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The Journal of Immunology, 2004, 173: 566-578.
Copyright © 2004 by The American Association of Immunologists

Phosphatidylserine Receptor-Mediated Recognition of Archaeosome Adjuvant Promotes Endocytosis and MHC Class I Cross-Presentation of the Entrapped Antigen by Phagosome-to-Cytosol Transport and Classical Processing1

Komal Gurnani, Jessica Kennedy, Subash Sad, G. Dennis Sprott and Lakshmi Krishnan2

National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4+ cells expressing OVA323–339 TCR) or OT1 (>90% CD8+ OVA257–264 cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2+) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1+ recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2KbOva257–264 CD8+ T cells into CD44highCD62Llow effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
New-generation vaccines are often subunit entities based on key protective soluble Ags and/or peptides. Aspects critical to the design of such vaccines include the ability to effectively deliver Ag to appropriate APCs, provide strong and safe immune stimulation, and evoke long-term immunity (1). Adjuvants in vaccines are used to bridge these aspects and, by definition, include immune-stimulating compounds that can augment robust immunity to a weak immunogen. However, only two adjuvants, alum and MF59 are currently approved for universal human use, and fail to elicit cell-mediated immunity. Thus, a wide range of substances such as particulate Ag carriers, attenuated bacteria and viruses, naked DNA, and bacterial ligands are being researched for their ability to evoke cell-mediated immunity (2, 3, 4, 5, 6).

CD8+ T cells are the critical cells for protection against intracellular infections and tumors, and are primed only when Ag is displayed on the surface of MHC class I molecules of APCs. Naturally, only endogenously generated peptides in the cytosol gain access to MHC class I (7). Thus, it was believed that recombinant attenuated intracellular viruses and bacteria that can enter the cytosol may constitute the best choice of Ag carriers for induction of CD8+ T cells (8, 9). In contrast, exogenous Ags in vaccines may gain access only to MHC class II and bias responses to CD4+ T cells (10). However, it is now clear that professional APCs often break the rule and present some exogenous Ags on MHC class I, even when they fail to gain access to the cytosol, by a process termed cross-presentation (11, 12). Furthermore, cross-priming often ensues after phagocytosis of particulate matter and uptake of apoptotic cells (13). Both macrophages and dendritic cells (DCs)3 are capable of cross-presentation, although only DCs prime naive CD8+ T cells (11, 14). Deciphering precisely the mechanisms by which exogenous Ags from the phagosome gain access to MHC class I will fundamentally aid vaccine design.

Cytosolic diversion is one route of cross-presentation based on passage of Ag from endosomes to cytosol. Examples in this category include bacteria such as Listeria monocytogenes, whose pore-forming toxins facilitate cytosolic entry of phagosomal Ag (15), and Ag carriers such as pH-sensitive liposomes that fuse with endosomal membrane at acidic pH, releasing cargo into the cytosol (16, 17, 18). For some particulate systems, the mechanisms of Ag escape into the cytosol are not evident and may include leakage of overloaded phagosomes (19) and/or transport through specialized endosomal channels on DCs (20). An alternate route of cross-presentation is based on processing of Ag within endosomes for loading onto MHC class I as well. Mechanisms proposed to accommodate this pathway include the ability of endosomal proteases to degrade appropriate-length peptides for MHC class I, loading onto recycled MHC I molecules, and fusion of endosome with plasma membrane that allows binding of peptides to surface MHC class I (21, 22, 23). Very recently, an endoplasmic reticulum (ER)-phagosome fusion pathway that brings together the cytosolic and phagosomal organelles into a single competent unit has also been proposed (24, 25).

We have developed an adjuvant system based on liposomal vesicles derived from the isopranoid-ether glycerolipids of various archaea. Ag entrapped within these vesicles termed archaeosomes elicits potent cell-mediated immunity and protective responses against intracellular infection and cancer (26, 27, 28, 29). For archaeosomes composed of Methanobrevibacter smithii lipids, the adjuvant activity can be attributed to their ability to recruit and activate DCs (30). However, the pathway of delivery is unclear for Ag internalized in stable vesicles rich in caldarchaeol membrane-spanning bipolar lipids. In this report, we elucidate the mechanism of intracellular processing and presentation of OVA entrapped in M. smithii-vesicles (OVA-archaeosomes) constituted with 40 mol% caldarchaeols. We show that the archaetidyl serine head groups of M. smithii lipids interact with phosphatidylserine (PS)-specific receptor on APCs facilitating receptor-mediated endocytosis. Subsequently, Ag follows an endosome-to-cytosol route, dependent on acidification of endosomes, and is processed by the proteasome and transported by TAP for MHC class I presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Sources for reagents were as follows: OVA (grade V), MTT, LPS (Escherichia coli 0152), cytochalasin B, chloroquine, monensin, antipain, leupeptin, N-acetyl-Leu-Leu-Norleu-al (AcLLnL), N-acetyl-Leu-Leu-Met-al (AcLLM), RBC-lysing solution, O-phospho-L-serine, mannopentaose, and dimyristoylphosphatidylglycerol (PG) from Sigma-Aldrich (Oakville, Ontario, Canada); {alpha}-PS from bovine brain (Larandon Fine Chemicals, Malmo, Sweden); lactacystin and epoxomicin from Calbiochem-Novabiochem (La Jolla, CA); purified murine GM-CSF and IL-2 from ID Laboratories (London, Ontario, Canada); flow cytometric Abs from BD Pharmingen (Mississauga, Ontario, Canada); H-2KbOva257–264 tetramers from Beckman Coulter (Fullerton, CA); RPMI 1640, IMDM, and gentamicin from Invitrogen Life Technologies (Carlsbad, CA); FBS from HyClone (Logan, UT); G418 from Rose Scientific (Edmonton, Alberta, Canada); and rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine from Molecular Probes (Eugene, OR). OVA257–264 (SIINFEKL) peptide was synthesized in-house.

