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Engrafting Costimulator Molecules onto Tumor Cell Surfaces with Chelator Lipids: A Potentially Convenient Approach in Cancer Vaccine Development¹

Christina L. van Broekhoven^*, Christopher R. Parish, Gerard Vassiliou and Joseph G. Altin^2,*

^* Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, Canberra, Australia; Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia; and Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200–300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or "engrafted" protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7.1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The emergence of techniques for introducing genetic material into cells recently has provided a powerful approach for modifying or engineering the surfaces of cells, and hence for developing cell-based vaccines. For example, in both animal and human tumor models, evidence suggests that the transfection of tumor cells with genes that induce the cells to express T cell costimulator molecules like B7-1 (CD80), B7-2 (CD86), CD40, and ICAM-1 on their surface may be effective in enhancing tumor immunity when the cells are used as vaccines in the tumor-bearing host (1, 2, 3, 4). In these situations, modification of the tumor cells is achieved by genetic manipulation of the tumor cells to induce them to express one or more specific protein(s) (i.e., costimulator molecules) on their surface (5). Unfortunately, such genetic manipulation of cells can be time consuming and inconvenient to perform in clinical situations for the treatment of human cancers; the frequency of transfection can be low, and successful transfection with multiple genes (to induce expression of multiple proteins on the tumor cell surface) can be difficult to achieve. Furthermore, transfection techniques, even when conducted by the use of seemingly harmless viral vectors, can be associated with risks to the patient due to the difficulty in precisely controlling the expression of the gene or its integration into the genome.

In view of the limitations associated with the genetic manipulation of tumor cells, novel strategies for modifying the cell surface are being sought. Of relevance to this, the engraftment onto tumor cell membranes of the extracellular region of certain receptors (e.g., the extracellular region of B7.1) linked to a glycosylphosphatidylinositol anchor is often sufficient to render the tumor cells more effective as vaccines (6, 7, 8). These observations suggest that techniques for modifying cell surfaces, for example, by anchoring molecules such as recombinant forms of the extracellular region of B7.1, may be a viable approach in the development of cell-based vaccines (6, 7). Immobilized metal chelators, such as nitrilotriacetic acid (NTA),³ have been used routinely for purifying recombinant proteins bearing polyhistidine tags by metal-ion affinity chromatography (9). Also, some studies report that metal-chelating head groups such as NTA can be covalently linked to acyl-like chains or lipids which can be used to anchor suitably tagged molecules onto planar lipid bilayer membranes (10). Therefore, since liposomal suspensions of lipids can often fuse with the plasma membrane of cells (11), a process commonly used in genetic transfections to introduce genetic material and other agents into cells (12), it is conceivable that the fusion and incorporation of metal chelator lipids into the plasma membrane of cells also can be used to anchor recombinant proteins bearing an appropriate metal affinity tag onto the cell surface.

This study describes the use of a novel chelator lipid, namely, NTA-ditetradecylamine (DTDA) consisting of an NTA head group covalently linked to two 14-carbon hydrocarbon chains, and the incorporation of this lipid into the membrane of P815 cells for the purpose of anchoring recombinant proteins onto the cell surface. Our results show that suspensions of this lipid can readily be formed in aqueous buffers such as PBS, and that the lipid can be incorporated into the plasma membrane of murine P815 mastocytoma cells and various other cultured cell lines. Furthermore, our data show that proteins bearing a hexahistidine (6His) tag, for example, recombinant forms of the extracellular region of B7.1 or CD40, can be conveniently anchored or "engrafted" onto the surface of P815 cells via the NTA metal-chelating linkage, and that cells bearing engrafted molecules are active in stimulating allogenic and syngeneic T cell proliferation in vitro and in generating tumor-specific effector T cells when used as vaccines in vivo. This study presents a convenient alternative to transfection for modifying cell surfaces for vaccine development and for manipulating immune function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Analytical grade reagents were used in all experiments. Paraformaldehyde was obtained from BDH Chemicals (Kilsyth, Victoria, Australia). ZnSO₄ was used for all additions of Zn²⁺ to buffers and growth media. RPMI 1640 and Eagle’s MEM (EMEM) both were obtained from Life Technologies (Melbourne, Australia). FCS was obtained from Trace Scientific (Noble Park, Victoria, Australia). Sulfo-N-hydroxysuccinimide (NHS)-LC-biotin was obtained from Pierce (Rockford, IL). Na⁵¹CrO₄, [³H]thymidine, and FITC-conjugated streptavidin were obtained from Amersham (Buckinghamshire, U.K.). Dioleoylphosphatidylethanolamine (DOPE), -palmitoyl-ß-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), Isopaque, Ficoll, propyl gallate, and the polyethylene glycol (PEG) preparations PEG₄₀₀, PEG₆₀₀, PEG₉₀₀, and PEG₁₅₀₀ were all obtained from Sigma-Aldrich (Castle Hill, New South Wales, Australia). MicroScint scintillation fluid and other items such as filters and seals for 96-well plates for use with the Topcount NXT microplate scintillation counter were obtained from Canberra Packard (Canberra, Australian Capital Territory, Australia).

Mice and cell lines

Female or male DBA/2J mice (H-2^d) were used for isolation of lymphoid tissue (spleen) for T cell proliferation, measurement of T cell cytotoxicity, and vaccination and monitoring of tumor growth in vivo. C57BL/6J mice (H-2^b) were used in experiments assessing allogenic stimulation of T cell proliferation. The mice were used at 6–8 wk of age and were obtained from the Animal Breeding Establishment, John Curtin School of Medical Research, Australian National University (Canberra). The murine cell lines P815 (murine DBA/2 (H-2^d) mastocytoma) and EL4 (murine C57BL/6 (H-2^b)) T cell lymphoma were obtained, respectively, from Drs. P. Waring (Division of Immunology and Cell Biology, John Curtin School of Medical Research) and H. O’Neill (Division of Biochemistry and Molecular Biology, Australian National University). Both cell lines were cultured in complete medium consisting of EMEM containing 10% FCS.

