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The Journal of Immunology, 2002, 168: 5709-5715.
Copyright © 2002 by The American Association of Immunologists

In Vivo Generation of Cytotoxic T Cells from Epitopes Displayed on Peptide-Based Delivery Vehicles1

Kim S. Kawamura, Ruey-Chyi Su, Linh T. Nguyen, Alisha R. Elford, Pamela S. Ohashi and Jean Gariépy2

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of nonviral, peptide-based constructs able to elicit protective in vivo CTL responses represents a major challenge in the design of future vaccines. We report the design of branched peptide delivery vehicles, termed loligomers, that facilitate the import, processing, and presentation of CTL epitopes onto nascent MHC class I molecules. These complexes are then effectively displayed on the surface of APCs. The intracellular delivery of CTL epitopes by loligomers prolonged the expression of Ag-MHC class I complexes on the surface of APCs in comparison with free CTL epitope alone. Furthermore, the injection of CTL epitope-containing loligomers into mice led to the generation of in vivo CTL responses and the induction of autoimmune disease in an animal model. Synthetic epitope-carrying, peptide-based delivery vehicles may represent useful components to be included in the formulation of future vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cellular arm of the immune system relies upon the efficient activation of lymphocytes through the recognition of Ag displayed on MHC molecules. Peptide Ags derived from endogenous protein or viral/mutant gene products are presented on MHC class I molecules and lead to either self-tolerance or the induction of a CTL response (1). Exogenous proteins, however, enter the MHC class II pathway through endocytosis and are degraded in lysosomes such that the resulting peptides generally do not contribute to MHC class I presentation (2, 3). Although cross-priming can result in CTL activation through the presentation of exogenous Ag on MHC class I molecules, it is not considered to be an efficient method of priming CTL responses (4). Surface loading of MHC class I molecules is also possible, but peptide immunization rarely generates a CTL response. Thus, the appropriate delivery of Ag into cells may represent a method of skewing the number of defined epitopes complexed with class I molecules and also increase the length of time these complexes are presented on APCs.

Over the past few years, there has been an increased interest in using various import signals to target CTL epitopes into cells. These import signals include penetratin from the antennapedia homeodomain (5) (residues 43–58 from the third helix) and the basic region of HIV-1 Tat protein (6) (residues 47–57). Despite the apparent abilities of these sequences to cross the cell membrane, the generation of an in vivo CTL response through the administration of peptide with adjuvant has proven inefficient (7, 8, 9). Multi-tasking delivery vehicles have recently been designed such that cellular import and nuclear localization signals were introduced into branched peptides to guide their routing into eukaryotic cells (10, 11, 12). These structures, termed loligomers, are easily assembled by solid phase approaches. The word loligomer is derived from the merger of two terms; the Latin root loligo, which refers to members of the squid family (tentacles), and the term oligomer, which defines a polymeric assembly of building blocks. The prototypic loligomer displayed an import signal (IS)3 and a nuclear localization sequence derived from the SV40 large T Ag (13, 14) on each branch of a lysine-based tentacular scaffold, and these were successfully used to target plasmids (15) and drugs into the nucleus of cells (16). The evolution of branched peptide designs into guided peptide constructs suggests that the efficient routing of CTL epitopes into cells could potentially present distinct advantages over surface loading of linear peptides. In this report, we demonstrate that the intracellular delivery of CTL epitopes into APCs enhances the presentation of Ag on MHC class I molecules and leads to the generation of an in vivo CTL response in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mice

The EL-4 cell line (American Type Culture Collection, Manassas, VA) and in vitro cultures of murine T cells were grown in {alpha}-MEM supplemented with 10% FCS, HEPES (10 mM), 50 µM 2-ME, and antibiotics. All cells were maintained at 37°C in 5% CO2. Eight- to 12-wk-old female C57BL/6 mice (B6, H-2b) were used in immunization strategies (The Jackson Laboratory, Bar Harbor, ME). P14 TCR transgenic mice that express a TCR specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein and MHC H-2Db and rat insulin promoter (RIP)-gp mice have been previously described (17, 18).

