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J. Biol. Chem., Vol. 282, Issue 29, 21145-21159, July 20, 2007
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From the
Departments of Immunohematology and Blood Transfusion, ¶Molecular Cell Biology, and **Clinical Oncology, Leiden University Medical Centre, P. O. Box 9600, 2300 RC Leiden, The Netherlands, Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands, and ||Immunology Laboratory, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands
Received for publication, February 27, 2007 , and in revised form, April 13, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pamconjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpGconjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8+ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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transcription factor and gene transcription leading to production of proinflammatory cytokines (3).
DCs are both initiators and regulators of T cell responses (4). Dendritic cells constantly screen the environment for potential foreign antigens by a variety of mechanisms such as phagocytosis, macropinocytosis, caveolin-mediated, or clathrin-dependent endocytosis. The manner of uptake is dependent on the size and nature of material to be internalized (5, 6). As specialized antigen-presenting cells, DCs have the capacity to efficiently process exogenous proteins and present the peptides in major histocompatibility complex (MHC) class I molecules, a process known as cross-presentation. In this scenario exogenously derived antigens are internalized and translocate from the endosomal route into the cytosol, where the proteasome complex processes the antigen. The generated peptides are transported from the cytosol into the endoplasmic reticulum via the peptide transporter TAP (7), after which the peptides undergo further trimming and are finally loaded onto MHC class I molecules, which translocate to the cell surface, where the peptide is presented to CD8+ T-cells.
The ability of DCs to cross-present peptides on MHC class I to CD8+ T-cells together with the capacity of TLR ligands to deliver maturation signals has inspired efforts to explore the use of DCs as a vaccine vehicle in the fight against infectious diseases and cancer (8, 9). Covalent linkage of immunogenic peptides to the TLR9 ligand, CpG DNA, or TLR2 ligands, like Pam3CysSS and Pam3CysSK4, induces a more prominent T-cell response than administration of free TLR2-L or TLR9-L mixed with protein (2, 10–16).
To explore the mode of action of TLR-L antigen conjugates, we have designed well defined synthetic vaccines composed of peptides containing the model antigen ovalbumin CD8+ cytotoxic T-lymphocyte (CTL) epitope (SIINFEKL) chemically linked to either the TLR2-ligand, Pam3CysSK4, or the TLR9 ligand, CpG. These conjugates were used to study the uptake, intracellular routing, and processing. We show that not only TLR9-L conjugates but also the TLR2-L conjugates are taken up independently of TLR expression albeit through two distinct internalization mechanisms. Down-stream processing route for MHC class I antigen presentation, however, were similar and requires endosomal acidification, TAP translocation, and proteasomal processing. Importantly, whereas the uptake of both types of TLR-L conjugates was independent of TLR expression, priming of specific CD8+ T-cell response required TLR signaling in dendritic cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-microglobulin-deficient mice strains was kindly provided by Prof. H. G. Ljunggren, Karolinska Institutet, Sweden. LPS of Escherichia coli (serotype026:B6), Monodansylcadaverine (MDC), and filipin were purchased from Sigma-Aldrich. Epoxomicin and chlorophenol red--D-galactopyranoside were from Calbiochem.
Cell Lines
Freshly isolated DCs were cultured from mouse bone marrow cells as described elsewhere (17). D1 cell line, a long term growth factor-dependent immature splenic DC line derived from B6 (H-2b) mice, was cultured as described (18). B3Z is a T-cell hybridoma specific for the H-2Kb CTL epitope SIINFEKL that expresses a -galactosidase construct under the regulation of the NF-AT element from the IL2 promoter (19). EG7 (EL4-OVA) (20) was cultured in complete medium with 400 µg of G418 (Invitrogen).
Generation of Pam3CysSK4- or CpG-conjugated Peptides and Labeling
Table 1 shows the conjugates and peptides used in this study.
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General Methods
Mass spectra were recorded on a PE SCIEX API 165 (PerkinElmer Life Sciences) mass spectrometer. Analytical liquid chromatography/mass spectroscopy was conducted on a Jasco system using an Alltima C18 analytical column (4.6 x 150 mm, 5-µm particle size, flow 1.0 ml/min) with detection at 214 and 254 nm. The solvent system was 100% water (A), 100% acetonitrile (B), and 1% trifluoroacetic acid (C). Gradients of A to B were applied over 15 min, keeping C isocratic at 10%. Purifications of the synthetic peptides were conducted on a BioCAD Vision automated HPLC system (PerSeptive Biosystems, Inc.) supplied with a Alltima C18 column (10 x 250 mm, 5-µm particle size, running at 4 ml/min). A Varian DMS 200 UV-visible spectrophotometer was used to measure UV absorption MALDI-time-of-flight spectra were recorded on a Voyager-DE PRO mass spectrometer (Perseptive Biosystems, Inc.).