Synthesis of NTA-DTDA

The chelator lipid NTA-DTDA, consisting of a NTA head group covalently linked to DTDA was synthesized by Dr. C. Easton (Research School of Chemistry, Australian National University) following a procedure similar to that described previously (13). Briefly, the DTDA was synthesized from bromotetradecane and ammonia. DTDA was then N-succinylated with succinic anhydride to produce N-succinyl-DTDA (DTDA-suc), which was reacted with NHS to produce DTDA-suc-NHS. The succinimidyl group of DTDA-suc-NHS was replaced with an N-tert-butyloxycarbonyl-lysine group, and the butyloxycarbonyl group was removed to produce N-[(DTDA) succinyl]-L-lysine. DTDA-suc-Lys was finally reacted with bromoacetic acid to produce N,N-bis[carboxymethyl]-N-[(DTDA)suc]-Lys, which will be referred to as NTA-DTDA. The purity of each product was measured by TLC, and the identity of the final product was confirmed by nuclear magnetic resonance spectroscopy, Fourier-transformed infrared spectroscopy, and mass spectroscopy. The purity of the final product was estimated to be in excess of 99%.

Preparation of NTA-DTDA liposome suspensions

For NTA-DTDA incorporation into cells, desiccated NTA-DTDA was suspended to a concentration of 0.5 mM in PBS containing 0.5 mM Zn²⁺ by sonication using a Tosco (Measuring and Scientific Ltd., London, UK) 100-W ultrasonic disintegrator at maximum amplitude for 2 min. The same procedure was used to produce suspensions of DMPC and mixtures of NTA-DTDA and DMPC, POPC, or DOPE. Stock suspensions of lipids were stored at -20°C and were always resonicated and diluted to the indicated concentration before use in experiments.

Monoclonal Abs

The mAbs and their sources were as follows: murine anti-CD40 (clone 3/23, Rat IgG2a) and murine anti-CD3 (clone 145-2C11, Armenian hamster IgG) mAbs were both obtained from PharMingen (San Diego, CA) and murine anti-B7.1 (clone 16-10A1, Armenian hamster IgG) mAb was kindly provided by Dr. K. Shortman (Walter and Eliza Hall Institute, Melbourne, Australia). Where indicated, mAbs were biotinylated by reacting with sulfo-LC-biotin (Pierce) as described previously (14).

Recombinant proteins

Recombinant forms of the extracellular regions of the murine T cell costimulatory molecules B7.1 (CD80) and CD40, and the extracellular region of the human erythropoietin receptor (EPOR), each with a hexahistidine (6His) tag and denoted B7.1-6H, CD40-6H, and EPOR-6H, respectively, were produced using the baculovirus expression system. Briefly, genes encoding the extracellular domains of murine B7.1, CD40, and EPOR were amplified by PCR, and the sequences for 6His tags were incorporated into the end of each gene (corresponding to the carboxyl-terminal of the protein) by PCR using primers containing the sequence of the tag. The constructs were then separately ligated into the pVL1393 plasmid baculovirus transfer vector and used to transform Escherichia coli. Appropriate transformants were selected, and recombinant pVL1393 plasmids from these transformants were cotransfected with the baclulovirus AcMNPV into SF9 insect cells. Cells infected with virus that had the pVL1393 plasmid incorporated into the viral genome were selected by plaque assays, further amplified, and these viral stocks were used to infect High-5 insect cells grown in Express-5 medium. Recombinant proteins were purified from the supernatants of recombinant virus infected High-5 cells by Ni²⁺-NTA affinity chromatography (using Ni²⁺-NTA Superflow from Qiagen, Cifton Hill, Victoria, Australia) followed by size exclusion gel filtration on fast protein liquid chromatography (Pharmacia Biotech, Uppsala, Sweden) using a Superdex-75 HR 10/30 column; the final purity of each protein was >95% as judged by SDS-PAGE analysis. For some experiments, recombinant proteins were biotinylated by reacting with sulfo-LC-biotin (Pierce) as described previously (14). The proteins were routinely stored at -20°C in PBS at a concentration of 0.2–0.6 mg/ml and then thawed at 37°C and vortexed gently before use in each experiment.

Incorporating and optimizing the incorporation of NTA-DTDA

Cultured P815 tumor cells were washed twice in PBS to remove proteins from the culture media and suspended to 1 x 10⁷ cells/ml in PBS. The cells were then aliquoted into 96-well V-bottom Serocluster plates (Costar, Corning, NY) at 1.8 x 10⁵ cells/well and incubated with 125 µM NTA-DTDA (alone or as a mixture with other lipids as indicated) or 125 µM DMPC (control) in PBS containing 125 µM Zn²⁺ for 40 min at 37°C. Following the incubation, unincorporated lipid was removed by washing three times with PBS containing 0.1% BSA (PBS-0.1% BSA). The relative level of NTA-DTDA incorporated was routinely assessed by FACS analysis (see below) after incubating the cells with biotinylated 6His peptide (B-6His) (0.2 mg/ml) for 30 min at 4°C, washing twice with PBS-0.1% BSA, and then staining with streptavidin-FITC. The cells were incubated with streptavidin-FITC (33 µg/ml) in PBS containing 1% BSA (PBS-1%-BSA) for 30 min at 4°C, washed three times with PBS-1% BSA, fixed with 2% paraformaldehyde in PBS, and then analyzed for FITC fluorescence by FACS.

To promote fusion of NTA-DTDA liposomes and hence incorporation of the NTA-DTDA into the membrane of cells, a number of agents previously reported to potentiate the fusion of cells and vesicles with lipid bilayers were tested. P815 cells aliquoted into 96-well V-bottom Serocluster plates as described above were incubated with 125 µM NTA-DTDA, DMPC, POPC, or DOPE, or with 125 µM NTA-DTDA plus DMPC, POPC, or DOPE (at the indicated molar ratio), in PBS containing 125 µM Zn²⁺ for 40 min at 37°C. For some experiments, the cells were treated with PEG following the incubation: the cells were pelleted, suspended in 15% PEG₄₀₀, mixed and diluted 10x with serum-free EMEM, and then washed once with serum-containing EMEM and twice with PBS-0.1% BSA before engrafting the cells with biotinylated recombinant protein (see below) and then staining with streptavidin-FITC as above for FACS analysis.

Engrafting recombinant proteins onto cells

Cells with incorporated NTA-DTDA were incubated with purified B7.1-6H and CD40-6H (or biotinylated forms of these as indicated), either alone (each at 50 µg/ml) or in combination (100 µg/ml total protein, with a B7.1-6H:CD40-6H molar ratio of 4:1) in 96-well V-bottom Serocluster plates for 1 h at 4°C. Unbound protein was then removed by washing twice with PBS-0.1% BSA before using the cells bearing the engrafted protein(s) either for immunizations or in assays of T cell proliferation. For experiments to determine the level of bound protein by FACS analysis, the cells were stained with streptavidin-FITC (for cells bearing engrafted biotinylated protein) or were first incubated with the appropriate biotinylated mAb (B-mAb) (4°C for 30 min), washed twice with PBS-0.1% BSA, and then stained with streptavidin-FITC.