Preparation and characterization of liposomes and archaeosomes

M. smithii ALI (DSM 2375) was grown in fermenters, total polar lipids (TPL) were extracted from frozen cell pastes, and the TPLs were collected as the acetone-insoluble fraction. Archaeosomes with entrapped OVA (lacking fragments) were prepared by the dried-reconstituted vesicle method, as described in detail previously (28). These vesicles are referred to as OVA-archaeosomes. The amount of protein incorporated into the vesicle was estimated by a modified SDS-Lowry method after lipid removal. The ratio of protein to lipid was based on the salt-free dry weights of the vesicles and was 50–70 µg of OVA per 1 mg of lipid. Rhodamine-labeled archaeosomes for phagocytosis assays were prepared by mixing TPL in chloroform with 0.5% (w/w) rhodamine B as described previously (31). For competitive studies, ester liposomes were constituted with PG containing 20 mol% PS (known to surface orient PS, PS liposomes) or PG alone and prepared as described before (31). All vesicles were unilamellar, and diameters were in the range of 110–170 nm, determined by number-weighted Gaussian size distributions using a Nicomp (Santa Barbara, CA) particle sizer. All glassware used in the preparation of archaeosomes was prebaked (6 h at 80°C) to render them pyrogen free, and endotoxin-free reagents were used throughout.

Mice

Inbred, 6- to 8-wk-old female C57BL/6 mice were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). OT1, D011.10, B6.PL, TAP–/– (B6.129S2-Tap1tm1arp), and C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in accordance with guidelines from the Canadian Council on Animal Care.

Cell lines and generation of bone-marrow DCs

IC21 (macrophages) were obtained from the American Type Culture Collection (Manassas, VA), whereas HT2 cells were obtained from Dr. T. Mosmann (University of Rochester, Rochester, NY) and cultured in RPMI 160 medium. CD8OVA1.3 hybridoma recognizing OVA257–264 was a gift from Dr. C. Harding (Case Western Reserve University, Cleveland, OH) and was grown in IMDM medium. Bone marrow-derived DCs were generated as described earlier (30). Briefly, bone marrow was flushed from the femurs and tibias of three to four C57BL/6 mice; single-cell suspensions were made; and cells were counted and stimulated at 1 x 106 cells/ml in medium supplemented with 5 ng/ml recombinant murine GM-CSF, in tissue culture flasks, for 6–8 days. Nonadherent cells were removed on days 2 and 4 of culture, and fresh medium with GM-CSF was added. DCs were harvested on days 6–8 as nonadherent cells and were consistently >90% CD11c+ (see Fig. 5c). All culture medium was supplemented with 8% FBS and included 10 µg/ml gentamicin. Cultures were maintained at 37°C under 8% CO2 in a humidified atmosphere.



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  		FIGURE 5. MHC class I presentation of OVA-archaeosomes by macrophages and DCs. IC21 macrophages or bone marrow-derived DCs were incubated with OVA-M. smithii archaeosomes (OVA-MS) for 4 or 17 h, as indicated in the figure, and then fixed, and their ability to stimulate OVA-specific CD8 hybridoma cells in an overnight assay was assessed. IL-2 production by the CD8OVA hybridoma is indicated as a readout of MHC I presentation. Means ± SD of triplicate cultures are indicated. a, Four-hour Ag presentation by macrophages of 1 or 10 µg of OVA entrapped in archaeosomes. b, Seventeen-hour Ag presentation by macrophages. c, Purity of DCs used based on CD11c expression. Plots are based on 20,000 events acquired. d, Four-hour Ag presentation by DCs. No Ag, Stimulation of CD8OVA cells by naive APCs. In all cases, controls included APCs treated with OVA257–264 peptide. Data are representative of three independent experiments conducted in each case.

 
In vitro Ag presentation and DC activation

For direct Ag presentation, spleen cells from D011.10 or OT1 mice were lysed for RBC, and cultured with 0.1, 1, and 10 µg of OVA entrapped in archaeosomes composed of M. smithii TPL, in 96-well microtiter plates, in triplicate. For 1 x 105 transgenic cells/well, additional autologous feeders from the wild-type mice were added to achieve a density of 5 x 105/well. Controls included soluble OVA, soluble OVA plus 25 µg/ml Ag-free archaeosomes, and OVA257–264 CTL epitope, as appropriate, as indicated in figure legends. Proliferation was assessed by 18-h [3H]thymidine incorporation, and IFN-{gamma} production in the supernatant was quantified by sandwich ELISA. In other studies, macrophages (IC21) or DCs (105/well) in 96-well microtiter plates, in triplicate, were incubated in the presence or absence of inhibitors, with 10 µg of OVA entrapped in archaeosomes (0.16–0.19 mg of lipid). Controls included untreated naive APCs and those treated with OVA257–264 CTL peptide. For competitive inhibition studies with synthetic ester liposomes, PS and PG liposomes were added at equivalent 1:1 lipid concentrations as archaeosomes. APCs were preincubated with inhibitor for 0.5 h before addition of OVA-archaeosomes. After 4 h, cells were washed and fixed, and used to stimulate either CD8OVA1.3 (106/well) or OT1 (3 x 105/well). Proliferation of CD8OVA 1.3 was based on IL-2 production determined by a bioassay on HT2 cells. To determine OT1 stimulation, IFN-{gamma} production was quantified. Inhibitors were used at optimized concentrations: 5 µg/ml cytochalasin B, 100 µM chloroquine, 10 µM monensin, 200 µg/ml leupeptin and antipain, 40 µM tripeptide aldehydes, 50 µM lactacystin, and 10 µM epoxomicin. O-phospho-L-serine and mannopentaose were used at 20 and 100 µg/ml concentrations. For determining activation, DCs (105/ml) were incubated in vitro with Ag-free archaeosomes or LPS. After 24 h, expression of CD40 was analyzed by flow cytometry (EPICS XL; Beckman Coulter). At 72 h, MTT uptake and cytokine production were determined. Sandwich ELISA was used to assay IL-12p40 production, whereas a bioassay using Wehi 164-13 cells measured TNF.