Peptide Synthesis
Fmoc-based solid-phase peptide synthesis was performed on a CS Bio 336 automated instrument (CS Bio Co.) starting from either preloaded Fmoc-Leu-PHB-Tentagel resin or from Tentagel -RAM resin. The synthesis was performed on a 50- or 250-µmol scale according to established methods (21). HCTU was used as coupling reagent. All peptides (see Table 1) were cleaved from the resin using trifluoroacetic acid/triisopropylsilane/H2O (95/2.5/2.5) for 2 h at room temperature, precipitated from diethyl ether, redissolved in 20% aqueous 1,1,1,3,3,3-hexafluoro-2-propanol, and purified by reverse phase HPLC and characterized using liquid chromatography/mass spectroscopy and MALDI mass spectroscopy (see "General Methods").
Oligonucleotide Synthesis
DMT-based solid-phase phosphorothioate oligonucleotide (ODN) synthesis was performed on an Expedite automated instrument (Perseptive Biosystems) starting from Control Pore Glass support with the 3'-thiol modifier. The syntheses were performed on a 10-µmol scale according to established methods (22). Elongation was performed using 3'-phosphoramidite derivatives of DMT-protected nucleosides 5'-DMT-A(TAC)-OH, 5'-DMT-C(TAC)-OH, 5'-DMT-G(TAC)-OH, and 5'-DMT-T-OH under the agency of dicyanoimidazole. After each coupling, remaining free 5'-hydroxyls were blocked using a capping solution (t-butylphenoxyacetic anhydride (Tac2O)/1-methylimidazole in tetrahydrofuran/pyridine) followed by sulfurization of the phosphite linkage to the phosphorothioate linkage using the Beaucage reagent. Next, the 5'-DMT protecting group was removed by trichloroacetic acid, after which elongation was continued. After final DMT removal the DNA oligomer was cleaved from the resin by 25% ammonium hydroxide solution to give a 3'-disulfide modified ODN (ODN-SS-propyl-OH). ODNs were purified on a Q-Sepharose column pre-equilibrated with 50 mM NaOAc applying a gradient of 2 M NaCl in 50 mM NaOAc. Fractions containing the pure product were combined and dialyzed three times with Millipore water using dialysis tubing with 1-kDa cut-off (Spectrum). Quantification was performed by UV absorbance at 260 nm. Sequences of the ODN prepared in this study were CpG, 5'-TCCATGACGTTCCTGACGTT-3'; GpC, 5'-TCCATGAGCTTCCTGATG-3'.
Maleimidopropionoyl Peptides Mal-OVA247–264 and Mal-OVA247–264A5K
Fmoc-deprotected peptide resin (200 mg, 30 µmol) was suspended in 2 ml of NMP, and 3-maleimidopropionic acid (5 eq, 250 µmol, 42.2 mg), HCTU (5 eq, 250 µmol, 103.2 mg), and N,N'-diisopropylethylamine (5 eq, 250 µmol, 42 µl) were added subsequently. The mixture was shaken for 2 h after which the resin was filtered, washed with NMP, dichloromethane, and dried. A 2,4,6-trinitrobenzenesulfonic acid test indicated complete coupling. The products were processed as described under "Peptide Synthesis."
ODN-Peptide Conjugates
For CpG-OVA247–264, 3'-disulfide modified CpG-SS-propyl-OH (266 nmol) was converted to 3'-SH-modified CpG-SH overnight with dithiothreitol (DTT)-containing buffer (35 mg of DTT, 26 mg of NaOAc·3H2O, 1 ml of water). Dithiothreitol was removed from the mixture using a PD-10 desalting column (Amersham Biosciences) that was pre-equilibrated with 25 ml of a 50 mM phosphate buffer (25 mM Na2HPO4, 25 mM NaH2PO4, 1 mM EDTA in water, continuously degassed with helium. Filtrate (3.25 ml) was directly transferred to a tube containing 5 mg of maleimidopropionyl peptide Mal-OVA247–264. The resulting solution was sonicated and placed under blanket of argon. The tube was sealed and shaken for 2 days at room temperature. The mixture was purified over a Superdex 75 gel filtration column using isocratic elution with 0.15 M triethylammonium acetate. Fractions containing the product were collected and lyophilized. Excess triethylammonium acetate was removed by lyophilization from water (3 times). Quantification was performed by UV absorbance (260 nm). CpG-OVA247–264A5K and GpC-OVA247–264 were prepared as described for CpG-OVA247–264 starting from the corresponding ODNs and maleimidopropionyl peptides.