Time courses

Cells with incorporated NTA-DTDA and DMPC, with or without engrafted CD40-6H, were suspended in EMEM containing 10% FCS and 50 µM added Zn²⁺ and incubated in 12-well flat-bottom tissue culture plates (Linbro; ICN Pharmaceuticals, Aurora, OH) for 2 min (time 0) or for 4 or 24 h at 37°C. After the indicated incubation time, cells were collected from the 12-well flat-bottom plates, transferred to 96-well V-bottom Serocluster plates, and washed twice in PBS-0.1% BSA before either staining with streptavidin-FITC (for cells with NTA-DTDA and engrafted B-CD40-6H), or first incubating with B-CD40-6H and then washing with PBS-0.1% BSA and staining with streptavidin-FITC (for cells with only NTA-DTDA).

Flow cytometry

FACS analysis was used to quantify the relative levels of NTA-DTDA incorporated into the membrane of cells following binding of B-6His and the levels of biotinylated recombinant proteins anchored to the cell surface via the incorporated NTA-DTDA. Flow cytometric analyses were performed using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mW argon ion laser. Cells were analyzed on the basis of forward light scatter, side light scatter, and FITC fluorescence; with the relative shift in fluorescence intensity above background providing a semiquantitative measure of the level of NTA-DTDA incorporation and the level of peptide or recombinant protein on the surface of cells. Typically, fluorescence information for 10,000 cells was collected for each condition using a log amplifier and the data processed using CellQuest (Becton Dickinson) software. Data were analyzed by gating live cells, as judged by forward light scatter vs side light scatter dot plots and plotting the fluorescence profile as a histogram. The fold increase in fluorescence intensity above background was determined by measuring the shift in fluorescence intensity using the control sample as background, from peak to peak. The results of independent experiments were then represented as the means ± SEM.

Confocal microscopy

The distribution of the NTA-DTDA on the surface of P815 cells was studied by laser scanning confocal microscopy using cells bearing incorporated NTA-DTDA engrafted with biotinylated CD40-6H and stained with streptavidin-FITC. Briefly, the cells were suspended in embedding medium (2% propyl gallate in 87% glycerol) and deposited into 0.05-mm deep chambers on microscope slides formed using perforated Scotch 465 adhesive transfer tape, and the chambers were then sealed with glass coverslips. The cells were examined for fluorescence at 520 nm with a MRC-500 laser scanning confocal imaging system (Bio-Rad, Richmond, CA) consisting of a Nikon confocal fluorescence microscope (x60 Nikon objective), with a Bio-Rad UV laser scanner and an ion laser technology laser head (model 5425; Bio-Rad) with an argon ion laser. The image was acquired by Kalman averaging of 10 successive laser scans and stored and analyzed using an Image Processor PC (Bio-Rad) and processed using NIH Image 1.61 software.

T cell proliferation assays

Murine T cells for use in T cell proliferation assays were isolated and purified from the spleens of either allogenic or syngeneic mice as described previously (15). Briefly, the spleens were dissociated into single-cell suspensions, and dead cells and RBC were removed by density gradient centrifugation using an Isopaque-Ficoll gradient. After centrifugation (20 min at 400 x g) the viable cells, mainly lymphocytes, were collected from the layer at the top of the gradient and suspended in RPMI 1640 containing 10% FCS, 5 x 10^-5 M 2-ME, 100 IU/ml penicillin, 100 µg/ml neomycin, 50 U/ml IL-2, and 10 mM HEPES. T cells were purified using an equilibrated nylon wool column (16). The purified T cells were then suspended in growth medium at a concentration of 2 x 10⁴ cells/50 µl/well in a 96-well flat-bottom plate (Cell Wells, Corning, NY) for culture at 37°C in an atmosphere of 5% CO₂.

T cell proliferation assays were conducted as described elsewhere 16). Syngeneic lymphocytes or responder cells were then cocultured with -irradiated (5000 rad) stimulator cells at a concentration of 2 x 10⁴ cells/50 µl/well. Stimulator cells included native P815 tumor cells, P815 cells with incorporated NTA-DTDA on their surface, and P815 cells with engrafted recombinant protein(s), as indicated. After 4 days of coculture at 37°C, the cells were pulsed with 1 µCi of [³H]thymidine/well for 6 h. The cells were then harvested using a Filtermate 196 cell harvester (Packard, Meriden, CT), and [³H]thymidine incorporation was assessed using a MicroScint scintillant and a Topcount NXT microplate scintillation counter (Packard) with Topcount software.

Cytotoxicity assays

Assays for in vivo tumor-specific CTL were performed using a procedure similar to that described (17). Syngeneic DBA/2 mice were immunized s.c. with either PBS (control) or 1 x 10⁵ -irradiated (5000 rad) P815 cells engrafted with recombinant protein(s). Spleens were removed from mice 14 days after immunization, and T lymphocytes (effector T cells) were isolated by density gradient centrifugation using Isopaque-Ficoll and nylon wool fractionation, as described above. Effector T cells were then suspended in incubation medium and aliquoted into 24-well flat-bottom plates at a concentration of 1 x 10⁵ cells/well and cocultured with 1 x 10⁵ -irradiated (5000 rad) native P815 cells. After 5 days of coculture, the cytolytic activity of the effector cells was assessed in a standard ⁵¹Cr release assay as described previously (17). Briefly, 2 x 10⁶ native P815 cells were labeled with 250 µCi ⁵¹Cr (Na⁵¹CrO₄) for 90 min. Labeled target cells were washed three times and resuspended in culture medium. Effector and target cells were coincubated with effector cells at different E:T ratios, as indicated, for 6 h at 37°C. Supernatants were harvested, and ⁵¹Cr release was assessed with a Topcount NXT microplate scintillation counter (Packard) using Topcount software (Packard). Percent specific lysis was calculated as follows: % specific lysis = 100 x(experimental cpm - spontaneous cpm)/(maximal cpm - spontaneous cpm).