Peptide synthesis and purification

Branched peptides were synthesized by classical solid phase peptide synthesis using tert-butoxycarbonyl chemistry and phenylacetamidomethyl resin supports (Novabiochem, San Diego, CA) on an Applied Biosystems 430A synthesizer (Foster City, CA). The synthesis, coupling efficiencies, purification, and characterization of loligomers were previously described (11). Linear peptides were synthesized on an Applied Biosystems 431A synthesizer using 9-fluorenylmethoxycarbonyl (F-moc) chemistry and p-hydroxymethyl phenoxymethyl polystyrene resin supports (Applied Biosystems, Mississauga, Canada). Peptides were dissolved in water containing 0.1% (v/v) trifluoroacetic acid and loaded on a C18 reverse phase column (Waters, Milford, MA) equilibrated in the same mobile phase. The peptides were eluted using an acetonitrile gradient from 20 to 60% acetonitrile in water in 30 min. Mass spectrometry and amino acid analyses were performed to verify the mass and amino acid composition of each peptide.

Derivatization of peptides with fluorescent probes

The single thiol group (cysteine residue) introduced in the C-terminal arm of peptides was derivatized with 5-maleimido forms of rhodamine, biotin, fluorescein, or Texas Red (Molecular Probes, Eugene, OR). Briefly, 3 mg of each peptide was dissolved in 1 ml of 0.1 M sodium citrate buffer (pH 6.5). The thiol-reactive moiety (0.3 mg) was then added as a solid to the peptide solution, and the resulting solution was mixed for 4 h at 4°C in the dark. Labeled peptides were desalted on a Sephadex G-25 column (33 x 2cm; Pharmacia, Baie d’Urfé, Canada) equilibrated with 50 mM ammonium bicarbonate, lyophilized, and stored at -70°C until used.

Confocal and electron microscopy

Peptides used for confocal microscopy were conjugated with fluorescein, Texas Red, or both chromophores. Biotinylated peptides (on the C-terminal arm) were used for electron microscopy. Briefly, EL-4 cells (5 x 105 cells) were incubated with 0.5 µM peptide (loligomerSIINFEKL+IS, loligomerSIINFEKL, loligomerIS, or linearSIINFEKL+IS) for various lengths of time. Cells were centrifuged, and the resulting pellets were washed with PBS. For confocal slides, a drop of resuspended cells was placed onto a glass slide. Slides were immediately examined on a Zeiss LSM 510 confocal microscope (argon-krypton lasers). For electron microscopy, cells were fixed with 0.2% paraformaldehyde in PBS, embedded in LRWhite resin (Marivac, Halifax, Canada) and cut to a thickness of 0.2 µm. Grids were then labeled with streptavidin-gold particles (15-nm diameter; Cedarlane, Mississauga, Canada) for 2 h at room temperature and subsequently labeled with uranyl acetate and lead citrate. Electron micrographs were collected on a Toshiba H-600 transmission electron microscope.

Internalization and retention of peptides by EL-4 cells

EL-4 cells (5 x 105 cells/ml) were incubated at 4 or 37°C with increasing amounts of fluorescein-labeled peptide for 4 h (concentration study) or with 0.5 µM peptide over a period of 6 h (time study). Cells were subsequently recovered by centrifugation (1000 rpm), washed with PBS to remove excess peptide, resuspended in 0.5 ml 1% BSA in PBS with 5 µl 7-amino-actinomycin D (7-AAD) (0.1 mg/ml; Molecular Probes), and placed on ice for 10 min. The fluorescence intensities of viable cell populations were recorded by flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ; gated on cells that excluded 7-AAD). For retention experiments cells were incubated with peptide for 4 h, washed, and recultured in 1 ml peptide-free medium. Cells were spun down at various time points during and after the removal of peptides from the medium, washed, and analyzed by flow cytometry as described above.

Expression of SIINFEKL:Kb complexes by EL-4 cells

EL-4 cells were incubated with either 0.5 µM SIINFEKL or loligomerSIINFEKL+IS at 37°C for increasing amounts of time. At each time point cells were spun down, washed with peptide-free medium, and resuspended in 1% BSA in PBS (100 µl) with fluorescein-labeled mAb 25D1.16.30 (1 µl). Samples were kept on ice for 30 min, washed twice with PBS, and subsequently resuspended in 0.5 ml 1% BSA in PBS. 7-AAD (5 µl) was added to each sample, and the fluorescence signal was measured by flow cytometry.