Lipopeptides
For Pam3CysSK4-OVA247–264, Fmoc-deprotected peptide resin (300 mg, 45 µmol) was suspended in 1.4 ml or 1:1 NMP/dichloromethane. Pam3Cys-OH (91 mg, 2 eq, 100 µmol) and PyBOP (benzotriazole-1-yl-oxytrispyrrolidinophosphonium hexafluorophosphate; 80 mg, 3 eq, 150 µmol) were added. N,N'-Diisopropylethylamine (30 µl, 175 µmol) was added in two portions of 15 µl with an interval of 15 min, and the mixture was shaken for 4 h.4 The resin was washed with NMP and dichloromethane and dried. A 2,4,6-trinitrobenzenesulfonic acid test indicated complete coupling. The product was cleaved from the resin as described under "Peptide Synthesis," dissolved in t-butyl alcohol/ACN/H2O (1:1:1), and purified on an Alltima CN column (10 x 250 mm, 5-µm particle size) with a gradient of A to B; C was kept isocratic at 10% (A, 1:1 CH3OH/H2O; B, ACN; C, 1% trifluoroacetic acid in 9:1 CH3OH/H2O). Pam3CysSK4-OVA247–264A5K and Pam3CysSK4C-OVA247–264 were prepared and purified as described above for Pam3CysSK4-OVA247–264 starting from the corresponding peptide resins.
Fluorescently Labeled Peptides and Conjugates
[Alexa488]OVA247–264—Fmoc-deprotected peptide resin (100 mg, 15 µmol) was treated twice with 2 ml of a capping reagent (0.5 M Ac2O, 0.125 M N,N'-diisopropylethylamine in NMP). A 2,4,6-trinitrobenzenesulfonic acid test indicated complete acetylation. The cleaved and purified peptide (1 mg) was dissolved into 50 µl of buffer (300 mM NaHCO3 in 30% acetonitrile/water), and Alexa Fluor 488 carboxylic acid succinimidyl ester (0.3 mg) was added. Another 50 µl of buffer was added, and the mixture was shaken overnight. The product was purified by reverse phase HPLC (see "General Methods").
CpG-[Alexa488]OVA247–264 and GpC-[Alexa488]OVA247–264—Conjugate CpG-OVA247–264 (296 nmol) or GpC-[Alexa488]OVA247–264 (296 nmol) was dissolved in 50 µl of buffer (300 mM NaHCO3, 30% ACN in H2O), and Alexa Fluor 488 carboxylic acid succidinimidyl ester (1.0 mg) was added. The bright green mixture was shaken overnight. The mixture was diluted 5 times with H2O before being subjected to reverse phase HPLC purification. (Alltima C18, gradient of A to B; C was kept isocratic at 10%; A, H2O; B, ACN; C, 100 mM aqueous NH4OAc).
Pam3CysSK4-C[BDP-FL]OVA247–264—Lipopeptide Pam3CysSK4C-OVA247–264 (0.46 µmol, 1.69 mg) and BODIPY-FL N-(2-aminoethyl)maleimide were transferred to a vial containing 0.5 ml of 50 mM phosphate buffer (25 mM NaH2PO4, 25 mM Na2HPO4, 1 mM EDTA in 2:1:1 H2O/CH3OH /ACN). The mixture was sonicated and shaken for 60 h under argon. The mixture was diluted 5 times with 40:30:30 H2O/ACN/t-butyl alcohol, 1% trifluoroacetic acid before being subjected to reverse phase HPLC purification as described for Pam3CysSK4C-OVA247–264.
SK4-C[BDP-FL]-OVA247–264—SK4-C[BDP-FL]-OVA247–264 was synthesized as described for Pam3CysSK4-C[BDP-FL]OVA247–264 using 50 mM phosphate in 3:2 H2O/ACN as a ligation buffer and SK4-C-OVA247–264 as the peptide substrate. Pam3CysSK4-C[Alexa488]OVA247–264 was synthesized as described for Pam3CysSK4-C[BDP-FL]OVA247–264 using Alexa Fluor 488 C5 maleimide as the reactive dye.
IL-12p40 Enzyme-linked Immunosorbent Assay—DCs (4 x 104) were plated into a 96-well round bottom plate and incubated for 24–48 h with the compounds indicated in the legend to Fig. 1. Supernatants were harvested and tested for IL-12 p40/p70 content using a standard sandwich enzyme-linked immunosorbent assay as previous described (23).