Immunization of animals and tumor challenge in vivo

Mice were immunized using a protocol similar to that described previously (17). Briefly, either PBS (control) or 1 x 10⁵ -irradiated P815 cells with the engrafted recombinant protein(s), as indicated, were suspended in a 0.2-ml volume of PBS and injected into the shaved right back of syngeneic DBA/2 mice using a 25-gauge needle and 1-ml syringe. After 14 days, the mice were either used in cytotoxicity assays using T cells isolated from the spleens of the mice, or were challenged with 1 x 10⁵ native P815 cells by s.c. injection in the shaved left back. For monitoring tumor growth, the mice were scored for tumor size once a week by measuring two perpendicular diameters in millimeters using a caliper (17). Survival data represent animals that were still alive when scored; animals that were near death were euthanized after scoring and were deemed to have died of the tumor. Data for a total of 10 or 12 mice in the group for each experimental condition are presented.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Incorporating NTA-DTDA into cell membranes

To explore the possibility that the chelator lipid NTA-DTDA could be useful for modifying cell surfaces, initial studies were aimed at incorporating the lipid into the membrane of cells. For these studies cultured P815 cells were incubated with the NTA-DTDA (1 mM liposomal suspension in PBS) for 30 min at 20°C, and the extent of incorporation of the lipid into the cell membrane was assessed by FACS analysis of the cells after they had been incubated with B-6His and stained with streptavidin-FITC. Preliminary experiments indicated that under these conditions the FITC fluorescence intensity of cells incubated with the NTA-DTDA was severalfold higher than that of control cells incubated with a similar concentration of DMPC instead of NTA-DTDA (data not shown). Further studies showed that significantly higher levels of the NTA-DTDA could be incorporated into the cell surface when the incubation with the lipid was conducted at 37°C instead of at 20°C and when the NTA-DTDA was preloaded with a divalent metal cation like Zn²⁺ or Ni²⁺._. Moreover, the inclusion of BSA during the NTA-DTDA incubation was found to significantly inhibit incorporation of the lipid into the membrane of P815 cells; but the inclusion of BSA (0.1%) during the wash steps was useful in maintaining cell viability (data not shown). In the experiments described below, therefore, the incorporation of NTA-DTDA into the cell surface was conducted at 37°C, in the absence of BSA during the incubation with NTA-DTDA, using a liposomal suspension of NTA-DTDA that had been preloaded with an equimolar concentration of Zn²⁺.

Representative fluorescence profiles of P815 cells that had been incubated with a liposomal suspension of either DMPC (controls) or NTA-DTDA, each ranging in concentration from 2.5 to 250 µM in PBS containing an equimolar concentration of Zn²⁺, and then stained with B-6His as outlined above are shown in Fig. 1A. P815 cells incubated with DMPC gave a background level of fluorescence (i.e., similar to that of cells without lipid treatment; data not shown) and was essentially the same for all concentrations of DMPC in the range 2.5–250 µM (250 µm DMPC is shown as the control in Fig. 1A). However, it can be seen that the fluorescence intensity of the cells incubated with NTA-DTDA increased with increasing concentrations of NTA-DTDA, and that the increase was saturable. Thus, although P815 cells incubated with 2.5 µM NTA-DTDA exhibited a 1.5-fold increase in fluorescence relative to control cells incubated with DMPC, cells incubated with 250 µM NTA-DTDA showed a 140-fold increase in fluorescence intensity above their respective control cells incubated with 250 µM DMPC; the fluorescence of cells incubated at the other concentrations of NTA-DTDA were intermediate. As shown in the dose curve in Fig. 1B, incorporation of NTA-DTDA was near-maximal at 62.5 µM NTA-DTDA. These findings indicate that the chelator lipid NTA-DTDA can associate/incorporate into the membrane of cells, and that the NTA head group of the cell-incorporated NTA-DTDA is available for binding of B-6His.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Incorporation of NTA-DTDA into the membrane of P815 cells. P815 cells were incubated with 250 µM DMPC (control) or with different concentrations of NTA-DTDA in the range of 2.5 to 250 µM (as indicated) in PBS containing 250 µM Zn2+ (40 min at 37°C). The cells were then incubated for 30 min (4°C) with B-6His peptide and stained with streptavidin-FITC. A, Representative fluorescence profiles of cells as analyzed by flow cytometry. B, The dose curve for the incorporation of NTA-DTDA into the plasma membrane of P815 cells. Each point represents the mean fold increase in fluorescence of cells incubated with the indicated concentration of NTA-DTDA relative to control (DMPC) cells as obtained from two experiments performed in duplicate. Error bars represent SEM, only errors 6% are shown.

To determine whether incorporated NTA-DTDA could be used to anchor proteins bearing a 6His tag onto the cell surface, P815 cells with membrane-incorporated NTA-DTDA were incubated for 1 h at 4°C with recombinant murine B7.1-6H or with CD40-6H (each at a concentration of 50 µg/ml), and the cells were then assayed for the level of bound protein by flow cytometry. For these experiments bound protein was detected by incubating the cells with either biotinylated 16-10A1 mAb (B-16-10A1) or biotinylated 3/23 mAb (B-3/23) for detection of B7.1 or CD40, respectively, and then staining with streptavidin-FITC. The data in Fig. 2 show that DMPC-treated cells (controls) exhibited only low levels of fluorescence, indicating that recombinant proteins and mAbs do not bind nonspecifically. Relative to the controls, however, P815 cells with incorporated NTA-DTDA exhibited a 200-fold increase in fluorescence intensity when incubated with B7.1-6H (Fig. 2A) and a 100-fold increase when incubated with CD40-6H (Fig. 2B). These results indicate that significant quantities of recombinant proteins bearing polyhistidine tags can be anchored to the cell surface via a metal-chelating linkage with NTA-DTDA.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Recombinant B7.1-6H and CD40-6H can be anchored onto P815 cells via incorporated NTA-DTDA. P815 cells with DMPC or NTA-DTDA incorporated into the plasma membrane were washed and incubated with 100 µg/ml total protein of either recombinant B7.1-6H, CD40-6H, or B7.1-6H plus CD40-6H (at equimolar ratio) for 1 h at 4°C. The cells were then washed, incubated with the appropriate biotinylated B7.1 or CD40 mAbs, and then stained with streptavidin-FITC. The fluorescence profiles reflect the level of recombinant protein B7.1-6H (A), CD40-6H (B), or B7.1-6H and CD40-6H (C) anchored to the cell surface via the NTA-DTDA. In each instance, the control represents the background binding of the respective protein(s) to P815 cells containing incorporated DMPC. Each result is representative of three experiments performed in duplicate.

