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The Journal of Immunology, 2001, 167: 6462-6470.
Copyright © 2001 by The American Association of Immunologists

Efficient Delivery of Antennapedia Homeodomain Fused to CTL Epitope with Liposomes into Dendritic Cells Results in the Activation of CD8+ T Cells

Ghania G. Chikh*,{dagger}, Spencer Kong*, Marcel B. Bally*,{ddagger}, Jean-Claude Meunier{dagger} and Marie-Paule M. Schutze-Redelmeier2,*,{ddagger}

* Systemic Therapy Program, Department of Advanced Therapeutics, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada; {dagger} Institut National Agronomique-Paris, Grignon, France; and {ddagger} Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The use of specific immunotherapy for treatment of patients with cancer has proven to be a challenging area of clinical and basic research. The goal of a cancer vaccine is to induce an immune response using tumor cells or tumor Ags that can protect the host. This protection depends mainly on activated tumor-specific CD8+ CTL (1, 2, 3). CTLs kill neoplastic cells through recognition of antigenic peptides presented by MHC class I molecules on the surface of the tumor target (4). These peptides are derived from tumor Ags that are synthesized by the affected cell and degraded in the cytosol of the tumor target (5).

In terms of generating an immune response to tumor-associated peptides, investigators have identified several obstacles. Peptide vaccination depends on the loading of empty MHC molecules on APCs in vivo. However, single administration of peptide without a means of targeting activating APCs can potentially lead to loading of MHC class I molecules on nonprofessional APCs, which could result in tolerance (6). In contrast, administration of dendritic cells (DCs)3 loaded with these peptides results in appropriate CTL generation (6). It is clearly established now that DCs pulsed with tumor Ags in vitro and then reinjected in vivo induce protective immune responses that block tumor growth (7, 8, 9). Although DCs pulsed with tumor peptides appear to be good candidates for a clinical use in humans, ex vivo approaches will suffer because of two problems related to 1) generation of high numbers of DCs in a clinically practicable manner, and 2) multiple rounds of immunization at fairly short intervals that can at times lead to the emergence of noncytolytic CD4+ T cells exhibiting the characteristic phenotype of Th2 cells (10).

There are many attempts being made to simplify immunization protocols; however, despite efforts to achieve delivery of tumor Ags to DCs, targeting the DC system in vivo has not been successfully achieved. Our laboratory has studied a novel approach for CD8+ peptide delivery in vitro and in vivo into DCs to activate CTL responses. This approach involves the combination of liposomes and a recombinant peptide that can deliver defined CTL peptides into the cytosol. Liposomes are potentially useful as drug carriers to deliver pharmacologically active agents into cells (11, 12, 13). Moreover, liposomes are known to be effective as immunoadjuvants and vaccine carriers (14, 15, 16). Numerous reports have presented induction of CTL by liposomal Ags (17) for review; however, the efficacy of the response has typically depended on the Ag and the presence of adjuvants (18). The pH-sensitive liposome-based approach developed a decade ago (19, 20) has shown detectable cytotoxic T cell response, but the response was dependent on the nature of the Ag. A number of approaches have been made to improve an immunoadjuvant action of liposomes, approaches that included modification of the liposome structure (21, 22). Small size and positively charged carriers have been shown to be preferentially taken up by phagocytic cells such as DCs/macrophages and to elicit a significant CTL response (23, 24). The mechanisms by which the liposomally encapsulated protein Ags are directed to the cytosol are believed to result from passive escape of the Ag from the endosomes into the cytoplasm and the access to the class I processing pathway (25). However, the amount of protein that enters the cytosol by this mechanism is limited. We believe that the Ag presentation would be more efficient if the Ag were directly delivered to the cytosol or if it were actively transported from the endosomes to the cytosolic compartment.

We have previously demonstrated that a peptide sequence, referred to as antennapedia homeodomain (AntpHD), can effectively introduce CTL epitopes into the class I processing pathway and induce CTL in vivo, a result that was dependent on use of SDS as a stabilizing factor (26). This is a very important property because the intracellular location of the Ag is considered as a major factor in determining the pathway in which the Ag is processed and presented. Further development of this technology has been limited because the recombinant peptide is very sensitive to degradation in serum. In the context of the present study we tested the hypothesis that encapsulation of AntpHD recombinant peptide in liposomes benefits from a mechanism that will allow the peptide to be protected from serum degradation and to be delivered into the cytosol of cells. Our vaccine design uses a recombinant peptide consisting of a CTL epitope, which binds MHC class I molecules (27), and a peptidic vector, AntpHD, that can deliver peptides into the cytosol of cells (26, 28). We have chosen for this study the CTL epitope Cw3(170–179), derived from HLA-Cw3, which is unable to induce a CTL response even in presence of adjuvants (26). The first aim of this study was to characterize the uptake of AntpHD recombinant peptide, presented as free peptide or encapsulated in neutral liposomes, by immature bone marrow-derived DCs and the subsequent cell activation of a CTL clone specific for Cw3(170–179). Our results demonstrated that the encapsulation of AntpHD recombinant peptide into liposomes allows delivery of the recombinant peptide into the endoplasmic reticulum via the classical MHC class I pathway, a processing pathway similar to that observed with the soluble AntpHD recombinant peptide. However, in contrast with free soluble AntpHD recombinant peptide, which is directly delivered to the cytosol, AntpHD recombinant peptide encapsulated into liposomes is initially delivered with the liposomal lipids in the endosomes of cells, and subsequently the peptide enters the cytosol from the endosomes. The second aim of this study was to evaluate the efficiency of a liposome formulation to improve the class I-restricted CD8+ T cell response to AntpHD-Cw3 in vivo. Based on a sensitive functional assay to measure IFN-{gamma} production, we found that AntpHD recombinant peptide can induce T cell responses which are greatly enhanced when AntpHD recombinant peptide is delivered by cationic liposomes.


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

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) lipids were purchased from Northern Lipids (Vancouver, Canada). 1,2-disteroyl-sn-glycero-3-phosphatidylethanolamine-n-(poly(ethylene glycol)2000) (DSPE-PEG2000) was purchased from Avanti Polar Lipids (Birmingham, AL). CHE [3H]cholesterol-hexadecyl ether and sodium [51Cr]chromate (1 mCi/ml) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cholesterol, chloroquine (CHL), and brefeldin A (BFA) were obtained from Sigma-Aldrich (St. Louis, MO). Lactacystin was purchased from Calbiochem (Darmstadt, Germany). Pico-Fluor-40 scintillation mixture was obtained from Canberra-Packard Canada (Missassauga, Ontario, Canada). RPMI 1640 and DMEM media were purchased from Stem Cell Technologies (Vancouver, Canada). Twelve 24-well plates and 96-well V-bottom plates were obtained from BD Labware (Franklin Lakes, NJ) and Costar (Corning, NY), respectively. Fluoraldehyde reagent and bicinchoninic acid protein assay kit were from Pierce (Rockford, IL). Abs and streptavidin-HRP were purchased from BD PharMingen (Mississauga, Canada). Biogel 1.5m was obtained from Bio-Rad (Hercules, CA). CpG ODN1668 (5'-TCC ATGACG TTC CTG CT) and peptide 170–179 HLA-Cw3 (RYLKNGKETL) were synthesized by Biotechnology Laboratory (University of British Columbia, Vancouver, Canada). The sequence coding for AntpHD-Cw3 fusion peptide initially inserted in pAH61S plasmid (26) was subcloned into the pET19 (Novagen, Madison, WI) between NdeI and BamHI restriction sites. The resulting plasmid encoding for 10 histidine residues plus a 13-aa spacer linked in 3' to AntpHD-Cw3 was expressed in Escherichia coli strain BL21 (DE3)LyS as previously described (26). The fusion peptide was purified by nickel-chelate affinity resin according to the recommendations of the supplier (Qiagen, Chatsworth, CA). The eluted fractions were analyzed by SDS-PAGE on 15% gel, Coomassie blue staining, and Western blot analysis. Purity was assessed at 80–90%.