Confocal Microscopy
DCs were plated out into glass-bottom Petri dishes (MatTek) 2 days before the experiment. Cells were incubated either with the fluorescence-labeled TLR-L peptide conjugate or the fluorescence-labeled peptide at 37 °C for different time periods at the concentrations indicated in the figure legends. In some experiments, as indicated, cells were coincubated with 1 µM Lysotracker for 5 min to stain endosomal/lysosomal compartments. In experiments with inhibitors, cells were preincubated for 30 min either with 50 µM MDC or 10 µg/ml filipin followed by extensively washing before incubation with the TLR-L conjugate either alone (pretreatment) or in the presence of inhibitors (coincubation). Cells were then washed and imaged using an inverted Leica SP2 confocal microscope. Dual color images were acquired by sequential scanning, with only one laser line per scan to avoid cross-excitation. The images were processed using the software program ImageJ.
Flow Cytometry
For analyzing the effect of the different compounds on dendritic cell phenotypic profile, DCs were incubated with the different compounds at a final concentration of 1 µM for 48 h. Subsequently, cells were harvested, resuspended in fluorescence-activated cell buffer (phosphate-buffered saline, 0.1% bovine serum albumin), and incubated for 20 min with the following panel of monoclonal antibodies: fluorescein isothiocyanate-anti-CD86 (clone GL-1), phycoerythrin-anti-I-Ab (clone M5/114 15.2), phycoerythrin-anti-CD40 (clone 3/23), allophycocyanin-anti-Kb (24). Cells were washed twice and fixed with 0.5% paraformaldehyde before being subjected to flow cytometry measurements.
MHC Class I-restricted Antigen Presentation Assay
DCs were incubated for 2 h (unless stated otherwise in the legends to Figs. 6 and 8) with 1) the parent peptide (DEVSGLEQLESIINFEKLAAAAAK, OVA247–264A5K, or DEVSGLEQLESIINFEKL, OVA247–264), 2) the peptide conjugate, or 3) the mixture of the parent peptide and the Pam3CySK4 or CpG at the indicated concentrations. Cells were washed five times with medium before the T-cell hybridoma B3Z cells were added and incubated for 16 h at 37 °C.
Antigen presentation of the ovalbumin cytotoxic T-cell epitope SIINFEKL (OVA257–264) in H-2Kb was detected by activation of B3Z cells measured by a colorimetric assay using chlorophenol red--D-galactopyranoside as substrate to detect lacZ activity in B3Z lysates, as described (23).
In some experiments cells were preincubated for 30 min with titrated amounts of epoxomicin ranging from 0.01 to 10 µM epoxomicin or with 3 mM NH4Cl or preincubated for 60 min with titrated amounts of monodansylcadaverine (25–50 µM) or with filipin (10–20 µg/ml) before adding the peptide compound still in the presence of the inhibitors. Cell viability was confirmed by tryphan blue exclusion at the indicated concentration range of inhibitors.
Priming of Endogenous Naïve CD8+ T-cells
To determine the endogenous CTL response, five nmol of the different compounds were injected s.c. into naïve C57BL/6 mice. After 10 days spleen cells were stimulated in vitro by plating 10 x 106 splenocytes with 1 x 106 mytomycin C (Kyowa)-treated (50 µg/ml for 1 h at 37 °C) and -irradiated (4000 rads) EG7 cell line (EL4-OVA) in the absence of additional cytokines. After 7 days viable splenocytes were isolated over a Ficoll gradient and stained for H-2Kb tetramer (TM)-OVA257–264, CD8b2 (clone 53-5.8) and propidium iodide to exclude dead cells as described previous (23).
Intracellular Cytokine Staining
An aliquot of spleen cells after re-stimulation and Ficoll purification (see above) were subjected to stimulation in vitro with or without 1 µg/ml OVA257–264 peptide (H2-Kb restricted SIINFEKL) overnight in the presence of GolgiPlug (BD Pharmingen). Cells were then washed twice with fluorescence-activated cell sorter buffer and stained with phycoerythrin-conjugated monoclonal rat anti-mouse CD8b2 antibody. Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Pharmingen). Intracellular interferon-
was stained with allophycocyanin-conjugated rat anti-mouse interferon-. All antibodies were purchased from BD Pharmingen. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software (BD Biosciences). Splenocytes without peptide stimulation were used as a negative control.