Confocal microscopy

Since the technique of flow cytometry does not distinguish between the fluorescence arising from recombinant protein bound to NTA-DTDA that is incorporated into the plasma membrane and that which is bound to NTA-DTDA liposomes bound to the cell surface, the distribution of cell-associated FITC fluorescence on cells containing incorporated NTA-DTDA that had been incubated with B-CD40-6H and stained with streptavidin-FITC also was examined by confocal microscopy. These studies were conducted using cells engrafted with B-CD40-6H since the fluorescence of these cells was generally higher than when using B-6His peptide. The studies showed that P815 cells treated with the NTA-DTDA (see Fig. 3B) displayed a much higher level of FITC fluorescence than P815 cells incubated with the control lipid DMPC (which exhibited little if any background fluorescence, see Fig. 3A). Although not resolved in the photograph shown in Fig. 3B, these studies showed that although some cell-associated FITC fluorescence was associated with distinct liposomal structures bound to the cell surface, a significant proportion of the fluorescence appeared as more diffuse staining of the surface of cells. The pattern of fluorescence thus indicates that a significant proportion of the NTA-DTDA liposomes become fused with the P815 cell membrane, resulting in incorporation of the NTA-DTDA.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Confocal microscopy of P815 cells with incorporated NTA-DTDA and engrafted with recombinant CD40. P815 cells were incubated with a suspension of 125 µM of total lipid of either DMPC (A), NTA-DTDA (B), or NTA-DTDA plus POPC (C) for 40 min at 37°C. After this incubation, some cells (those in C) were treated with 15% PEG400. All cells were then washed, incubated with B-CD40-6H, and then stained with streptavidin-FITC before imaging by confocal scanning laser microscopy. Images were acquired by Kalman averaging of 10 successive laser scans and then analyzed and processed using Image Processor PC software. A representative image for each condition is shown.

Our observation that a significant proportion of the cell-associated NTA-DTDA liposomes were not fused but appeared bound to the cell surface suggested that conditions for the fusion of the NTA-DTDA liposomes with the cells, and hence incorporation of the NTA-DTDA lipid into the cell membrane, could be further optimized. Experiments were conducted in which the liposomal suspension of the NTA-DTDA was prepared as an equimolar mixture of NTA-DTDA and another phospholipid, namely, DMPC, POPC, or DOPE, to see whether any of these lipids could promote NTA-DTDA incorporation. Control cells incubated with liposomal suspensions of DMPC, POPC, or DOPE alone did not exhibit significant levels of fluorescence when assayed for B-CD40-6H binding by flow cytometry (data not shown). However, cells incubated with NTA-DTDA exhibited a 140-fold increase in fluorescence above control cells, and cells incubated with NTA-DTDA plus POPC and NTA-DTDA plus DMPC exhibited a 270-fold and 255-fold increase, respectively, in fluorescence intensity above that of control cells (see Fig. 4A). Conversely, cells incubated with a suspension of NTA-DTDA plus DOPE exhibited a slight decrease in fluorescence (122-fold above background) relative to cells incubated with NTA-DTDA alone (Fig. 4A). Additional studies performed using a different molar ratio (e.g., 2:1, 1:2, and 1:4) of NTA-DTDA to each of the three phospholipids indicated that cells incubated with liposomes composed of a mixture of NTA-DTDA and POPC (at 1:1 molar ratio) gave the highest level of fluorescence and hence incorporation of the NTA-DTDA into the membrane of P815 cells.

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Effect of phospholipids and PEG treatment on the incorporation of NTA-DTDA into the membrane of P815 cells. A, P815 cells were incubated (for 40 min at 37°C) with a 125 µM suspension of DMPC, DOPE, or POPC (each used as control) or with a suspension made from a mixture of 125 µM NTA-DTDA or 125 µM NTA-DTDA plus an equimolar ratio of DOPE (DOPE:NTA-DTDA), DMPC (DMPC:NTA-DTDA), or POPC (POPC:NTA-DTDA), as indicated. The cells were then washed, incubated with B-CD40-6H, and stained with streptavidin-FITC before being analyzed for fluorescence by flow cytometry. Each column represents the mean fold increase in fluorescence of cells incubated for each condition relative to that of the respective control cells (incubated with DMPC, DOPE, DMPC, or POPC). B, P815 cells were incubated for 40 min at 37°C with a suspension of either 125 µM DMPC (as control; data not shown), 125 µM NTA-DTDA or with 125 µM NTA-DTDA plus equimolar POPC (POPC:NTA-DTDA). Some cells were then treated with 15% PEG400, as indicated (POPC:NTA-DTDA + 15% PEG). All cells were then washed, incubated with B-CD40-6H, and stained with streptavidin-FITC before being analyzed for fluorescence by flow cytometry. Each column represents the mean fold increase in fluorescence (±SEM) of the P815 cells for each condition relative to control (DMPC-treated) cells. Data were obtained from three experiments performed in duplicate.

Cells that had been incubated with NTA-DTDA, or with NTA-DTDA plus POPC and treated with PEG₄₀₀, and then engrafted with biotinylated CD40-6H and stained with streptavidin-FITC also were examined by confocal microscopy. Consistent with the flow cytometric data above, confocal microscopy indicated that cells incubated with NTA-DTDA plus POPC followed by treatment with PEG₄₀₀ exhibited not only higher levels of fluorescence, compared with cells incubated with NTA-DTDA without the PEG treatment, but also fluorescence which was more evenly distributed on the cell surface (compare Fig. 3, B and C).

Retention of engrafted molecules

The stability or length of time for which the engrafted molecules can be maintained on the cell surface is an important factor in determining the usefulness of the engrafting technique. Clearly, stability of cell-engrafted protein will depend on the conditions to which the modified cells are subjected, the rate at which the NTA-DTDA is lost from the cell surface after its incorporation, and by the stability of the protein-6H-NTA interaction. To begin to explore these factors, P815 cells treated with NTA-DTDA plus POPC and with or without PEG₄₀₀ were assessed for B-CD40-6H binding after they had been incubated for different periods of time in complete growth medium at 37°C. The flow cytometric profiles in Fig. 5A indicate that cells that had been incubated with NTA-DTDA plus POPC, treated with 15% PEG₄₀₀, and assayed for B-CD40-6H binding immediately after suspension in complete medium (time 0) exhibited a 400-fold increase in fluorescence, whereas the fluorescence of cells assayed after a 4-h and 24-h incubation in complete medium was reduced to 90-fold and 20-fold above background, respectively. These findings suggest that the level of NTA-DTDA on the cell surface decreases with time, but that significant levels of the lipid can still be detected on the cell surface after culturing the cells for 24 h.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Retention of incorporated NTA-DTDA and engrafted protein on the P815 cell surface. A, P815 cells with DMPC (control) or POPC:NTA-DTDA incorporated into the plasma membrane in the presence of 15% PEG400 were washed, suspended in EMEM containing 10% FCS, and then incubated at 37°C for 0, 4, or 24 h. After each indicated incubation time, the cells for the condition were washed, incubated with B-CD40-6H (1 h at 4°C), and stained with streptavidin-FITC before being analyzed for fluorescence by flow cytometry. B, P815 cells with DMPC (control) or POPC:NTA-DTDA incorporated into the plasma membrane in the presence of 15% PEG400 were washed and incubated with B-CD40-6H (1 h at 4°C). Following this incubation, the cells were washed, suspended in EMEM containing 10% FCS, and then incubated for 0, 4, or 24 h at 37°C. After each indicated incubation time, the cells for the condition were washed and stained with streptavidin-FITC before being analyzed by flow cytometry. Fluorescence profiles reflecting the level of NTA-DTDA and CD40 protein on the cell surface with time are shown; each result is a representative obtained from three experiments performed in duplicate.

