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[Cancer Research 64, 1502-1508, February 15, 2004]
© 2004 American Association for Cancer Research

Immunology

Enhanced Efficacy of Tumor Cell Vaccines Transfected with Secretable hsp70

Chiara Massa, Cristiana Guiducci, Ivano Arioli, Mariella Parenza, Mario P. Colombo and Cecilia Melani

Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor immunotherapy has exploited the ability of heat shock proteins to chaperone precursors of antigenic peptides to antigen-presenting cells and to activate efficiently an immune response against tumor-associated antigens. The most common strategy is based on the purification of heat shock protein-peptide complexes from tumor cell lines or from tumor surgical samples for in vivo administration. In this article, we have modified the murine-inducible hsp70 into a secreted protein and engineered tumor cells to secrete constitutively their antigenic repertoire associated with the hsp70 protein. In vitro studies showed that the relocalization of hsp70 from the cytoplasm to the secretory pathway did not modify the ability of hsp70 to interact with peptides derived either from natural tumor-associated antigens or model antigens, and that antigen-presenting cells specifically took up the secreted hsp70 and presented the chaperoned epitopes to T cells. In vivo studies showed that tumors secreting hsp70 displayed increased immunogenicity, with induction of a strong and specific CTL response. Mice injected with hsp70-secreting tumors showed increased survival and impaired tumor take compared with mice bearing parental tumors. More than 70% of mice rejected tumor cells secreting hsp70 through mechanisms that involve T lymphocytes and natural killer cells, with the induction of a memory response in the case of T lymphocytes. Moreover, hsp70 secretion increased the immunogenic potential of tumor cell vaccines.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chaperone proteins are key elements in the protein-folding machinery of the cell. Constitutively expressed chaperone proteins help newly synthesized proteins to fold properly and to reach their terminal destination inside the cell, whereas inducible chaperones act to avoid protein denaturation and aggregation during stress, thus preventing the triggering of an apoptotic pathway. The ability of chaperone proteins, commonly grouped as heat shock proteins (HSP), to interact with polypeptide chains provides them with a protective role not only at the single-cell level but also for the entire organism in response to pathologic situations such as viral infection and malignancy. De Leo and Srivastava (1) first identified HSP as the component of tumor lysates underlying the protection from a subsequent tumor challenge in mice immunized with the lysates. Subsequent studies correlated the specificity of HSP immunostimulatory activity with the chaperone activity of these proteins inside the cell (2) ; cellular peptides that remained associated with the HSP during the purification and fractionation steps were responsible for the antitumor immune response elicited by the HSP preparations (3, 4, 5) . The extreme efficiency with which HSP vaccines induce an immune response is caused by the presence of two types of specific receptors on antigen-presenting cells (APCs; Ref. 6 ); CD91 (7) and LOX1 (8) receptors are involved in the uptake and release of the HSP-chaperoned peptides into the MHC presentation pathways of the APC, whereas receptors such as TLR4 (9 , 10) and CD14 (11) transduce a danger signal through nuclear factor {kappa}B to up-regulate costimulatory molecules and induce secretion of proinflammatory cytokines. The adjuvant activity of HSP has been associated with the appearance of these proteins in the extracellular milieu in response to stress stimuli or cell necrosis (12) .

A common approach used in cancer immunotherapy to activate the host immune system uses tumor cells transfected with one of two different types of molecules: (a) molecules that enhance the direct interaction between tumor cells and lymphocytes, such as components of the MHC presentation pathways (13 , 14) or costimuli (15 , 16) ; and (b) molecules that recruit professional APC and promote antigen presentation to T lymphocytes (17 , 18) . The dual role of HSP as antigenic peptide chaperone and danger signal makes them especially useful in this second type.

In this study, we evaluated the possibility of enhancing the immunogenicity of tumor cell vaccines by inducing secretion of HSP from tumor cells, which allows direct interaction and loading of APC with antigenic peptides. Several tumor cell lines were transduced to express a secretable form of murine hsp70 constitutively. Despite the relocalization of hsp70 from the cytosol to the secretory pathway, engineered hsp70 retained its chaperone activity and specificity for interaction with APC. Tumor cells secreting hsp70 showed reduced tumorigenicity and increased immunogenicity in vivo as result of activation of innate and acquired immune responses. Our data show that secretion of hsp70, together with the associated peptides, improves the potency of tumor cell vaccines by increasing the availability of the antigenic peptides to APC. Such an increase induces a specific and robust T-cell response and an innate immune response to tumors by enhancing their susceptibility to natural killer (NK) cell recognition.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Cell Lines.
Female C57BL/6 (H-2b) and wild-type and BALB/c-nu/nu (H-2d) mice were obtained from Charles River Laboratories (Calco, Italy) and used at 8–10 weeks of age. Mice were maintained at the Istituto Nazionale per lo Studio e la Cura dei Tumori under standard conditions according to institutional guidelines.