In Vivo Uptake Studies
To monitor the uptake of the TLR-L-conjugated peptides and the free peptide in vivo, mice were injected s.c. either with 5 nmol of Alexa488 Fluor-labeled CpGconjugated peptide, 5 nmol of BODIPY-FL Pamconjugated peptide, CpG mixed with Alexa488 Fluor-labeled peptide, or Pam mixed with BODIPY-FL-labeled peptide. Three days later mice were sacrificed, and a single cell suspension of the draining lymph nodes was stained for CD11c (clone HL3) before being subjected to flow cytometry analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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OVA247–264A5K. Crucially, all of our compounds (listed in Table 1) are produced synthetically and are, therefore, chemically well defined, of high purity, and of constant quality, avoiding potential contamination with other TLR ligands such as LPS, which commonly occur in purified TLR ligand preparations.
Robust Induction of Naïve CD8-specific T-cells Mediated by the Conjugates—To establish the quality of our generated TLR-L-peptide conjugates, we first investigated the induction of an endogenous T-cell response following s.c. injection of either TLR-L-conjugated peptide or free peptide into naïve C57BL/6 mice. After 10 days, the induction of OVA247–264-specific CD8+ T-cells was analyzed. As is shown in Fig. 1A, the magnitude of the OVA257–264-specific T-cell response induction by either CpGOVA247–264A5K or PamOVA247–264A5K was significantly higher than that in mice injected with non-conjugated OVA247–264A5K peptide mixed together with either free CpG or free Pam. To address whether the induction of specific T-cells depended on activation of the DCs, we injected GpCOVA247–264 conjugate, which is a non-stimulatory oligonucleotide, as shown by its lack of capacity to induce IL-12 production by DCs (Fig. 1C). Injection of the GpCpeptide conjugate into naïve mice led to a significantly lower induction of specific CD8+ T-cells than of CpGconjugated peptide but was still as high as the response obtained after mixing of peptide with the CpG (Fig. 1A). Importantly, only when a stimulatory TLR-L conjugate was given, were the majority of CD8+ T-cells able to produce interferon- (Fig. 1B), indicating that signaling via the TLR is essential for the generation of large numbers of functional T-cells in vivo. These results suggest that the enhanced induction of specific T-cells is primarily the result of efficient delivery of the TLR-L-conjugated peptide into the antigen-presenting cell.
TLR Conjugates Activate Dendritic Cells—Next we analyzed the ability of the different conjugates to induce maturation of DCs. As evident from Table 2, increased surface marker expression of CD40, CD86, MHC I, and MHC II was observed in dendritic cells after treatment with the different TLR-L conjugates to a similar extent as the free TLR-Ls. To confirm the involvement of the TLRs in DC activation, we isolated bone marrow-derived dendritic cells (BMDCs) from wild type (WT) mice, TLR2-deficient mice, and TLR9-deficient mice and stimulated these cells with the different conjugates followed by phenotypic characterization by staining for different surface markers associated with DC maturation (25). No up-regulation of the cell surface markers CD86 and MHC class II was detected when BMDCs from TLR2-deficient mice or TLR9-deficient mice were stimulated with Pamconjugated peptide or CpGconjugated peptide, respectively (Fig. 2). This impaired up-regulation was not due to a general defect in the maturation signaling pathway, because stimulation with LPS could induce up-regulation of CD86 and MHC II in BMDCs derived from both TLR2- and TLR9-deficient mice to a similar extent as observed for BMDCs derived from wild type mice. Taken together, these results demonstrate that the conjugates are as effective as free TLR ligand in activating the DCs and show that the expression of the cognate TLR is required for activation of DCs by the TLR-L-peptide conjugates.
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conjugated peptide or free peptide. Both compounds were labeled with a fluorophore (attached on the peptide backbone), which allowed us to monitor the internalization of these compounds by confocal microscopy analysis. Introduction of the fluorophore (either Alexa488 or BODIPY-FL) into the conjugates did not alter the ability of the conjugates to activate DCs as comparable levels of the IL-12 cytokine were produced by the fluorescent conjugates and the dark conjugates.5 As indicated by the increased intensity of fluorescence inside the cells, the Pamconjugated peptides were taken up far more efficiently than the non-conjugated peptide (Fig. 3A). Interestingly, the Pamconjugated peptide was found to accumulate in hot spots (indicated by arrows in Fig. 3A), whereas a more diffuse pattern was observed in DCs incubated with the peptide alone. Quantification of the mean fluorescence intensity (MFI) revealed a more than 4-fold higher fluorescence in DCs incubated with the Pamconjugate compared with DCs incubated with the peptide (Fig. 3B). Similarly, CpGconjugated peptide was internalized more efficiently than the unconjugated peptide by DCs (Fig. 3C). In addition, the non-stimulatory GpCconjugate (MFI 63 ± 7.3) was internalized to a similar extent as the stimulatory CpGconjugate (MFI 72 ± 9.1) (Fig. 3D).
