Article pubs.acs.org/molecularpharmaceutics
Octaarginine-Modified Liposomes Enhance Cross-Presentation by Promoting the C‑Terminal Trimming of Antigen Peptide Takashi Nakamura,† Kouhei Ono,† Yoshiteru Suzuki,† Rumiko Moriguchi,† Kentaro Kogure,‡ and Hideyoshi Harashima*,† †
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
‡
ABSTRACT: Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class I (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cellpenetrating peptide, the production of the mature MHC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length clearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production. KEYWORDS: liposomes, antigen presentation, cell-penetrating peptide, trimming of antigen peptide, proteasomes
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INTRODUCTION Dendritic cells (DCs) are antigen presenting cells, which take up, process, and present exogenous antigens on MHC molecules to activate an adaptive immune response.1 The efficient induction of cross-presentation of exogenous antigens to the MHC class I (MHC-I) pathway is required to induce cytotoxic T cell responses against infected cells or cancer cells. For this purpose, various antigen delivery systems have been developed with the goal of enhancing the internalization and cytosolic delivery of antigens.2−7 We recently reported that liposomes modified with octaarginine (R8), a type of cellpenetrating peptide (CPP), significantly enhance crosspresentation at lower doses than either soluble ovalbumin (OVA) or OVA contained within pH-sensitive or conventional cationic liposomes in mouse bone-marrow-derived dendritic cells (BMDCs).8 Moreover, in mice that were subcutaneously immunized with R8-modified liposomes (R8-Lip), the growth of the antigen-expressing tumors in vivo, was significantly reduced. These results suggest that R8-Lip has promise for use as a carrier for inducing antigen cross-presentation in vaccinations. In this study, to clarify the mechanism of R8Lip mediated cross-presentation, we used octalysine-modified liposomes (K8-Lip) as a comparison. The results show that K8Lip was much weaker in inducing cross-presentation compared to the R8-Lip (Figure 1A). This result prompted us to examine © XXXX American Chemical Society
the difference in cross-presentation machinery between R8-Lip and K8-Lip. On the other hand, our previous studies showed that R8-Lip encapsulating plasmid DNA stimulated uptake via macropinocytosis and enhanced endosomal escape, resulting in a high degree of transgene expression in NIH3T3 cells.9,10 In contrast, gene expression by K8-Lip was 1 order of magnitude less than that for R8-Lip, although both particles are cationic.11 Further analysis revealed that, in fibroblasts, R8-Lip and K8-Lip were taken up primarily via macropinocytosis with comparable efficiency; however, R8-Lip was able to escape from the endosomes at both acidic pH (5.5) and neutral pH (7.4) conditions. On the contrary, the K8-Lip escaped from endosomes only at a neutral pH. It thus appears that the difference in gene expression between the two liposomes can be attributed to R8 on the liposome surface, which stimulates the efficient escape from endocytic vesicles. On the basis of these results, we hypothesized that the mechanism by which R8 induced efficient cross-presentation was the enhanced endosomal escape of encapsulated antigens in BMDCs. During our Received: February 20, 2014 Revised: April 24, 2014 Accepted: June 5, 2014
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dx.doi.org/10.1021/mp500147y | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
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Figure 1. Comparison of MHC-I antigen presentation, cellular internalization and intracellular trafficking between R8-Lip and K8-Lip. (A) and (B) BMDCs were incubated with the indicated concentrations of liposomes containing OVA for 5 h at 37 °C, and cocultured overnight with B3Z T-cell hybridoma cells. β-Galactosidase activity was then measured at an absorbance of 595 nm as an indicator of antigen presentation activity. The absorbance of the B3Z cells incubated with untreated BMDCs was subtracted as background. (A) MHC-I presentation induced by R8-Lip and K8Lip. Data are the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (B) BMDCs were incubated with 80 μM liposomes containing OVA (gray) or with 80 μM liposomes containing OVA and 80 μM empty R8-Lip (black) or with 80 μM liposomes containing OVA and empty K8-Lip (white), and the antigen presentation activities were evaluated using B3Z cells. Data are the means ± SD of at least three independent experiments (*P < 0.05, **P < 0.01). (C) BMDCs were incubated with indicated concentrations of sulforhodamine B-encapsulated R8-Lip or K8Lip for 2 h at 37 °C. Subsequently, cells were washed and analyzed using flow cytometry. The vertical axis shows fluorescence intensity (FI) relative to the mean fluorescence of the untreated BMDCs, which was set equal to 1. Data are the mean ± SD of three independent experiments. (D) The cells were visualized using confocal laser scanning microscopy 3 h after incubation with liposomes which encapsulated tetramethylrhodamine-labeled neutral dextran (red signal) and were labeled with NBD-DOPE (green signal) as a lipid marker. Nuclei were stained with Hoechst 33342 (blue) for 10 min prior to visualization. Bars = 10 μm.
