Effects of Localization of Antigen Proteins in Antigen-Loaded

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Effects of localization of antigen proteins in antigenloaded exosomes on efficiency of antigen presentation Yuta Arima, Wen Liu, Yuki Takahashi, Makiya Nishikawa, and Yoshinobu Takakura Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

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Molecular Pharmaceutics

Effects of localization of antigen proteins in antigen-loaded exosomes on efficiency of antigen presentation Short Title: Effects of localization of loaded protein in exosomes

Yuta Arima1†, Wen Liu1†, Yuki Takahashi1*, Makiya Nishikawa2, & Yoshinobu Takakura1.

Author Affiliations: 1; Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

2; Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan † These authors contributed equally to this work.

*Corresponding Author: Yuki Takahashi, Ph.D.

ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. Tel: +81-75-753-4615; FAX: +81-75-753-4614; E-mail: [email protected]

Abbreviations LA, lactadherin; Gag, group-specific antigen; HEK293, human embryonic kidney cells 293; GFP, green fluorescent protein; DC, dendritic cell; OVA, ovalbumin; BMDCs, bone marrow derived dendritic cells; APC, antigen presenting cell; MHC, major histocompatibility complex; FBS, fetal bovine serum; DMEM, Dulbecco's modified eagle medium; pDNA, plasmid DNA; PBS, phosphate buffered saline; TEM, transmission electron microscope; ALIX, apoptosis-linked gene 2-interacting protein X; MFI, mean fluorescent intensity; FACS, fluorescence-activated cell sorting; MVEs, multivesicular endosomes.


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Abstract

Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively (Takahashi et al. J Biotechnol. 2013; Charoenviriyakul et al. Mol Pharm. 2018), were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-innerloaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with that of OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular


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uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.

Keywords: exosomes, antigen presentation, lactadherin (LA), group-specific antigen (Gag)


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1. Introduction Exosomes are secretory lipid bilayer membrane vesicles with a diameter of 40-120 nm that are produced by various types of cells1-4. Exosomes enclose proteins and nucleic acids derived from their cells of origin and play important roles as carriers in cell-cell communication by delivering enclosed cargo to recipient cells5-6. Because of their intrinsic nature as endogenous delivery carriers, exosomes are considered as promising candidate delivery systems for proteins and nucleic acids. Antigen proteins are one of the most common cargoes for exosome-based delivery systems. It has been hypothesized that antigen-loaded exosomes can be used as effective vaccines by delivering antigen proteins to antigen presenting cells (APCs) 7-8. Upon administration, antigen-loaded exosomes are engulfed by the dendritic cells (DCs) patrolling the body; the DCs then swim to the secondary lymphoid organs and present the epitopes of the antigen to naive T cells through the major histocompatibility complex (MHC). MHC molecules belong to two classes based on origins of epitopes they present: MHC class I molecules present epitope peptides derived from proteins in the cytoplasm of APCs, and MHC class II molecules present epitope peptides generated in the endosome/lysosome compartment of APCs. MHC class I and II molecules present epitope peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. Antigen proteins taken up by APCs via


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endocytosis are presented to immune cells by MHC class I molecules via a process called cross-presentation, in which exogenous antigen proteins translocated to the cytoplasm are processed to produce peptides that are then presented on the surface of APCs by MHC class I molecules9. Antigen proteins can be loaded into either outer surface or inner surface of exosomes. Considering that the intracellular location where antigen proteins are primed, affects the efficacy of MHC class I and II antigen presentation, the localization of the antigen proteins loaded into exosomes might also affect their behavior upon cellular uptake, and the efficacy of MHC class I and II antigen presentation. However, there is limited data regarding the effect of localization of antigen proteins loaded in exosomes on the efficacy of antigen presentation, as well as the intracellular behavior of the loaded proteins after cellular uptake of exosomes. It is possible to load an antigen protein in or onto exosomes by designing a fusion protein composed of the antigen protein and an exosome-tropic protein. In our previous study, we used lactadherin (LA) to load proteins to the outer surface of exosomes. LA is an exosometropic protein that binds to the phosphatidylserine present in the outer membrane of exosomes through C-terminal lectin-type domains (C1/C2 domains) of LA10. In addition, we also used virus-derived group-specific antigen (Gag) to load proteins inside exosomes. Gag is an


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exosome-tropic protein that binds to the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] present in the inner membrane of exosomes11. Therefore, we can load antigens either to the inner surface or outer surface of exosomes by using these proteins. In this study, we investigated the effects of localization of the proteins loaded in the exosomes on the efficacy of antigen presentation, as well as on the intracellular behavior of the loaded protein in APCs. To evaluate the intracellular behavior of loaded proteins, green fluorescent protein (GFP) was loaded to the inner surface and outer surface of exosomes using Gag and LA, respectively. To investigate the effects of the localization of antigen proteins in exosomes on antigen presentation efficiency, ovalbumin (OVA), a model antigen protein, was loaded to the inner surface and outer surface of exosomes. An antigen presentation assay was performed using the two types of OVA-loaded exosomes.