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conjugate or peptide labeled with Alexa488 Fluor mixed with dark CpG. Three days later draining lymph node cells were stained for the DC surface marker CD11c. In line with the in vitro results, a significantly higher proportion of CD11c+ cells had taken up the CpGconjugated peptide (2.5%) when compared with the population of DCs that ingested unconjugated peptide (0.3%) or non-injected mice (0.1%; Fig. 3E). A similar tendency was observed upon comparison of BOPIPY-FL-labeled Pamconjugate with peptide labeled with BODIPY-FL mixed with dark Pam (Fig. 3F). Collectively, these results indicate that it is the covalent linking of the peptide to the TLR-L that is responsible for the enhanced uptake by the DCs.
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conjugates to a similar extent as BMDCs from wild type mice, as reported previously (2).
Surprisingly, similar experiment carried out with Pamconjugate and BMDCs from WT mice and TLR2-/- mice showed that also the Pam conjugate, despite the fact that the receptor for Pam (TLR2) is located on the cell surface (1, 26), were internalized equally well by both types of DCs (Fig. 4, B and D).
To monitor the intracellular localization of the conjugates, we performed a co-localization study between the conjugates (green) and the endosomes (red). As seen in Fig. 4E, both the CpG and Pamconjugates are co-localized partially with an endosomal tracker (Lysotracker) in a pattern characteristic for the endosomal vehicles. Moreover, no overall difference in the uptake kinetic or in the trafficking of the compounds could be detected when comparing BMDCs from wild type mice to mice deficient for either TLR9 or TLR2 expression.5 These results indicate that TLR expression is not required for uptake and that the conjugate re-locates to the endosomal compartment.
Conjugation of Peptide Leads to Pronounced Enhancement in Antigen Presentation in Vitro—Having established that the conjugates were taken up much more efficiently than the free peptide harboring the OVA CTL epitope SIINFEKL, we next addressed the effect of conjugation of peptide to TLR-L on antigen presentation. DCs were loaded with either the CpGconjugated peptide, Pamconjugated peptide, the CpG or Pam mixed with the peptide, or the peptide alone (Fig. 5, A and B) before incubation with the peptide-specific T-cell hybridoma B3Z cells that recognize the H-2Kb, SIINFEKL CTL epitope (27). As the concentration of the compounds decreased, antigen recognition was rapidly lost when DCs were incubated either with the peptide alone or with the peptide mixed with CpG but not when the CpGconjugated peptide was used. This indicates that the conjugation of peptides to TLR-L enhances antigen presentation (Fig. 5A). Likewise, an increased antigen presentation was observed for the Pamconjugated peptide (Fig. 5B). In this case the difference in antigen presentation between conjugate and non-conjugate was even more prominent. Incubation with a mixture of free TLR-L (Pam or CpG) and the peptides resulted in a decreased antigen presentation by DCs when compared with loading with peptide alone or with conjugated peptide. This might be related to decreased uptake, since it has previously been reported that the endocytotic capacity of DCs declines upon encountering maturation signals (28, 29).
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The confocal microscopy results indicated that uptake of the conjugates occurred independently from the expression of the respective TLRs. Therefore, we next evaluated the impact of TLR expression upon antigen presentation. To this end BMDCs from WT, TLR2-/-, and TLR9-/- mice loaded with the conjugates were incubated in vitro together and subsequently incubated with the peptide-specific T-cell hybridoma B3Z cell line. In line with the confocal uptake studies, BMDCs derived from WT or the TLR2- or TLR9-deficient mice strains were recognized to the same extent (Fig. 6, A and B).
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conjugates as well as CpGconjugates in a dose-dependent manner, whereas antigen presentation of the CTL epitope OVA257–264 was only marginal affected (Fig. 6C). Because lipid raft formation is involved both in clathrin-dependent endocytosis as well as caveolae-dependent internalization (32), we analyzed the impact of MDC, a specific inhibitor of clathrin formation (33, 34), upon antigen presentation. Interestingly, whereas antigen presentation of CpGconjugated peptides was not affected by MDC, antigen presentation of Pamconjugated peptides was abrogated in a dose-dependent fashion (Fig. 6D). To further explore the distinct uptake mechanisms used by the two types of conjugates, confocal microscopy was performed on DCs treated with the two inhibitors. As evident from Fig. 7, both MDC and filipin abolished the internalization of the Pam conjugate in terms of mean fluorescence per cell, whereas inhibition of clathrin formation had a less pronounced effect on internalization of the CpGconjugate (Fig. 7C). On the other hand, inhibition of caveolin formation by filipin reduced the mean fluorescence of cells incubated with the CpGconjugate. The selective effect of the inhibitors was not due to a direct effect on one conjugate, as preincubation of cells with the inhibitor followed by extensive washing before incubation with the conjugates (indicated as preincubation in Fig. 7) in the absence of inhibitors led to similar results. Thus, these results indicate that the two TLR-L conjugates are internalized by distinct uptake receptors.
