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The Journal of Immunology, 2006, 177: 5946-5955.
Copyright © 2006 by The American Association of Immunologists, Inc.

Enhanced Immunogenicity of Heat Shock Protein 70 Peptide Complexes from Dendritic Cell-Tumor Fusion Cells1

Yutaka Enomoto2,*, Ajit Bharti2,*, Ad Abdul Khaleque{dagger}, Baizheng Song*, Chunlei Liu*, Vasso Apostolopoulos{ddagger}, Pei-xiang Xing{ddagger}, Stuart K. Calderwood{dagger} and Jianlin Gong3,*

* Department of Medicine, Boston University School of Medicine, Boston, MA 02118; {dagger} Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and {ddagger} Austin Research Institute, Heidelberg, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have developed a molecular chaperone-based tumor vaccine that reverses the immune tolerance of cancer cells. Heat shock protein (HSP) 70 extracted from fusions of dendritic (DC) and tumor cells (HSP70.PC-F) possess superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells. More importantly, immunization of mice with HSP70.PC-F resulted in a T cell-mediated immune response including significant increase of CD8 T cells and induction of the effector and memory T cells that was able to break T cell unresponsiveness to a nonmutated tumor Ag and provide protection of mice against challenge with tumor cells. By contrast, the immune response to vaccination with HSP70-PC derived from tumor cells is muted against such nonmutated tumor Ag. HSP70.PC-F complexes differed from those derived from tumor cells in a number of key manners, most notably, enhanced association with immunologic peptides. In addition, the molecular chaperone HSP90 was found to be associated with HSP70.PC-F as indicated by coimmunoprecipitation, suggesting ability to carry an increased repertoire of antigenic peptides by the two chaperones. Significantly, activation of DC by HSP70.PC-F was dependent on the presence of an intact MyD88 gene, suggesting a role for TLR signaling in DC activation and T cell stimulation. These experiments indicate that HSP70-peptide complexes (PC) derived from DC-tumor fusion cells have increased their immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Heat-shock proteins (HSP)4 play a primary role as intracellular molecular chaperones in the pathways of protein folding in the cell (1, 2). They play a role in the immune response when released from cells in complex with chaperoned antigenic peptides. When antigenic peptides are complexed with HSP, either 1) noncovalently associated with the substrate binding domain or 2) when covalently bound to HSP70, and injected into mice, they can prime CTL responses (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Immunization with HSP-peptide complexes purified from cancer cells provides protection against tumors derived from the cancer cells from which the HSP are purified (13, 14, 15, 16, 17). Moreover, treatment of mice bearing established cancers or residual tumors with such vaccines is effective in reducing the tumor burden and metastasis and in prolonging their survival (16, 18). In a number of clinical trials, immunization of patients with advanced malignancies with autologous HSP peptides has resulted in induction of CTL against autologous tumor cells. Response to the immunization and prolonged stabilization of disease have been observed (19, 20, 21, 22, 23, 24). These results indicate that HSP-peptide complexes have the qualities for a tumor vaccine. However, the effectiveness of the vaccine needs to be increased. Whereas immunization with HSP-peptide complexes derived from tumor cells usually elicits CTL response and provides partial protection, it fails to eliminate the tumors (10, 13, 17). These results indicate the need to improve the potency of chaperone protein-based vaccine. We have attempted to produce an improved HSP70-based vaccine based on the use of HSP70 purified from dendritic cell (DC)-tumor fusion cells. We took this approach as our preliminary investigation of the mechanism underlying the potency of DC-tumor fusion cells showed a role for HSP70. We found that DC fused to MC38/MUC1 tumor cells and infected with anti-HSP70 siRNA virus showed a reduced proimmune activity and compromised the antitumor immunity compared with controls (our unpublished data). These results suggest that HSP70 is involved in Ag processing in DC-tumor fusion cells and may constitute a part of therapeutic component in such fusion cells. Although DC-tumor fusion vaccines have shown effectiveness, we aim to produce a more molecularly designed vaccine, susceptible to rationale development. We have therefore examined whether HSP70-peptide complexes (PC) derived from DC-tumor fusion cells (HSP70.PC-F) per se constitutes a tumor vaccine with effective antitumor immunity. Indeed, such HSP70-PC stimulates an enhanced immune response to tumor and provides protection of mice against the challenge with tumor cells than those immunized with HSP70-PC from tumor cells (HSP70.PC-Tu). We further discovered that HSP70-PC from fusion cells is associated with the molecular chaperone HSP90, and carries immunogenic tumor Ag MUC1 peptides, resulting in maturation of DC and stimulation of T cells in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cells