2. Materials and methods 2.1 Mice and cells. Eight weeks-old male C57BL/6 mice were purchased from Shimizu Laboratory Supplies Co., Inc. (Shizuoka, Japan). Protocols for all animal experiments were approved by the Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences, Kyoto University. Human Embryonic Kidney cells 293 (HEK293) purchased from American Type


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Culture Collection (ATCC; CRL-1573) were cultured in 10 % fetal bovine serum (FBS)containing Dulbecco's modified Eagle’s medium (DMEM; Nissui Co., Ltd., Tokyo, Japan). Mouse dendritic DC2.4 cells were kindly provided by Dr. K. L. Rock (University of Massachusetts Medical School) and cultured in 10 % FBS-containing RPMI 1640 (Nissui Co., Ltd., Tokyo, Japan), supplemented with 0.5 mM monothioglycerol and 0.5 mM nonessential amino acids. Mouse T-cell hybridoma CD8-OVA1.3 cells, generously gifted by Dr. C. V. Harding (Case Western Reserve University), were cultured in complete 10 % FBScontaining DMEM, supplemented with 0.5 mM monothioglycerol and 0.1 mM non-essential amino acids.

2.2 Generation of bone marrow derived dendritic cells (BMDCs). To determine the antigen presentation capacity of exosomes, we collected BMDCs as described previously

12.

Briefly, bone marrow cells were isolated from C57BL/6 mouse

femurs and tibias and were filtered through a 40 μm cell strainer (BD Falcon, Franklin Lakes, NJ) to eliminate bone and debris. After filtration, bone marrow cells were suspended in 0.86 % ammonium chloride for 1 min to lyse erythrocytes; the remaining cells were cultured for 6 days in complete 10 % FBS-containing RPMI 1640, supplemented with 20 ng/ml


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recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ). The culture medium was changed every 2 days. Finally, non-adherent cells were harvested and used as BMDCs.

2.3 Designing of fusion proteins and construction of plasmid DNA (pDNA). LA and Gag coding sequences were obtained as described in previous reports10-11. The OVA-encoding sequence was synthesized along with the C1C2 domain sequences of mouse LA and Gag using PCR, to obtain OVA-LA and Gag-OVA, respectively. The enhanced green fluorescent protein (GFP)-encoding sequence was derived from the pEGFP-N1 vector (BD Biosciences Clontech, Palo Alto, CA, USA) along with the C1C2 domain of LA and Gag using PCR, to obtain GFP-LA and Gag-GFP fusion sequences, respectively. Information regarding the primers used to synthesize the fusion proteins used in this study is available upon request. The cDNAs of OVA-LA, Gag-OVA, GFP-LA, or Gag-GFP were inserted into the BamHI/XbaI site of the pcDNA 3.1 vector (Invitrogen, Carlsbad, CA, USA).

2.4 Preparation of exosomes. GFP-outer and GFP-inner-loaded exosomes were isolated from HEK293 cells transfected with GFP-LA and Gag-GFP-pDNAs, respectively, using PEI Max (Polysciences, Inc., Warrington, PA, USA) as described previously13. OVA-outer and OVA-inner-loaded


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exosomes were isolated from HEK293 cells transfected with OVA-LA and Gag-OVAexpressing pDNAs, respectively, using PEI Max. Exosomes were isolated from culture supernatants of non-transfected and transfected cells using sequential centrifugation (300 × g 10 min, 2000 × g 20 min, 10000 × g 30 min) followed by ultracentrifugation (100000 × g 1 h), according to the method previously described (ref 14). Exosomes thus collected were resuspended in phosphate buffered saline (PBS). The amounts of exosomes collected were estimated by quantifying protein concentration using the Bradford assay.