C26 murine colon adenocarcinoma cells were derived from BALB/c mice treated with N-nitroso-N-methylurethane (19) . MCA38 is a colon adenocarcinoma induced in mice on a C57BL/6 background by treatment with 1,2-dimethylhydrazine dihydrochloride (19) . F1 is a fibrosarcoma cell line that developed spontaneously in vitro from BALB/c newborn mouse fibroblasts and does not express known antigens. This cell line has been transfected with model tumor antigens such as the human {alpha}-folate receptor ({alpha}FR) (20) and the murine leukemia virus (MuLV) env genes (21) . Tumor cell lines were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD), 100 units/ml of penicillin, 100 units/ml of streptomycin, and 2 mM L-glutamine and kept at 37°C in a 5% CO2 atmosphere.

RAW264.7 is a macrophage cell line (clone TIB-71; American Type Culture Collection, Manassas, VA) cultured in complete DMEM medium. Bone marrow (BM)-derived dendritic cells (DC) were prepared as described (22) .

E88 is an H-2Ld-restricted T-cell clone specific for the AH1 epitope of the MuLV env protein (23) , whereas the T-cell line IF1 recognizes the {alpha}FR antigen (24) . Lymphocytes were cultured in complete RPMI medium (RPMI supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, 100 units/ml of streptomycin, 2 mM L-glutamine, and 50 nM ß-mercaptoethanol) and stimulated weekly with 25 units/ml of recombinant interleukin 2 (rIL-2; Chiron, Emeryville, CA) and irradiated tumor cells.

Construction of Expression Vector for Secreted hsp70.
The complete coding sequence of the murine-inducible hsp70 was obtained from the pcDNA3-hsp70Myc plasmid vector (provided by Dr. R. Vile; Ref. 25 ). The genomic sequence of the constant domain of the murine immunoglobulin light chain kappa (C{kappa}) was derived from the pHPCR{gamma}III vector (26) . PCR was used to amplify the hsp70 and C{kappa} coding sequence and to introduce the following modifications: a HindIII site upstream of the start codon of hsp70 introduced by the hsp70 forward primer (5'-cgaagcttggcgccatggccaagaac-3') and a BamHI site with removal of the stop codon introduced by the hsp70 reverse primer (5'- cgtggatccacctcctcgatggtg-3'); the C{kappa} forward primer (5'-cgggatccaggggctgatgctgca-3') removes the splicing acceptor site of the C{kappa} sequence and introduces a BamHI site in the correct reading frame, whereas the C{kappa} reverse primer (5'-ccaatgcatgtctctaacactcatt-3') introduces an NsiI site after the stop codon. The commercial vector Signal pIg Plus (R&D System, Minneapolis, MN), which contains the leader sequence of the human CD33 antigen upstream of the multiple cloning sites, was used to clone sequentially the C{kappa} (pSignalC{kappa}) and hsp70 coding sequence (pSignalhsp70C{kappa}). The resulting vectors were fully sequenced using the ABI Prism kit (PerkinElmer, Boston, MA) according to the manufacturer’s instructions.

Construct Characterization by Transient Transfection of COS-7 Cells.
COS-7 cells were transfected transiently with the pSignalhsp70C{kappa} plasmid using the DEAE-dextran technique. Briefly, cells were seeded in 10-cm tissue culture plates and incubated with 0.5 mg/ml DEAE-dextran and 10 µg of plasmid DNA for 30 min, and 100 ng/ml chloroquine were added. After 2 h, medium was replaced with DMEM containing 10% DMSO; incubation continued for 2 min, and medium was replaced with fresh DMEM. Supernatants were collected 48 h later, and cells were lysed in 10 mM Tris HCl, 0.15 M NaCl, 1 mM EDTA, and 1% NP40, which was added to a mixture of protease inhibitors (10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride).

Cell lysates (50 µg of total protein) and supernatants (30 µl) were denatured by heating at 100°C for 5 min, loaded on an 8% SDS-PAGE gel, separated under reducing conditions, and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Filters were blocked overnight with 4% BSA and incubated with polyclonal rabbit anti-hsp70 antibody (Ab; 1:10,000; StressGene, Victoria, Canada) followed by antirabbit immunoglobulin horseradish peroxidase- conjugated Ab (1:20,000; Calbiochem, San Diego, CA). Blots were developed by incubation in enhanced chemiluminescence reagent and exposed to film (both from Amersham Pharmacia Biotech, Piscataway, NJ).

