + gp96 + CpG-B. Supernatants were collected and analyzed by CBA analysis. The arrow indicates the inhibition of IL-8 secretion upon gp96 stimulation. IFN-{alpha} release was quantified by ELISA. Three independent experiments on different healthy donors were performed. C, Freshly purified pDC (5 x 105 cells/sample) were matured as in A, stained for BDCA-2 and CD91 (BD Pharmingen) as previously described, and analyzed by flow cytometry. *, p ≤ 0.05. Four independent experiments on different healthy donors were performed.

 
Although gp96 did not directly promote phenotypic or functional maturation of pDC, it did have a direct effect in up-regulating its own receptor. gp96 treatment of purified pDC mediated a 2-fold up-regulation of CD91, as shown in Fig. 8C. This level of increase was similar to that achieved by CpG treatment. These data suggest that gp96 does not directly regulate maturation of pDC, as maturation markers and cytokine release analyses clearly point out. However, gp96 does stimulate increase of CD91 expression, further favoring a HSP receptor-specific interaction of pDC.

pDC limit gp96-induced maturation of MoDC

Since our data show that gp96 or CpG treatment strongly enhances CD91 cell surface expression and consequently gp96 binding capacity of pDC, we addressed whether this functional response could have any role in limiting the immunostimulatory effects of gp96. To this purpose, MoDC were induced to maturation by gp96 in the absence or presence of pDC either freshly isolated from PBMC or previously exposed for 24 h to gp96 or CpG, then washed and PFA fixed to prevent cytokine release. Cocultures were performed in transwell plates to avoid cell-to-cell contact, and after 24 h media and MoDC were collected for analysis. As shown in Fig. 9A, purified pDC strongly reduced the production of the proinflammatory cytokines TNF-{alpha}, IL-6, and IL-8 induced by gp96 in MoDC. This effect was also evident using CpG- or gp96-treated pDC, which completely abrogated the release of these cytokines by MoDC. In line with these data, both CpG and gp96-treated pDC significantly inhibited the gp96-mediated up-regulation of the costimulatory markers CD80 and CD86 in MoDC. Indeed, a >50% reduction and 25% reduction of the mean fluorescence intensities of CD80 and CD86, respectively, were observed when MoDC were treated with gp96 in the presence of pDC (Fig. 9B). Differently from what was obtained for cytokine secretion, fresh pDC, expressing less CD91 and having lower capacity to bind gp96 than gp96- or CpG-treated pDC (Fig. 5B), did not affect the cell surface expression of MoDC maturation markers. This finding is in line with the notion that phenotypic and functional maturation of DC are differently regulated compared with the cytokine production requiring a stronger and sustained stimulation (33). Thus, it is entirely possible that gp96 neutralization operated by fresh pDC was enough to bring the free gp96 molecules below the level required for a functional activation of MoDC, a level that was nonetheless still sufficient to induce the cell surface modulation of costimulatory markers.


Figure 9
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  		FIGURE 9. gp96-treated pDC inhibit MoDC maturation. A, Immature MoDC (MoDC only) cultured with IL-4 (20 ng/ml) + GM-CSF (50 ng/ml) were then induced to mature by LPS (2 µg/ml) or by gp96 (50 µg/ml). Coculture of MoDC (4 x 105 cells/well) and pDC were performed at a 1:1 ratio in a transwell plate in complete medium supplemented with gp96 (50 µg/ml). Blood-purified pDC were used fresh or previously treated for 24 h with gp96 (50 µg/ml) or CpG-B (1.5 µg/ml), washed, and then fixed with PFA. After 24 h of incubation at 37°C, conditioned media and MoDC were collected for analysis. Cytokine content was assessed by CBA assay. Cytokines released from LPS-treated MoDC were over the maximum assay detection limit. Levels of IL-12p70 and IL-10 cytokines were below the assay detection limit. B, MoDC treated as in A were analyzed for the cell surface expression of CD80 and CD86 using PE- or FITC-conjugated mAb. Values of mean fluorescence intensity are indicated in the figure.