2.5 Confocal microscopy. DC2.4 cells were seeded onto glass coverslips at a density of 1.5 × 104 cells/well and incubated for 24 h. The culture medium was replaced with fresh medium containing 100 nM LysoTracker Red DND-99 (Molecular Probes, Eugene, OR, USA) and then GFP-loaded exosomes were added to it. Cells were washed using PBS for 30 min, 1 h, 2 h or 4 h after exosome-addition and fixed with 4 % paraformaldehyde for 20 min to stop the cellular uptake of exosomes. Then, 300 nM of 4′,6′-diamidino-2-phenylindole (DAPI) was added and cells were incubated for 5 min. After washing with PBS, the coverslips were mounted using SlowFade Gold (Thermo Fisher Scientific) to prevent fluorescent fading. The prepared


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samples were observed using a confocal microscope (A1R MP, Nikon Instech Co., Ltd., Tokyo, Japan).

2.6 Transmission electron microscopy. The exosome suspension was added to an equal volume of 4 % paraformaldehyde (Nacalai Tesque, Kyoto, Japan), and the mixture was added to a Carbon/Formvar film coated transmission electron microscope (TEM)-grid (Alliance Biosystems, Osaka, Japan). After washing with PBS, the sample was incubated with 1 % glutaraldehyde for 5 min and then with 1 % uranyl acetate for 5 min. The sample was observed using TEM (Hitachi H-7650; Hitachi High-Technologies).

2.7 Measuring of zeta potential of exosomes. The HEK293-derived exosome suspension was added to a disposable folded capillary cell and the zeta potential of exosomes was measured using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).

2.8 Fluorescent labeling of exosomes.


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The PKH67 green fluorescent cell linker kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Exosomes were labeled with PKH67, as described previously13. Briefly, exosomes were mixed with 2 μM PKH67 dye dissolved in Diluent C from the kit and were incubated for 5 min at 25 ℃. Then, exosomes were added to phosphate-buffered saline (PBS) supplemented with 5 % bovine serum albumin in order to stop the staining reaction and were centrifuged at 100,000 × g for 1 h and were resuspended in PBS for washing.

2.9 Western blotting. HEK293 cell lysates were collected by centrifugation at 12,000 × g for 10 min after four freeze-thaw cycles. The exosome samples, cell lysates, and OVA protein were separated on a 10 % sulfate-polyacrylamide gel using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (Merck Millipore Corporation, Billerica, MA, USA). OVA, 70 kDa heat-shock protein (HSP70), CD81, apoptosis-linked gene 2-interacting protein X (Alix) and Calnexin were detected by incubating the respective blots with the following primary antibodies for 1 h at 25 ℃: rabbit anti-OVA antibody (dilution, 1:4000; Ab186717; Abcam, Cambridge, UK), rabbit anti-HSP70 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CD81 antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Alix


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antibody (1:200; Santa Cruz Biotechnology) and rabbit anti-Calnexin antibody (1:1000; Santa Cruz Biotechnology). Then, the blots were incubated with the following secondary antibodies for 1 h at 25 ℃: rabbit anti-mouse IgG-HRP (1:2000; Thermo Fisher Scientific), goat anti-rabbit IgG-HRP (1:5000; Santa Cruz Biotechnology). After washing with tris buffered saline with 0.05 % Tween 20 (TBS-T), the blots were treated with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore), and chemiluminescence was detected using the Fujifilm LAS-3000 Imager (FUJIFILM, Tokyo, Japan).

2.10 Flow cytometry analysis of exosome-uptake by cells. BMDCs were seeded in a 96-well culture plate at a density of 5.0 × 104 cells/well for 24 h before the addition of exosomes. PKH67-labeled non-transfected (NT) exosomes, OVA-LA and Gag-OVA-loaded exosomes were added to the cells and BMDCs were incubated for 24 h. Then, BMDCs were washed with PBS three times and harvested in PBS. The cellular uptake of PKH-67-labeled exosomes was determined using the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA), according to the manufacturer’s instructions. Data were analyzed using the Kaluza software (Beckman Coulter).

2.11 Flow cytometry analysis of MHC class I-OVA complex on the surface of DC.


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DC2.4 cells were seeded in a 24-well culture plate at a density of 2.0 × 105 cells/well for 24 h before the addition of exosomes. OVA-LA and Gag-OVA-loaded exosomes were added to the cells at a concentration of 10 μg/mL and DC2.4 cells were incubated for 24 h. Then, DC2.4 cells were washed with PBS three times and harvested in PBS. DC2.4 cells were stained with OVA257-264 (SIINFEKL) peptide bound to H-2Kb Monoclonal Antibody, PE, (Thermo Fisher Scientific) in a final concentration of 30 uM at 37°C for 2 hours. After washed with flow stain buffer, DC2.4 cells were measured using the Gallios Flow Cytometer, according to the manufacturer’s instructions. Data were analyzed using the Kaluza software.