C57BL/6 wild-type (WT) mice were obtained from The Jackson Laboratory. MUC1 transgenic (Tg) mice on a C57BL/6 background have been obtained from Dr. S. J. Gendler (Mayo Clinic, Scottsdale, AZ) (25). PCR was performed routinely to identify MUC1.Tg-positive mice in the colony. MyD88 knockout (MyD88 KO) mice were obtained from Dr. S. Levitz (Boston Medical School, Boston, MA). The mice were maintained in microisolator cages under specific pathogen-free conditions.

Murine MC38 colon adenocarcinoma cells were stably transfected with a MUC1 cDNA, resulting in MC38/MUC1 (26, 27). DC were generated from bone marrow cultures of C57BL/6 WT mice using the method previously described (28). DC were fused to MC38/MUC1 cells using previously described method (29, 30). The fusion efficiency of DC-tumor fusion cells (FC/MUC1) was ~20–30%.

Immunoprecipitation of HSP70-associated complexes from DC-tumor fusion cells

MC38/MUC1 tumor cells and FC/MUC1 (30, 31) were incubated in lysis buffer (PBS, 1% Triton X-100, 1 mg/ml BSA, 0.2 U/ml aprotinin, 1 mM PMSF) with 20 U/ml apyrase (Sigma-Aldrich) for 1 h on ice (apyrase is used to deplete ATP in the extracts and stabilize HSP70 associations). The lysates were clarified by centrifugation, and the aqueous phase were collected. The Ab46 Ab against HSP70 (32) at concentration of 1/100 were added and incubated overnight at 4°C. Then, 50 µl of protein A/G (1/1) agarose were added and incubated at 4°C for an additional 1 h. After extensive wash with lysis buffer, the immunoprecipitates were eluted with PBS with high salt (250 mM NaCl). The elute of HSP70.PC-F (derived from DC-tumor cells) or HSP70.PC-Tu (derived from tumor cells) were diluted to bring the salt concentration to 150 mM, quantified and aliquoted at 100 µg/ml for in vivo immunization. For protein analysis, the immunoprecipitates were dissolved in SDS sample buffer (0.1 Tris-Cl, 4% SDS, 20% glycerol, 0.05% bromphenol blue, 5% 2-ME), and analyzed by immunoblotting. For in vivo and in vitro experiments, the preparations were routinely checked by Limulus amebocyte lysate (LAL kit; Cambrex Bio Science) assay to ensure no contamination of endotoxin.

Immunoblotting

The proteins were subjected to SDS-PAGE and transferred on nitrocellulose membrane. The membranes were incubated with anti-HSP25 (SPA-801) anti-HSP40 (SPA-450), anti-HSP90 (SPA-830), or anti-HSP110 (SPA-1103) Abs (StressGen Biotechnologies) or anti-MUC1 Ab (HMPV; BD Pharmingen) and Ag/Ab complexes were visualized by ECL (ECL Detection System; GE).

In vivo tumor rejection

Seven-week-old WT mice were immunized s.c. with 1.5 or 3 µg of HSP70.PC-F or HSP70.PC-Tu on days 0 and 7. A group of mice immunized with 2 x 105 FC/MUC1 or injected with PBS were used as controls. On day 14, the mice were challenged by s.c. injection in the flank with 2 x 105 syngeneic MC38/MUC1 tumor cells. In a separate experiment, MUC1.Tg mice (six per group) were immunized twice with 1.5 µg of HSP70.PC-F or HSP70.PC-Tu and then challenged with 2 x 105 tumor cells in both sides of the flank of the mice 1 wk after second immunization. All the mice were followed for 30 days to determine the tumor incidence. Tumor volume was measured by caliper for twice a week, and tumors with a diameter of ≥2 mm were designated as positive.