 
These data suggest that specific gp96-pDC interaction, by inducing a strong up-regulation of CD91, initiates a loop mechanism that, preventing the interface of gp96 with other APC, neutralizes the immunostimulatory capacity of gp96.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
gp96, released upon cell damage into the extracellular milieu, is an alerting signal for the immune system. However, gp96 can be released also during tissue damage not mediated by pathogenic insults. Thus, it is likely that mechanisms limiting gp96-mediated local immune activation should also exist and be active in controlling the inflammatory response during tissue remodeling and healing. Negative roles of gp96 in immune responses have been also evidenced. In certain vaccination settings, gp96 administration prevents the onset of autoimmune diseases (34) and improves survival of allogenic skin grafts in mice (35), suggesting that gp96 may have a role in tolerance induction. Moreover, transgenic mice, expressing gp96 at the cell surface, display regulatory T cells with enhanced suppressive functions (36). Among APCs, pDC have been often associated to tolerance and to the induction of regulatory T cells. pDC, in addition to being one of the components of blood DC, are located in lymph nodes and can be also found in inflamed tissues (12). It is therefore entirely possible that at the site of inflammation gp96 released by damaged cells can directly encounter pDC.

Our study explored the relationships between gp96 and pDC, and it proved that human gp96 directly targets this cell subset. gp96 binds pDC in a saturable and competitive manner, demonstrating the specificity and receptor dependence of this interaction. Competition studies identified CD91 as a gp96 receptor on pDC. Using two sovereign Abs raised independently against CD91 and recognizing various regions on the molecule, we showed that this scavenger receptor is expressed by human pDC freshly isolated from blood, albeit strong differences in its expression level were evident among the analyzed healthy donors. Importantly, no extensive analysis on the expression of CD91 by human pDC has been previously reported, although human leukemia cells, whose normal counterpart were identified in pDC, have been described to be CD91+ (37). However, a heterogeneous expression of CD91 has been already observed in monocytes (38, 39).

In accordance with what has been reported in murine studies (9) and in human platelets (32), we found that in pDC competition for gp96 binding with anti-CD91 Abs was not complete. This observation suggests the possibility that other receptors on pDC could be involved in gp96 binding, as has been shown to occur in mice (40), or that CD91 at the cell surface is subjected to a rapid turnover or to active mechanisms of up-regulation. Interestingly, we showed that gp96 treatment itself strongly up-regulated the cell surface expression of its own receptor. Moreover, CD91 expression can be increased in response to IL-3 and TLR9 triggering. CD91 up-regulation occurs with an equal extent also in pDC displaying a low, basal level of receptor, leading to a more homogeneous pDC population expressing a high percentage of CD91+ cells. Positive regulation of CD91 expression by maturation stimuli has been also observed for human platelets treated with thrombin (32). However, while the regulatory elements of the CD91 gene are unknown, the regulation of CD91 expression in response to maturation stimuli offers intriguing insights into pathogen- and tumor-driven immune responses. In this view, up-regulation of CD91 receptor induced by pDC maturation through the binding to cognate ligands may initiate an autocrine loop ensuring an efficient uptake of circulating gp96.

In our study we showed that gp96-CD91 interaction is a productive event that triggers functional responses in pDC. By laser confocal imaging, we provided evidence that one important consequence of CD91 engagement is the active and efficient endocytosis of gp96. Our analysis on the trafficking of internalized gp96 then showed that gp96 is initially localized into early endosomes, as already shown for mice macrophages (41), and that it does not reach lysosomes LAMP-2-expressing compartment for degradation, even at later observation times. Surprisingly, the internalized gp96 appears in the calnexin-positive ER. As the precise vesicular pathway involved in the endosome-ER traffic of gp96 could not be identified, it is unclear whether gp96 is retrieved to the ER by its KDEL sequence. The "out-in" pathway of other exogenous proteins, such as the epidermal growth factor receptor and its ligand, have been shown to gain access to ER and from here, exploiting the Sec61 translocon, to reach the cytoplasm (42, 43). Additionally, gp96 and hsp70, which share at least one receptor CD91 among a list of five other reported receptors (44), have been previously shown to mediate the cross-presentation of their chaperoned peptides by delivering the peptides to the cytosol for proteasomal processing, following loading on MHC class I occurring into ER (45, 46). This might suggest an endosome-cytosol-ER traffic pattern for gp96, with dissociation of the chaperoned peptides from the gp96 into the endosome and the subsequent targeting of the two molecules to different final destinations. Studies with Sec61 inhibitors and KDEL mutant-gp96 molecules should help decipher these pathways.