2.12 In vitro antigen presentation assay. BMDCs were seeded in a 96-well culture plate at a density of 5.0 × 104 cells/well for 24 h before addition of exosomes. Indicated concentrations of HEK293-derived exosomes were added to BMDCs, which were then co-cultured with CD8-OVA1.3 cells (1.0 × 105 cells/well) for 24 h. CD8-OVA1.3 cells co-cultured with BMDCs treated with OVA257-264, MHC class I-restricted peptide epitope of ovalbumin (OVA) were used as a positive control, and CD8OVA1.3 cells cultured with BMDCs treated with SIINFEKL (InvivoGen, San Diego, CA, USA), and Opti-modified Eagle's Medium (Opti-MEM), were used as negative controls. The medium used for the co-culture was collected, and the concentration of IL-2 in the medium


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was measured using mouse IL-2 enzyme-linked immunosorbent assay (ELISA) OptEIATM sets (Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions.

2.13 Statistical analysis. Differences among groups were evaluated using the Tukey-Kramer method, and P < 0.05 was considered statistically significant.

3.

Results　

3.1 GFP was detected in the cytoplasm of DC2.4 cells after cellular uptake of GFPinner-loaded exosomes, but not after that of GFP-outer-loaded exosomes. GFP-outer-loaded (GFPOUT) and GFP-inner-loaded (GFPIN) exosomes were isolated from cells transfected with pDNA encoding GFP-LA or Gag-GFP, respectively. DC2.4 cells, to which GFPOUT or GFPIN exosomes were added, were observed using confocal microscopy to visualize the intracellular distribution of GFP (Fig. 1). Confocal microscopy results showed that the green signal derived from GFP was detected in DC2.4 cells to which GFPIN exosomes were added. The green signal of GFP did not co-localize with the red signal of LysoTracker Red, which indicates that GFP was not present in the endosome and might exist in the cytoplasm. Moreover, the intensity of the green signal coming from GFP in the cells


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gradually increased with time. On the other hand, the green signal was barely detected in DC2.4 cells to which GFPOUT exosomes were added. 30 min

1h

2h

4h

GFPOUT exosomes

GFPIN exosomes

Fig. 1 Confocal microscopy showing DC2.4 cells, to which GFPOUT or GFPIN exosomes were added. DC2.4 cells, to which GFPOUT or GFPIN exosomes and LysoTracker Red were added, were observed using confocal microscopy. Green, GFP. Blue, DAPI. Scale bar = 20 μm.

3.2 Preparation of OVA-outer and OVA-inner-loaded exosomes. pDNAs encoding OVA-LA and Gag-OVA were designed (Fig. 2a). OVA-outer-loaded (OVAOUT) and OVA-inner-loaded (OVAIN) exosomes were isolated from cells transfected with the pDNA encoding OVA-LA or Gag-OVA, respectively. Western blotting analysis of


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the exosomes collected showed that OVA was loaded to the exosomes (Fig. 2b). Moreover, to degrade proteins outer surface exosomes, OVAOUT and OVAIN exosomes were treated with Protease K and were subjected to western blotting analysis. OVA was detected in OVAIN exosomes treated with protease K (Fig 2c) whereas, OVA was not detected in OVAOUT exosomes after the treatment, which suggests that OVA loaded to the inner surface of exosomes was protected from degradation by the protease.

a OVA-LA (76 kDa) N’

Signal peptide

OVA

C’

C1C2 domain (LA)

Gag-OVA (101 kDa) N’

Gag

C’

OVA

b

c Exosomes OVAOUT

OVAIN

OVAOUT NT

OVA

Protease

100 kDa ➡

100 kDa ➡

75 kDa ➡

75 kDa ➡

(−)

(+)

OVAIN (−)

(+)

48 kDa ➡ 35 kDa ➡

Fig. 2 Design of pDNAs encoding OVA-LA or Gag-OVA, and detection of OVA loaded to exosomes. (a) Design of pDNAs encoding fusion proteins consisting of OVA-LA (76 kDa)


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or Gag-OVA (101 kDa). (b) Western blotting analysis of 5 μg of OVAOUT and OVAIN exosomes using anti-OVA antibody. Arrows indicate positions of the bands of molecular weight markers. (c) After treatment with or without protease, 5 μg of OVAOUT and OVAIN exosomes were analyzed using western blotting using the anti-OVA antibody. Arrows indicate positions of the bands of molecular weight markers.