mechanistic investigations, we unexpectedly observed that the efficiency of cellular uptake and endosomal escape between R8Lip and K8-Lip was comparable (Figure 1C and D). These results indicate that R8-Lip facilitates the subsequent cytosolic delivery of an antigen, although CPPs generally function as enhancers of cellular internalization and endocytic escape. MHC-I presentation of cytosolic antigens requires the generation of mature MHC-I peptides in a length (8−9 amino acids) suitable to permit them to be present on MHC-I molecules. The proteasome is mainly responsible for the degradation of cytosolic antigens and the trimming of antigen peptides. The proteolysis of antigens by proteasome results in the production of several peptide fragments with different sizes, including mature MHC-I peptides and extended MHC-I peptides. The extended MHC-I peptides are nearly Nterminally extended peptides because the proteasome preferentially cleaves at the C-terminus of antigen peptides.12 Peptide fragments produced by proteasomes are transported by the transporter associated with antigen presentations (TAPs) into the endoplasmic reticulum (ER). In the ER, peptide fragments, especially N-terminally extended peptides, are additionally trimmed by the action of ER aminopeptidases (ERAPs) to produce the necessary lengths for binding to the MHC-I molecule.13 The mature MHC-I peptides then bind to the peptide binding groove of the MHC-I molecule in the ER. The MHC-I/peptide complex is transported to the cell surface so that they can be specifically recognized by cytotoxic T
lymphocytes. On the other hand, the nature of machinery for peptide generation and selection by proteasomes and cytosolic peptidases remain controversial and constitute a serious issue.14 Recently, controversial issues about immunoproteasomes that are constitutively expressed in immune cells are reported.15−17 Needless to say, the involvement of CPPs in antigen processing is completely unknown. In this study, we report on an investigation of the effect of R8-Lip in antigen processing in addition to the cellular internalization and intracellular trafficking of antigens by comparing the findings for the corresponding data for K8Lip. Quantitative comparisons were performed in the following steps in the MHC-I presentation pathway: cellular uptake, endosomal escape, antigen degradation, trimming of an antigen peptide and the maturation of BMDCs.
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MATERIALS AND METHODS Materials. Egg phosphatidylcholine (EPC) was purchased from NOF Corporation (Tokyo, Japan). Dioleoylphosphatidylethanolamine (DOPE), N-(7-nitro-2-1,3-benzoxadiazol-4-yl)DOPE (NBD-DOPE) and cholesterol were obtained from AVANTI Polar Lipids, Inc. (Alabaster, AL). Cholesteryl hemisuccinate (CHEMS), Hoechst 33342, OVA (grade VI), chloroquine and lactacystin were purchased from SIGMAAldrich (St. Louis, MO). Stearylated R8 (STR-R8) and stearylated K8 (STR-K8) were synthesized by KURABO industries (Osaka, Japan). Mouse recombinant granulocyteB
dx.doi.org/10.1021/mp500147y | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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trimming, BMDCs were incubated with peptide encapsulated liposomes for 30 min at 37 °C and 5% CO2 in serum-free RPMI1640 medium containing 10 ng/mL GM-CSF. Liposomal doses were optimized to induce similar MHC-I antigen presentation between peptide encapsulated liposomes. After 30 min incubation, the BMDCs were washed twice with PBS to remove the direct binding of peptide to MHC-I on cell surface. In the case of SIINFEKL encapsulated liposomes, moreover, SIINFEKL encapsulated liposomes were treated with 100 μg/ mL proteinase K (MERCK, Darmstadt, Germany) to prevent SIINFEKL peptide from directly binding to MHC-I on cell surface, before BMDCs were incubated with SIINFEKLencapsulated liposomes. RPMI1640 medium containing GMCSF was then added to the cells, followed by an additional 4.5 h incubation. After incubation, BMDCs were washed twice with RPMI1640 medium and cocultured with B3Z T-cell hybridoma cells (2 × 105 BMDCs and 1 × 105 B3Z cells) in 96-well plates for 16 h. The cells were then washed with PBS and incubated with 100 μL of CPRG buffer (5 mM CPRG, 0.125% NP-40 and 9 mM MgCl2 in