Animals

BALB/c (H2d) mice were obtained from the joint animal facility at the British Columbia Cancer Research Center (Vancouver, Canada). All animal studies were completed using protocols that were approved by the institution’s animal care committee and the methods used are consistent with the current guidelines of the Canadian Council of Animal Care.

Cell culture

P815 (DBA/2, H2d) mouse mastocytoma cells were cultured in DMEM supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% glutamine.

Cloned DBA/2 CTL clone, termed CAS20, was raised in vivo against HLA-Cw3 P815 transfectants and isolated after in vitro restimulation by limiting dilution (27). CAS20 cells were maintained in RPMI 1640 medium supplemented by T cell growth factors (TCGF) obtained from rat splenocytes stimulated with Con A. CAS20 cells were stimulated every week with irradiated HLA-Cw3 P815 transfectants or with Cw3(170–179) peptide.

Liposome preparation

Liposomes were prepared using the method described in Ref. 29 . Lipid mixtures used were DOPC/C (55/45 mol%) and DOPC/DOTAP/C at (45/10/45 mol%). When DSPE-PEG2000 was incorporated into these liposomes it was done by substitution of 5 mol% DOPC with 5 mol% of the polyethylene glycol (PEG)-modified lipid. The lipids were dissolved in chloroform and a trace of [3H]cholesteryl hexadecyl ether (1–5 µCi/100 µmol total lipid) was added as a nonexchangeable liposomal lipid label (30, 31). A lipid film was formed following removal of solvents under a stream of nitrogen gas. The lipid film was then placed under high vacuum for at least 3 h before hydration with HBS (100 mM HEPES (pH 7.4), 150 mM NaCl). The resulting multilamellar vesicles were subjected to freeze-thaw cycles (32) and then extruded through 100-nm polycarbonate filters (Nucleopore, Pleasanton, CA) using an extrusion device (Lipex Biomembranes, Vancouver, Canada). Liposome size was determined by quasi-elastic light scattering using Nicomp submicron particle size analyzer (Pacific Scientific, Santa Barbara, CA). All liposomes exhibited average diameters of 100–120 ± 25 nm. For AntpHD-Cw3 incorporation into liposomes, 800 µg of AntpHD-Cw3 were incubated with 50 µmol of liposomes for 30 min at room temperature under rotary shaking. The mixture was then loaded onto a chromatography column (Biogel 1.5m) to separate free peptide from liposome-associated peptide. The ratio of peptide:liposome was determined by quantifying liposomal lipid by measuring [3H]CHE radioactivity by liquid scintillation counting on the Canberra-Packard Scintillation beta counter (1900 TR Tri Carb), using Pico-fluor 40 scintillation mixture. The peptide was quantified by a spectrophotometeric assay using bicinchoninic acid protein assay kit (Pierce).

Preparation of DCs

Bone marrow DCs were prepared as described by Inaba et al. (33). Briefly, bone marrow cells were harvested from femurs and tibias of BALB/c mice. After lysis of red cells with ammonium chloride, 106 cells were placed in 24-well plates in 1 ml of RPMI 1640 medium supplemented with 10 ng/ml mouse recombinant GM-CSF and IL-4 (Sigma-Aldrich). The cultures were fed every 2 days by gently swirling the plates, aspirating 75% of the medium to remove the nonadherent cells (lymphocytes and other granulocytes); fresh medium containing the cytokines was then added. At day 7 the resulting cells were considered to be immature DCs. The DC purity was assessed by FACS analysis using mAbs for the following markers: anti-CD4-FITC (Life Technologies, Rockville, MD), anti-Mac-3-FITC (BD PharMingen), anti-CD80 (B7.1)-PE (Immunotech, Marseille, France), and CD86 (B7.2)-FITC (Immunotech). More than 70% of the generated cells showed a phenotype consistent with DCs (highly positive for CD80 and CD86 and negative for CD4 and Mac-3).

Ag presentation assay

DC (106 cells per 22-mm well) or P815 cells (0.5 x 106) were incubated in 12-well plates at 37°C with different AntpHD-Cw3 peptide formulations at the indicated amounts of peptide and time periods. DCs (105) were used to stimulate 2 x 106 CAS20 in the presence of 105 irradiated splenocytes during the 5-day incubation. Cytolytic activity of the stimulated CTL clone was assessed. P815 cells were used as target cells in cytotoxicity assay performed as described (34).

To study the Ag processing pathway, 0.5 x 106 P815 cells were preincubated with 0.2 µg of BFA, 10 µM CHL, or 25 µM lactacystin for 1 h, then AntpHD-Cw3 (50 µg) or Cw3(170–179) (5 µg) free or encapsulated in liposomes were added in the continuous presence of these drugs. After an overnight incubation, P815 cells were washed and used as target cells in cytotoxicity assay.

Induction of primary CTL in vitro

Following incubation of DCs with Ag, 5 x 105 irradiated DCs were used to stimulate 5 x 106 BALB/c naive splenocytes for 7 days. The live collected cells were then stimulated for 5 days using 10 µM Cw3(170–179) peptide in the presence of 105 irradiated splenocytes as feeder cells. Chromium release assay was then completed with harvested spleen cells.

Immunization and CTL induction

For immunization studies with DCs, 106 DCs were incubated for 3 h at 37°C with free Cw3(170–179), or with AntpHD-Cw3 in free form or encapsulated in DOPC/PEG liposomes. The cells were then washed and irradiated before i.v. injection of 1.5 x 106 cells into BALB/c mice (three per group) on day 0. For comparison, groups of mice were injected s.c. with AntpHD-Cw3 in presence or absence of SDS, or with AntpHD-Cw3 encapsulated in DOPC/PEG liposomes. On day 7, spleens were harvested and 30 x 106 splenocytes were cultured in 10 ml of RPMI 1640 supplemented with 10% Con A supernatant containing TCGF, 10% FCS, 1% glutamine, 1% penicillin/streptomycin, and 5 x 10-5 M 2-ME and stimulated for 5 days with 10 µM Cw3(170–179). A CTL assay was then performed.

Immunization and ELISPOT assay

Groups of BALB/c mice (six per group), 6–8 wk of age, were immunized s.c. on days 0 and 7 as indicated in Fig. 7Go. On day 14, spleens were harvested and stimulated under conditions described above. An ELISPOT assay was then performed.



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  		FIGURE 7. A, Frequency of Cw3-specific T cells as determined by an IFN-{gamma} ELISPOT assay. BALB/c mice were immunized s.c. on days 0 and 7 with either AntpHD-Cw3 free form in SDS, entrapped in DOTAP liposomes in the presence or absence of 10 nmol of CpG ODN1668, or with Cw3(170 179) entrapped in DOTAP liposomes. On day 14, spleen cells from individual mice were stimulated for 5 days with 10 µM Cw3(170 179) and then assayed for the number of Cw3-specific cells. The Ag-specific cell number was quantitated by an ELISPOT assay, as described in Materials and Methods. B, Effects of CD4+ or CD8+ T cell depletion on the response to Cw3 Ag. BALB/c mice were immunized s.c. on days 0 and 7 with either a free form of AntpHD-Cw3 in SDS or entrapped in DOTAP-containing liposomes in presence of 10 nmol of CpG ODN1668. One group received saline solution as negative control. On day 14, spleen cells from individual mice were stimulated for 5 days with 10 µM Cw3(170 179). Following the 5-day culture, effector cells were depleted of CD8+ ({square}) or CD4+ T cells () as described in Materials and Methods or treated with complement alone ({blacksquare}). They were then assayed for the number of Cw3-specific cells. The Ag-specific T cell number was quantitated by an ELISPOT assay.