P815 cells bearing engrafted costimulatory molecules stimulate T cell proliferation

A potential application for the technique of modifying cell surfaces is in the area of vaccine development, including cell-based vaccines to enhance tumor immunity. In preliminary studies to explore the functionality (18) of the engrafted molecules in vitro, we examined the ability of P815 cells bearing engrafted molecules to stimulate the proliferation of purified murine responder T cells, when the cells were cocultured in a standard T cell proliferation assay. Initial studies of [³H]thymidine incorporation by allogenic (C57BL/6) T cells when cocultured with either native or modified P815 cells (-irradiated) indicated that relative to P815 cells engrafted with a control protein, P815 cells engrafted with B7.1 and/or CD40 were 20–30-fold more effective in stimulating [³H]thymidine incorporation and hence T cell proliferation in this system (data not shown). Subsequent studies explored whether the modified P815 cells also were effective in stimulating T cell proliferation in a syngeneic system. As shown in Fig. 6, only low levels of [³H]thymidine incorporation were observed in control cultures, incorporation being 100 cpm for cultures of DBA/2 T cells alone (T cells) and 800 cpm for cocultures of syngeneic (DBA/2) T cells with -irradiation: native P815 cells (T + P815), P815 cells with incorporated NTA-DTDA (T + P815-NTA), and P815 cells with incorporated NTA-DTDA and engrafted EPOR as control protein (T + P815–NTA-EPOR). Conversely, a higher level of [³H]thymidine incorporation (2500 cpm) was observed when the syngeneic T cells were cocultured with anti-CD3 mAb (1:1000 dilution) as a positive control (T + anti-CD3 mAb). Moreover, [³H]thymidine incorporation was increased from 800 cpm in cocultures of P815 cells engrafted with control protein to 2000 cpm (2.5-fold increase) in cocultures of syngeneic T cells and P815 cells bearing engrafted B7.1-6H (T + P815–NTA-B7) or CD40-6H (T + P815–NTA-CD40). [³H]Thymidine incorporation was 3200 cpm when P815 cells were engrafted with both B7.1-6H and CD40-6H (T + P815–NTA-B7–CD40), being 4-fold higher than that seen in cocultures of P815 cells engrafted with the control protein (see Fig. 6). In parallel experiments, the addition of soluble B7.1-6H and CD40-6H proteins (at concentrations of 100 µg/ml) to T cells coincubated with either native P815 or EL4 cells did not induce any significant increase in [³H]thymidine incorporation relative to their respective controls (data not shown). Therefore, since neither P815 cells bearing incorporated NTA-DTDA alone, nor P815 cells bearing NTA-DTDA and engrafted with the control protein, had any effect on [³H]thymidine incorporation, the results suggest that the increase in [³H]thymidine incorporation is specific for the engrafted protein, and hence that the costimulatory molecules B7.1 and CD40 when engrafted via the NTA-DTDA lipid onto the surface of P815 cells are functionally active in stimulating T cell proliferation in both the allogenic and syngeneic system.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Stimulation of T cell proliferation by P815 cells engrafted with the costimulatory molecules B7.1 and CD40. Syngeneic splenic T lymphocytes (1 x 105) were incubated with 100 µg/ml of soluble B7.1-6H and CD40-6H (T cells) or coincubated with 1 x 105 -irradiated stimulator P815 cells, which included P815 cells in the presence of both B7.1-6H and CD40-6H in solution (T + P815), P815 cells with incorporated NTD-DTDA (T + P815-NTA), P815 cells with engrafted EPOR (T + P815-NTA-EPOR), P815 cells with engrafted B7.1 (T + P815-NTA-B7.1), P815 cells with engrafted B7.1 and CD40 (T + P815-NTA-B7.1-CD40), and P815 cells with added CD3 mAb (T + P815 + CD3 mAb). After 4 days of coculture at 37°C in the presence of 5% CO2, the cells were pulsed with 1 µCi of [3H]thymidine for 6–8 h, harvested, and then assessed for [3H]thymidine incorporation. Results are cpm ± SEM.