Consistent with the murine data with myeloid cell targets (30), gp96-pDC interaction activates the NF-{kappa}B pathway, leading to phosphorylation and nuclear translocation of the NF-{kappa}B complex. However, the activation of this pathway does not lead to pDC maturation as it occurred in other APC upon gp96 stimulation. Although no direct up-regulation of costimulatory molecules or release of inflammatory cytokines following gp96 stimulation could be detected, in our hands gp96 was able to strongly up-regulate the expression of its own receptor CD91.

Although Scatchard analysis revealed that pDC do have a lower affinity for gp96 binding than do DC of myeloid origin, the strong up-regulation of CD91 induced by the gp96 binding itself can enable pDC to adsorb and neutralize the majority of extracellular gp96, thus preventing the activation of myeloid DC or monocytes present at tissue level and limiting the initiation of an immune response. Indeed, maturation leads to two opposite outcomes in pDC and in DC of the myeloid lineage. In fact, at variance to what we found for pDC, it has been shown that the gp96 receptor is down-regulated in matured MoDC, which are no longer able to bind gp96 (47). This mechanism of gp96 adsorption by pDC may be of significance in tissue remodeling and wound healing where chronic inflammation and response to self Ags should be avoided.

By in vitro coculture experiments, we were able to show that pDC, either freshly purified from blood or treated with gp96 or CpG, were able to inhibit the gp96-induced activation of MoDC, thus counteracting the immunostimulatory capacity of gp96. Interestingly, nonactivated pDC abrogated the stimulatory capacity of gp96 on MoDC only partially, leading to "semimature" DC that did not release any inflammatory cytokine while still displaying up-regulated expression of costimulatory molecules CD80 and CD86. Notably, these partially or semimature DC have been often associated with tolerogenic functions (48).

Additional evidence supporting the role of gp96 in a negative regulation of the immune response is provided by the gp96 ability to down-modulate the release of IL-8. The primary function of IL-8 is to recruit cells, such as neutrophils and macrophages, helping the initiation of immune responses. By down-modulating its secretion, gp96 helps to prevent systemic inflammation and harmful immune reactions.

In our system we did not address the cross-presentation capacity of gp96; however, pDC have been recently shown to be able to phagocytose and cross-present viral lipopeptides and HIV-1 Ags to CD8+ T cells (17). Thus, it is tempting to speculate that gp96 with its interaction with pDC may be also involved in inducing peripheral tolerance to self Ags by promoting the cross-presentation of their client peptides.

Taken together, our data show that gp96 directly, specifically, and functionally interacts with human blood pDC. In the absence of pDC maturation, the gp96 effect could be more related to a down-modulation of the immune response, working as a control mechanism to limit the strength of the response to its extracellular localization. The fact that CpG treatment induced an up-regulation of CD91 as well may indicate that TLR9 and CD91 work together. On this account, the gp96-pDC relationship may be a link relating innate immune responses, involving TLRs, to adaptive ones determined by the ER processing and presentation on MHC class I. However, the role of human pDC in regulating the immune response still remains puzzling, and further studies are needed to understand how pDC functions are modulated according to the stimulation provided, including activation mediated by gp96.


   Acknowledgments
 
We thank Alessio Palini (Goccia Laboratory, San Raffaele Foundation, Scientific Institute, Milano, Italy) for supervising FACS analysis, Pramod K. Srivastava (Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut Health Center, Farmington, CT) for helpful discussion, Mrs. Francesca Rini (Units of Immunotherapy of Human Tumor, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy) for expert technical help. We gratefully acknowledge Grazia Barp for editing assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano) and European Community (Cancerimmunotherapy, contract 518234). Back

2 Current address: Unit of Immuno-Biotherapy of Solid Tumors, San Raffaele Scientific Institute, Milano, Italy. Back

3 Address correspondence and reprint requests to Dr. Chiara Castelli, Unit of Immunotherapy of Human Tumors, Fondazione Istituto Di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Nazionale dei Tumori, Via G Venezian 1, 20133 Milano, Italy. E-mail address: chiara.castelli{at}istitutotumori.mi.it Back