Tumor Cell Line Transfection and Characterization.
Tumor cell lines were transfected with pSignalhsp70C{kappa} and pSignalC{kappa} by standard calcium phosphate coprecipitation, and different colonies were selected in medium containing G418 (0.8 mg/ml; Life Technologies). Hsp70C{kappa} secreted from 106 cells/ml/24 h was assayed by sandwich ELISA using antibodies against the C{kappa} tag (Southern Biotechnologies Associates Inc., Birmingham, AL). Similarly, tumor cells were transfected with the model tumor antigen {alpha}FR or MuLV env, cloned in the expression vector pcDNA3.1H, and selected in 1 mg/ml hygromycin (Calbiochem). To detect expression of the model antigens, tumor cells were analyzed by immunofluorescence using the monoclonal MOV18 Ab (27) followed by antimouse immunoglobulin-FITC Ab for the {alpha}FR, whereas MuLV env expression was tested by the monoclonal 35/299 Ab (provided by Dr. D. Pardoll) followed by antirat immunoglobulin-FITC Ab. A minimum of 10,000 events was acquired using fluorescent activated cell sorter (FACScan) cytometer and analyzed with CellQuest software (both from BD Bioscience, Mountain View, CA).

In Vitro Interaction between hsp70C{kappa} and APC: Uptake and Chaperoning.
To evaluate hsp70C{kappa} interaction with APCs, 5 x 105 BM-DCs were incubated with 1 ml of supernatant collected from transfected tumor cells. After 1 h of incubation at 4°C, the APCs were washed extensively and stained with the biotinylated polyclonal anti-C{kappa} Ab (1:5000) followed by streptavidin-phycoerythrin (1:100; PharMingen, San Diego, CA) to evaluate binding.

For uptake experiments, BM-DC and RAW264.7 macrophage cell lines were cultured overnight with tumor supernatant at 37°C. After extensive washing, cells were fixed in 2% formaldehyde and permeabilized with 0.5% saponin before staining.

To evaluate chaperone activity of hsp70C{kappa}, pulsed APCs were incubated with specific T-cell lines, and IFN-{gamma} release as an indicator of T-cell activation was evaluated in the supernatant by sandwich ELISA (PharMingen) after 20 h of coincubation.

Cell-Mediated Cytotoxicity Assay.
Naive mice of the appropriate haplotype were inoculated into the footpad with 5 x 106 {gamma}-irradiated (15,000 rad) transfected or parental tumor cells. After 5 days, popliteal lymph nodes were removed aseptically, and single-cell suspensions were obtained by mechanical disruption. Lymphocytes were restimulated in a mixed lymphocyte tumor culture with the irradiated parental tumor cells (1:10 ratio) and 20 units/ml of rIL-2 in complete RPMI medium. After 5 days, cytotoxic activity was tested in a standard 4-h 51Cr release assay against antigen-positive and -negative cells. Blast cells obtained from splenocytes cultured for 72 h with 2 µg/ml concanavalin A were pulsed with 1 µg of specific peptides while labeling with 51Cr. Percent-specific lysis was calculated as 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release), where spontaneous release (never exceeding 10%) was obtained from target cells incubated in medium alone, and maximum release was obtained from incubation in 1% NP40.

In Vivo Tumorigenesis Assay.
Parental and hsp70C{kappa}- or C{kappa}-transfected tumor cells were injected into groups of syngeneic immunocompetent or immunodeficient mice. The percentage of surviving mice among total injected mice was monitored at the indicated time points. Each experiment was performed with seven mice/group (five mice when nu/nu mice were used) and repeated at least three times.

Lymphokine-Activated Killer Cell Assay.
Splenocytes from naive BALB/c mice were cultured in complete RPMI medium with 500 units/ml of rIL-2. After 2 days, lymphokine-activated killer cells were used as effectors in a standard 51Cr release assay against hsp70C{kappa}-secreting or parental tumor cells.

Cell-Based Immunotherapy of Experimental Lung Metastases.
To induce lung metastases, BALB/c mice were injected i.v. with 104 {alpha}FR-transfected C26 tumor cells (C26{alpha}FR) on day 0. Immunotherapy was started on day 1 and repeated on days 3, 8, and 10 by s.c. injection of 2 x 106 irradiated F1{alpha}FR or F1hsp70C{kappa}{alpha}FR cells. Mice were killed when they displayed respiratory symptoms; surviving mice were considered cured 3 months after the end of the treatment. Statistical analysis was performed using the log-rank test and considered significant at P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction and Characterization of a Secreted Form of hsp70C{kappa}.
The sequence encoding the inducible form of murine hsp70 was cloned downstream of the human CD33 leader sequence and upstream the sequence of the constant domain of the murine immunoglobulin C{kappa} that is used as a nonimmunogenic tag for easier identification of the chimeric protein. The resulting pSignalhsp70C{kappa} vector (Fig. 1A)Citation was transfected into COS-7 cells to characterize the chimeric protein. Western blot hybridization of COS-7 cell supernatants confirmed the retargeting of hsp70C{kappa} into the secretory pathway (Fig. 1B)Citation . No other forms of hsp70 were detected in the supernatant, whereas the endogenous form and the chimeric hsp70C{kappa} were identified in COS-7 cell lysates (Fig. 1C)Citation . ELISA performed on COS-7 supernatants using a capture Ab against hsp70 and a detection Ab against C{kappa} allowed detection of the protein, suggesting the correct conformation of both moieties (data not shown).