Immunization of mice and in vitro restimulation

Peptides (500 µg) were dissolved in 100 µl Quil A (100 µg/ml; Cedarlane) and injected s.c. into the hind leg of C57BL/6. Mice were sacrificed 11 days later, and their spleens were resected. Three days before restimulation B6 spleen cells were activated with Con A (2 mg/ml; ICN Pharmaceuticals Canada, Montréal, Canada), and peptides (loligomerSIINFEKL+IS and SIINFEKL, 0.5 µM) were added to the culture the night before restimulation. Con A blasts were harvested using Lympholyte M (Cedarlane, Hornby, Canada), irradiated (50 Gy), and used as stimulators for the lymphocytes obtained from the immunized mice at a ratio of 7 x 107 spleen cells to 7 x 106 stimulators. After 5 days CTLs were harvested with Lympholyte M and used in a standard 51Cr release assay at ratios of 100:1, 30:1, and 10:1 against target cells.

Chromium 51 release assay

EL-4 cells were preincubated with 0.5 µM peptide (loligomerSIINFEKL+IS, SIINFEKL, or loligomerIENKSFLI+IS) overnight (18 h before assay) as well as during the labeling procedure with sodium [51Cr]chromate for 1 h. The isotope (Na)251CrO4 (360 µCi; NEN Life Science, Boston, MA) was added to ~5 x 106 cells suspended in 100 µl FCS. The cells were counted after three washes with medium containing 1% FCS. Target cells (2 x 103; 100 µl) were dispensed with CTLs (100 µl) into wells of 96-well plates and incubated at 37°C for 4 h. Cells were centrifuged (800 rpm) for 5 min, and 100 µl of each supernatant was collected for radioactivity measurements. Specific lysis was calculated as: % specific lysis = ((release by CTL - spontaneous release)/(total release - spontaneous release)) x 100.

Induction of diabetes

Double-transgenic RIP-gp/P14 mice were immunized with 20 µg loligomerKAVYNFATM+IS and 100 µg anti-CD40 (FGK45) i.v. (19). The loligomer activates the transgenic P14 T cells, while the anti-CD40 activates APCs (20). Blood glucose was monitored regularly using an Accu-Chek monitor from Roche (Indianapolis, IN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design and preparation of peptide and loligomer constructs harboring CTL epitopes

Branched peptide delivery vehicles (CTL loligomers) were designed to carry a CTL epitope, either SIINFEKL (H-2Kb restricted epitope from chicken OVA (21); residues 257–264) or KAVYNFATM (H-2Db restricted epitope derived from the LCMV glycoprotein (22), p33; residues 33–41) placed on the N terminus of a pentalysine IS (Fig. 1Go). Cleavage within the IS domain by resident endoplasmic reticulum (ER)/Golgi proteases would then release the epitope from the delivery vehicle. Various control peptides were also constructed to demonstrate the necessity of the IS for efficient cellular entry and the advantage of displaying multiple epitopes on one delivery vehicle compared with a linear peptide carrying the same domains (Fig. 1GoB).



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  		FIGURE 1. A, Structure of a CTL loligomer that carries the chicken OVA CTL epitope, SIINFEKL. Peptides and loligomer constructs harboring the p33 epitope (KAVYNFATM) were also prepared for this study. Fluorescein, Texas Red, and biotin were covalently attached to the peptide using the single thiol group (cysteine side chain labeled with an asterisk) present within the C-terminal analytical arm of peptides. For dual-labeling experiments, fluorescein was introduced at the {epsilon}-amino group of lysine 263 (labeled Fl) of SIINFEKL during solid phase synthesis. B, List of synthetic peptides and associated acronyms used in the present study.