4 Abbreviations used in this paper: HSP, heat shock protein; AF488, Alexa Fluor 488; BDCA-2, blood dendritic cell Ag 2; CBA, cytometric bead array; DC, dendritic cell; ER, endoplasmic reticulum; IKK2, I{kappa}B kinase 2; LAMP-2, lysosome-associated membrane protein 2; MoDC, monocyte-derived DC; pDC, plasmacytoid DC; PFA, paraformaldehyde; RT, room temperature. Back

Received for publication May 6, 2008. Accepted for publication August 19, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Srivastava, P.. 2002. Roles of heat-shock proteins in innate and adaptive immunity. Nat. Rev. Immunol. 2: 185-194. [Medline]
  2. Rivoltini, L., C. Castelli, M. Carrabba, V. Mazzaferro, L. Pilla, V. Huber, J. Coppa, G. Gallino, C. Scheibenbogen, P. Squarcina, et al 2003. Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells. J. Immunol. 171: 3467-3474. [Abstract/Free Full Text]
  3. Podack, E. R., L. E. Raez. 2007. Allogeneic tumor-cell-based vaccines secreting endoplasmic reticulum chaperone gp96. Expert Opin. Biol. Ther. 7: 1679-1688. [Medline]
  4. Srivastava, P. K., R. J. Amato. 2001. Heat shock proteins: the "Swiss Army Knife" vaccines against cancers and infectious agents. Vaccine 19: 2590-2597. [Medline]
  5. Binder, R. J., D. K. Han, P. K. Srivastava. 2000. CD91: a receptor for heat shock protein gp96. Nat. Immunol. 1: 151-155. [Medline]
  6. Basu, S., R. J. Binder, T. Ramalingam, P. K. Srivastava. 2001. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14: 303-313. [Medline]
  7. Binder, R. J., P. K. Srivastava. 2004. Essential role of CD91 in re-presentation of gp96-chaperoned peptides. Proc. Natl. Acad. Sci. USA 101: 6128-6133. [Abstract/Free Full Text]
  8. Vabulas, R. M., S. Braedel, N. Hilf, H. Singh-Jasuja, S. Herter, P. Ahmad-Nejad, C. J. Kirschning, C. da Costa, H.-G. Rammensee, H. Wagner, H. Schild. 2002. The endoplasmic reticulum-resident heat shock protein gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J. Biol. Chem. 277: 20847-20853. [Abstract/Free Full Text]
  9. Binder, R. J., M. L. Harris, A. Ménoret, P. K. Srivastava. 2000. Saturation, competition, and specificity in interaction of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CD11b1 cells. J. Immunol. 165: 2582-2587. [Abstract/Free Full Text]
  10. Singh-Jasuja, H., R. E. M. Toes, P. Spee, C. Münz, N. Hilf, S. P. Schoenberger, P. Ricciardi-Castagnoli, J. Neefjes, H.-G. Rammensee, et al 2000. Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis. J. Exp. Med. 191: 1965-1974. [Medline]
  11. Radsak, M. P., N. Hilf, H. Singh-Jasuja, S. Braedel, P. Brossart, H.-G. Rammensee, H. Schild. 2003. The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions. Blood 101: 2810-2815. [Abstract/Free Full Text]
  12. Colonna, M., G. Trinchieri, Y. L. Liu. 2004. Plasmacytoid dendritic cells in immunity. Cell 106: 259-262.
  13. Fonteneau, J.-F., M. Gilliet, M. Larsson, I. Dasilva, C. Münz, Y.-J. Liu, N. Bhardwaj. 2003. Activation of influenza virus-specific CD4+ and CD8 T+ cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood 101: 3520-3526.
  14. Benitez-Ribas, D., G. J. Adema, G. Winkels, I. S. Klasen, C. J. A. Punt, C. G. Figdor, I. J. M. de Vries. 2006. Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after Fc{gamma}RII-mediated uptake. J. Exp. Med. 203: 1629-1635. [Abstract/Free Full Text]
  15. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Günther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon induction. J. Exp. Med. 194: 1823-1834. [Abstract/Free Full Text]