PBS) for 4 h at 37 °C. The absorbance at 595 nm of each well was measured using a microplate reader (Benchmark Plus, Bio-Rad Laboratories, Inc., Hercules, CA). Quantification of Cellular Uptake of Liposomes. To quantify liposomal uptake, BMDCs were incubated for 2 h with liposomes encapsulating sulforhodamine B in serum-free RPMI1640 medium containing GM-CSF. To evaluate the effect of endocytosis inhibitors, BMDCs were preincubated with amiloride (1 mM) or chlorpromazine (CPZ, 5 μg/mL) for 30 min or 1 h, respectively, followed by incubation with liposomes for 1 h. To remove cell-surface-bound liposomes, the cells were washed once with colic acid buffer (4 mM colic acid in PBS) and three times with FACS buffer (0.5% BSA, 0.1% NaN3 in PBS). Cell-associated fluorescence was measured using FACSCalibur (BD Biosciences, Mountain View, CA) and were then analyzed using Cell Quest software (BD Biosciences, Mountain View, CA). Confocal Laser Scanning Microscopic Analysis. To analyze intracellular liposomal trafficking, BMDCs were incubated with liposomes, which contained NBD-DOPE (AVANTI Polar Lipids, Inc., Alabaster, AL) in the lipid membrane and tetraethylrhodamine-labeled neutral dextran (70 kDa, Molecular Probes, Inc.) in the aqueous phase, in serumfree RPMI1640 medium containing GM-CSF for 1 h. The cells were then washed with RPMI1640 medium and incubated for 2 h in fresh RPMI1640 medium. Nuclei were stained with Hoechst 33342 for 10 min before visualization by confocal laser scanning microscopy (CLSM) (LSM510, Carl Zeiss, Inc., Oberkochen, Germany). To evaluate the effects of chloroquine, BMDCs were incubated with chloroquine at a concentration of 25 μM for 30 min prior to incubation with liposomes. Flow Cytometric Analysis of Surface Molecules of BMDCs. BMDCs were incubated with liposomes (100 μM lipid) or LPS (1 μg/mL, Sigma-Aldrich, St. Louis, MO) for 2 h in serum-free RPMI1640 medium. RPMI1640 medium was then added to cells, followed by an additional 22 h incubation. FITC-labeled antimouse CD80 (clone 16-10A1) and CD86 (clone GL-1) for flow cytometric analysis were purchased from BioLegend (San Diego, CA). Their isotype controls, FITClabeled Armenian hamster IgG and Rat IgG2a, were obtained from eBioscience, Inc. (San Diego, CA). After blocking nonspecific Fc-mediated binding, BMDCs were incubated with each antibody (5 μg/mL) for 30 min on ice and washed in FACS buffer. Fluorescence intensities of the cells were
macrophage colony-stimulating factor (GM-CSF) was purchased from R&D Systems, Inc. (Minneapolis, MN). Chlorophenol red β-D-galactopyranoside (CPRG) was obtained from Roche Diagnostics (Basel, Schwiez). Peptide SIINFEKL, 5+SIINFEKL (LEQLESIINFEKL), SIINFEKL+5 (SIINFEKLTEWTS), and 5+SIINFEKL+5 (LEQLESIINFEKLTEWTS) were synthesized by the Toray Research Center (Tokyo, Japan). Mice. C57BL/6 (H-2b, 6−8 weeks old) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were maintained under specific pathogen-free conditions. The use of mice was approved by the Pharmaceutical Science Animal Committee of Hokkaido University. Cells. The B3Z cell is a CD8+ T cell-hybridoma-specific for the OVA257-264 epitope in the context of H-2Kb.18 B3Z cells were kindly provided by Dr. Nilabh Shastri (University of California, Berkeley, CA, U. S. A.). These cells were cultured in RPMI1640 medium containing 50 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/ml penicillin− streptomycin and 10% fetal calf serum. BMDCs were generated by culturing bone marrow cells in the presence of GM-CSF (10 ng/mL) as described previously.8,19 On days 2 and 4, nonadherent cells were removed and adherent cells were cultured in fresh medium containing GM-CSF. On days 6−8, nonadherent and loosely adherent cells were harvested and used in experiments as immature DCs. Preparations of Liposomes. OVA-encapsulated liposomes and peptide-encapsulated liposomes were prepared as described in our previous report.8 Peptide SIINFEKL, LEQLESIINFEKL, SIINFEKLTEWTS, and LEQLESIINFEKLTEWTS were used as OVA-associated peptides. In a typical preparation, lipid films composed of DOPE, CHEMS and EPC (75:1.25:23.75, molar ratio) were hydrated with an OVA solution (5 mg/mL, 10 mM HEPES, pH7.4) or OVA peptides solution (0.5 mg/mL, 10 mM HEPES, pH7.4) for 10 min at room