 
Internalization and processing of CTL loligomers by EL-4 cells

The internalization of CTL-containing loligomers by cells had to be established before demonstrating their utility as intracellular Ag delivery vehicles. EL-4 cells were treated with increasing concentrations of fluorescein-labeled loligomers. The mean fluorescence intensity of cells exposed to the labeled constructs was analyzed by flow cytometry and represented a measure of the amount of peptide internalized by EL-4 cells (Fig. 2Go). Both loligomerSIINFEKL+IS and loligomerIS were taken up into EL-4 cells at 37°C in a time- and peptide concentration-dependent manner (Fig. 2Go, A and B). Fluorescence signal inside cells was not detected for cells incubated with these peptides at 4°C (Fig. 2Go, A and B), which verified the mechanism of internalization (absorptive endocytosis) observed for previous constructs (11). The uptake of loligomers reached saturation well before 4 h, and this time point was used in subsequent experiments to maximize the amount of peptide within the cell. The addition of the epitope onto the N-terminal end of the molecule decreased the efficiency of import of loligomerSIINFEKL+IS in relation to loligomerIS alone (Fig. 2Go, A and B). However, adequate internalization levels were still achieved as observed by confocal microscopy (Fig. 3Go). The minimal uptake of loligomerSIINFEKL at either 4 or 37°C (Fig. 2GoC) demonstrated the necessity for the IS domain to cross the cell membrane, while the exclusion of linearSIINFEKL+IS illustrated the need for multiple targeting domains (Fig. 2GoC). The increased internalization of loligomerSIINFEKL+IS and loligomerIS (Fig. 2GoD) within the 0–0.5 µM range also illustrates that uptake remains a concentration-dependent event. Cationic peptides are moderately cytotoxic (loligomerSIINFEKL+IS has a CD50 value of 8 µM; data not shown), and for this reason a concentration of 0.5 µM (Fig. 2Go, A–C) and an incubation time of 4 h were used in assays to ensure the optimal loading of EL-4 cells with minimal toxicity.



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  		FIGURE 2. Internalization of loligomers by EL-4 cells is dependent upon time and concentration. EL-4 cells were incubated with 0.5 µM fluorescein-labeled loligomerSIINFEKL+IS (A) at 37°C ({blacksquare}) and 4°C ({square}), 0.5 µM fluorescein-labeled loligomerIS (B) at 37°C (•) and 4°C ({circ}), 0.5 µM fluorescein-labeled loligomerSIINFEKL (C) at 37°C ({blacktriangleup}) and 4°C ({triangleup}), or 0.5 µM fluorescein-labeled linearSIINFEKL+IS (C) 37°C ({diamondsuit}) and 4°C ({diamond}) as a function of time. Autofluorescence was minimal for EL-4 cells ({triangledown}, all panels). Relative mean fluorescence intensities were measured by flow cytometry. D, Cells were incubated with increasing concentrations of peptide (loligomerSIINFEKL+IS ({blacksquare}), loligomerIS (•), loligomerSIINFEKL ({blacktriangleup}), and linearSIINFEKL+IS ({diamondsuit})) for 4 h at 37°C before flow cytometry measurements. Each symbol represents the average mean fluorescence signals derived from three separate experiments.

 


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  		FIGURE 3. Intracellular routing of loligomers to the cytoplasm and ER/Golgi compartments of APCs. EL-4 cells were exposed to Texas Red-labeled loligomerSIINFEKL+IS for 1 h (A) or 4 h (B). ER and Golgi compartments were stained with DiOC6 (green). Phase contrast (C) and confocal (D) images of EL-4 cells incubated for 4 h with a dual-labeled loligomerSIINFEKL+IS at 37°C. Fluorescein was attached to lysine 273 of SIINFEKL, while Texas Red was linked to the thiol group present in the C-terminal arm of the construct. The color yellow indicates the presence of intact loligomer, while regions of green and red suggest the detachment of arms from the branched peptide scaffold. Electron micrographs highlight the location of biotinylated loligomers within EL-4 cells 4 h (E) and 10 h (F) after peptide incubation. The cellular location of loligomerSIINFEKL+IS was visualized using streptavidin-gold particles (15-nm diameter).

 
The proper routing of CTL loligomers within cells is necessary for the success of this intracellular Ag delivery strategy. EL-4 cells (murine thymoma cells) were used as APCs in the context of our experiments, as previous studies have shown that they effectively present both SIINFEKL and KAVYNFATM epitopes (23). Biotinylated or fluorescently labeled peptides were used to establish the internalization of various peptides into EL-4 cells. Confocal and electron microscopy results (Fig. 3Go) illustrate that loligomerSIINFEKL+IS was readily imported and processed by EL-4 cells, events that were not observed for the linearSIINFEKL+IS peptide. Colocalization of DiOC6 (a fluorescent label for ER and Golgi compartments, green) with Texas Red-labeled peptides (red) suggested the partial routing of peptide constructs to the Golgi and ER vesicular compartments within 4 h (Fig. 3GoB). Electron microscopy further revealed the internalization and compartmentalization of loligomerSIINFEKL+IS. A 4-h incubation of EL-4 cells with CTL loligomers and subsequent visualization with streptavidin gold particles localized the peptides to vesicles found close to the cell membrane (Fig. 3GoE). Ten hours later, CTL loligomers were present in both the cytoplasm and within vesicles (Fig. 3GoF). Evidence of loligomerSIINFEKL+IS within the ER lumen of cells was not conclusive by this method of staining.