 
The ELISPOT assay described by Murali-Krishna et al. (35) was modified to detect Cw3-specific CD8+ T cells. ELISPOT plates (Multiscreen-IP Clear plates; Millipore, Bedford, MA) were coated overnight at 4°C with 2 µg/ml capture anti-IFN{gamma} Ab (clone R4-6A2; BD PharMingen). The plates were then blocked with 1% BSA in PBS for 2 h at room temperature. After three washes, responder cells in RPMI 1640 medium supplemented with 10% Con A supernatant containing TCGF, 10% FCS, 1% glutamine, 1% penicillin/streptomycin, and 5 x 10-5 M 2-ME were added to the wells along with 5 x 105 irradiated syngeneic feeder cells. Cells were incubated for 36 h in the presence or absence of 10 µM Cw3(170–179) peptide. After culture, the plates were washed, and biotinylated anti-IFN-{gamma} detection Ab (clone XMG1.2; BD PharMingen) was added (1 µg/ml), and the plates were then incubated for 1 h at room temperature. Spots developed following addition of freshly prepared HRP diluted 1/2000 in PBS/Tween containing 1% BSA, followed by repeated (five times) washes with PBS/Tween and addition of 200 µl of HRP substrate (Opti-4CN substrate kit; Bio-Rad). The frequency of peptide-specific T cells was calculated based on the percentage of cells present in the responding population.

For CD4+ and CD8+ T cell depletion experiment, two rat mAbs were used. The clone 53-6.7 (anti-mouse CD8, rat IgG2a{kappa}) and the clone GK1.5 (anti-mouse CD4, rat IgG2a{kappa}) were obtained from Southern Biotechnology Associates (Birmingham, AL). They were used in vitro in presence of low-tox-M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada). Control individual mice were treated with complement alone for background toxicity. Following depletion treatments, an ELISPOT assay was performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the presentation by DCs of AntpHD-Cw3 encapsulated in liposomes

Ags in the extracellular fluids can be internalized into macrophages and DCs by two distinct mechanisms. Generally, soluble Ags are internalized by endocytosis while particulate Ags and liposomes are taken up through phagocytosis. An important feature of the recombinant AntpHD peptide is its capacity to be internalized by spontaneously crossing the plasma membrane into the cytoplasm of cells. Because liposomes tend to deliver protein Ags more efficiently for the class II-restricted presentation than for the class I-restricted presentation, we took advantage of the property of AntpHD to deliver peptide into the class I pathway. It was anticipated that either AntpHD-Cw3 would be released from the liposomes at the vicinity of cells and subsequently enter the cell directly through the plasma membrane or AntpHD-Cw3 in liposomes would be endocytosed and AntpHD-Cw3 would then cross the endosomal membranes and penetrate into the cytosol. Liposomes prepared from DOPC, cholesterol, and 5 mol% DSPE-PEG2000 (DOPC/C/PEG) were used to evaluate the capacity of neutral liposomes to introduce the Cw3 epitope fused to AntpHD into the intracellular MHC class I presentation pathway. To assess the ability of AntpHD-Cw3 to enter the cells through spontaneous membrane transfer, DSPE-PEG was included in the formulation to decrease liposomes-DC interactions, including binding and internalization. It is important to note that the liposomal formulation containing AntpHD-Cw3 does serve to protect AntpHD-Cw3 from hydrolysis by proteases (36). DCs from bone marrow were isolated and cultured in the presence of GMCSF and IL-4. On day 7, generated DCs were incubated with AntpHD-Cw3 free in solution or encapsulated in DOPC/C/PEG liposomes for 3 h, then washed and used as APCs to stimulate a CAS20 CTL clone. The CAS20 CTL clone recognizes the Cw3(170–179) CTL epitope complexed to Kd molecules on the surface of APCs and is able to lyse syngeneic target cells expressing the Cw3(170–179) synthetic peptide. DCs pulsed with Cw3(170–179) peptide were used as positive control for the presentation of the epitope on the surface of APCs. As shown in Fig. 1GoA, AntpHD-Cw3 is presented efficiently by DCs whether it is added as the free protein or encapsulated in DOPC/C/PEG liposomes. These results indicate that AntpHD-Cw3 can be internalized by immature DCs and activate T cells for lysis and that AntpHD-Cw3 encapsulated in DOPC/C/PEG liposomes can be directed with a similar efficiency to the class I processing pathway of DCs. The mechanism by which the Ag is taken up by APCs and then processed is very efficient, because the level of sensitization of CTL observed with AntpHD-Cw3 either soluble or in liposomes is similar to the sensitization obtained with DCs pulsed with the synthetic CTL epitope Cw3(170–179).



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  		FIGURE 1. Presentation of Cw3 by DC to CAS20 CTL clone. A, Immature DCs were incubated for 3 h at 37°C with 50 µg of AntpHD-Cw3 soluble or encapsulated in DOPC/C/PEG liposomes. DCs pulsed with Cw3(170 179) for 30 min were used as a positive control. After several washes, 105 DCs were irradiated and used as stimulators for 2 x 106 CAS20 during a 5-day culture, and CAS20 CTL clone was generated from immunization by HLA-Cw3 P815 transfectants and recognizes Cw3(170 179)-loaded target. Following 5 days of stimulation, CTL activity was measured in standard 51Cr release assay on P815 target cells untreated () or pulsed with Cw3(170 179) peptide ({blacksquare}). Data are expressed as percentage of lysis at 10:1 and 3:1 E:T ratios. B, Immature DCs were incubated for 3 h at 37°C with 50 µg of AntpHD-Cw3 soluble or encapsulated in DOPC/C liposomes and used as stimulator for CAS20 as described in A. CTL assay was performed as in A.

 
In the next experiment, we tested whether the uptake of AntpHD-Cw3 recombinant peptide is directly associated with the internalization of liposomal lipids in DCs; therefore, DSPE-PEG lipids were not included in the formulation. The results presented on Fig. 1GoB show that DCs incubated with AntpHD-Cw3 encapsulated in DOPC/C liposomes were able to sensitize CTL to lysis similarly to AntpHD-Cw3 encapsulated in a formulation with PEG indicating that PEG did not influence the uptake of AntpHD-Cw3. These results suggest that, under the conditions used in this study, encapsulated AntpHD-Cw3 may readily dissociate from the liposomes in the extracellular medium before cell membrane crossing. Alternatively, PEG-mediated inhibition of cell binding and uptake is perhaps not complete. To address this further, we characterized the CTL response induced in vivo and in vitro following administration of AntpHD-Cw3-loaded liposomes.

Induction of primary CTL responses by DCs loaded with AntpHD-Cw3 in liposomes

To determine whether in vitro delivery of DOPC/C/PEG liposomes containing AntpHD-Cw3 peptide into DCs could induce a CTL response, day 7 immature DCs were pulsed for 3 h with AntpHD-Cw3 in DOPC/C/PEG liposomes, washed, and tested for their ability to stimulate naive spleen cells. Six days later, live cells were harvested and assayed for CTL activity against P815 target cells pulsed with Cw3(170–179) peptide. Fig. 2Go shows vigorous CTL response with the DCs treated with AntpHD-Cw3 in DOPC/C/PEG liposomes as stimulators indicating that AntpHD-Cw3 encapsulated in DOPC/C/PEG can be delivered to the MHC class I presentation pathway of DCs and be processed and properly presented to naive T cells.