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conjugates to 70% for the CpGconjugated peptide. Similarly, inhibition of the proteasome activity resulted in an overall decrease in antigen presentation of both the CpGconjugated and Pamconjugated peptide (up to 50% inhibition) in a dose-dependent manner (Fig. 8C). To ascertain that the inhibitory effect observed was not due to an overall decrease in the surface expression of MHC class I, DCs that had been pretreated with either epoxomicin or NH4Cl were incubated with the minimal CTL epitope OVA257–264. As expected, the inhibitors did not cause a major affect upon antigen presentation of exogenous loaded OVA257–264 peptide (Fig. 8, B and C).
After proteasomal processing, CTL peptide epitopes need to be translocated into the luminal side of the endoplasmic reticulum via the transporter complex TAP to be loaded onto MHC class I molecules. To address the involvement of TAP for cross presentation of the TLR-L conjugates, BMDCs from either wild type mice or TAP-deficient mice were loaded with the TLR-L conjugates or the minimal CTL epitope OVA257–264. As evident in Fig. 8D, antigen presentation of both the TLR2L and TLR9L conjugates depended upon TAP activity, as the presentation was abrogated in TAP-deficient mice. Importantly, antigen presentation of the minimal CTL epitope OVA257–264 was not affected in the TAP-deficient DCs, showing that the observed TAP dependence of the conjugates was not due to an overall reduced surface expression of MHC class I in the TAP-deficient DCs. In contrast, when using BMDCs from mice deficient in both TAP and 2-microglobulin expression (TAP-/- 2m-/-) completely lacking MHC class I surface expression (39), antigen presentation was completely lost for all peptides (Fig. 8D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Our immunofluorescence analysis revealed that conjugates of both types of TLR ligands were taken up very efficiently compared with unconjugated peptides (Fig. 3). Internalization was a very rapid process since uptake studies showed that already within 15–30 min, the major part of the conjugates could be found in endosome/lysosome compartments (Fig. 4).5 For this we have used fluorescent conjugates; these may have slightly different properties from those of the unmodified conjugates, which could influence the uptake and function. However, the fluorescent conjugates induced DC maturation to a similar extent as the unmodified conjugates.5 In line with our findings, CpG linked to fluorescein isothiocyanate-labeled ovalbumin protein was recently shown to translocate to LAMP-1-positive endosomal-lysosomal compartments (10). Further support of enhanced uptake mediated by the conjugates was provided from our in vivo uptake analysis, which revealed a 6–8-fold increase in uptake of the CpG-conjugated peptide by CD11c+ cells and a 2-fold increase in uptake of the Pam-conjugated peptide by CD11c+ cells compared with uptake of non-conjugated peptide (Fig. 3, E and F). We found that CpGconjugated peptides were internalized independently from the expression of TLR9 and could also support antigen presentation in vitro independently of TLR9 expression (Fig. 4). Accordingly, Wagner and co-workers (2) showed that cross-presentation of OVA-linked CpG occurred independently from TLR9 expression but that TLR9 expression nevertheless was essential for activation of the DCs. At first sight these findings might seem paradoxical; however, TLR9 are mainly expressed in the endoplasmic reticulum followed by recruitment to the endosomes upon dendritic cell maturation (40).
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conjugate was taken up independently from the expression of TLR2. BMDCs isolated from TLR2-deficient mice internalized the Pamconjugate to a comparable level as BMDCs from wild type mice, and antigen recognition was unaffected in BMDCs from TLR2-deficient mice. This could not be attributed to a side effect mediated by the peptide part, since TLR2-independent internalization was also observed when incubating the cells with free Pam.5 Importantly, inhibition of clathrin-dependent endocytosis or caveolae formation abrogated both uptake and antigen presentation of Pam conjugates (Figs. 6 and 7), whereas internalization of CpG conjugates was independent of clathrin-coated pits but dependent on caveolae formation. These results indicate that other (distinct) receptors than the TLRs are involved in the uptake of the TLR conjugates, although the exact nature of these receptors remains to be established. Other TLR2 ligands have been reported to be internalized independently from the expression of TLR2. Outer membrane protein A, a conserved major component of the outer membrane of Enterobacteriaceae family that triggers cytokine production in macrophages and DCs (41), was recently shown to be internalized by the scavenger receptor LOX-1 independently from the expression of TLR2 (42). Moreover, lipoteichoic acid has also been reported to be internalized independently from TLR2 expression (43), although the receptor involved in the uptake of lipoteichoic acid still remains to be identified. Therefore, the contribution of TLR2 and other receptors expressed on the cell surface to the uptake of Pam and other TLR ligands remains to be established.