Appropriate length of peptide is essential for Ag binding to the MHC class I groove. The cleavage of SIINFEKL from the branched carrier was visualized through the use of a dual-labeled loligomerSIINFEKL+IS (Fig. 3Go, C and D). A Texas Red chromophore (red) was introduced at the C-terminal arm of this construct, while the {epsilon}-amino group of the unique lysine residue on each SIINFEKL sequence was derivatized with a fluorescein probe (Fig. 1Go, green). Areas of yellow fluorescence indicated the presence of intact or partially processed loligomers after 4 h (Fig. 3GoD, bottom cell), while distinct regions of both red and green fluorescence (observed in both cells) illustrated the cleavage of epitopes from the branched vehicle. The shuttling of the epitope into different cellular compartments from the branched core suggests that one or more host proteases selective for Arg/Lys dipeptide sequences or proteasomes associated with the cytosolic face of vesicular membranes are able to process the pentalysine import sequences, thus leading to the release of the CTL epitope. The phase contrast image confirmed the cytosolic location of the loligomers with no nuclear staining visible (Fig. 3GoC). Images depict representative events observed for all cells in the field of view.

Extending the cellular half-life and presentation of CTL epitopes by APCs

The duration of antigenic stimulation is an important factor in committing T cells to activation (24). Thus, CTL loligomers were evaluated for their ability to 1) extend the biological half-life of the epitopes within cells, and 2) lengthen and/or skew the processing and presentation of CTL epitopes by APCs. The incubation of EL-4 cells with loligomerSIINFEKL+IS for 4 h and subsequent removal of peptide from the medium illustrated the ability of cells to retain CTL loligomers while the initial uptake of linearSIINFEKL+IS was minimal and therefore resulted in very little retention of this peptide within cells (Fig. 4Go). Confocal images also verified the presence of CTL loligomers within EL-4 cells >20 h after its removal from the medium (Fig. 4GoB).



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  		FIGURE 4. LoligomerSIINFEKL+IS is retained within EL-4 cells for a prolonged period of time. A, EL-4 cells were incubated with fluorescein-labeled linearSIINFEKL+IS (•), loligomerSIINFEKL+IS ({blacksquare}), or no peptide ({triangleup}) for 4 h. Cells were subsequently washed and recultured in fresh medium for the remainder of the experiment. Mean fluorescence intensities were monitored by flow cytometry. Each time point represents the average of two experiments. B, Confocal images depicting the presence of Texas Red-labeled loligomerSIINFEKL+IS (red) within EL-4 cells at the indicated time points. DiOC6 (green) was used to stain Golgi and ER compartments inside cells.

 
We then looked at the expression time of SIINFEKL:Kb complexes on the cell surface using a fluorescein-labeled mAb (25) (25D1.16.30, Fig. 5GoA) that specifically recognized such complexes. Both SIINFEKL and loligomerSIINFEKL+IS led to the presence of surface SIINFEKL:Kb molecules after incubation of EL-4 cells with peptide. However, the initial generation of complexes by SIINFEKL alone decreased more rapidly than loligomerSIINFEKL+IS-generated complexes as a function of time.