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  		FIGURE 2. Primary Cw3-specific CTL response induced by stimulation with DCs treated with AntpHD-Cw3 encapsulated in DOPC/C/PEG liposomes. Immature DCs were incubated for 3 h at 37°C with AntpHD-Cw3 encapsulated in DOPC/C/PEG liposomes or left untreated. After treatment, cells were washed and 5 x 105 irradiated DCs were used as stimulators for 5 x 106 naive spleen cells during 6 days. The effectors were harvested and tested for Cw3 CTL response in a cytotoxic assay on P815 target cells untreated ({square}) or pulsed with Cw3(170 179) peptide ({blacksquare}).

 
Characterization of the CTL response induced by AntpHD-Cw3 encapsulated in DOPC/C liposomes.

Because PEG does not influence the uptake of AntpHD-Cw3, we have continued the in vitro study with a formulation without PEG to simplify the liposome preparation. We determined whether the encapsulation of AntpHD-Cw3 in DOPC/C liposomes modifies the time course of delivery and processing of the Ag in DCs compared with soluble AntpHD-Cw3. Fig. 3Go, left panel, shows that the level of lysis was dependent on the time of incubation of DCs with AntpHD-Cw3 in liposomes (Fig. 3GoA) or as the free protein (Fig. 3GoB) with a maximum of cell lysis observed following 5 h of incubation. No significant difference in terms of cell lysis was observed between CAS20 stimulated with AntpHD-Cw3 added in free or encapsulated form. We then examined the ability of varying doses of AntpHD-Cw3 to sensitize DCs to stimulate CAS20 CTL clone when delivered either free or in liposomal form. Fig. 3Go, right panel, shows that encapsulation of the recombinant peptide does not modify the dose required by DCs to sensitize CAS20 (Fig. 3GoC). DCs sensitized with AntpHD-Cw3 in free or liposomal form can activate CAS20 efficiently because the CAS20 response is comparable to the response obtained after stimulation with DCs pulsed with an equimolar amount (0.3 µg) of the synthetic CTL epitope Cw3(170–179) (Fig. 3GoD, open symbols).The ability of the epitope Cw3(170–179) encapsulated in liposomes (Fig. 3GoC, open symbols) to sensitize the DCs is not dramatically different from AntpHD-Cw3 in liposomes but is somewhat lower (40% lysis compared with 60% lysis at an E:T ratio of 10:1).



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  		FIGURE 3. Left panel, Cw3 CTL response is dependent on the incubation time of DCs with AntpHD-Cw3 soluble or encapsulated in DOPC/C liposomes. Immature DCs were incubated with 3 µg of either AntpHD-Cw3 encapsulated in DOPC/C liposomes (A) or AntpHD-Cw3 soluble (B) during 3 ({blacktriangleup}), 5 (•), and 8 ({blacksquare}) h. Right panel, AntpHD-Cw3 in liposomes sensitizes DCs to activate CAS20 when added at similar levels as soluble AntpHD-Cw3. Immature DCs were incubated for 3 h at 37°C either with peptides encapsulated in DOPC/C liposomes (C), 30 µg (•) or 3 µg ({blacksquare}) of Antp-Cw3, and 3 µg ({circ}) or 0.3 µg ({square}) of Cw3(170 179) peptide or with soluble peptides (D). After incubation and wash, DCs were used as stimulators for CAS20 CTL clone, and CTL response against Cw3 was tested on P815 target loaded or not with Cw3(170 179) peptide. Cw3 CTL response of CAS20 stimulated with DCs left untreated during 3 ({triangleup}), 5 ({circ}), and 8 ({square}) h as negative controls (left and right panels). Data are expressed as percentage of specific lysis

 
Intracellular processing of liposomal AntpHD-Cw3 requires endosomal and endoplasmic reticulum-Golgi trafficking

The mechanism leading to the internalization of AntpHD-Cw3 encapsulated in liposomes and the intracellular delivery could not be elucidated on the basis of the studies summarized above. We have previously demonstrated that AntpHD facilitates Ag presentation by delivering the epitope to the cytosolic compartment and this does not require endocytic acidic processing for presentation of the peptide (26). We investigated whether AntpHD-Cw3 peptide encapsulated in DOPC/C liposomes is released at the vicinity of cells and directly transported to the cytosol or is internalized with liposomes through endocytosis. In this study, we used P815 cells instead of DCs to investigate the capacity of liposomes and AntpHD to deliver CTL peptide into the cytoplasm independently of endosome-cytosol transport processes unique to DCs (37). Thus the use of DCs, although the primary target cell population of interest, would not discriminate between inherent transport of AntpHD-peptide and endosomal-cytosol processes. P815 cells were treated with BFA before and during the incubation with the Ag. BFA blocks Ag presentation by inhibiting membrane trafficking between the endoplasmic reticulum and the Golgi apparatus, which is necessary for delivery of nascent MHC class I molecules to the cell surface (38). The results, presented in Fig. 4GoA, indicate that incubation of P815 target cells with AntpHD-Cw3 peptide encapsulated in DOPC/C liposomes in the presence of BFA prevents the presentation of Cw3(170–179) peptide to the CAS20 CTL clone. This is similar to the results obtained when the target cells are incubated with free AntpHD-Cw3 protein. Control experiments indicated that BFA did not inhibit the lysis of Cw3(170–179)-pulsed cells, ruling out the possibility that the influence of BFA was related to down-regulation of MHC class I molecules. The presence of lactacystin, a specific proteasome inhibitor, during the incubation of cells with the recombinant peptide in liposomes prevented the presentation of Cw3(170–179) (Fig. 4GoB). This was not the case when lactacystin was present during the incubation with the soluble recombinant peptide; in contrast, an enhanced presentation of Cw3 Ag was observed (Fig. 4GoB). As a control of these experiments, the presentation of synthetic Cw3(170–179) was not blocked by the presence of lactacystin (Fig. 4GoB).



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  		FIGURE 4. Cw3 presentation of AntpHD-Cw3 encapsulated in DOPC/C liposomes requires both endosomal and cytosolic intracellular processing. A, P815 cells were treated for 1 h with 10 µM of CHL or 0.2 µg of BFA before and during the 3-h incubation with 5 µg of Cw3(170 179) either free in solution or encapsulated in DOPC/C (PC) liposomes as well as 50 µg of free AntpHD-Cw3 protein or encapsulated protein in DOPC/C liposomes. After washing, the P815-treated cells were used as targets in a CTL assay in the presence of CAS20 CTL clone. P815 untreated and HLA-Cw3 transfected P815 cells were used respectively as negative and positive control cells. The results are expressed as percentage of Cw3-specific lysis at 10:1 E:T ratio after substraction of lysis measured against P815. The cytotoxicity observed on P815 negative control cells was usually less than 15%. B, P815 cells were treated for 1 h with 25 µM lactacystin before and during overnight incubation with either 50 µg of free AntpHD-Cw3 protein or encapsulated in DOPC/DOTAP/C liposomes, or with 5 µg of Cw3(170 179). After washing, the P815-treated cells (in presence of the inhibitors) were used as targets in a CTL assay in the presence of CAS20 CTL clone. The results are expressed as percentage of Cw3-specific lysis at a 30:1 E:T ratio.