To test the ability of P815 cells bearing engrafted costimulatory molecules to induce antitumor responses in vivo, mice were immunized with P815 cells bearing the engrafted molecules to see whether this could stimulate CTL activity and/or affect tumor growth in syngeneic animals. Separate groups of DBA/2 mice were immunized with either PBS or with -irradiated P815 cells bearing engrafted EPOR-6H, B7.1-6H, or B7.1-6H plus CD40-6H. Two weeks after immunization, spleens were removed from the mice, and splenic T cells were isolated and assessed for their ability to kill native P815 cells in a standard ⁵¹Cr release assay. The data in Fig. 7 show that at all of the effector target cell ratios indicated (0.5:1, 1:1, 5:1), only a low level (2–5%) of lysis was induced by T cells from mice immunized with PBS (as control). The lytic activity of T cells from mice immunized with P815-EPOR (as control protein) was also low, ranging from 7 to 16%. Interestingly, at all E:T cell ratios tested, the level of tumor cell-specific lysis was higher for conditions where the effector T cells were derived from mice immunized with P815 cells bearing one or more engrafted costimulatory molecule(s) (see Fig. 7). The highest cytolytic activity was observed at the E:T cell ratio of 5:1, for which the specific lysis induced by T cells obtained from mice immunized with P815 cells bearing engrafted B7.1 and P815 cells with engrafted B7.1 and CD40 was 3- and 5-fold higher, respectively, than that for T cells obtained from mice immunized with P815 cells engrafted with control protein (see Fig. 7). Parallel experiments using native EL4 cells instead of P815 cells showed only background levels of lysis (data not shown), indicating that the cytolytic response was specific for P815 cells as targets. The results indicate that CTL responses against P815 cells can be generated in mice immunized with P815 cells bearing engrafted B7.1/CD40.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Induction of tumor-specific cytotoxicity in T lymphocytes isolated from mice vaccinated with tumor cells bearing engrafted costimulator molecules. Syngeneic DBA/2 mice were immunized s.c. with either PBS or 1 x 105 -irradiated P815 cells engrafted with the recombinant proteins EPOR-6H, B7.1-6H, and B7.1-6H plus CD40-6H, as indicated. Spleens were removed from the mice 14 days after immunization, and T lymphocytes (effector T cells) were isolated, suspended in incubation medium, and aliquoted into 24-well flat-bottom plates at a concentration of 1 x 105 cells/well, and then cocultured with 1 x 105 -irradiated native P815 cells. After 5 days of coculture at 37°C in the presence of 5% CO2, the cells were incubated with 51Cr-labeled native P815 cell targets for 6 h at 37°C at the indicated E:T ratio, before harvesting the supernatants and determining the amount of 51Cr released through specific lysis. Results are expressed as the percentage of specific lysis ± SEM, calculated as described in Materials and Methods.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Induction of tumor immunity by immunization with P815 tumor cells engrafted with recombinant costimulatory molecules. Mice were immunized by injection of either PBS or 1 x 105 -irradiated P815 cells engrafted with recombinant protein(s) including EPOR-6H, B7.1-6H, and B7.1-6H plus CD40-6H, as indicated. Two weeks after injection, the mice in each group were challenged with 1 x 105 native P815 cells by s.c. injection and then monitored for tumor growth and survival. Each point in A represents the mean tumor diameter for each group of mice as a function of time for the first 5 wk. The data in B show the percentage survival of the animals with time.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Experiments to optimize the incorporation of the NTA-DTDA lipid into the membrane of P815 cells indicated that incorporation was greater at 37°C (compared with 20°C) and when the NTA head group was chelated with a divalent metal cation; under these conditions, near-maximal incorporation of the lipid into P815 cells occurred at concentrations of 60 µM. Since the lipid was added to the cells in the form of a liposome suspension, these observations are consistent with the knowledge that cell membranes and the NTA-DTDA liposomes are more fluid at the higher temperatures (membrane transition temperature of NTA-DTDA is 25°C), and hence more likely for the NTA liposomes to fuse with the cell surface (11, 12). Also, the increased incorporation of the NTA-DTDA into the cell membrane when the lipid was loaded with divalent metal cations may be attributable to a reduction in the repulsive force between negative charges on the cell surface and the net negative charge on the NTA head group when not chelating metal ions like Zn²⁺ or Ni²⁺. Therefore, an equimolar concentration of Zn²⁺ and NTA-DTDA was used routinely in preparing liposomes for incorporation of NTA-DTDA into cells. Although no noticeable difference in the stability of the engrafted B7.1-6H and CD40-6H proteins was observed when using Ni²⁺ instead of Zn²⁺ as the chelated species (data not shown), the use of nickel salts was avoided in view of their purported toxicity.

Despite the fact that significant levels of NTA-DTDA lipid could be incorporated into P815 cells by a simple incubation of a suspension of the lipid with the cells in PBS, an examination of P815 cells bearing incorporated NTA-DTDA and engrafted B-CD40-6H by confocal microscopy indicated that a significant proportion of the cell-associated fluorescence (after staining with streptavidin-FITC) was bound to the cell surface in the form of liposomes or vesicles. An 2-fold increase in the incorporation of NTA-DTDA lipid into cells was achieved by preparing the NTA-DTDA liposomes as a mixture of NTA-DTDA and POPC. The reasons for this are unclear, but may reflect a greater fluidity of the NTA-DTDA-POPC liposomes (membrane transition temperature approximately -2°C for POPC and approximately 25°C for NTA-DTDA) and the fact that the oleic acid chain in POPC contains a double carbon-carbon bond which may facilitate membrane fusion by introducing stress points on the liposome surface (19). Further increases in the incorporation of the NTA-DTDA into cell membranes was achieved by treating the cells containing bound NTA-DTDA with the synthetic fusogenic polymer PEG, an agent widely used to induce cell-cell and vesicle-cell fusion (20). The mechanism(s) by which PEG induces membrane fusion is not well understood, but is thought to involve changes in membrane fluidity through alterations in osmotic gradients across the membranes, which upon resuspension of the cells in isotonic buffer may allow cell-bound liposomes to fuse with the cell membrane (20, 21).

The studies presented show clearly that membrane incorporated NTA-DTDA can be used to anchor one or more recombinant proteins bearing a suitable tag onto the surface of P815 cells. In studies not shown, we found that essentially the same procedure can be used for engrafting recombinant proteins onto the surface of other cell types including murine EL4 tumor cells and yeast cells, suggesting that the approach can be used to anchor suitably tagged molecules onto a number of different biological membrane systems. The approach thus should have wide applicability in the area of vaccine development, and in this paper is exemplified by the engrafting of costimulatory molecules onto P815 cells to enhance tumor immunity as discussed below.

A vast body of evidence suggests that the immune system, particularly T cells, can actively recognize and eliminate tumor cells, but an effective response against the tumor requires that tumor-specific T cells first become activated (22, 23). Efficient T cell activation requires two distinct signals (24), namely, the recognition by the TCR of antigenic peptides presented by MHC molecules on APCs and the engagement of costimulatory molecules, like B7.1 and CD40 on the APCs, by counter receptors on the T cell (25). The failure of some tumors to elicit an immune response may in part be explained by the failure of the tumor cells to express adequate levels of these costimulatory molecules (26). It is well established that the immunogenicity of tumor cells may be enhanced by genetically engineering them to express B7.1 and CD40 (5). Thus, in several murine tumor models, tumor cells transfected to express B7.1 and/or CD40 induce proliferation of tumor-specific T cells (3, 24). Moreover, the use of such transfected tumor cells as vaccines in syngeneic animals can induce protection of the host from subsequent challenge with the parental tumor and often eliminate the primary tumor in vivo (8).