CTL assay

CTL assay was performed as described previously (33, 34). Briefly, splenocytes were isolated from vaccinated or control mice. MC38/MUC1 tumor cells (1–2 x 106 cells) were labeled with 100–200 µCi Na251CrO4 for 60 min at 37°C, and then washed to remove unincorporated isotope. Splenocytes or tumor targets were resuspended in culture medium (RPMI 1640 medium supplemented with 15 mM HEPES (pH 7.4), 5% heat-inactivated-FCS, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10–5 M 2-ME), and then combined at various E:T ratios in 96-well V-bottom plates. In the Ab-blocking assay, the target cells were incubated with anti-MHC class I mAb (M1/42.3.9.8) before addition of the effector cells. The plates were centrifuged at 100 x g for 5 min to initiate cell contact and incubated for 5 h at 37°C with 5% CO2. After incubation, supernatants were collected and radioactivity was quantitated in a gamma counter. Spontaneous release of 51Cr were determined by incubation of targets in the absence of effectors, and maximum or total release of 51Cr by incubation of targets in 0.1% Triton X-100. Percentage of specific release of 51Cr is calculated by the following equation: percentage-specific release = ((experimental – spontaneous)/(maximum – spontaneous)) x 100.

T cell proliferation

Splenocytes and/or lymph node cells (LNC) were isolated from naive mice or mice immunized with HSP70.PC-F or HSP70.PC-Tu. Erythrocytes and dead cells were removed by centrifugation over a Ficoll-Hypaque gradient. The cells were washed and resuspended at the appropriate concentration in RPMI 1640 medium supplemented with 15 mM HEPES (pH 7.4), 5% heat-inactivated-FCS, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10–5 M 2-ME. Cells were incubated in the presence or absence of HSP70.PC-F or HSP70.PC-Tu as indicated concentration. Incubations were performed in 96-well, U-bottom plates for 5 days. T cell proliferation were assessed by [3H]thymidine incorporation after an additional 12-h incubation with 1 µCi/well [3H]thymidine.

Phenotype of T cells

MUC1.Tg mice were immunized s.c. on days 0 and 7 with 1.5 µg of HSP70.PC-F or HSP70.PC-Tu in posterior flank near the base of tail. One week after second immunization, mice were sacrificed and inguinal lymph node (LN) collected and stained with anti-CD4 (L3T4), CD8 (Ly-2), IFN-{gamma} (XMG1.2), CD69 (H1.2F3), CD44 (IM7), and/or IL-15R (TM-beta1) Abs (BD Pharmingen) and analyzed by flow cytometry using CellQuest software (BD Biosciences).

DC generation and maturation

DC were generated using the method described previously (28) with minor modification. Briefly, bone marrow cells were selected by lysis of red cells and depletion of lymphocytes and Ia+ cells by series of treatments with panels of mAbs followed by rabbit complement, and then cultured in the presence of GM-CSF (20 ng/ml; Sigma-Aldrich). On third day of culture, the nonadherent cells were collected and cultured in medium containing GM-CSF overnight (ON). Then the loosely adherent DC were collected and cultured in the presence or absence of HSP70.PC-F or HSP70.PC-Tu (10 µg/ml) for 6 or 24 h in a 24-well plate. The DC were collected, stained with anti-MHC class II (M5/114), CD86 (GL1), B7-DC (TY25), CD40 (3/23), or ICAM (3E2) Abs (BD Pharmingen) and analyzed by FACS. In a separate experiment, immature DC were cocultured with 10 µg/ml LPS (Sigma-Aldrich), HSP70.PC-F, boiled HSP70.PC-F, HSP70.PC-Tu, HSP70-PC derived from fusions of MC38/MUC1 tumor cells in the presence of polyethylene glycol (PEG; Sigma-Aldrich) or the mixture of DC and MC38/MUC1 tumor cells, and then analyzed for the expression of indicated molecules.

To determine the DC maturation in vivo, inguinal LN were obtained from mice immunized twice with 1.5 µg of HSP70.PC-F, HSP70.PC-Tu, or injected with PBS and snap-frozen. Cryosections were obtained, stained with anti-CD86 mAb, and then examined under microscopy.

HSP70-PC pull down from fusion cells by GST-HSP70 fusion proteins

MC38/MUC1 carcinoma cells were transfected with a total of 7.5 µg of pCMG plasmid vector encoding GST-HSP70 fusion proteins per 100-mm diameter dish using Lipofectamine (Invitrogen Life Technologies). The transfected MC38/MUC1 were fused with DC. The cells, 2 x 107 each, were lysed in lysis buffer (50 mM Na2HPO4, 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM DTT, 200 µM benxamidine, 40 µg/ml leupeptin, 300 µM PMSF). The detergent-solubilized cell lysates were incubated with glutathione Sepharose beads ON at 4°C. Beads were washed twice in lysis buffer and twice in lysis buffer with 150 mM NaCl. Proteins bound to the glutathione Sepharose beads were eluted with glutathione elution buffer (50 mM Tris (pH 8.5) and 20 mM reduced glutathione).