 
An important finding was that P815 target cells incubated with AntpHD-Cw3 peptide encapsulated in DOPC/C liposomes in presence of CHL, an inhibitor of endosomal proteolysis, were not lysed by CAS20 cells (Fig. 4GoA). These data, when considered with our observations using lactacystin, provide evidence that the liposomal form of the protein is processed differently from the free protein. Importantly, CHL did not influence the ability of target cells to present Cw3(170–179) peptide when P815 cells were treated with free AntpHD-Cw3 (Fig. 4GoA), confirming that AntpHD shuttles the Cw3 peptide directly in the cytosol. These results show that AntpHD-Cw3 peptide is processed through the classical class I pathway when it is added in a liposomal form. In addition, it can be suggested that the Ag follows two sequential intracellular routes, one that is mediated by the endosomal/lysosomal pathway and the other that requires the endoplasmic reticulum-Golgi trafficking. The results obtained with the inhibitors BFA and CHL strongly indicate that liposomal AntpHD-Cw3 enters the endocytic compartment before being shuttled to the cytosol, where it is processed by the proteasome complex. An important result from this experiment was that CHL but not BFA inhibits the Ag presentation when Cw3(170–179) alone (without AntpHD) is delivered under an encapsulated form. This result established clearly the role of AntpHD to transport CTL peptide in the cytosol of cells.

Induction of Cw3-specific CTL response

Although in vitro data demonstrate efficient uptake of soluble AntpHD-Cw3 by cells and subsequent presentation of Cw3 antigenic peptide to CTLs (Fig. 1Go), in vivo immunization with AntpHD-Cw3 is not very efficient because a CTL response can be induced only in presence of SDS (26). Our data have shown that liposomal formulations are efficient stimulators of CTL induction in vitro; therefore, we tested whether liposomes could be used alternatively to enhance Cw3-specific CTL response to AntpHD-Cw3 in vivo (Fig. 5Go). The CTL response of BALB/c mice was evaluated following the s.c. injection of AntpHD-Cw3 either in free form, in the presence of SDS, or encapsulated in DOPC/C/PEG liposomes. These responses were compared with the responses generated in vivo following i.v. injection of DCs which had been pulsed with AntpHD-Cw3 in free form or associated with DOPC/C/PEG liposomes. Spleens of immunized mice were collected 1 wk after the immunization and stimulated in vitro with Cw3(170–179) peptide. The CTL bulk cultures were harvested after 5 days and tested in a cytotoxicity assay for recognition of syngeneic P815 target cells loaded with the Cw3(170–179) epitope. The results (Fig. 5Go) show that direct in vivo immunization with AntpHD-Cw3 in saline elicited poor reactivity toward the target cells (Fig. 5Go, lane d). However, when AntpHD-Cw3 is given in SDS (Fig. 5Go, lane e) or encapsulated in DOPC/C/PEG liposomes (Fig. 5Go, lane f), cultures of splenocytes from immunized mice show recognition of the peptide-loaded target, an activity that was comparable to that observed following i.v. injection of loaded DCs (Fig. 5Go, lane c). Encapsulation of AntpHD-Cw3 in liposomes has improved the efficiency of the response against Cw3 compared with the response elicited by free AntpHD-Cw3, even when it is administered as a mixture with SDS. As indicated above, s.c. injection of AntpHD-Cw3 in DOPC/C/PEG liposomes was able to achieve the same level of response as that obtained in mice immunized i.v. with 1.5 x 106 DCs pulsed with AntpHD-Cw3 in DOPC/C/PEG liposomes (Fig. 5Go, lane c). However, it should be noted that the highest level of target-specific cell lysis was obtained using DCs pulsed with Cw3(170–179) administered i.v. (Fig. 5Go, lane a). The differences were not statistically significant due to the large variability in response observed in this group of mice.



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  		FIGURE 5. Priming of Cw3-specific CTL responses induced by immunization with AntpHD-Cw3 encapsulated in DOPC/C liposomes. BALB/c mice (four per group) were immunized by i.v. injection of 1.5 x 106 DCs incubated with either 5 µg of Cw3(170 179), 50 µg of AntpHD-Cw3 soluble, or 50 µg of AntpHD-Cw3 encapsulated in DOPC/C/PEG liposomes. In comparison, three groups of BALB/c mice were immunized by s.c. injection of 50 µg of AntpHD-Cw3 either in saline or in SDS (500 µg), and 50 µg of AntpHD-Cw3 encapsulated in DOPC/C/PEG liposomes. Seven days later, spleen cells from individual mice were stimulated in vitro with 10 µM of Cw3(170 179) and the CTL generated were tested in 51Cr release assay with P815 target cells pulsed with Cw3(170 179) or left untreated. The results are expressed as percentage of Cw3-specific lysis at 40:1 E:T ratio after substraction of lysis measured against P815. The cytotoxicity observed on P815 negative control cells was <15%.

 
It is generally assumed that neutral liposomes are not highly immunogenic (22, 39). However, we show in this work that neutral liposomes significantly increase the immunogenicity of AntpHD-Cw3. It is anticipated that other liposomal formulations may provide better immune response; therefore, an effort to optimize the liposomal formulation was made. This formulation effort was guided by the assumption that a more active liposomal carrier, in terms of uptake efficiency, would bring AntpHD-Cw3 inside the cells through endocytosis more efficiently. We examined whether positively charged liposomes would have this ability based on studies showing that positive liposomes containing soluble proteins are potent inducers of CTLs (23, 24, 40). It is believed that this adjuvanticity is due in part to the fact that these cationic liposomes are taken up more efficiently, when compared with neutral liposomes, by both DCs and macrophages (36). To test the ability of positively charged liposomes to introduce AntpHD-Cw3 into the class I processing pathway, we examined the presentation of Cw3 by cells to CAS20 CTLs following the incubation of AntpHD-Cw3 in DOTAP-containing liposomes with target cells for 3 h. As shown in Fig. 6Go, CAS20 CTLs are able to lyse target cells after incubation with the AntpHD-Cw3 peptide in positively charged liposomes. The level of cell lysis was similar to that observed when cells were incubated with soluble AntpHD-Cw3. This suggests that AntpHD-Cw3 in DOTAP-containing liposomes enters the class I Ag presentation pathway and that the mechanism of delivery is independent of liposome charge. In the next set of experiments, we evaluated whether the encapsulation of AntpHD-Cw3 in DOTAP liposomes improved its immunogenicity. In addition, we explored the use of the influence of oligodeoxynucleotides (ODN) containing unmethylated CpG as a means to achieve even better responses. We used CpG-DNA ODN1668 in this experiment because of its ability to activate in vivo T cell epitope-presenting DCs to generate CTL responses (41). BALB/c mice were immunized s.c. with AntpHD-Cw3 mixed with SDS or incorporated in DOTAP liposomes on days 0 and 7. One week after the last immunization, spleen cells were stimulated in vitro with Cw3(170 179). An ELISPOT assay was performed on stimulated spleen cells. IFN-{gamma} is currently the most widely used choice of readout for ELISPOT assay, and we selected this assay because it provides a measure of actual T cell frequency (35) and it is also more sensitive than the standard 51Cr-release assay (42). Fig. 7GoA shows that mice immunized with AntpHD-Cw3, as protein mixed with SDS, led to a marginal response. In comparison, AntpHD-Cw3 entrapped in DOTAP-containing liposomes gave a much higher (10 times) frequency (200 spots/105 cells) as compared with the <20 spots/105 cells observed for the free peptide.



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  		FIGURE 6. Presentation of Cw3(170 179) epitope to CAS20 CTL clone after introduction in target cells of AntpHD-Cw3 in cationic DOTAP liposomes. P815 cells were incubated with 50 µg of AntpHD-Cw3 either soluble or entrapped in DOTAP liposomes and used as targets for CAS20 in a cytotoxic assay. P815 cells left untreated represent the negative control. Data are expressed as percentage of cell lysis at 60:1 and 30:1 E:T ratio.