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  		FIGURE 5. Loligomers extend the presentation time of Ags on MHC class I molecules. A, The presence of SIINFEKL:Kb complexes on the cell surface of EL-4 cells was monitored using the fluorescein-labeled mAb 25D1.16.30, an Ab that specifically recognize SIINFEKL:Kb complexes (25 ). Cells were incubated with 0.5 µM loligomerSIINFEKL+IS ({blacksquare}) or SIINFEKL (•) or with no peptide ({blacktriangleup}) for the indicated time. B, EL-4 target cells pulsed once ({square}) or twice ({blacksquare}) with loligomerSIINFEKL+IS, once ({circ}) or twice (•) with SIINFEKL, twice with loligomerIENKSFLI+IS ({blacktriangleup}), or with no peptide ({triangleup}) were recognized by loligomerSIINFEKL+IS-raised T cells in a 51Cr release assay. C, EL-4 cells incubated either once ({triangleup}) or twice ({blacktriangleup}) with loligomerKAVYNFATM+IS, once ({diamond}) or twice ({diamondsuit}) with KAVYNFATM, or with no peptide ({triangledown}) were killed by loligomerKAVYNFATM+IS-raised T cells. Chromium release assays were performed with loligomer-raised CTLs, and each data point represents the average of results from three separate experiments.

 
To determine whether this prolonged expression was still biologically relevant, a chromium release assay was performed on target EL-4 cells that had been incubated with peptide either once (18 h before the assay was performed) or twice (18 h before the assay and during the labeling procedure). CTLs were capable of killing EL-4 cells that had been incubated with SIINFEKL-containing peptides (Fig. 5GoB). More importantly, recognition of loligomer-loaded EL-4 cells (loaded once or twice) was still greater than that of EL-4 cells that had been loaded twice with SIINFEKL. Unexpectedly, SIINFEKL-loaded Kb complexes (loaded once) were still weakly recognized after 18 h, which was attributed to either the high concentration of peptide used to load cells (0.5 µM) or the longer half-life of the Ag on Kb molecules. EL-4 cells in the absence of peptide or loaded with a loligomer harboring a scrambled SIINFEKL epitope (loligomerIENKSFLI+IS) were not targets for CTL lysis, illustrating the specificity of the CTL population (Fig. 5GoB).

Interestingly, when the LCMV p33 epitope was presented by EL-4 cells, the amount of KAVYNFATM:Db complexes recognized by CTLs on targets that had been loaded once with KAVYNFATM (0.5 µM, 18 h prior) had diminished to background levels (Fig. 5GoC). In addition, CTLs recognized targets that had been loaded twice with either loligomerKAVYNFATM+IS or KAVYNFATM and EL-4 cells that had been pulsed only once with loligomerKAVYNFATM+IS to the same extent. Thus, loligomers were, in fact, capable of prolonging the expression of peptide:MHC complexes on the cell surface, and this phenomenon was not restricted to one epitope. The half-life of peptide:MHC class I complexes on the surface of EL-4 cells was significantly shorter when these APCs were exposed to the free peptide KAVYNFATM instead of SIINFEKL.

In vivo priming of CTLs by loligomers

The demonstration that loligomers can prolong the expression of peptide:MHC class I complexes on APCs led us to test its ability to stimulate in vivo CTL responses. Administration of loligomer or SIINFEKL (500 µg in Quil A) to mice led to the priming of CTLs that recognized EL-4 targets that had been preloaded with loligomerSIINFEKL+IS (0.5 µM; Fig. 6GoA). Loligomer-raised CTLs were capable of killing targets ~3 times better than SIINFEKL-raised CTLs could kill the same targets. Administration of SIINFEKL or loligomerSIINFEKL+IS (500 µg) i.v. in PBS also produced similar results with a higher recognition of peptide-loaded targets by loligomer-raised T cells than SIINFEKL-raised T cells (data not shown). Thus, CTL-epitope containing loligomers are able to produce a specific in vivo immune response that was better than immunization with the linearSIINFEKL peptide alone. The results obtained using the CTL epitope, KAVYNFATM, led to a similar outcome in which CTLs generated from loligomerKAVYNFATM+IS-injected mice resulted in a higher specific lysis of targets compared with KAVYNFATM-raised CTLs (Fig. 6GoB). Therefore, the ability of loligomers to generate an in vivo CTL response to different epitopes could potentially lead to a generalized model of Ag delivery by branched peptide delivery vehicles.