Detection of MUC1 peptide from GST-pull down HSP70-PC

The GST-pull down of HSP70.PC-F or HSP70.PC-Tu or purified HSP70.PC-F or HSP70.PC-Tu were precoated to the ELISA plates (Nunc) ON. After washing five times with Tween 20/PBS, the plates were blocked with 5% horse serum/PBS (100 µl/well) for 1 h, and then anti-MUC1 peptide Abs BCP8 (anti-DTRPAPGST) or BCP9 (anti-GSTAPPAHG) (5 µg/ml in PBS, 100 µl/well) (35) were added into the individual well for 2 h at room temperature (RT). After washing with PBS, the plates were blocked with 1% horse serum/PBS (100 µl/well) for 1 h. The plates were washed four times with PBS and incubated for 2 h at RT with HRP-conjugated anti-mouse IgG (1/5000; Amersham Biosciences). Ab complexes were detected by development with o-phenylenediamine (Sigma-Aldrich) and measured in a microplate autoreader EL310 (Bio-Rad) at OD492 nm.

Statistical analysis

Statistical significance was analyzed using {chi}2 and Student’s t test. Percentage of positive cells in phenotype assay of T cells and DC was derived from two or three independent experiments and presented as means ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enhanced antitumor immunity induced by HSP70-PC derived from fusion cells

We have examined the ability of HSP70-PC from either tumor cells or DC-tumor fusion cells to immunize mice against tumor cells. HSP70-PC were immunoprecipitated from either fusion cells (HSP70.PC-F) or MC38/MUC1 tumor cells (HSP70.PC-Tu), using anti-HSP70 Ab Ab46 and the complexes were used as a tumor vaccine against MUC1 expressing MC38 cells (MC38/MUC1). Mice developed tumors in 100% of cases if untreated or sham treated with injection of buffer. Groups of WT mice were next vaccinated with HSP70.PC-F or HSP70.PC-Tu and then challenged with MC38/MUC1 tumor cells. Immunization with vaccine derived from tumor cells alone, at doses of 1.5 or 3.0 µg HSP70.PC-Tu resulted in partial protection and led to a reduction of tumor incidence to, respectively, 83% at the lower dose (five of six) and 33% (two of six) at the higher dose of mice with tumor growth (Fig. 1A). By contrast, immunization with 3.0 µg of immunoprecipitate derived from the fusion cells protected all the mice from tumor challenge and only one mouse developed a tumor when immunized with the lower dose of 1.5 µg of HSP70.PC-F (Fig. 1A). Extraction of HSP70-PC from DC fused to MC38/MUC1 tumor cells (FC/MUC1), therefore, increased efficacy in inhibiting tumor growth compared with mice immunized with HSP70.PC-Tu (p < 0.05 at the dose of 1.5 µg). These tumor responses correlated well with the results obtained in the CTL assay which showed that immunization of mice with HSP70.PC-F resulted in the highest CTL activity against these tumor cells that express MUC1 (Fig. 1B). Moreover, CTLs from WT mice immunized with HSP70.PC-F induces lysis of MC38/MUC1, B16/MUC1 and, to a lesser extent, MC38 cells, but not B16 cells (Fig. 1C). These results indicate that immunization with HSP70.PC-F induced immunity against MUC1 and other unknown Ags on MC38. Thus, the demonstration that B16/MUC1, and not B16, cells are lysed by the CTLs confirms that HSP70.PC-F induces MUC1-specific response. In addition, the lysis of MC38/MUC1, B16/MUC1, and MC38 cells was inhibited by anti-MHC class I mAb (Fig. 1C), suggesting MHC class I-restricted CTL activity. These experiments show that although both types of HSP70-based vaccine induce antitumor immunity, improved efficacy is observed in mice immunized with HSP70.PC-F, suggesting enhanced immunogenicity of these preparations.