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  		Fig. 1. Construction and characterization of secreted hsp70C{kappa}. A, schematic of the vector pSignalhsp70C{kappa} with the cytomegalovirus (CMV) promoter, the leader sequence of the human CD33 antigen, the complete coding sequence of the murine-inducible hsp70, and the constant domain of the immunoglobulin light chain kappa (C{kappa}). B and C, Western blot analyses of COS-7 supernatant (B) and lysates (C) hybridized with polyclonal anti-hsp70 antibody (Ab). Lane 1, molecular size marker (kDa); Lane 2, 50 ng rhsp70; Lane 3, pSignalhsp70C{kappa}-transfected COS-7 cells; and Lane 4, mock-transfected COS-7 cells. D, levels of hsp70C{kappa} secreted by the different tumor cell lines. Supernatant from 106 cells cultured for 24 h in 1 ml medium was assayed using ELISA against the C{kappa} tag. E, fluorescence-activated cell sorter (FACS) analysis of F1{alpha}FR (gray line) and F1hsp70C{kappa}{alpha}FR (bold line) clones for the expression of transfected model antigen stained with the MOV18 Ab. F, FACS analysis of murine leukemia virus env antigen expression in F1env (gray line) and F1hsp70C{kappa}env (bold line) detected with 35/299 Ab. Broken lines represent isotype control Ab.

 
Transfection of Tumor Cell Lines with pSignalhsp70C{kappa}.
C26, MCA38, and F1 tumor cells were transfected with pSignalhsp70C{kappa} or, as control, with the same vector lacking hsp70 (pSignalC{kappa}). Using antibodies against the C{kappa} tag, G418-resistant colonies were screened by ELISA. The levels of hsp70C{kappa} expression (evaluated as ng/ml of C{kappa} secreted by 106 cells in 24 h) differed greatly among the three cell lines (Fig. 1D)Citation . Because F1 fibrosarcoma cells secreted the highest amounts of hsp70C{kappa} but did not express known antigens, F1, F1hsp70C{kappa}, and F1C{kappa} cells were transfected with the model antigens human {alpha}FR, which is overexpressed commonly in human ovarian carcinoma and acts as an antigen in a murine setting (20) , and MuLV env, which is the common tumor-associated antigen (TAA) of C26 and MCA38 carcinomas (21) . Different {alpha}FR- and MuLV env-expressing clones were isolated by limiting dilution and assayed for antigen expression as described in "Materials and Methods." As shown for the two clones mostly used throughout the experiments, namely, F1hsp70C{kappa}{alpha}FR/1g12 and F1hsp70C{kappa}env/2a5, double transfectants secreted lower levels of hsp70C{kappa} (Fig. 1D)Citation and had reduced expression of the model antigen compared with singly transfected cells (Fig. 1, E and F)Citation .

APCs Internalize Secreted hsp70C{kappa} in Vitro.
To determine whether secreted hsp70C{kappa} interacts specifically with APC and can be internalized, different cell types were incubated in vitro with supernatants from transfected cells and stained as described in "Materials and Methods." Hsp70C{kappa} from cell supernatant, but not an unrelated protein containing the same C{kappa} tag (IL2MOV19C{kappa}; Ref. 26 ), bound to BM-DC after 1 h of incubation at 4°C (Fig. 2A)Citation and was internalized after an 18-h incubation at 37°C (Fig. 2B)Citation . Uptake experiments also were performed with RAW264.7 macrophage cell line that showed specific uptake of hsp70C{kappa} in its native conformation (Fig. 2C)Citation but not after heat denaturation (Fig. 2D)Citation . Because the anti-C{kappa} polyclonal Ab recognizes native and denatured antigen, this lack of RAW264.7 cell staining reflects the inability of denatured hsp70C{kappa} to interact with specific receptors on APCs.



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  		Fig. 2. Interactions between secreted hsp70C{kappa} and antigen-presenting cells in vitro. A and B, bone marrow dendritic cells were incubated with supernatant from F1hsp70C{kappa} (bold line) or with IL2MOV19C{kappa} (dotted line) for 1 h at 4°C (A) or for 18 h at 37°C (B) and then were stained with the anti-C{kappa} antibody (Ab) as described in the "Materials and Methods." C and D, RAW264.7 cells were incubated for 18 h at 37°C with naive (C) or heat-denatured supernatant (D) and stained with the anti-C{kappa} Ab (bold line) or isotype-matched Ab control (dotted line). E and F, RAW264.7 cells previously incubated with supernatant from the indicated tumors or medium only were cultured with T-cell line IF1 (E) or T-cell clone E88 (F). After 18 h, supernatants were collected and assayed for IFN-{gamma} production by ELISA.