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  		FIGURE 6. In vivo generation of CTLs by loligomer recognize targets 3-fold higher than peptide-raised CTLs. A, C57BL/6 mice were given either 500 µg loligomer ({blacksquare}) or SIINFEKL (•) mixed with Quil A (10 µg). Lymphocytes were restimulated in vitro with the same peptide. CTLs derived from injecting loligomerSIINFEKL+IS ({diamondsuit}) or SIINFEKL ({blacktriangleup}) into mice were restimulated without peptide as controls. Target EL-4 cells were preloaded twice with loligomerSIINFEKL+IS (0.5 µM), and a standard chromium release assay was performed. B, LoligomerKAVYNFATM+IS (500 µg; {blacksquare}) or KAVYNFATM (500 µg; •) mixed with Quil A (10 µg) was injected into C57BL/6 mice. Restimulation and chromium release assays were performed as described above, except for the substitution of loligomerKAVYNFATM+IS or KAVYNFATM peptides. CTLs derived from injecting loligomerKAVYNFATM+IS ({diamondsuit}) or KAVYNFATM ({blacktriangleup}) into mice were also restimulated in the absence of peptide to serve as controls. C, RIP-gp transgenic mice were injected with 20 µg loligomerKAVYNFATM+IS with 100 µg anti-CD40 ({blacksquare}, {blacktriangleup}, •) or with 100 µg of anti-CD40 ({square}, {triangleup}, {circ}) alone. Each symbol represents an individual mouse.

 
To determine whether loligomers were capable of inducing effective CTL function in vivo, we examined its ability to induce autoimmunity in a transgenic animal model. RIP-gp transgenic mice express the LCMV glycoprotein in the {beta}-islet cells of the pancreas under the direction of the rat insulin promoter. LCMV-specific T cells remain in the T cell repertoire and upon infection with LCMV become activated to destroy the islet cells expressing the LCMV-gp, leading to diabetes. The administration of peptides together with adjuvant does not lead to the induction of diabetes in the single transgenic model. However, the presence of a large number of LCMV-specific T cells in the double-transgenic RIP-gp/P14 model permits the induction of diabetes with peptide and adjuvant to activate the APC. The onset of diabetes is dependent upon the activation of CD8+ cytotoxic T cell function (18, 26). To determine whether loligomers were capable of inducing CD8 effector function and diabetes in this model, RIP-gp/P14 double-transgenic mice were immunized with loligomer KAVYNFATM+IS and anti-CD40 and monitored for hyperglycemia (Fig. 6GoC). RIP-gp/P14 transgenic mice developed diabetes with similar kinetics and severity as virus-infected mice (18), indicating that loligomers were effective at inducing CTL function in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was undertaken to evaluate whether peptide-delivery vehicles known as loligomers (10, 11, 12, 15, 16) could present distinct advantages over linear peptides in terms of routing CTL epitopes into APCs and eliciting a CTL response in mice. Loligomers were constructed to carry a CTL epitope, either SIINFEKL or KAVYNFATM, fused to the N terminus of a pentalysine import sequence. Such linear sequences were assembled on each branch of an eight-arm peptide (Fig. 1Go). Flow cytometry and confocal and electron microscopy results (Figs. 2Go and 3Go) demonstrated that loligomerSIINFEKL+IS was readily imported and processed by EL-4 cells, events that were not observed for linearSIINFEKL+IS. Loligomers were also capable of extending the cellular half-life and presentation of CTL epitopes by APCs. Because loligomers were imported and processed internally by APCs in a pathway distinct from the surface loading of peptide, the guided delivery of Ags into a cell may result in the prolongation of a CTL response. This hypothesis was proven by the ability of EL-4 cells to retain loligomerSIINFEKL+IS (Fig. 4Go) and extend the expression time of SIINFEKL:Kb complexes on the cell surface, as detected by a specific fluorescein-labeled mAb (25D1.16.30, Fig. 5GoA). Both SIINFEKL and loligomerSIINFEKL+IS led to the presence of SIINFEKL:Kb molecules after incubation of EL-4 cells with peptide. However, the initial generation of complexes by SIINFEKL alone decreased more rapidly than that of loligomerSIINFEKL+IS-generated complexes as a function of time. Furthermore, lysis of peptide-loaded target cells by epitope-specific CTLs verified that the surface complexes were still biologically relevant. Results from chromium release assays indicated that recognition of loligomer-loaded targets (loaded once or twice) was greater than that of EL-4 cells that had been loaded twice with SIINFEKL (Fig. 5GoB). This time-dependent event was even more dramatic for the p33 epitope (Fig. 5GoC). Recognition of complexes generated by EL-4 cells loaded once with loligomerKAVNFATM+IS (18 h before assay) by peptide-specific CTLs was equivalent to that of EL-4 cells that had been incubated either twice with the same CTL loligomer or twice with KAVYNFATM. This demonstrated that a significant number of KAVYNFATM:Db complexes were still present on the cell surface 18 h after exposure to loligomers, whereas the number of complexes present on targets loaded once with KAVYNFATM had diminished to background levels (Fig. 5GoC). The half-life of peptide:MHC class I complexes on the surface of EL-4 cells was significantly shorter when these APCs were exposed to the free peptide KAVYNFATM instead of SIINFEKL. Stability differences associated with selected Ags and their complexes with class I molecules may explain temporal variations in terms of target recognition by CTLs. The generation of a functional T cell response in the absence of CD28 (27) and the commitment of naive T cells to proliferation (24) are events that are regulated by the prolongation of peptide:MHC class I complexes on the surface of APCs.