Flow cytometric analysis of phagocytosis and receptor binding

DCs (106) were incubated with rhodamine-archaeosomes (25 µg of lipid) in the presence or absence of various competitive inhibitors, in a volume of 100 µl of RPMI 1640 medium, in 3-ml Falcon FACS tubes (BD Biosciences, Franklin Lakes, NJ). Parallel incubations were conducted at 4 and 37°C for each assay condition. After 45-min incubation, cells were washed with 3 ml of cold RPMI 1640, fixed in 1 ml of 1% formaldehyde, and analyzed by flow cytometry. Fluorescent intensity of rhodamine observed at 4°C was considered surface binding of archaeosomes, whereas that observed at 37°C was considered active phagocytosis/uptake. Inhibitors used included the following: PS-liposomes (100 µg of lipid), soluble phospho-L-serine (400 µM), mannopentaose ({alpha}-Man-(1,3)({alpha}-Man-[1,6])-{alpha}-Man-(1,6)({alpha}-Man-[1,3])-Man; 100 µM), and anti-CD16 Ab.

In vivo MHC class I Ag presentation

Spleen cells (25 x 106) from donor OT1 transgenic mice (Thy1.2+) were labeled with CFSE (32), and injected i.v. into recipient B6.PL (Thy1.1+), TAP–/–, or C57BL/6J mice that had been immunized with OVA-M. smithii archaeosomes (25 µg of OVA in 0.2 mg of lipid) 3 days earlier. Four days after adoptive cell transfer, recipient mice were euthanized, and the numbers of donor origin (Thy1.2+) and H-2KbOva257–264 tetramer-positive CD8+ T cells that were cycling (based on reduction in CFSE) were determined by flow cytometry. Analysis of CD44+CD62L+ on H-2KbOva257–264 tetramer-positive CD8+ T cells was also done.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OVA entrapped in M. smithii archaeosomes is processed for both MHC class II and class I presentation

To address the ability of archaeosomes to gain access to both MHC class I and class II presentation pathways, we stimulated spleen cells from OVA-TCR transgenic DO11.10 (CD4+ cells express the TCR for OVA323–339) or OT1 (>90% of the CD8+ cells express TCR for OVA257–264), with OVA-archaeosomes. Because whole spleen cells were used, we surmised that OVA-archaeosomes would be phagocytosed and presented by the APC population to stimulate the naive Ag-specific T cells. Both proliferation and IFN-{gamma} production were monitored. Fig. 1 indicates the proliferation and IFN-{gamma} production of DO11.10 cells at 24–72 h after stimulation with various doses of OVA-archaeosomes or soluble exogenous OVA. At both cell densities, OVA-archaeosomes stimulated a strong dose-dependent stimulation of OVA-specific CD4+ T cells, peaking at 72 h, indicating that Ag entrapped in archaeosomes was being presented on MHC class II. Stimulation by OVA-archaeosomes was often more pronounced than equivalent amounts of soluble OVA, indicative of increased uptake and/or processing efficiency.



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  		FIGURE 1. MHC class II presentation of OVA-archaeosomes. In vitro stimulation of OVA-TCR transgenic spleen cells (105 or 5 x 105 cells/well concentration) from D011.10 mice is indicated. Proliferation (a) and IFN-{gamma} production (b) of D011.10 cells at 24, 48, and 72 h. Stimulation with OVA entrapped in M. smithii archaeosomes (OVA-MS) at three Ag concentrations, and soluble OVA at two concentrations tested, is indicated. Data represent means ± SD of triplicate cultures of two such experiments conducted.

 
In similarly designed experiments, spleen cells from OT1 mice were incubated with various concentrations of OVA-archaeosomes or OVA257–264 CTL epitope peptide, to stimulate naive CD8+ T cells. Proliferation (Fig. 2a) and IFN-{gamma} production (b) were assessed at 24–72 h, at two different OT1 spleen cell concentrations. Peak response occurred at 48 h after OVA-archaeosome presentation. Often 1–10 µg of whole OVA entrapped in archaeosomes evoked equivalent cytokine production as observed after direct external loading of surface MHC class I with 1 µM OVA257–264 CTL peptide. Next, we tested whether soluble OVA admixed with Ag-free archaeosomes would be cross-presented on MHC class I to stimulate OT1 cells. Although 1–10 µg of OVA entrapped in archaeosomes (1.6–160 µg of lipid) yielded strong stimulation of OT1 cells, neither soluble OVA alone (1–10 µg) nor OVA admixed with Ag-free archaeosomes (25 µg/ml lipid) were effective to promote cross-presentation (Fig. 3). The concentration of Ag-free archaeosomes used for these experiments was based on our earlier observation that this dose acts efficiently as an adjuvant for DC recruitment and activation (30). We also tested admixing soluble OVA at 1- to 10-µg dose with a varying concentration range of Ag-free archaeosomes (1.6–160 µg of lipid), relative to the concentrations of lipid in the OVA-archaeosome preparation used above. In this scenario as well, very little stimulation of OT1 cells was seen even at high lipid doses (data not shown). Thus, physical association (entrapment) of soluble Ag with archaeosomes was instrumental at efficiently directing even small amounts of Ag (<1 µg) to the processing pathway.