 
From these experiments, we conclude that interaction of secreted hsp70C{kappa} with APCs is specific, conformation-dependent, and independent of the C{kappa} tag.

Cross-Presenting Activity of hsp70C{kappa}.
One consequence of HSP interaction with APCs is the introduction of the chaperoned peptides into the MHC presentation pathway. To evaluate the cross-priming activity of APCs following hsp70C{kappa} uptake, RAW264.7 cells were pulsed with supernatants from tumors secreting hsp70C{kappa} and expressing or not expressing {alpha}FR or MuLV env, and incubated with specific T-cell clones or lines recognizing such antigens. Only APCs pulsed with supernatant from antigen-expressing and hsp70C{kappa}-secreting cells activated a T-cell response, as evaluated based on IFN-{gamma} production; no T-cell activation was detected when hsp70C{kappa} was secreted by antigen-negative tumor cells (Fig. 2, E and F)Citation . Thus, retargeting of hsp70 into the secretory pathway did not alter its ability to chaperone immunogenic peptides.

Hsp70C{kappa} Secretion Increases Tumor Immunogenicity.
The immunogenicity of tumors secreting hsp70C{kappa} was evaluated based on the induction of a cytotoxic response in the draining lymph node of mice immunized with irradiated cells either secreting or not secreting hsp70C{kappa}. Mice immunized with hsp70C{kappa}-secreting tumor cells displayed a stronger and specific cytotoxic response than did mice immunized with parental cells, as evaluated using antigen-positive and -negative target cells (Fig. 3)Citation . Blast cells pulsed with peptides corresponding to the known epitopes of the MuLV env protein restricted to the H-2Ld (AH1) and H-2Kb (KSP) molecules were used to detect the response against specific epitopes of the endogenous TAA of the two colon carcinoma cell lines (Fig. 3, A and BCitation , respectively). The specificity of the response to the {alpha}FR antigen, which contains two different epitopes restricted to the H-2Kd and the H-2Dd molecules (24) , was evaluated using two different congenic fibroblast cell lines transfected with the {alpha}FR gene and expressing one of the two H-2d molecules, namely, (B6x5R){alpha}FR cells for H-2Kd and (B6xHTG){alpha}FR for H-2Dd, as target cells (Fig. 3C)Citation . F1hsp70C{kappa}{alpha}FR induced a strong CTL response against H-2Kd and H-2Dd target cells (Fig. 3C)Citation , suggesting that secreted hsp70C{kappa} chaperoned {alpha}FR-associated peptides restricted by both MHC molecules.



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  		Fig. 3. Secreted hsp70C{kappa} primes an enhanced CTL response. Syngeneic mice were immunized in the footpad with irradiated parental ({square}) or hsp70C{kappa}-transfected ({bullet}) C26 (A), MCA38 (B), or F1{alpha}FR (C) cells, and lymphocytes from the draining popliteal lymph node were tested for cytotoxic activity against the indicated target cells in a standard 4-h 51Cr release assay. The results shown are representative of one of three independent experiments.

 
Transfected Tumor Cell Lines Secreting hsp70C{kappa} Have Reduced Tumorigenicity.
To determine whether hsp70C{kappa} secretion affects tumorigenicity in vivo, mice were injected with live, transfected or nontransfected tumor cells by different routes, and survival of mice was monitored (Fig. 4)Citation . Despite the C26hsp70C{kappa}-induced CTL response in vitro, the survival of mice injected with C26hsp70C{kappa} was not significantly different from that of mice injected with the parental C26 tumor (Fig. 4A)Citation , probably because of the aggressiveness of the C26 carcinoma and the low amount of secreted hsp70. The take of MCA38hsp70C{kappa}, which secretes higher amounts of hsp70C{kappa}, was reduced, and 15% of mice injected s.c. (Fig. 4B)Citation and 50% of mice injected intradermally survived (Fig. 4C)Citation , whereas mice injected i.v. had statistically significant prolonged survival compared with control mice (P < 0.005; Fig. 4DCitation ). Four different clones of F1hsp70C{kappa}{alpha}FR, releasing the highest levels of hsp70C{kappa}, were tested for tumorigenicity in BALB/c mice. All of the clones, as shown for clone 1g12, had reduced tumorigenicity in vivo; at least 50% of mice survived after s.c. injection (Fig. 4E)Citation and after i.v. injection in the tail vein (Fig. 4F)Citation . These data indicate that the reduced tumorigenicity of hsp70C{kappa}-secreting cell lines correlates with the amount of secreted hsp70C{kappa} and varies according to the site of tumor growth. We disregard the possibility of transfection artifacts because all of the transfected cells grew equally in vitro (data not shown), and control cells transfected with pSignalC{kappa} showed the same tumorigenicity of parental cells in vivo (data not shown).