  16. Meyer-Wentrup, F., D. Benitez-Ribas, P. J. Tacken, C. J. A. Punt, C. G. Figdor, I. J. M. de Vries, G. J. Adema. 2008. Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-{alpha} production. Blood 111: 4245-4253. [Abstract/Free Full Text]
  17. Hoeffel, G., A.-C. Ripoche, D. Matheoud, M. Nascimbeni, N. Escriou, P. Lebon, F. Heshmati, J.-G. Guillet, M. Gannagé, S. Caillat-Zucman, et al 2007. Antigen crosspresentation by human plasmacytoid dendritic cells. Immunity 27: 481-492. [Medline]
  18. Kuwana, M.. 2002. Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets. Hum. Immunol. 63: 1156-1163. [Medline]
  19. Kawamura, K., N. Kadowaki, T. Kitawaki, T. Uchiyama. 2006. Virus-stimulated plasmacytoid dendritic cells induce CD4 cytotoxic regulatory T cells. Blood 107: 1031-1038. [Abstract/Free Full Text]
  20. Ito, T., M. Yang, Y.-H. Wang, R. Lande, J. Gregorio, O. A. Perng, X.-F. Qin, Y.-J. Liu, M. Gilliet. 2007. Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J. Exp. Med. 204: 105-115. [Abstract/Free Full Text]
  21. Ochando, J. C., C. Homma, Y. Yang, A. Hidalgo, A. Garin, F. Tacke, V. Angeli, Y. Li, P. Boros, Y. Ding, et al 2006. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat. Immunol. 7: 652-662. [Medline]
  22. Treilleux, I., J. Y. Blay, N. Bendriss-Vermare, I. Ray-Coquard, T. Bachelot, J. P. Guastalla, A. Bremond, S. Goddard, J. J. Pin, C. Barthelemy-Dubois, S. Lebecque. 2004. Dendritic cell infiltration and prognosis of early stage breast cancer. Clin. Cancer Res. 10: 7466-7474. [Abstract/Free Full Text]
  23. Farkas, L., K. Beiske, F. Lund-Johansen, P. Brandtzaeg, F. L. Jahnsen. 2001. Plasmacytoid dendritic cells (natural interferon-{alpha}/β-producing cells) accumulate in cutaneous lupus erythematosus lesions. Am. J. Pathol. 159: 237-243. [Abstract/Free Full Text]
  24. Middel, P., D. Raddatz, B. Gunawan, F. Haller, H.-J. Radzun. 2006. Increased number of mature dendritic cells in Crohn’s disease: evidence for a chemokine mediated retention mechanism. Gut 55: 220-227. [Abstract/Free Full Text]
  25. López de Padilla, C. M., A. N. Vallejo, K. T. McNallan, R. Vehe, S. A. Smith, A. B. Dietz, S. Vuk-Pavlovic, A. M. Reed. 2007. Plasmacytoid dendritic cells in inflamed muscle of patients with juvenile dermatomyositis. Arthritis Rheum. 56: 1658-1668. [Medline]
  26. Novak, N., W. Peng, C. Yu. 2007. Network of myeloid and plasmacytoid dendritic cells in atopic dermatitis. Adv. Exp. Med. Biol. 601: 97-104. [Medline]
  27. Zou, W., V. Machelon, A. Coulomb-L'Hermin, J. Borvak, F. N. T. Isaev, S. Wei, R. Krzysiek, I. Durand-Gasselin, A. Gordon, T. Pustilnik, et al 2001. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat. Med. 7: 1339-1346. [Medline]
  28. Faith, A., E. Peek, J. McDonald, Z. Urry, D. F. Richards, C. Tan, G. Santis, C. Hawrylowicz. 2007. Plasmacytoid dendritic cells from human lung cancer draining lymph nodes induce Tc1 responses. Am. J. Respir. Cell Mol. Biol. 36: 360-367. [Abstract/Free Full Text]
  29. -Tabarkiewicz, J., P. Rybojad, A. Jablonka, J. Rolinski. 2008. CD1c+ and CD303+ dendritic cells in peripheral blood, lymph nodes and tumor tissue of patients with non-small cell lung cancer. Oncol. Rep. 19: 237-243. [Medline]
  30. Basu, S., R. J. Binder, R. Suto, K. M. Anderson, P. K. Srivastava. 2000. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-{kappa}B pathway. Int. Immunol. 12: 1539-1546. [Abstract/Free Full Text]
  31. Srivastava, P. K.. 1997. Purification of heat shock protein-peptide complexes for use in vaccination against cancers and intracellular pathogens. Methods 12: 165-171. [Medline]