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  		FIGURE 2. MHC class I presentation of OVA-archaeosomes. In vitro stimulation of OVA-TCR transgenic OT1 spleen cells is indicated. Proliferation (a) and IFN-{gamma} production (b) at 24, 48, and 72 h at two different OT1 concentrations (105 or 5 x 105 cells/well). Stimulation with OVA-M. smithii archaeosomes (OVA-MS) tested at three OVA concentrations, and stimulation with OVA257–264 CTL peptide tested at two concentrations, are indicated. Data represent means ± SD of triplicate cultures of two such experiments conducted.

 


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  		FIGURE 3. Requirement for encapsulation of OVA in archaeosomes for MHC class I presentation to OT1 cells. In vitro stimulation of OVA-TCR transgenic OT1 spleen cells is indicated. Proliferation (a) and IFN-{gamma} production (b) at 48 h at two different OT1 concentrations (105 or 5 x 105 cells/well). Stimulation with 0.1, 1, and 10 µg/ml OVA-M. smithii archaeosomes (OVA-MS), OVA257–264 CTL peptide, soluble OVA (Sol. OVA), and soluble OVA in the presence of Ag-free M. smithii archaeosomes (Sol. OVA + 25 µg/ml MS) are indicated. Data represent means ± SD of triplicate cultures.

 
OVA-archaeosomes elicit profound in vivo Ag presentation

We next evaluated the in vivo Ag presentation by OVA-archaeosomes using an adoptive transfer model, wherein naive OT1 spleen cells were injected into mice previously immunized with OVA-archaeosomes, and proliferation of Ag-specific CD8+ T cells was tracked 4 days later based on H-2KbOva257–264 tetramer staining and CFSE reduction. Because hosts and donors differed in their expression of Thy1.2 allele, the proliferation of donor cells could be tracked specifically using an anti-Thy1.2 Ab. When OT1 cells were transferred into PBS-injected mice, <4% of donor CD8+ T cells were detected, of which <5% were cycling. In contrast, in OVA-archaeosome-immunized mice, there was substantial accumulation of OVA-specific CD8+ cells, because ~40% of all splenic CD8+ T cells were of donor origin (Fig. 4a). Furthermore, these cells exhibited profound cycling (95%) based on CFSE reduction profile, indicating efficient Ag presentation and stimulation (Fig. 4b). Additionally, we also measured the phenotype of CD8+ T cells in OVA-archaeosome-immunized mice. Within 4 days of transfer, the OT1 cells underwent differentiation characterized by increased percentages of OVA-specific CD44highCD62LlowCD8+ effector T cells and equal proportions of CD44highCD62Lhigh resting memory cells (Fig. 4c).



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  		FIGURE 4. The intensity of in vivo MHC class I presentation and stimulation of CD8+ T cells by OVA-archaeosomes. Thy1.1+ B6.PL mice were immunized s.c. with 25 µg OVA entrapped in M. smithii (0.2 mg of lipid) archaeosomes (OVA-MS). On day 3, naive and OVA-MS-immunized Thy1.1+ recipients (n = 3) were injected with CFSE-labeled OT1 (Thy1.2+) cells. Four days later, the number of CD8+ T cells of donor origin (Thy1.2+) was evaluated (a). Furthermore, the reduction in CFSE of gated CD8+ T cells of donor origin was determined as a measure of cycling and proliferation of the transferred cells (b). The expression of CD62L vs CD44 on gated CD8+ donor cells was evaluated (c) as a measure of T cell differentiation.

 
PS-specific receptor-mediated uptake and MHC class I presentation of OVA-archaeosomes

We then traced the intracellular trafficking pathway of archaeosomes that leads to MHC class I presentation of Ag. APCs (H-2Kb) were incubated in vitro with OVA-archaeosomes in the presence or absence of inhibitors of Ag processing (11, 33), and then their ability to stimulate IL-2 production by OVA257–264-specific hybridoma cells (CD8OVA1.3) was determined. In the absence of any inhibitors, OVA-archaeosomes were processed for MHC class I presentation by IC21 macrophage cells within 4 h, in a dose-dependent fashion, resulting in the stimulation of CD8OVA1.3 cell IL-2 production (Fig. 5a). At 10-µg dose of OVA (0.25 µM) entrapped in archaeosomes, the stimulation observed was similar to that seen upon direct surface loading of MHC class I with 1–10 µM (1–10 µg) OVA257–264 peptide. Incubation of IC21 macrophages with OVA-archaeosomes for longer time (17 h) before addition of CD8OVA1.3 responders did not enhance further the Ag presentation (Fig. 5b). Nevertheless, CD8OVA1.3 stimulation was still substantial, indicating prolonged presentation. In similarly designed experiments, when bone marrow-derived DCs (>90% pure; Fig. 5c) were used as APCs, profound MHC class I presentation and stimulation of CD8+ T cells were observed after incubation with OVA-M. smithii archaeosomes for 4 h (d). Indeed, DCs presented even 1 µg (25 nM) of OVA entrapped in archaeosomes effectively and stimulated nanogram per milliliter quantities of IL-2 by CD8OVA1.3 cells, reiterating their potent Ag-presenting capacity. Cytochalasin B, which prevents polymerization of actin filaments, completely blocked MHC class I presentation of OVA-archaeosomes by macrophages (Fig. 6a) and DCs (b), indicative of phagocytic uptake. Cytochalasin-treated macrophages and DCs incubated with OVA257–264 peptide were able to fully activate CD8OVA1.3 cells, indicating lack of nonspecific toxicity of the inhibitor.