Optimal presentation of the peptide antigen cargo in the conjugates required endosomal acidification (Fig. 8), although it cannot be ruled out that the fusion of early endosomal vesicles with late endosomal vesicles is hampered by the lysosomotropic agent NH4Cl (37, 44–46). These results imply that endosomal proteases, such as cathepins (45, 47), might be involved in the processing of the TLR-L-peptide conjugate. Furthermore, proteasomal cleavage was required because proteasome inhibition greatly decreased antigen presentation of the TLR-L conjugates (Fig. 8C). Because proteasomes are mainly located in the cytosol (48, 49), our results indicate that the peptide/conjugate, after being released in the endosomal compartment, translocate to the cytosol to undergo proteasomal processing (45). In this regard it was recently reported that the translocon complex SEC61 could be involved in facilitating the translocation of peptide from the endosomes to the cytosol (50). Moreover, abrogation of translocation of peptides from the cytosol to the endoplasmic reticulum completely abrogated antigen presentation of both types of TLR-L-peptide conjugates (Fig. 8D). Aside from being internalized efficiently, we found that all of the TLR-L conjugates retained their capacity to activate DCs to a comparable level as the free TLR-L both in terms of production of the Th1-favoring cytokine, IL-12,5 and up-regulation of DC maturation surface markers (Table 2). Importantly, TLR expression was required for DC activation, since BMDCs lacking TLR2 or TLR9 were not able to up-regulate co-stimulatory molecules upon stimulation with either Pamconjugate or CpGconjugate, respectively (Fig. 2). In addition, conjugation of the non-stimulatory GpC oligonucleotide to the peptide antigen resulted in inefficient CTL priming (Fig. 1), showing that the DC activation of TLR-L peptide conjugates is essential. Therefore, intracellular signaling of TLR is crucially important for effective CTL priming by the conjugates.
In summary, we demonstrate that well defined synthetic TLR-L-peptide conjugates induce a robust and systemic response of specific T-cells due to the combined action of enhanced antigen uptake, improved MHC class I antigen presentation, and dendritic cell maturation. Our data show that targeting to two different TLRs requires a distinct uptake mechanism independent of TLR expression but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming. The chemical properties of these conjugates, which ensure that the same DCs that take up the antigen receives simultaneously a proper maturation signal, is likely to be the mechanism behind the superior activity of the peptide conjugates. Accordingly, Medzhitov and Blander (51) recently reported that synchronous entrance of TLR-L and antigen enhanced MHC class II presentation, although in their system antigen and TLR-L was delivered on microspheres in a non-covalent manner. The collective features of our TLR-L peptide conjugates together with their convenient manufacture and handling makes synthetic peptide-TLR-ligand conjugates an attractive novel vaccine modality.
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FOOTNOTES |
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1 These authors contributed equally.
2 To whom correspondence should be addressed. Tel.: 31-71-5263843; Fax: 31-71-5265267; E-mail: f.a.ossendorp{at}lumc.nl.
3 The abbreviations used are: TLR, Toll-like receptor; TLR-L, TLR ligand; DC, dendritic cell; BMDC, bone marrow-derived DC; CTL, cytotoxic T-cell lymphocyte; MDC, monodansylcadaverine; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; ODN, oligonucleotide; Pam, Pam3CysSK4; s.c., subcutaneous; TM, tetramer; MALDI, matrix-assisted laser desorption ionization; LPS, lipopolysaccharide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; ACN, actonitrile; IL, interleukin; WT, wild type; TAP, transporter associated with antigen processing; HCTU, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate; DMT, 4,4'-dimethoxytrityl; NMP, N-methylpyrrolidone; FACS, fluorescence-activated cell sorter; TAC, tert-butylphenoxyacetyl.
4 The use of a large excess of N,N'-diisopropylethylamine (4 eq relative to Pam3Cys-OH) proved to be detrimental for the purity of the product as judged by liquid chromatography/mass spectroscopy analysis.
5 S. Khan, M. S. Bijker, J. J. Weterings, H. J. Tanke, G. J. Adema, T. van Hall, J. W. Drijfhout, C. J. M. Melief, H. S. Overkleeft, G. A. van der Marel, D. V. Filippov, S. H. van der Burg, and F. Ossendorp, unpublished data.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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