3.3 Properties of OVAOUT and OVAIN exosomes are almost identical to those of antigen-unloaded exosomes. Western blotting for the three exosome marker proteins, HSP70, CD81, and Alix, was performed to verify the properties of OVA-loaded exosomes (Fig. 3a). HSP70, CD81, and Alix were detected in all exosomes isolated from HEK293 cells. All exosomes showed negative results for Calnexin, an endoplasmic reticulum marker, which suggests little contamination from cell-derived debris in the exosome samples collected. Then, exosomes were observed using TEM (Fig. 3b). TEM data revealed that the size of all exosomes was approximately 100 nm in diameter. Zeta potential values for exosomes OVAOUT and OVAIN, and exosomes collected from non-transfected cells (NT exosomes) were -39.2 ± 2.6, -37.3 ± 2.6 and -38.8 ± 1.3 (mV), respectively (Fig. 3c). These results suggested that OVA loading scarcely altered the properties of exosomes.


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Exosomes

a OVAOUT

OVAIN

NT

Cell Lysates

HSP70 CD81 Alix Calnexin

b OVAOUT exosomes

OVAIN exosomes

NT exosomes

c OVAOUT exosomes

OVAIN exosomes

NT exosomes

0

Zeta potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-20

-40

-60

Fig. 3 Properties of exosomes were scarcely affected by OVA-loading. (a) Western blotting for HSP70, CD81, Alix, and Calnexin in OVAOUT, OVAIN exosomes, NT exosomes and HEK293 cell lysates. (b) TEM images of OVAOUT, OVAIN exosomes and NT exosomes.


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Scale bar = 100 nm. (c) Zeta potential values for OVAOUT, OVAIN exosomes and NT exosomes. Results are expressed as the mean ± standard deviation (n = 4).

3.4 Cellular uptake of exosomes by BMDCs was not affected by the localization of OVA in the exosomes. To evaluate the uptake of OVA-loaded exosomes by BMDCs, NT exosomes and OVAloaded exosomes fluorescently labeled with PKH67 were added to BMDCs, and the BMDCs were analyzed using flow cytometry (Fig. 4). Flow cytometry results showed that there was no significant difference in mean fluorescent intensity (MFI) between BMDCs containing NT exosomes, OVAOUT and OVAIN exosomes, which suggests that localization of OVA in exosomes had little influence on cellular uptake of exosomes.


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120

100 Mean Fluorescent Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80

1μg/mL

60

5μg/mL

40

20

0 Control

NT exosomes

OVAOUT exosomes

OVAIN exosomes

Fig. 4 Cellular uptake of exosomes by BMDCs was almost identical between NT exosomes, OVAOUT and OVAIN exosomes. NT exosomes, OVAOUT and OVAIN exosomes were labeled using the PKH67 green fluorescent cell linker kit. C57BL/6 mice derived BMDCs were seeded at a density of 5.0 × 105 cells/ml. After 24 h of culture, 1 and 5 μg/mL of PKH-67-labelled NT exosomes, OVAOUT or OVAIN exosomes were added to each well 4 h before eliminating the supernatant. The uptake of exosomes by BMDCs was measured using fluorescence-activated cell sorting (FACS) analysis. Results are expressed as the mean ± standard deviation (n = 4).


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3.5 BMDCs containing OVAIN exosomes cross-presented OVA-epitope to CD8OVA1.3 cells through MHC class I. To evaluate the efficiency of MHC class I antigen presentation, two experiments were performed. First, MHC class I-OVA complex displayed on the surface of DC was evaluated. DC treated with OVAIN exosomes showed a higher expression of MHC class I-OVA complex than DC treated with OVAOUT exosomes (Fig.5A). Second, NT exosomes, OVA and OVAOUT or OVAIN exosomes were added to BMDCs co-cultured with CD8-OVA1.3 cells. T cells secrete IL-2 upon cross-presentation of an antigen by DCs to CD8 positive T cells through MHC class I. ELISA analysis showed that the concentration of IL-2 secreted by CD8-OVA1.3 cells with OVAIN exosomes was significantly higher as compared to that secreted by cells with NT exosomes and OVAOUT exosomes in a concentration-dependent way (Fig. 5B).