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  		FIGURE 6. MHC class I presentation of OVA-archaeosomes in the presence of an inhibitor of phagocytosis. Ag presentation by macrophages exposed to 10 µg of OVA entrapped in M. smithii archaeosomes (OVA-MS), in the presence or absence of cytochalasin B (a). Ag presentation by DCs exposed to 10 µg of OVA entrapped in archaeosomes, in the presence or absence of cytochalasin B (b). Stimulation with OVA257–264 peptide in the presence of inhibitor in each case is also indicated. In all cases, the ability of APCs to stimulate IL-2 production by CD8OVA hybridoma was taken as the readout of MHC class I presentation. Values indicate means ± SD of triplicate cultures, and data are representative of three experiments conducted for each inhibitor.

 
Next, to determine receptor-mediated endocytosis, we incubated APCs with OVA-archaeosomes in the presence of PS liposomes that are known to surface orient PS and inhibit uptake by the PS-specific receptor (31, 34). These vesicles competitively inhibited MHC class I presentation and consequent CD8OVA1.2 cell stimulation by both macrophages and DCs incubated with OVA-M. smithii archaeosomes (Fig. 7, a and b). Control liposomes composed of PG alone had little effect. In a complementary experiment, the ability of DCs incubated with OVA-archaeosomes to stimulate OT1 cells, in the absence or presence of PS liposomes, soluble O-phospho-L-serine (soluble PS), or mannopentaose was examined (Fig. 7c). Soluble PS is known to bind and block uptake by the PS receptor (PSR) (34), similar to PS liposomes. In contrast, soluble mannopentaose blocks uptake by the mannose receptor (35). As seen earlier, PS liposomes blocked presentation by OVA-archaeosomes. Furthermore, even 20 µg/ml O-phospho-L-serine completely blocked presentation of OVA-archaeosomes, whereas mannopentaose (even at 100 µg/ml dose) lacked effect. Soluble PS, as well as PS liposomes, had no effect on OVA257–264 peptide and soluble OVA presentations. To further confirm PS-specific recognition resulting in binding and phagocytosis of M. smithii archaeosomes by APCs, a flow cytometric phagocytic assay was used. DCs were incubated with fluorescent rhodamine archaeosomes in the presence or absence of various competitors of binding, and uptake based on increase in number of fluorescent cells was evaluated by flow cytometry. At 37°C, 30–35% DCs indicated rhodamine-archaeosome uptake (Fig. 8). This uptake was strongly inhibited by PS liposomes and soluble PS, but not by anti-CD16 (specific for FcR) and soluble mannopentaose (Fig. 8). At 4°C, which potentially measures only surface binding and not active phagocytosis, ~10% positive binding of rhodamine archaeosomes was observed. Nevertheless, even this binding was strongly inhibited by blocking PS-specific receptor interaction, but not mannose receptor or FcR (data not shown). Thus, our results clearly indicate that APCs use PS-specific receptor for uptake of M. smithii archaeosomes.



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  		FIGURE 7. PS-specific receptor-mediated endocytosis of OVA-archaeosomes. Ag presentation by macrophages (a) and DCs (b) exposed to 10 µg of OVA entrapped in M. smithii archaeosomes (0.19 mg of lipid) in the absence or presence of competing liposomes containing 20 mol% PS with PG (PS-liposomes) or PG alone (0.19 mg of lipid in each case). PS- and PG-liposomes were added 0.5 h before addition of OVA-archaeosomes. After 4 h, the APCs were fixed, and their ability to stimulate CD8OVA1.3 cell IL-2 production was determined as the measure of MHC class I presentation. Similarly, the ability of DCs to stimulate IFN-{gamma} production by OT1 cells after exposure to 1 µg of OVA entrapped in M. smithii (MS) archaeosomes (16 µg of lipid) was tested in the absence or presence of various competitors (c). Soluble O-phospho-L-serine (Sol. PS) tested at 20 and 100 µg/ml; PS-liposomes (PS-lipo.) tested at two ratios relative to MS, 1:1 (16 µg of lipid) and 5:1 (100 µg of lipid); and mannopentaose (MP) tested at 20 and 100 µg/ml concentrations. Negative controls included soluble OVA (Sol. OVA) in the presence or absence of PS-liposomes, Sol. PS, and MP. Positive controls included OVA257–264 peptide in the absence or presence of PS-lipo., Sol. PS, and MP. Values in each case indicate means ± SD of triplicate cultures, and data are representative of three experiments conducted for each inhibitor.

 


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  		FIGURE 8. PS-specific inhibition of phagocytosis. DCs were incubated with 25 µg of rhodamine-M. smithii (MS) archaeosomes for 45 min, in the presence or absence of various competitors, and uptake of fluorescent archaeosomes was analyzed by flow cytometry. The solid line indicates positive staining for rhodamine, and dotted line indicates autofluorescence of DCs. Percentage within each panel denotes positive fluorescence for each condition, which is indicative of uptake of archaeosomes. Inhibitors used included PS-liposomes (4:1 ratio; 100 µg lipid) relative to rhodamine-MS archaeosomes, O-phospho-L-serine (Sol. PS) at 400 µM, anti-CD16, and mannopentaose (Sol. MP; 100 µM).