  32. Hilf, N., H. Singh-Jasuja, P. Schwarzmaier, C. Gouttefangeas, H.-G. Rammensee, H. Schild. 2002. Human platelets express heat shock protein receptors and regulate dendritic cell maturation. Blood 99: 3676-3682. [Abstract/Free Full Text]
  33. Macagno, A., G. Napolitani, A. Lanzavecchia, F. Sallusto. 2007. Duration, combination, and timing: the signal integration model of dendritic cell activation. Trends Immunol. 28: 227-233. [Medline]
  34. Chandawarkar, R. Y., M. S. Wagh, J. T. Kovalchin, P. K. Srivastava. 2004. Immune modulation with high-dose heatshock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis. Int. Immunol. 16: 615-624. [Abstract/Free Full Text]
  35. Kovalchin, J. T., C. Mendonca, M. S. Wagh, R. Wang, R. Y. Chandawarkar. 2006. In vivo treatment of mice with heat shock protein, gp96, improves survival of skin grafts with minor and major antigenic disparity. Transpl. Immunol. 15: 179-185. [Medline]
  36. Dai, J., B. Liu, S. M. Ngoi, S. Sun, A. T. Vella, Z. Li. 2007. TLR4 hyperresponsiveness via cell surface expression of heat shock protein gp96 potentiates suppressive function of regulatory T cells. J. Immunol. 178: 3219-3225. [Abstract/Free Full Text]
  37. Gopcsa, L., A. Banyai, K. Jakab, L. Kormos, J. Tamaska, A. Matolcsy, P. Gogolak, E. Rajnavolgyi, K. Paloczi. 2005. Extensive flow cytometric characterization of plasmacytoid dendritic cell leukemia cells. Eur. J. Haematol. 75: 346-351. [Medline]
  38. Kebba, A., J. Stebbing, S. Rowland, R. Ingram, J. Agaba, S. Patterson, P. Kaleebu, N. Imami, F. Gotch. 2005. Expression of the common heat-shock protein receptor CD91 is increased on monocytes of exposed yet HIV-1-seronegative subjects. J. Leukocyte Biol. 78: 37-42. [Abstract/Free Full Text]
  39. Stebbing, J., M. Bower, B. Gazzard, A. Wildfire, H. Pandha, A. Dalgleish, J. Spicer. 2004. The common heat shock protein receptor CD91 is up-regulated on monocytes of advanced melanoma slow progressors. Clin. Exp. Immunol. 138: 312-316. [Medline]
  40. Berwin, B., J. P. Hart, S. Rice, C. Gass, S. V. Pizzo, S. R. Post, C. V. Nicchitta. 2003. Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells. EMBO J. 22: 6127-6136. [Medline]
  41. Wassenberg, J. J., C. Dezfulian, C. V. Nicchitta. 1999. Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages. J. Cell Sci. 112: 2167-2175. [Abstract]
  42. Cresswell, P., E. A. Hughes. 1997. Protein degradation: the ins and outs of the matter. Curr. Biol. 7: R552-R555. [Medline]
  43. Liao, H.-J., G. Carpenter. 2007. Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression. Mol. Biol. Cell 18: 1064-1072. [Abstract/Free Full Text]
  44. Binder, R. J., R. Vatner, P. K. Srivastava. 2004. The heat-shock protein receptors: some answers and more questions. Tissue Antigens 64: 442-454. [Medline]
  45. Castellino, F., P. E. Boucher, K. Eichelberg, M. Mayhew, J. E. Rothman, A. N. Houghton, R. N. Germain. 2000. Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J. Exp. Med. 191: 1957-1964. [Medline]
  46. Binder, R. J., N. E. Blachere, P. K. Srivastava. 2001. Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules. J. Biol. Chem. 276: 17163-17171. [Abstract/Free Full Text]
  47. Singh-Jasuja, H., H. U. Scherer, N. Hilf, D. Arnold-Schild, H. G. Rammensee, R. E. Toes, H. Schild. 2000. The heat shock protein gp96 induces maturation of dendritic cells and down-regulation of its receptor. Eur. J. Immunol. 30: 2211-2215. [Medline]
  48. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature, and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]



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