 
The role of AntpHD as a shuttle vector of Ag in the class I pathway in vivo was clearly demonstrated in this study because Cw3(170 179) itself in liposomes was not able to induce a T cell reactivity (frequency of <50 spots/105 cells). The most striking response was obtained in the group of mice immunized with AntpHD-Cw3/DOTAP liposomes in the presence of CpG ODNs. The frequency of T cells reacting to Cw3 was enhanced three times compared with the T cell frequency obtained in the group immunized with AntpHD-Cw3/DOTAP in saline. Fig. 7GoB shows that the anti-Cw3 response induced by AntpHD-Cw3 in DOTAP-containing liposomes in mice in the presence of the immunostimulatory CpG is mostly mediated by the activation of Cw3-specific MHC class I-restricted CD8+ cytotoxic T cells, because the response was almost completely blocked by anti-CD8 mAb (Fig. 7GoB).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is a great interest in developing efficient modes of delivery of T cell epitopes into the MHC class I processing pathway of DCs, with the objective of eliciting an effective therapeutic antitumor immunity. Several vectors have been reported to have this ability, including toxins (43, 44), the protein transduction domain from the HIV, TAT protein (45), and AntpHD (26). Their common attribute is their capacity to deliver peptides directly in the cytosol in a receptor-dependent manner for toxins (44) or following spontaneous crossing of cell membranes in the case of TAT (46) and AntpHD (47). They all differ from exogenous proteins in terms of their internalization mechanisms, which commonly use phagocytosis/endocytosis. Despite this important property, the utility of these vectors for clinical applications may fail because of inherent toxicity or because of their sensitivity to protease degradation. In this work, we show that immunization of mice with a liposomal formulation of AntpHD recombined to a poorly immunogenic T cell epitope primes APCs for an epitope-specific T cell response. This response is greater than that observed following injection of the T cell epitope alone, encapsulated in liposomes or fused to AntpHD in free form. Our results are consistent with the results of Ludewig et al. (48), which demonstrated that incorporation of the LCMV-GP33, a peptide in liposomes, improved the CTL response observed. However, importantly, our studies have demonstrated the value of using a hybrid carrier system, which relies on the liposome formulation to protect the protein from degradation while shuttling the AntpHD peptide vector linked to CTL epitope into the APCs. Ludewig et al. (48) demonstrated that liposomes functioned to enhance the immunogenicity of LCMV-GP33 peptide that was already immunogenic. Our data suggest that the hybrid carrier system can be used to elicit an immune response to a nonimmunogenic or poorly immunogenic Ag.

The efficacy of the AntpHD recombinant peptide encapsulated in liposomes as a vaccine was assessed in vitro. The results clearly show that AntpHD is capable of delivering CTL epitopes in the MHC class I processing pathway of immature DCs. This can be accomplished efficiently regardless of whether the protein is encapsulated in liposomes or given free in solution. Although the capacity of AntpHD to spontaneously cross cell membranes has been already established (26), the potential value of the method for CTL epitope delivery is limited as a consequence of peptide instability in vivo. Therefore, the purpose of using liposomes in our study is to limit the degradation of the peptide in vivo. Importantly, the results also demonstrate that the intracellular processing of the liposomal formulation is unique when compared with the free protein. It was important to distinguish whether encapsulation of the recombinant peptide in liposomes would allow direct targeting in the cytosol or whether liposomes would deliver the recombinant peptide in the endosomal pathway in a manner that might result in only a moderate class I-restricted response. It is demonstrated that even though AntpHD recombinant peptide is endocytosed with liposomes, the majority of AntpHD recombinant peptide is subsequently transferred into the cytosol, entering the class I processing pathway. In this regard, an inhibition by lactacystin was observed, although not seen with the free AntpHD-Cw3 recombinant peptide. Lack of inhibition by lactacystin was reported elsewhere for different peptides, including a recent study on AntpHD recombinant peptide and MG132, another proteasome inhibitor (49, 50, 51). It can be suggested that lactacystin inhibition of the proteasome is not significant, due to the ability of AntpHD peptide vector to escape from the proteasome. In contrast, when encapsulated in liposomes and endocytosed, the AntpHD peptide is routed toward the proteasome. It appears that the soluble and the liposomal AntpHD recombinant peptides are using different intracellular routes to enter the cytosol, directing them toward different degradation processes.

The processing pathway followed by liposome-associated AntpHD-Cw3 differs from the typical processing of endocytosed proteins, where the processed peptides bind recycling class I molecules in vacuolar compartment (52, 53). However, when the CTL epitope alone is delivered with the liposomal carrier, it can be presented using this pathway. This result does not support a recent study demonstrating that lipid-protein complexes escaping into the cytosol of cells can associate with the class I molecules in the trans Golgi (54). Whether AntpHD-Cw3 associated with liposomes escapes degradation in the endosomal compartment and enters the endoplasmic reticulum in a transporter-associated peptide-dependent manner following proteasome degradation or is directly delivered to the Golgi apparatus remains to be clarified. Similar to the active egress of bacterial Ags, such as those derived from Listeria monocytogenes, from phagosomes into the cytosol (55), we propose that the AntpHD/liposome combination would dissociate in the acidic environment of the endosomes, releasing the AntpHD recombinant peptide, which would then be able to cross endosomal membranes and shuttle the fusion peptides to the cytosol (Fig. 8Go). Considering that a low local pH is required for AntpHD to cross membranes optimally (56), the complete blockade of peptide presentation in presence of CHL could possibly be due to the retention of AntpHD-Cw3 in the endosomal/lysosomal compartment.



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  		FIGURE 8. Schematic representation of the proposed liposomally encapsulated AntpHD-Cw3 trafficking in the MHC class I presentation pathway. AntpHD/liposome combination (A) dissociates in the acidic environment of the endosomes and the liposomal AntpHD recombinant peptide (B) transported to the cytosol, where it is degraded in a proteasome-dependent manner. The free form of AntpHD (C) is able to cross plasma membranes and shuttle directly the fusion peptide to the cytosol

 
We should point out that we did not observe any inhibition of the Ag presentation when PEG was incorporated in the liposome formulation, and this seems inconsistent with our observation with CHL. Although it is known that PEG-modified lipids inhibit to some extent the uptake of liposomes by cells (57), we have recently demonstrated that this effect is less pronounced when evaluating bone marrow-derived DCs as compared with the macrophage J774 cell line (36).

The data described in this work define the immunogenicity of a CTL epitope achieved by the combination of lipid-based carriers and a recombinant protein carrier which is capable of delivering peptides into the class I processing pathway. We have shown that liposomes, and in particular those prepared with positively charged lipids, enhance the in vivo response to a nonimmunogenic CTL peptide when fused to AntpHD peptide. The absolute requirement of AntpHD was shown by the absence of response to the CTL peptide encapsulated in liposomes. We believe that this liposomal formulation can be further optimized to achieve controlled release of entrapped contents to allow optimal retention of AntpHD recombinant peptide before it has reached DCs. It is believed that liposomes can be designed to contain attributes that maximize their stability to biological fluids, to facilitate controlled distribution to regions where target cells are localized, and to engender specific targeting to define cell types following localization (58). The mannose receptor is an attractive candidate for targeting to DCs. It has been localized in vesicular structures distinct from MHC class II compartments, suggesting that in immature DCs the mannose receptor functions as a reusable Ag receptor for concentration of predominantly non-self Ags for processing and presentation. Liposomes designed with specific targeting for the DC system in vivo will have an important impact for vaccine applications, and we are currently investigating the ability of mannosylated liposomes to target the mannose receptor present on DCs.