 
OVA-archaeosome processing requires vacuolar acidification but not endosomal protease hydrolysis

Endocytosis of particulate matter by APCs is followed by fusion with lysosomes, and acidification of the endosome-lysosome compartment is an important event that activates lysosomal proteases (10). The effect of inhibitors of endosomal acidification (chloroquine, a weak base; and monensin, a Na+ ionophore that inhibits transport between endocytic compartments) were tested. Macrophages incubated with OVA-archaeosomes in the presence of chloroquine were completely unable to process OVA for MHC class I (Fig. 9a). Similarly, DCs incubated with chloroquine were severely inhibited in their ability to process OVA-archaeosomes for MHC class I (Fig. 9e). Furthermore, macrophages and DCs incubated with monensin were also unable to process OVA-archaeosomes for MHC class I (Fig. 9, b and f). In all cases, no effect of the inhibitors was noted when surface MHC class I was directly loaded by incubating with OVA257–264 peptide. We then used serine and cysteine protease inhibitors (leupeptin and antipain) and analyzed the participation of endosomal proteases in the processing of OVA-archaeosomes. Incubation of macrophages with these inhibitors failed to block MHC class I OVA-archaeosome presentation (Fig. 9, c and d). Similarly, leupeptin failed to block DC presentation of OVA-archaeosomes on MHC class I (Fig. 9g). Because acidification of the endosomal compartment was necessary for OVA-archaeosome MHC class I processing, even though endosomal hydrolysis was not, it appeared that acidification of the late endosomes facilitated molecular transport of Ag into the cytosol.



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  		FIGURE 9. MHC class I Ag presentation by macrophages and DCs in the presence of inhibitors of endosomal function. IC21 macrophages (a–d) or DCs (e–g) were incubated with OVA-M. smithii archaeosomes (OVA-MS) or OVA257–264 peptide in the presence and absence of various inhibitors of endosomal function (chloroquine, monensin, leupeptin, or antipain, as indicated in each panel). Then, the ability to stimulate CD8OVA hybridoma cells was assessed, and IL-2 production by the CD8OVA cells was taken as a readout of MHC class I presentation. Values indicate means ± SD of triplicate cultures. Data are representative of three experiments for each inhibitor.

 
Proteasomal proteases process OVA-archaeosomes for MHC class I

In contrast to the above results, a calpain inhibitor, tripeptide aldehyde AcLLnL, which also modulates proteasome activity (33), substantially diminished MHC class I presentation of OVA-archaeosomes by macrophages (Fig. 10a). The lack of inhibition of OVA-archaeosomes by control peptide AcLLM, known to have little effect on proteasomal proteases (33), confirmed the specificity of inhibition observed by AcLLnL (Fig. 10a). Similarly, two other proteasome inhibitors, lactacystin and epoxomicin, which are highly specific and modify the catalytic subunits of the proteasome (33, 36), were tested. These inhibitors severely blocked MHC class I presentation of OVA-archaeosomes by both macrophages (Fig. 10b) and DCs (c and d). Overall, from the range of inhibitors studied, it was clear that OVA entrapped in archaeosomes translocates to the cytosol, and peptide breakdown for MHC class I presentation occurred classically in the proteasome. Optimized doses of inhibitors were used, and toxic effects were ruled out, because direct surface loading of MHC class I with OVA257–264 was unaffected.



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  		FIGURE 10. MHC class I Ag presentation by macrophages and DCs in the presence of proteasomal inhibitors. IC21 macrophages (a and b) or bone marrow-derived DCs (c and d) were incubated with OVA-M. smithii archaeosomes (OVA-MS) or OVA257–264 peptide in the presence of inhibitors of proteasome function. Then, the ability to stimulate CD8OVA hybridoma cells was assessed, and IL-2 production by the CD8OVA cells was taken as a readout of MHC class I presentation. a, AcLLnL, A tripeptide aldehyde calpain inhibitor with effects on the proteasome; AcLLM, a control peptide with lack of effect on proteasome. b and c, Lactacystin, a specific inhibitor of proteasomal catalytic unit assembly. d, Epoxomicin, a specific inhibitor of proteasomal catalytic units. Values indicate means ± SD of triplicate cultures. Data are representative of three experiments for each inhibitor.

 
OVA-archaeosomes are presented by DCs in a TAP-dependent manner both in vitro and in vivo

Most proteasome-degraded peptides use the TAP transporter for entry into the ER. In vitro, although wild-type DCs efficiently presented OVA-archaeosomes on MHC class I, DCs from TAP–/– mice lacked this ability (Fig. 11a). Although TAP–/– cells have a diminished expression of MHC class I, they could present OVA257–264 peptide on H-Kb. In vivo, wherein CFSE-labeled OT1 transgenic cells were adoptively transferred into OVA-archaeosome-immunized recipients, proliferation of CD8+ OVA257–264 donor cells (assessed based on CFSE reduction and tetramer staining) occurred only in wild-type but not in TAP–/– recipients (Fig. 11b).



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  		FIGURE 11. Requirement of TAP in the MHC class I Ag presentation of OVA-archaeosomes. a, In vitro Ag presentation. DCs generated from wild-type C57BL6/J (W/T) and TAP–/– mice were treated with OVA-archaeosomes, and then the ability to stimulate IL-2 production by OVA-specific CD8 hybridoma was determined as a measure of MHC I presentation. No-Ag, Stimulation with naive APC; OVA-MS, stimulation with OVA entrapped in M. smithii archaeosomes; and OVA257–264, stimulation with peptide. Two doses of Ag (1 and 10 µg) were used. Means + SD of triplicate cultures are indicated. Data are representative of two independent experiments. b, In vivo Ag presentation. W/T or TAP–/– mice were immunized with 25 µg of OVA entrapped in M. smithii archaeosomes. On day 3, they were injected with CFSE-labeled OT1 spleen cells. Four days later, the reduction in CFSE fluorescence intensity of H-2KbOva257–264 tetramer-positive CD8+ T cells was evaluated as a measure of intensity of Ag presentation. Data indicate substantial reduction in CFSE intensity of cells in W/T but not TAP–/– mice. In vivo data are representative of two independent experiments conducted with two mice per group in each trial.