It was demonstrated that the administration of loligomers harboring the SIINFEKL epitope into mice elicited a CTL response in the presence of adjuvant (Fig. 6GoA). The results obtained using the CTL epitope, KAVYNFATM, led to a similar outcome, in which CTLs generated from loligomerKAVYNFATM+IS-injected mice resulted in a higher specific lysis of targets compared with KAVYNFATM-raised CTLs (Fig. 6GoB). The induction of effective CTL function in vivo was also demonstrated by the ability of the loligomers to induce diabetes in a transgenic model (Fig. 6GoC). The mouse studies presented in Fig. 6Go did not, however, determine whether the higher specific lysis of CTL derived from loligomer-treated mice originated from an in vivo immune response or from a potentially more efficient in vitro restimulation event. Therefore, the ability of loligomers to generate an in vivo CTL response to different epitopes could potentially lead to a generalized model of Ag delivery by branched peptide delivery vehicles.

Peptides derived from the third helix of the antennapedia homeodomain and the basic region of HIV Tat (Tat) represent two other peptide-based cellular import signals that have been recently used to deliver CTL epitopes into cells (7, 8, 9). The antennapedia peptide constructs have been moderately successful in eliciting in vivo T cell responses requiring that the constructs be injected twice into mice and only yielded a response when SDS was added to the vaccine preparation (9). Studies with Tat fusion peptides indicated that such constructs were not able to produce a T cell response even with the addition of CFA (7). The simple fusion of CTL epitopes to these import signals led to their delivery into cells. However, subsequent steps to enable Ag loading onto MHC molecules were not addressed. For example, peptide length (28, 29, 30) as well as C-terminal amino acids of the Ags (31, 32) are important factors for peptide loading onto MHC class I molecules. One would expect that tailoring these vehicles to meet these requirements could potentially enhance their ability to load MHC class I molecules with peptide. The results presented in this study suggest that the incorporation of Ags into loligomer scaffolds sustains the presentation of peptide:MHC I complexes on the surface of cells with a limited requirement for adjuvant. Finally, loligomers are ideal for the incorporation of signals that may enhance other mechanistic arms associated with a productive immune response. For example, the addition of a CpG motif (33) or a lipid tail (34) to these constructs may enhance their ability to target a specific immune response.


   Acknowledgments
 
We thank Jim Ferguson and Eleonora Bolewska-Pedyczak for synthesizing peptides, Steven Doyle, and Battista Calvieri for their help with microscopy studies, and Drs. Richard Miller and Tania Watts for helpful comments. mAb 25D1.16.30 was a gift from Dr. Ronald Germain.


   Footnotes
 
1 This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and by the Canadian Network for Vaccines and Immunotherapeutics. K.S.K. was the recipient of Ontario Graduate and Canadian Institutes of Health Research doctoral studentships. Back

2 Address correspondence and reprint requests to Dr. Jean Gariépy, University Health Network, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. E-mail address: gariepy{at}uhnres.utoronto.ca Back

3 Abbreviations used in this paper: IS, import signal; ER, endoplasmic reticulum; LCMV, lymphocytic choriomeningitis virus; RIP, rat insulin promoter; 7-AAD, 7-amino-actinomycin D. Back

Received for publication September 7, 2001. Accepted for publication March 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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