The results of this study demonstrate the successful conversion of nonimmunogenic Ag into an immunogenic Ag and may represent a relevant model for poorly immunogenic tumor Ags. The high sequence homology of AntpHD with mammalian homeodomains of Hox 7 group (59) represents an additional advantage, and no adverse immune response against AntpHD has been observed. We anticipate that a recombinant peptide containing AntpHD fused to a series of human tumoral epitopes can be prepared and may have important implications for the development of tumoral vaccines.


   Acknowledgments
 
We thank Dr. F. Lemonnier for the kind gift of P815HLA-Cw3-transfected cells and CAS20 CTL clone. We thank Dr. Jan Dutz for helpful discussions and critical reading of the manuscript and Dana Masin and Rebecca Ng for excellent technical assistance.


   Footnotes
 
1 This research has been funded by a Canadian Institute of Health Research operating grant. M.-P.M.S.-R. is a Research Scholar of the British Columbia Health Research Foundation. Back

2 Address correspondence and reprint requests to Dr. Marie-Paule M. Schutze-Redelmeier, Department of Advanced Therapeutics, British Columbia Cancer Research Centre, 601 W 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada. E-mail address: mpredelm{at}bccancer.bc.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; AntpHD, Antennapedia homeodomain; BFA, brefeldin A; CHL, chloroquine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; ODN, oligodeoxynucleotide; PEG, polyethylene glycol; DSPE-PEG2000, 1,2-disteroyl-sn-glycero-3-phosphatidylethanolamine-n-(PEG2000); TCGF, T cell growth factor. Back