 
Ag-free archaeosomes act as adjuvant for DC activation

A second aspect of Ag presentation requires that sufficient activation of APCs occur, resulting in costimulatory marker up-regulation and cytokine production to facilitate optimal T cell stimulation. We have previously shown that M. smithii archaeosomes recruit and activate DCs in vivo (30). Herein, we further extend this observation to show that that Ag-free M. smithii archaeosomes up-regulated CD40 (~2-fold activation), a key costimulatory marker expressed on DCs (Fig. 12a). Furthermore, inflammatory cytokines IL-12 and TNF production were augmented by archaeosomes in a dose-dependent fashion (Fig. 12b). Interestingly, although CD40 up-regulation by archaeosomes matched levels of expression evoked by LPS, induction of inflammatory cytokine production by archaeosomes was substantially weaker.



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  		FIGURE 12. Activation of DCs (105/ml). a, CD40 expression on DCs was determined after 24 h in vitro culture with no activation, or treatment with 25 µg of Ag-free M. smithii archaeosomes (MS) or 10 µg of LPS. Solid line indicates positive staining with specific Ab, whereas the dotted line indicates isotype control Ab staining. Data are derived from 20,000 events acquired for each sample, and the percentage of CD40high cells beyond the vertical gate is indicated. b, MTT incorporation and cytokine production by triplicate cultures of DCs treated for 48 h with various concentrations of Ag-free M. smithii archaeosomes (MS) and LPS are indicated. Means ± SD of triplicate cultures are indicated. Data are representative of three experiments conducted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Generation and maintenance of T cell immunity to vaccines involves numerous, highly orchestrated events that act in concert. While Ag identification is a key component of vaccine development, adjuvant technologies broadly categorize into those that can deliver Ag to APCs and those that can provide immune activation. In the category of Ag delivery systems, nonreplicating, particulate carriers such as iron-coated beads (37) are unsuitable for vaccines, and others such as polymeric microspheres and liposomes often require coadjuvants (2, 38, 39). Among replicating systems, viral vectors are biased toward induction of CD8+ T cell responses (40) with limited CD4+ T cell help, whereas bacterial vectors are biased toward CD4+ T cells (8). Replicating vectors, although attenuated, also come with a risk of toxicity. A promising approach is to use recombinant virus-like particles that are nonreplicating but mimic viral Ag delivery pathways (41). We have previously demonstrated the ability of archaeosomes to activate DC costimulation and augment CTL responses to entrapped Ag. This study provides a mechanistic insight into the Ag uptake and processing pathway targeted by archaeosomes. By definition, archaeosomes may consist of any of the many archaeal polar lipids with expected differences in adjuvant activity, as we reported in other studies (27, 31, 42). However, we consider in this study only archaeosomes prepared from the TPL of M. smithii.

We have shown that M. smithii archaeosomes can stimulate both CD4+ and CD8+ Ag-specific T cells. This ability of archaeosomes cannot be attributed to the efficient Ag-presenting ability of DCs alone, because strong activation of CD8+ T cells is seen even when macrophages are used as APCs. More importantly, archaeosomes augment profound Ag presentation in vivo. Because strong clonal burst of Ag-specific T cells promotes memory generation (43), our observation that OVA-archaeosome immunization can result in cycling of >90% of Ag-specific cells suggests considerable clonal expansion. Such profound cycling is observed after i.v. challenge with live L. monocytogenes (44). Further induction of both CD44highCD62Llow effector and CD44highCD62Lhigh resting memory cells by archaeosomes suggests balanced memory differentiation. Indeed, this correlates to our previous observation of sustained CTL immunity in mice immunized with Ag entrapped in archaeosomes (27). Furthermore, we have shown that a peptide vaccine composed of Listeriolysin peptide 91–99 incorporated into archaeosomes protects against live L. monocytogenes challenge even at 9-mo postimmunization (29). Similarly, in tumor models, archaeosome-Ag formulations provide striking protection (28).

In the context of lipid vesicles, possible routes of entry into the cell include direct fusion with the plasma membrane or phagocytosis. We show that archaeosomes are actively phagocytosed. Endocytosis mediated by receptors such as mannose receptor, FcRs, and those recognizing apoptotic cells, like PS-specific receptors, is often the efficient route for uptake and signaling (35, 45, 46, 47). The polar lipids from M. smithii are fully characterized and comprise 30 mol% of the archaetidyl equivalent of PS (42) and are striking in forming archaeosomes with surface-exposed PS head groups (31). We have earlier observed that PS containing synthetic liposomes competed for phagocytic uptake of fluorescent-labeled M. smithii archaeosomes in a qualitative microscopic analysis (31). This study provides a quantitative assessment of phagocytosis by flow cytometry and demonstrates competitors that block PS-specific receptors strongly and specifically inhibit binding/uptake. Furthermore, our Ag presentation results corroborate PS-specific recognition as triggering archaeosome entry into the phagosomal compartment, leading to Ag delivery for MHC class I. Several mammalian receptors have been implicated in the recognition of PS; however, a two-step tether-and-tickle requirement, which primarily relies on phagocytic uptake via the PSR has been suggested (47). Because PS-specific recognition of archaeosomes resulted in binding and uptake, as measured by MHC class I presentation of encapsulated Ag, it appears that archaeosomes efficiently harness a natural cross-presentation pathway that ensues after PSR-mediated clearance of apoptotic cells by APCs (34).