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A OVAOUT exosomes Count

OVAIN exosomes

744

B 200 180 160 140

1μg/mL

120 IL-2(pg/mL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5μg/mL

100 *

80

10μg/mL 20μg/mL

60

*

40 20 0 Control

Peptide

OVA

NT exosomes

OVAOUT exosomes

OVAIN exosomes

Fig. 5 OVA epitope was presented by BMDCs added with OVAIN exosomes but not OVAOUT exosomes. (A) OVAOUT and OVAIN exosomes were added to DC2.4 cells and the cells were incubated for 24 h. The expression level of MHC class I-OVA complex was measured by FACS. (B) BMDCs derived from C57BL/6 mice were seeded (5.0 × 105 cells/ml). After 24 h of culture, OVA, NT exosomes, OVAOUT, OVAIN exosomes, or MHC


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class I specific peptide, and CD8-OVA1.3 (1.0 × 106 cells/ml) were added to the BDMC culture for a 24 h incubation. IL-2 concentration of the co-culture supernatants was measured using ELISA. *P < 0.05 compared with the other exosome groups in the same concentration. Results are expressed as the mean ± standard deviation (n = 4).

4. Discussion To elucidate the influence of the localization of antigen proteins in exosomes on their cytoplasmic distribution and antigen presentation, LA and Gag were selected to design fusion proteins according to the previous study10-11. Subsequently, we demonstrated that proteins of interest, GFP and OVA, were loaded to the inner surface or outer surface of exosomes, respectively as seen in the physiological scenario. In the present study, MHC Class I presentation by DCs added with antigen-loaded exosomes was evaluated because induction of cellular immune response is more important than that of humoral immune response in cancer vaccination, which is one of the most expected application of exosome-based vaccine. However, considering that more broad application of exosome as vaccine, evaluation of MHC class II-mediated presentation should be considered in the further research. Exosomes may be taken up by clathrin-mediated or clathrin-independent endocytosis such as phagocytosis, micropinocytosis, and endocytosis via caveolae and lipid rafts14. After


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cellular uptake, exosomes are transported into lysosomes through the formation of multivesicular endosomes (MVEs) and are then degraded by low pH in the endosome and by proteases in the lysosome. Protease treatment of OVAOUT exosomes degraded the loaded OVA protein, whereas, the treatment hardly degraded OVA from OVAIN exosomes, thus, proteins loaded outer surface of exosomes were more susceptible to intracellular degradation as compared to proteins loaded inside exosomes, results obtained using GFP-loaded exosomes suggest that GFP in GFPIN exosomes was protected by the membranes of exosomes from degradation in the endosome/lysosome. MHC class I presentation of OVA was higher in BMDCs with OVAIN exosomes than that in BMDCs with OVAOUT exosomes, whereas, cellular uptake of OVA was comparable between these two groups. These results imply that antigen processing efficiency after cellular uptake of exosomes is different between DCs with OVAIN exosomes and OVAOUT exosomes. As OVA loaded in OVAIN exosomes was protected by the membranes of exosomes from degradation after cellular uptake, OVA loaded in OVAIN exosomes might be transferred to the cytoplasm more efficiently than that in OVAOUT exosomes. A previous study reported that lymphoid organ-resident CD8+ DCs efficiently perform MHC class I antigen presentation by inhibiting degradation of antigens in endosomes/lysosomes by maintaining an alkaline pH in the endosome and phagosome15. Another study showed that


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efficient antigen presentation was promoted by loading antigen proteins onto cationic liposomes, which alkalinized the pH in the lysosomes of DCs16. The results obtained by the current study are in agreement with the results of previous studies and suggest that protection of antigen proteins from degradation in endosomes/lysosomes plays a significant role in increasing the efficacy of MHC class I antigen presentation. In conclusion, this study demonstrates that the localization of antigen proteins loaded in exosomes, inner surface or outer surface, alters their fate in cells. It was demonstrated that proteins loaded inside of exosomes were delivered to the cytoplasm of the cells that took up exosomes more efficiently than the proteins loaded outside of exosomes. Therefore, innerloading is effective for immunotherapy using exosomes loading antigen proteins.

Acknowledgement This work was supported by JSPS KAKENHI (grant number JP17K19390 and JP18H02562) from the Japan Society for the Promotion of Science (JSPS). All authors declare no conflict of interest.

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Effects of Localization of Antigen Proteins in Antigen-Loaded

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