Received for publication April 23, 2001. Accepted for publication September 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ciernik, I. F., J. A. Berzofsky, D. P. Carbone. 1996. Induction of CTLs and antitumor immunity with DNA vaccines expressing single T cell epitopes. J. Immunol. 156:2369.[Abstract]
  2. Tjoa, B. A., A. A. Elgamal, G. P. Murphy. 1999. Vaccine therapy for prostate cancer. Urol. Clin. North Am. 26:365.[Medline]
  3. Ollila, D. W., M. C. Kelley, G. Gammon, D. L. Morton. 1998. Overview of melanoma vaccines: active specific immunotherapy for melanoma patients. Semin. Surg. Oncol. 14:328.[Medline]
  4. Jaeger, E., H. Bernhard, P. Romero, M. Ringhoffer, M. Arand, J. Karbach, C. Ilsemann, M. Hagedorn, A. Knuth. 1996. Generation of cytotoxic T-cell responses with synthetic melanoma-associated peptides in vivo: implications for tumor vaccines with melanoma-associated antigens. Int. J. Cancer 66:162.[Medline]
  5. Boon, T., P. G. Coulie, B. Van den Eynde. 1997. Tumor antigens recognized by T cells. Immunol. Today 18:267.[Medline]
  6. Toes, R. E., E. I. van der Voort, S. P. Schoenberger, J. W. Drijfhout, L. van Bloois, G. Storm, W. M. Kast, R. Offringa, C. J. Melief. 1998. Enhancement of tumor outgrowth through CTL tolerization after peptide vaccination is avoided by peptide presentation on dendritic cells. J. Immunol. 160:4449.[Abstract/Free Full Text]
  7. Mayordomo, J. I., T. Zorina, W. J. Storkus, L. Zitvogel, C. Celluzzi, L. D. Falo, C. J. Melief, S. T. Ildstad, W. M. Kast, A. B. Deleo, et al 1995. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat. Med. 1:1297.[Medline]
  8. Tarte, K., B. Klein. 1999. Dendritic cell-based vaccine: a promising approach for cancer immunotherapy. Leukemia 13:653.[Medline]
  9. Dallal, R. M., M. T. Lotze. 2000. The dendritic cell and human cancer vaccines. Curr. Opin. Immunol. 12:583.[Medline]
  10. Chakraborty, N. G., L. Li, J. R. Sporn, S. H. Kurtzman, M. T. Ergin, B. Mukherji. 1999. Emergence of regulatory CD4+ T cell response to repetitive stimulation with APCs in vitro: implications in designing APC-based tumor vaccines. J. Immunol. 162:5576.[Abstract/Free Full Text]
  11. Kim, S.. 1993. Liposomes as carriers of cancer chemotherapy. Current status and future prospects. Drugs 46:618.[Medline]
  12. Bally, M. B., D. Masin, R. Nayar, P. Cullis, L. D. Mayer. 1994. Transfer of liposomal drug carriers from the blood to the peritoneal cavity of normal and ascitic tumor-bearing mice. Cancer Chemother. Pharmacol. 34:137.[Medline]
  13. Tardi, P. G., E. N. Swartz, T. O. Harasym, P. R. Cullis, M. B. Bally. 1997. An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin. J. Immunol. Methods 210:137.[Medline]
  14. White, W. I., D. R. Cassatt, J. Madsen, S. J. Burke, R. M. Woods, N. M. Wassef, C. R. Alving, S. Koenig. 1995. Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen. Vaccine 13:1111.[Medline]
  15. Babu, J. S., S. Nair, P. Kanda, B. T. Rouse. 1995. Priming for virus-specific CD8+ but not CD4+ cytotoxic T lymphocytes with synthetic lipopeptide is influenced by acylation units and liposome encapsulation. Vaccine 13:1669.[Medline]
  16. Guan, H. H., W. Budzynski, R. R. Koganty, M. J. Krantz, M. A. Reddish, J. A. Rogers, B. M. Longenecker, J. Samuel. 1998. Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses. Bioconjugate Chem. 9:451.[Medline]
  17. Alving, C. R., V. Koulchin, G. M. Glenn, M. Rao. 1995. Liposomes as carriers of peptide antigens: induction of antibodies and cytotoxic T lymphocytes to conjugated and unconjugated peptides. Immunol. Rev. 145:5.[Medline]
  18. Richards, R. L., M. Rao, N. M. Wassef, G. M. Glenn, S. W. Rothwell, C. R. Alving. 1998. Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS, S malaria antigen. Infect. Immun. 66:2859.[Abstract/Free Full Text]
  19. Reddy, R., F. Zhou, L. Huang, F. Carbone, M. Bevan, B. T. Rouse. 1991. pH sensitive liposomes provide an efficient means of sensitizing target cells to class I restricted Ctl recognition of a soluble protein. J. Immunol. Methods 141:157.[Medline]
  20. Zhou, F., B. T. Rouse, L. Huang. 1992. Prolonged survival of thymoma-bearing mice after vaccination with a soluble protein antigen entrapped in liposomes: a model study. Cancer Res. 52:6287.[Abstract/Free Full Text]
  21. Reddy, R., F. Zhou, S. Nair, L. Huang, B. T. Rouse. 1992. In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes. J. Immunol. 148:1585.[Abstract]
  22. Nakanishi, T., A. Hayashi, J. Kunisawa, Y. Tsutsumi, K. Tanaka, Y. Yashiro-Ohtani, M. Nakanishi, H. Fujiwara, T. Hamaoka, T. Mayumi. 2000. Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway. Eur. J. Immunol. 30:1740.[Medline]
  23. Oussoren, C.. 1997. Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid composition and lipid dose. Biochim. Biophys. Acta 1328:261.[Medline]
  24. Nakanishi, T., J. Kunisawa, A. Hayashi, Y. Tsutsumi, K. Kubo, S. Nakagawa, M. Nakanishi, K. Tanaka, T. Mayumi. 1999. Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J. Controlled Release 61:233.[Medline]
  25. Zhou, F., S. C. Watkins, L. Huang. 1994. Characterization and kinetics of MHC class I-restricted presentation of a soluble antigen delivered by liposomes. Immunobiology 190:35.[Medline]
  26. Schutze-Redelmeier, M. P., H. Gournier, F. Garcia-Pons, M. Moussa, A. H. Joliot, M. Volovitch, A. Prochiantz, F. A. Lemonnier. 1996. Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo. J. Immunol. 157:650.[Abstract]
  27. Gournier, H., S. Pascolo, C. A. Siegrist, J. Jehan, B. Perarnau, Z. Garcia, T. Rose, J. Neefjes, F. A. Lemonnier. 1995. Restriction of self-antigen presentation to cytolytic T lymphocytes by mouse peptide pumps. Eur. J. Immunol. 25:2019.[Medline]
  28. Perez, F., P. M. Lledo, D. Karagogeos, J. D. Vincent, A. Prochiantz, J. Ayala. 1994. Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells. Mol. Endocrinol. 8:1278.[Abstract]
  29. Hope, M. J., M. B. Bally, G. Webb, P. R. Cullis. 1985. Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812:55.
  30. Derksen, J. T., H. W. Morselt, G. L. Scherphof. 1987. Processing of different liposome markers after in vitro uptake of immunoglobulin-coated liposomes by rat liver macrophages. Biochim. Biophys. Acta 931:33.[Medline]
  31. Bally, M. B., L. D. Mayer, M. J. Hope, R. Nayar. 1993. Pharmacodynamics of liposomal drug carriers: methodological considerations. G. Gregoriadis, ed. In Liposome Technology Vol. III:27. CRC Press, Boca Raton, FL.
  32. Mayer, L. D., M. J. Hope, P. R. Cullis. 1986. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858:161.[Medline]
  33. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  34. Schutze, M. P., P. Langlade-Demoyen, G. Przewlocki, C. Leclerc. 1989. Alloantigen pretreatment induces a down-regulation of the in vivo subsequent development of cytotoxic T lymphocytes directed against linked alloantigens. [Published erratum appears in 1989 Eur. J. Immunol. 19:1978.]. Eur. J. Immunol. 19:1365.[Medline]
  35. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
  36. Chikh, G., M. Bally, M. Schutze-Redelmeier. 2001. Characterization of hybrid CTL epitope delivery systems consisting of the Antennapedia homeodomain peptide vector formulated in liposomes. J. Immunol. Methods 254:119.[Medline]
  37. Rodriguez, A., A. Regnault, M. Kleijmeer, P. Ricciardi-Castagnoli, S. Amigorena. 1999. Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells. Nat. Cell Biol. 1:362.[Medline]
  38. Yewdell, J. W., J. R. Bennink. 1989. Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes. Science 244:1072.[Abstract/Free Full Text]
  39. Serre, K., P. Machy, J. C. Grivel, G. Jolly, N. Brun, J. Barbet, L. Leserman. 1998. Efficient presentation of multivalent antigens targeted to various cell surface molecules of dendritic cells and surface Ig of antigen-specific B cells. J. Immunol. 161:6059.[Abstract/Free Full Text]
  40. Ishii, N., J. Fukushima, T. Kaneko, E. Okada, K. Tani, S. I. Tanaka, K. Hamajima, K. Q. Xin, S. Kawamoto, W. Koff, et al 1997. Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res. Hum. Retroviruses 13:1421.[Medline]
  41. Vabulas, R. M., H. Pircher, G. B. Lipford, H. Hacker, H. Wagner. 2000. CpG-DNA activates in vivo T cell epitope-presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses. J. Immunol. 164:2372.[Abstract/Free Full Text]
  42. McKinney, D. M., R. Skvoretz, M. Qin, G. Ishioka, A. Sette. 2000. Characterization of an in situ IFN-{gamma} ELISA which is able to detect specific peptide responses from freshly isolated splenocytes induced by DNA minigene immunization. J. Immunol. Methods 237:105.[Medline]
  43. Fayolle, C., D. Ladant, G. Karimova, A. Ullmann, C. Leclerc. 1999. Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J. Immunol. 162:4157.[Abstract/Free Full Text]
  44. Haicheur, N., E. Bismuth, S. Bosset, O. Adotevi, G. Warnier, V. Lacabanne, A. Regnault, C. Desaymard, S. Amigorena, P. Ricciardi-Castagnoli, et al 2000. The B subunit of shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J. Immunol. 165:3301.[Abstract/Free Full Text]
  45. Schwarze, S. R., A. Ho, A. Vocero-Akbani, S. F. Dowdy. 1999. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569.[Abstract/Free Full Text]
  46. Nagahara, H., A. M. Vocero-Akbani, E. L. Snyder, A. Ho, D. G. Latham, N. A. Lissy, M. Becker-Hapak, S. A. Ezhevsky, S. F. Dowdy. 1998. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4:1449.[Medline]
  47. Joliot, A., C. Pernelle, H. Deagostini-Bazin, A. Prochiantz. 1991. Antennapedia homeobox peptide regulates neural morphogenesis. Proc. Natl. Acad. Sci. USA 88:1864.[Abstract/Free Full Text]
  48. Ludewig, B., F. Barchiesi, M. Pericin, R. M. Zinkernagel, H. Hengartner, R. A. Schwendener. 2000. In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity. Vaccine 19:23.[Medline]
  49. Tirosh, B., M. Fridkin, E. Tzehoval, E. Vadai, F. A. Lemonnier, L. Eisenbach. 2000. Antigenicity and immunogenicity of an intracellular delivery system of major histocompatibility complex class I epitopes that bypasses proteasome processing. J. Immunother. 23:611.
  50. Fu, T. M., L. M. Mylin, T. D. Schell, I. Bacik, G. Russ, J. W. Yewdell, J. R. Bennink, S. S. Tevethia. 1998. An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope. J. Virol. 72:1469.[Abstract/Free Full Text]
  51. Luckey, C. J., G. M. King, J. A. Marto, S. Venketeswaran, B. F. Maier, V. L. Crotzer, T. A. Colella, J. Shabanowitz, D. F. Hunt, V. H. Engelhard. 1998. Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol. J. Immunol. 161:112.[Abstract/Free Full Text]
  52. Pfeifer, J. D., M. J. Wick, R. L. Roberts, K. Findlay, S. J. Normark, C. V. Harding. 1993. Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361:359.[Medline]
  53. Harding, C. V., R. Song. 1994. Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules. J. Immunol. 153:4925.[Abstract]
  54. Rao, M., S. W. Rothwell, N. M. Wassef, A. B. Koolwal, C. R. Alving. 1999. Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein. Exp. Cell Res. 246:203.[Medline]
  55. Brunt, L. M., D. A. Portnoy, E. R. Unanue. 1990. Presentation of Listeria monocytogenes to CD8+ T cells requires secretion of hemolysin and intracellular bacterial growth. J. Immunol. 145:3540.[Abstract]
  56. Derossi, D., S. Calvet, A. Trembleau, A. Brunissen, G. Chassaing, A. Prochiantz. 1996. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271:18188.[Abstract/Free Full Text]
  57. Ishiwata, H., S. B. Sato, S. Kobayashi, M. Oku, A. Vertut-Doi, K. Miyajima. 1998. Poly(ethylene glycol) derivative of cholesterol reduces binding step of liposome uptake by murine macrophage-like cell line J774 and human hepatoma cell line HepG2. Chem. Pharm. Bull. 46:1907.
  58. Wasan, E. K., A. Fairchild, M. B. Bally. 1998. Cationic liposome-plasmid DNA complexes used for gene transfer retain a significant trapped volume. J. Pharm. Sci. 87:9.[Medline]
  59. Gehring, W. J., M. Affolter, T. Burglin. 1994. Homeodomain proteins. Annu. Rev. Biochem. 63:487.[Medline]



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