Subsequent to phagocytosis, our data indicate cytosolic proteasome processing and TAP-dependent presentation of archaeosomal-Ag on MHC class I. However, it is intriguing that endosomal acidification blocked the MHC class I processing of OVA-archaeosomes, because most pathways involving cytosolic diversion of Ag, such as processing of Ag bound to latex beads and TLR-mediated cross-presentation, are independent of endosomal acidification (37, 48). Therefore, it appears that endosomal acidification specifically aids cytosolic diversion of archaeosomal Ag. In the case of latex beads as Ag carriers, such particles are often 1–3 µm in range, and Ag escape from phagosome to cytosol may be due to leakage of overloaded phagosomes. This is unlikely for archaeosomes that are only ~100 nm and considering that essentially most of the Ag associated with the vesicles is internalized (data not shown and Ref.26). DCs possess a unique translocation system for small-molecule movement from endosome to cytosol (20). Recently, recombinant parvovirus-like particles have been reported to use this unique endosome-cytosol pathway of DCs for Ag delivery to MHC class I (41). However, M. smithii archaeosomes are presented by both DCs and macrophages by the same pathway, and macrophages are considered to be less efficient at routing endosomal Ag to the cytosol. More recently, a new cross-presentation pathway involving ER-phagosome fusion that involves proteasomal Ag breakdown and TAP translocation has been discovered (24, 25). The key component of this pathway is loading of MHC class I within an ER-phagosome compartment. Such a pathway may be considered for OVA-archaeosomes, but we have observed that brefeldin A (an inhibitor of post-ER transport of MHC class I-peptide complex) fully blocks archaeosome-Ag MHC class I presentation (27), suggesting cytosolic loading of MHC class I. Regardless, it is not clear how Ag is released from within M. smithii archaeosomes that are rich in membrane-spanning caldarchaeols (49), rendering them highly stable (50). OVA leakage in vitro is not seen even after prolonged storage (our unpublished data). Synthetic ester liposomes sensitive to pH deliver their cargo to the cytosolic compartment through acid-dependent fusion of liposomal and endosomal membranes under physiologically relevant conditions (16, 17). However, it is unclear whether stable archaeal lipids can be rendered fusogenic under physiological conditions (51). Another possibility is that fluctuations in cationic/calcium gradients between intracellular compartments following acidification, promotes fusion (52) and/or Ag leakage.

Interestingly, although archaeosomes evoked inflammatory cytokine production including IL-12 and TNF by DCs, the levels produced were substantially less than LPS. Indeed, we have previously observed that M. smithii archaeosomes induce CTL responses in IL-12-deficient mice (28). Furthermore, our results are in line with the suggestion that a key consequence of engaging the PSR is suppression of inflammation (46). However, archaeosomes directed strong costimulation benefiting potent T cell activation, in the absence of overt inflammation, and no noticeable toxicity (53, 54). The key signaling mechanisms that ensue after engagement of PSR are still not fully understood (46), and whether the dichotomous ability of archaeosomes to minimize inflammation while promoting costimulation is related to PSR engagement will need further study. Indeed, DCs charged with apoptotic cells as a vaccine strategy for evoking long-lived protective T cell immunity have been recently reported (55). However, because cross-presentation of self-Ags can lead to autoimmunity, additional inflammatory danger signals may be required to sufficiently activate DCs for adaptive immunity (12). Pattern recognition receptors such as Toll constitute the primary receptors that activate inflammatory immunity (56), and many new adjuvants function by activating such receptors (3, 4). Whether such innate receptors recognize archaeal lipids is not known. However, archaea are nonpathogenic, and M. smithii is a resident of human gut (57). Furthermore, our preliminary studies indicate noninvolvement of TLR-4 with archaeosomes (our unpublished data).

We surmise that the adjuvant ability of archaeosomes is likely attributable not only to head group-receptor interaction but also to the archaeal lipid structures composed of branched saturated isopranoid chains linked by ether bonds to the glycerol (58). Two major classes of lipid cores exist: the archaeol with sn-2,3 C-20 chains, and the dimeric form of the same, the membrane-spanning caldarchaeol. The TPL of M. smithii comprise 40% caldarchaeol, and are dominated by head groups of archaetidylserine and caldarchaetidylserine (42). Because caldarchaeol lipids confer stability to archaeosomes (50), this feature may facilitate sustained Ag delivery. Indeed, vesicles composed of nonarchaeal eubacterial ester lipids elicit only a short-lived CTL response, even when they share remarkable similarity to archaeal lipids in other structural properties (59).

Naturally, only DCs have the ability to bridge the three components of an immune response, namely, to respond innately, to trigger adaptive immunity, and to promote memory (12, 46). Burgeoning of genomics and proteomics has made identification of protective Ags of cancer and intracellular infections increasingly possible. Identification of technologies that effectively augment the natural response of DCs for induction and maintenance of CD8+ T cell response to subunit Ags is critical. This study mechanistically suggests that archaeosomes represent a promising single-component, particulate, nonreplicating vaccine delivery modality for naturally harnessing cross-presentation.


   Acknowledgments
 
We thank Dr. Girish Patel for fermenter growth of archaea, and Dr. Gordon Willick for peptide synthesis. We acknowledge the technical assistance provided by Chantal Dicaire and Lise Deschatelets for archaeosome/liposome preparations, and Henk van Faassen for tetramer staining.


   Footnotes
 
1 This is Publication No. 42489 of the National Research Council of Canada. Back

2 Address correspondence and reprint requests to Dr. Lakshmi Krishnan, National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Room 3016, Ottawa, Ontario, Canada K1A 0R6. E-mail address: Lakshmi.Krishnan{at}nrc-cnrc.gc.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; ER, endoplasmic reticulum; OVA-archaeosome, OVA entrapped in vesicle composed of Methanobrevibacter smithii polar lipids; PS, phosphatidylserine; AcLLnL, N-acetyl-Leu-Leu-Norleu-al; AcLLM, N-acetyl-Leu-Leu-Met-al; PG, dimyristoylphosphatidylglycerol; TPL, total polar lipids; PSR, PS receptor. Back

Received for publication December 23, 2003. Accepted for publication April 27, 2004.


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