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  		Fig. 4. Tumor cells secreting hsp70C{kappa} display reduced tumorigenicity. Parental ({square}) and hsp70C{kappa}-transfected cells ({bullet}) were injected into syngeneic mice (n = 7) at the minimum lethal dose by different routes, and mouse survival was monitored. A, 5 x 104 C26 cells injected s.c. B, 105 MCA38 cells injected s.c. C, 105 MCA38 cells injected intradermally. D, 105 MCA38 cells injected i.v. E, 5 x 103 F1{alpha}FR injected s.c. F, 5 x 103 F1{alpha}FR injected i.v. The data shown are representative of one of three independent experiments with similar results.

 
Role of T Lymphocytes in the Control of hsp70C{kappa}-Secreting Tumors.
To evaluate the role of specific effector cells in reducing the tumorigenicity of F1hsp70C{kappa}{alpha}FR tumor, parental and transfected cells were injected either into wild-type or BALB/c-nu/nu mice. Whereas the parental tumor grew similarly in the two strains (Fig. 5A)Citation , F1hsp70C{kappa}{alpha}FR was no longer rejected in nu/nu mice (Fig. 5A)Citation . However, the prolonged survival observed in immunodeficient mice injected with F1hsp70C{kappa}{alpha}FR compared with F1{alpha}FR suggests that effectors of the innate immunity may participate in the rejection of hsp70-secreting tumor.



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  		Fig. 5. Tumor cells secreting hsp70C{kappa} induce an antitumor memory response and show enhanced susceptibility to natural killer-like lysis. A, BALB/c wild-type ({square}; n = 7) and nu/nu ({triangleup}) mice (n = 5) were injected s.c. with 5 x 103 F1{alpha}FR (open symbols) or F1hsp70C{kappa}{alpha}FR (filled symbols), and mouse survival was monitored. B, splenocytes from two naive mice (open symbols) and from six mice that had rejected F1hsp70C{kappa}{alpha}FR tumor cells s.c. (filled symbols) were assayed for cytotoxic activity against F1{alpha}FR cells in a standard 4-h 51Cr release assay. C, BALB/c mice were injected s.c. with 5 x 103 F1{alpha}FR (open symbols) or F1hsp70C{kappa}{alpha}FR (filled symbols; n = 7). Mice that survived were challenged with 104 F1{alpha}FR live cells and monitored for survival. D, lymphokine-activated killer cells obtained from BALB/c splenocytes cultured with recombinant interleukin 2 (500 units/ml) for 2 days were tested for cytotoxicity against F1{alpha}FR (open symbols) or F1hsp70C{kappa}{alpha}FR (filled symbols) or Yac cells (cross symbols). Results are from one of three experiments with similar results.

 
F1hsp70C{kappa}{alpha}FR Induces a Memory Response against the {alpha}FR Antigen.
To characterize further the role of T lymphocytes in the rejection of hsp70C{kappa}-secreting tumor, we evaluated whether mice that rejected the F1hsp70C{kappa}{alpha}FR had developed a memory response assayed as presence of memory CTL in their spleen and an ability to reject a secondary challenge with live F1{alpha}FR cells. Spleens were collected 70–90 days after primary challenge, restimulated in vitro, and tested for CTL activity against F1{alpha}FR. Two-thirds of the mice showed specific CTL lysis (Fig. 5B)Citation , although with different intensity among individual mice. Seventy percent of another group of mice that survived the primary F1hsp70C{kappa}{alpha}FR tumor rejected a challenge of live F1{alpha}FR cells (Fig. 5C)Citation . Mice surviving this secondary challenge were tested for CTL activity, and all of them displayed {alpha}FR-specific CTL in their spleen 60 days after the challenge (data not shown). Thus, in ~70% of mice that survived the primary F1hsp70C{kappa}{alpha}FR tumor injection, the rejection was associated with the development of memory lymphocytes that persisted for >2 months and was able to protect them from a subsequent tumor challenge. No evidence of memory response was found in the remaining 30% of mice.

Susceptibility of F1hsp70C{kappa}{alpha}FR Cells to NK-Like Lysis.
Recent reports have indicated that hsp70 on the tumor cell surface can be recognized by NK cells (28, 29, 30) . FACS analysis of F1hsp70C{kappa}{alpha}FR cells revealed a positive membrane staining for C{kappa}, indicating that some of the hsp70C{kappa} is retained at the cell surface (data not shown). Analysis to determine whether F1hsp70C{kappa}{alpha}FR cells were more susceptible to NK-like lysis showed that lymphokine-activated killer cells, obtained from naive BALB/c splenocytes cultured with high levels of rIL-2, did display an increased ability to lyse F1hsp70C{kappa}{alpha}FR compared with F1{alpha}FR cells (Fig. 5D)Citation . Because both cell types express similar levels of MHC-I molecules on their surface, these data suggest that membrane-associated hsp70C{kappa} can improve tumor cell recognition by the effector cells of the innate immune response (data not shown).