Despite its usefulness, a number of factors limit the use of gene transfer techniques in a clinical setting. First, the gene transfer approach can be time consuming and inconvenient in terms of the production of the gene, its insertion into a foreign vector, and the selection of viable clones. Second, many cells, especially tumor cells, are not permissive to foreign DNA, thus making it difficult to achieve adequate levels of expression of the desired protein; a factor that may be critical in successfully eliciting an immune response. Third, through the process of insertional mutagenesis, the transfection process can potentially alter the expression of other genes, including those involved in oncogenic transformation or those important for the immunogenicity of the tumor cell (27). Fourth, unwanted immunological responses against the vector being used for gene transfection, similar to that reported against the vaccinia and adenovirus-based vectors (28), also is a possibility.

The studies described in this paper demonstrate that recombinant proteins bearing 6His tags can be engrafted onto the surface of cells after incorporation of the chelator lipid NTA-DTDA. The functionality and potential for use of the engrafted molecules to induce physiologically relevant responses was demonstrated by our showing that P815 cells bearing engrafted B7.1-6H and/or CD40-6H can be used to stimulate allogenic and syngeneic T cell proliferation in vitro (Fig. 6). That the stimulation of T cell proliferation observed in these studies reflects a specific effect of the P815 cell-anchored B7.1 and CD40 proteins is demonstrated by the fact that no significant stimulation of T cell proliferation occurred in cocultures of T cells with native P815 cells when these proteins were added in solution, either singly or in combination, at concentrations equivalent to those used to engraft the proteins onto P815 cells. That is, the B7.1-6H and CD40-6H were effective in stimulating T cell proliferation only when they were engrafted onto P815 cells via the NTA-DTDA, and there was no increase in T cell proliferation using P815 cells bearing the engrafted control protein (EPOR-6H). The results suggest that the effects are specific for the engrafted costimulatory molecules, that the engrafted recombinants B7.1-6H and CD40-6H are functionally active, and that tumor Ags on the modified P815 cells are still available for stimulating T cell proliferation in a syngeneic system. In addition, the extracellular region of B7.1 linked to a GPI anchor has been reported to be functionally active (6, 7), but previous studies have shown only that the extracellular region of murine CD40 (i.e., CD40-6H) can bind to activated T cells when in a multimeric complex with fluoresceinated dextran (29). The present work, therefore, also constitutes the first demonstration that the extracellular region of murine CD40 (when engrafted onto tumor cell membranes) is sufficient for enhancing T cell proliferation and other functional responses.

The present work also shows that P815 cells bearing engrafted B7.1 and CD40 can elicit antitumor responses in vivo. Thus, splenic T cells obtained from mice that had been immunized with -irradiated P815 cells bearing engrafted costimulator molecules induced a higher level of specific lysis toward native P815 targets in standard cytotoxicity assays. The data show that P815 cells engrafted with T cell costimulatory molecules, in addition to being able to stimulate T cell proliferation in vitro (Fig. 6), can stimulate the differentiation of T cells into functional CTL in vivo (Fig. 7). Interestingly, the level of cytolytic activity was significantly higher for T cells obtained from mice vaccinated with P815 cells engrafted with both B7.1 and CD40 than with B7.1 alone. It seems that the potentiating effect of CD40 observed may be attributable not only to increased costimulatory activity, but also to a prolongation of the activation state of reactive T cells after CD40-CD40 ligand ligation (30). Consistent with these findings, mice immunized with P815 cells engrafted with B7.1 with or without CD40 also showed enhanced tumor immunity as reflected by a slower rate of tumor growth and an increase in the time for which the animals survived after tumor challenge, with P815 cells bearing both B7.1 and CD40 being the most effective strategy for protection against challenge with native P815 cells.

The proportion of mice surviving a challenge with native P815 tumor cells following immunization with B7.1 gene-transfected P815 cells is reported to be up to 100%. Because transfected P815 cells often exhibit different size and growth properties compared with native P815 cells, a direct comparison between the two systems (i.e., between using P815 cells bearing engrafted costimulator molecules and gene-transfected P815 cells) is not straightforward and is dependent on the number of native tumor cells used for the tumor challenge, the number of modified cells used in the immunizations, the level of costimulator gene expression by the transfected P815 cells, and the level of costimulator molecules engrafted onto the P815 cells used. Although a direct comparison was not conducted in our study, a comparison of published data with the results presented herein would suggest that under the conditions used the survival of syngeneic mice immunized with P815 cells bearing engrafted B7.1 and/or B7.1 and CD40 may be somewhat less effective than that of mice immunized with P815 cells transfected to express B7.1. We acknowledge that the loss of costimulatory molecules from the P815 cell surface, as reflected by the loss observed following 24-h in vitro culture (see Fig. 5), could contribute to the apparently lower response observed in our studies (compared with studies using P815 cells transfected to express costimulatory molecules). Furthermore, our results indicate that the loss of engrafted molecules on the cell surface is likely to be due to receptor internalization. Therefore, it would seem that one way to overcome possible difficulties associated with the loss of costimulatory molecules may be to carry out the immunizations using P815 cell plasma membranes engrafted with the costimulatory molecules (rather than using intact P815 cells engrafted with the molecules); this is one area we are presently investigating.

In summary, the results presented in this paper suggest that the incorporation of chelator lipids like NTA-DTDA into tumor cell membranes, followed by the engraftment of recombinant costimulatory and/or other molecules (or combinations of molecules) with an appropriate tag, may be a convenient approach in the development of cell-based vaccines to enhance tumor immunity. Therefore, analogous to its demonstrated ability to alter tumor immunity, the technique also can be expected to provide a convenient approach to engraft specific costimulatory and/or other cell surface molecules (or combinations of such molecules) onto other cell types, including T cells, B cells, and dendritic cells, to see what role such molecules might play in regulating immune function. Therefore, in addition to its potential use in cancer immunotherapy, the technique described herein will have application to areas that could significantly enhance our understanding of immune function.

	Acknowledgments

 
We thank Robyn Watts for her expert assistance in the production of recombinant proteins.

	Footnotes

 
¹ This work was supported by Project Grant 971019 to J.G.A. from the National Health and Medical Research Council of Australia.

² Address correspondence and reprint requests to Dr. J. G. Altin, Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra ACT, 0200, Australia. E-mail address:

³ Abbreviations used in this paper: NTA, nitrilotriacetic acid; DTDA, ditetradecyl-amine; EMEM, Eagle’s MEM; NHS, N-hydroxysuccinimide; DOPE, ditetradecylamine; POPC, -palmitoyl-ß-oleoyl-phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; PEG, polyethylene glycol; suc, succinyl; EPOR, erythropoietin receptor; B, biotinylated.

Received for publication October 18, 1999. Accepted for publication December 23, 1999.

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