temperature. For uptake measurements, the lipid film of EPC, Cholesterol and CHEMS (70:28.75:1.25, molar ratio) was hydrated with sulforhodamine B solution (10 mM, Life Technologies Co., Carlsbad, CA). The suspension was then briefly sonicated and subjected to six freeze−thaw cycles followed by extrusion through a 400 nm filter (Nucleopore, AVANTI Polar Lipids, Inc., Alabaster, AL). Unencapsulated OVA, peptide or sulforhodamine B were removed by ultracentrifugation at 80 000g for 30 min at 4 °C. After dividing the purified liposomal solution into two equal portions, the surface of the liposomal membrane was modified with either STR-R8 or STR-K8 (5 mol % of total lipid). The concentrations of lipid and OVA were determined using a phospholipid assay kit (Wako Pure Chemical Industries, Osaka, Japan) and a BCA protein assay kit (Thermo Scientific, Rockford, IL), respectively, after protein precipitation. OVAencapsulation efficiency was 10.0 ± 2.5%. Diameter and ζpotential of the liposomes were measured using a ZETASISER Nano (ZEN3600, Malvern Instruments, Ltd., UK). Antigen Presentation Assay. BMDCs were incubated with liposome-encapsulated OVA, either alone or in the presence of empty liposomes at different lipid concentrations for 2 h at 37 °C and 5% CO2 in serum-free RPMI1640 medium containing 10 ng/mL GM-CSF. RPMI1640 medium containing GM-CSF was then added to the cells, followed by an additional 3-h incubation. To evaluate the effects of chloroquine, BMDCs were preincubated with chloroquine for 30 min prior to incubation with liposomes. In the case of the analysis of peptide C
dx.doi.org/10.1021/mp500147y | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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concentrations. After an incubation for 2 h, the fluorescent intensities of the BMDCs were measured using flow cytometry. As shown in Figure 1C, the internalization of both liposomes increased in a dose-dependent manner. Cellular internalization was not significantly different between R8-Lip and K8-Lip. This result suggests that the amount of cellular uptake had little effect on MHC-I presentation. We also confirmed the intracellular localization of an encapsulated substance in the liposomes by CLSM. BMDCs were pulsed with NBD-labeled liposomes (green) containing rhodamine-labeled dextran (red). Three hours after the treatment, dextran red signals were widely distributed in the cytosolic space; the intracellular distributions of dextran with R8-Lip and K8-Lip were similar (Figure 1D). The efficiency of endosomal escape between R8Lip and K8-Lip were comparable. These results indicate that the efficient MHC-I presentation by R8-Lip/OVA is not due to an enhancement in cellular internalization and antigen delivery to the cytosol. Analysis of Internalization Pathway of Liposomes. The mechanism of endocytosis affects the subsequent antigen processing in DCs.22,23 Therefore, the uptake mechanism was investigated in greater detail. As shown in Figure 2, the uptake
measured using a FACSCalibur and were then analyzed using Cell Quest software. Quantification of OVA Degradation. To analyze OVA processing by the BMDCs, DQ-OVA (Life Technologies Co., Carlsbad, CA), which exhibits green fluorescence upon proteolytic degradation, was encapsulated in liposomes.20,21 BMDCs were pulsed with liposomes containing DQ-OVA (100 μM lipid) or free DQ-OVA (50 μg/mL) either in the absence or presence of lactacystin (10 μM) or NH4Cl (20 mM) for 1 h. The dose of free DQ-OVA (50 μg/mL) was the detectable dose of the MHC-I antigen presentation. After washing with colic acid buffer, the cells were incubated in fresh RPMI1640 medium with or without inhibitors for 3 h. Fluorescence intensity was then measured using a FP750 Jasco spectrofluorometer (Tokyo, Japan). Inhibitor concentrations were determined according to cell viability and antigen presentation of free OVA257−264 peptide on MHC-I. To quantify OVA degradation, fluorescence intensity measured before incubation was subtracted from that measured after the 3 h incubation. Statistical Analysis. Comparisons between two treatments were performed by unpaired t test. Comparisons between multiple treatments were made using one-way ANOVA, followed by Dunnett test or Tukey−Kramer test. A P-value of