Secretion of hsp70C{kappa} Improves the Efficacy of Cell-Based Immunotherapy.
To evaluate the increased immunogenicity of tumor cells secreting hsp70C{kappa} in a therapeutic setting, experimental lung metastases were induced in BALB/c mice by i.v. injection of 104 C26{alpha}FR cells, and vaccination therapy was carried out by s.c. injection of irradiated cells. Whereas treatment with F1 cells expressing the shared model antigen {alpha}FR did not improve mouse survival, immunotherapy with F1hsp70C{kappa}{alpha}FR cells cured 60% of treated mice (P < 0.005; Fig. 6Citation ).



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  		Fig. 6. Therapy of mouse lung metastases with F1hsp70C{kappa}{alpha}FR. BALB/c mice (n = 14) were injected i.v. with 104 cells of C26{alpha}FR on day 0 and vaccinated s.c. on days 1, 3, 8, and 10 with 2 x 106 irradiated F1{alpha}FR ({square}) or F1hsp70C{kappa}{alpha}FR cells ({bullet}) or were left untreated (solid line). *P < 0.005.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The discovery that chaperone proteins have immunostimulatory activity has led to several efforts to exploit HSP for tumor immunotherapy. Initial studies focused on endogenous HSP purified from tumor cells and their injection in vivo as vaccine. Despite encouraging results with this strategy in the experimental setting (31 , 32) and in clinics (33 , 34) , HSP purification from tumor cells is a labor-intensive and time-consuming procedure. Other strategies have involved purification of HSP from bacterial cells or normal tissue followed by association with an antigenic peptide in vitro (35 , 36) , or direct fusion into a chimeric protein with an antigenic molecule or peptide (37 , 38) . Drawbacks of these strategies include the requirement for known antigens to be associated with the HSP and a consequent reduction of the antigenic repertoire to peptides derived from a few selected proteins, with possible consequences in case of antigen loss or mutation. Moreover, bacterial products potentially contaminating recombinant and purified HSP may be responsible for part of the immunologic activity of the vaccine (39 , 40) .

Modification of HSP content in tumors through gene therapy approaches preserves the primary advantage of purified HSP (i.e., the broad spectrum of chaperoned peptides that includes unknown antigens) without the cumbersome procedure of HSP purification. However, transfection of tumor cell lines with HSP coding sequences has given contradictory results. In some experimental models, overexpression of HSP increased the immunogenicity of transfected tumor cells (25 , 41) , whereas in other models, it increased tumorigenicity (42) . This discrepancy can be explained by the protective activity of HSP, which helps to prevent cells from triggering apoptosis in response to stressful stimuli (43) , and indeed tumors frequently display increased HSP content (44 , 45) . Studies showing that a reduction in HSP content of a tumor by antisense techniques favors the induction of apoptosis (46) and impairs tumor progression (47, 48, 49) support this explanation. To avoid the possible protumorigenic activity of transfected HSP, their intracellular localization has been modified. The endoplasmic reticulum-resident gp96 has been engineered to be secreted (50 , 51) or surface associated (52) , and cytosolic hsp70 has been converted into a transmembrane protein (53) ; these modified forms of HSP maintain the ability to activate the immune response against parental tumor cells.

We modified murine hsp70, one of the most potent HSPs (54) , such that it is secreted constitutively by tumor cells in the extracellular milieu, where it fosters direct interaction of the antigenic repertoire with immunocompetent cells without enhancing tumor survival. Introduction of a leader sequence upstream of the cDNA for murine-inducible hsp70 and a C{kappa} tag at the other end resulted in constitutive secretion of the chimeric protein. Molecular characterization revealed that the retargeting of hsp70 from the cytosol to the endoplasmic reticulum did not change the biochemical and immunologic properties of the protein. In vitro analysis indicated that the hsp70 moiety of chimeric hsp70C{kappa} maintained the ability to interact with APC in a specific manner, thus allowing protein internalization. Most importantly, chimeric hsp70 has maintained the chaperone activity essential for its immunologic properties. The macrophage line RAW264.7 stimulated specific T-cell clones only when pulsed with supernatant from tumor cells expressing the specific antigen and secreting hsp70C{kappa}.

With a view toward using hsp70 secretion as a means to improve the potency of a cellular vaccine, we initially evaluated the ability of parental and transfected tumor cells to induce a CTL response. Immunization with irradiated tumor cells secreting hsp70C{kappa} induced a stronger CTL response against the TAA than did the parental tumor. Despite the lower antigen expression levels, F1hsp70C{kappa}{alpha}FR induced a stronger CTL response than F1{alpha}FR, underlining the efficiency of hsp70C{kappa} to export {alpha}FR peptides and to provide them to APC. Analysis of tumors expressing endogenous TAA, such as C26hsp70C{kappa} and MCA38hsp70C{kappa}, gave analogous results.