Figure 1
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  		FIGURE 1. Antitumor immunity induced by immunization with HSP70 complexes derived from DC-tumor fusion cells. A, C57BL/6 wild-type mice (WT mice, six mice per group) were immunized twice with 1.5 or 3 µg of HSP70-associated complexes from FC/MUC1 fusion cells (HSP.70-PC-F, {square}) or MC38/MUC1 tumor cells (HSP.70-PC-Tu, Figure 1) at the posterior flank near the base of tail on days 0 and 7, respectively. The mice injected with PBS (three per group, {blacksquare}) were used as control. On day 14, mice were challenged s.c. with 2 x 105 MC38/MUC1 tumor cells. Tumor incidence (≥2 mm in diameter) was monitored up to 30 days. B, Splenocytes isolated on day 30 from WT mice immunized with HSP.70-PC-F ({square}), HSP70-PC-Tu (Figure 1) or PBS ({blacksquare}) were incubated with MC38/MUC1 target cells at an E:T ratio of 50:1. C, MUC1-positive and -negative targets were incubated with anti-MHC class I mAb (Figure 1) or IgG (Figure 1) before addition of splenocytes isolated on from WT mice immunized with HSP.70-PC-F. D, MUC1.Tg mice (six mice per group) were vaccinated twice with 1.5 µg of HSP70.PC-F ({oplus}) or HSP70.PC-Tu ({square}). The MUC1.Tg mice immunized with 2 x 105 FC/MUC1 fusion cells ({circ}) or PBS ({triangleup}) were used as control. One week after the second immunization, mice were challenged s.c. with 2 x 105 MC38/MUC1 tumor cells. Tumor volume was measured by caliper at twice per week for 30 days. E, CTL activity induced by immunization with HSP70 complexes derived from FC/MUC1. Splenocytes isolated from mice immunized with HSP70.PC-F ({oplus}), HSP70.PC-Tu ({square}), 2 x 105 FC/MUC1 fusion cells ({circ}) or PBS ({triangleup}) at 20 days after tumor challenge were incubated with MC38/MUC1 target cells at the indicated E:T ratios. F, MUC1.Tg mice (six mice per group) were vaccinated twice with 1.5 µg of HSP70 peptides from FC/MUC1 fusion cells (HSP70-peptide-F, {oplus}) or MC38/MUC1 tumor cells (HSP70-peptide-Tu, {square}) purified by ADP-agarose column. The MUC1.Tg mice immunized with DC pulsed with purified HSP70 peptides ({circ}) or PBS ({triangleup}) were used as control. Tumor volume was measured by caliper at twice a week up to 30 days. G, CTL activity induced by immunization with purified HSP70 peptides derived from FC/MUC1. Splenocytes isolated from mice immunized with HSP70-peptide-F ({oplus}), HSP70-peptide-Tu ({square}), 2 x 105 DC pulsed with HSP70-peptide-Tu ({circ}) or PBS ({triangleup}) at 20 days after tumor challenge were incubated with MC38/MUC1 target cells at the indicated E:T ratios. (A, D, and F), the statistical significance of in vivo data was determined using {chi}2 analysis (* and *** indicate p < 0.05 and p < 0.005, respectively). B, C, E, and G, Percentage of cytotoxicity was determined by standard 51Cr release assay. In B and C, the data was presented as mean ± SD of three replicates.

 
We next tested the efficacy of HSP70.PC-F in MUC1.Tg mice which have been engineered to overexpress MUC1. Because the model Ag we used in the present study is human MUC1, a tumor-associated Ag expressed in ~72% of epithelial cancers (36), and it is possible that the immune response we observed may be derived from the reaction to human MUC1 as a xenoantigen in WT mice, this was included as an essential control. The MUC1.Tg mouse has been shown to express MUC1 as a self-Ag (37), to develop tolerance in both B and T cell compartments and to be refractory to immunization with the MUC1 protein (25). This model was therefore used to determine whether HSP70 vaccines could break tolerance to nonmutated tumor Ags. MUC1.Tg mice were vaccinated twice with 1.5 µg of HSP70-associated complexes derived from DC-tumor fusion cells or from tumor cells. Vaccination with HSP70-associated complexes derived from FC/MUC1 fusion cells generated strong CTL activity (Fig. 1E) and protected all but one mouse from challenge with tumor cells, an effect comparable to those vaccinated with DC-tumor fusion cells (Fig. 1D). By contrast, vaccination with the same dose of HSP70.PC-Tu generated low levels of CTL and failed to protect the mice from challenge with MUC1-positive tumor cells (Fig. 1, D and E). In control mice injected with PBS, tumors grew progressively. The preventive effect of the HSP70-FC vaccine is also statistically significant between mice immunized with HSP70.PC-F and those immunized with HSP70.PC-Tu (p < 0.005). These results indicate that HSP70-PC derived from DC-tumor fusion vaccine are able to overcome the tolerance to a nonmutated tumor Ag and trigger immune response that provided protection of a host expressing MUC1 as a self-Ag. These findings have significant implication because most tumor Ags are nonmutated self-Ags.