The ability of hsp70C{kappa} to induce a CTL response appeared to be dose independent, and a low level of secreted hsp70C{kappa} was sufficient; despite the 3-log difference between C26 and F1{alpha}FR cells in hsp70C{kappa} secretion, both cell types were more potent than their corresponding parental counterparts in inducing a CTL response in vitro.

Conversely, the level of tumor-secreted hsp70C{kappa} was a determinant of the in vivo growth of live tumor cells. Tumorigenicity was comparable in C26hsp70C{kappa} cells, which produced low levels of secreted hsp70C{kappa} but nonetheless showed increased immunogenicity, and in parental C26 cells. A modest reduction in tumorigenicity of MCA38hsp70C{kappa} cells was detected after s.c. and i.v. injection and was more pronounced after injection into the dermal compartment, where a higher number of APCs is likely available to interact with extracellular HSP (55 , 56) . In F1hsp70C{kappa}{alpha}FR cells, which secrete higher levels of hsp70C{kappa}, tumorigenicity was reduced drastically not only after s.c. injection but also after i.v. injection, a site where purified HSPs are unable to induce protection against a tumor challenge (55) . The reduced tumorigenicity of the hsp70C{kappa}-secreting tumor was immunomediated because the effect was lost when the injection was performed in immunodeficient mice. Like parental cells, F1hsp70C{kappa}{alpha}FR cells produced tumors in 100% of injected BALB/c-nu/nu mice, although with slower kinetics. The increased susceptibility of F1hsp70C{kappa}{alpha}FR to NK-like lysis in vitro and the previous report of enhanced NK cell recognition of tumor cells expressing membrane-associated hsp70C{kappa} molecules suggest a role for effectors of the innate immune response also in vivo (29 , 30) . NK cells may contrast the initial tumor take, but in the absence of specific effector T cells, they are unable to induce a complete rejection. The important role of T lymphocytes was confirmed by the presence of a TAA-specific memory response in a percentage of mice surviving the primary injection with hsp70C{kappa}-secreting tumor cells.

Thus, a scenario can be envisioned whereby tumor-secreted hsp70C{kappa}, mimicking a necrotic event (12) , acts as a danger signal and concurrently chaperones TAA into APC, whereas cell surface-associated hsp70C{kappa} recruits and activates NK cells to kill the tumor (29) and additionally provides tumor cell debris and cytokines to APCs (57 , 58) to allow CTL activation. However, interaction with NK cells has been shown to amplify cytokine release by DC or to mediate DC killing, depending on the relative number of the two cell types, thus determining the possibility of inducing memory T cells (59) .

The ability of secreted hsp70C{kappa} to induce a specific immune response may provide a new tool for tumor immunotherapy, particularly for cell-based vaccination strategies. One such strategy aims to introduce into the cellular vaccine molecules that can overcome the immunosuppressive milieu induced by the tumor. Before in vivo injection, tumor cell vaccines must be irradiated, a procedure that induces apoptosis. In light of the physiologic role of apoptosis, "true" apoptotic bodies are not associated with "danger signals" (60, 61, 62) and thus may induce tolerance or even immunosuppression rather than activation of the immune system (63) . However, a specific immune response against antigens derived from apoptotic cells can be obtained when apoptosis is associated with a danger signal such as viral infection (64) , stress (65) , or dying cells previously transfected with a potent immunostimulatory molecule(s). Thus, transfection of tumor cells with a gene encoding secreted HSP may eliminate the need to purify the chaperone protein from tumors while conferring a danger signal to an apoptotic cell vaccine to favor immune activation. Moreover, transfected tumor cells can combine the potential of HSP complexes and cell-based vaccines. HSPs and whole tumor cells require different immune effector cells for their immunologic activity, and their combination in therapy may produce synergistic effects even in patients with immunologic deficit (66) .

In our therapeutic application of F1hsp70C{kappa}{alpha}FR to treat mice bearing C26{alpha}FR lung metastases, up to 60% of treated mice were cured, whereas F1{alpha}FR-irradiated cells failed to have a curative effect.

Thus, our data demonstrate that hsp70C{kappa} secretion leads to potent activation of the innate and the acquired immune response, with possible development of a memory response able to counter tumor relapse, as well as its potential to improve the efficacy of cell-based vaccine in a therapeutic setting. Because of the possible technical difficulties to transfect tumor cells ex vivo, we currently are investigating adenoviral vector to introduce the gene for secreted hsp70C{kappa} directly into primary tumors.


   FOOTNOTES
 
Grant support: Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondo per gli Investimenti della Ricerca di Base (FIRB).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Mario P. Colombo, Immunotherapy and Gene Therapy Unit, Istituto Nazionale Tumori, via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390-2252; Fax: 39-02-2390-2630; E-mail: mcolombo@istitutotumori.mi.it.

Received 9/17/03. Revised 11/10/03. Accepted 12/10/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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