As HSP70 vaccines are commonly prepared using HSP70 isolated from tumors by ADP-affinity chromatography, we examined this mode of HSP70 preparation also. We obtained purified HSP70-peptide complexes from tumor and fusion cells using ADP-agarose affinity chromatography (38). Immunization of MUC1.Tg mice with such purified HSP70-peptide complexes from fusion cells (1.5 µg), although significantly reducing the rate of tumor growth resulted in suboptimal protection against tumor challenge although the growth rate was less than in mice immunized with purified HSP70-PC from tumor cells alone or PBS (Fig. 1F). Corresponding results were obtained using the CTL assay. Immunization with column purified HSP70 peptides induced moderate CTL activity against MC38/MUC1 (Fig. 1G). These results suggest the importance of isolation methodology in HSP70-PC preparation. The rationale behind these differences is not defined here but could include a number of mechanisms such as 1) the nature of the HSP70 isoforms isolated by the respective methods, 2) relative association with/dissociation from MUC1 peptides, and 3) the potential presence of other proteins coprecipitated with HSP70-PC. HSP70-PC obtained by rapid immunoprecipitation may preserve association with chaperoned peptides and other molecules, whereas HSP70-PC purification by sequential ATP agarose affinity chromatography, DEAE cellulose, and Sephadex G25 chromatography (39), although highly effective in yielding a pure HSP70 preparation, may lead to dissociation of peptides and proteins especially considering the relatively low affinity of HSP70 polypeptide binding (Kd 10–6) and the prolonged nature of the isolation procedure (40).

Taken together, these results indicate that HSP70-PC preparations derived by immunoprecipitation from fusion cell lysates constitute an improved tumor vaccine. The immunogenicity of HSP70-PC from FC is significantly enhanced as determined by its ability to reverse T cell tolerance to a self-Ag and provide protection against challenge with tumor cells.

Enhanced stimulation of T cells by HSP70-PC from fusion cells

The studies described above demonstrate that HSP70.PC-F is superior to its counterpart from tumor cells in the induction of CTL and antitumor immunity. However, they do not indicate the potential mechanisms underlying the increased immunogenicity. For example, we do not know whether the enhanced antitumor immunity is achieved by more robust induction and expansion of effector and memory T cells. To address these issues, we next assessed the ability of HSP70.PC-F to stimulate naive T cells in vitro. The naive LNC and splenocytes were isolated and cocultured with HSP70.PC-F or HSP70.PC-Tu at indicated concentrations (Fig. 2, A and B). Naive LNC and splenocytes were activated by the HSP70 preparations and proliferated more in the presence of HSP70.PC-F than those in the presence of HSP70.PC-Tu (Fig. 2, A and B). In addition, the CD4 and CD8 T cells from vaccinated MUC1.Tg mice were cocultured with either HSP70.PC-F or HSP70.PC-Tu in the presence of DC. The CD4 and CD8 T cells proliferated when restimulated by HSP70.PC-F and, to a lesser extent, by HSP70.PC-Tu (Fig. 2, C and D). These experiments indicate the ability to stimulate T cells is enhanced in HSP70 from fusion cells.


Figure 2
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  		FIGURE 2. Proliferation and activation of CD4 and CD8 T cells by HSP70 complexes derived from DC-tumor fusion cells. A and B, Stimulation of naive T cells in vitro. Whole LNC (A) and splenocytes (B) were isolated from naive MUC1.Tg mice and cocultured with HSP70.PC-F ({circ}) or HSP70.PC-Tu ({square}) at indicated concentration. After 5 days, cells were pulsed with 1 µCi/well [3H]thymidine and harvested on filters. Radioactivity (mean ± SD of triplicates) was measured by liquid scintillation counting. The measurement of cells cultured with medium alone was used as baseline. Results were obtained in three separate experiments. C and D, Stimulation of primed CD8 or CD4 T cells in vitro. T-LNC were isolated from vaccinated MUC1.Tg mice and then sorted to CD8 or CD4 T cells. CD8 (C) or CD4 (D) T cells from HSP70.PC-F immunized mice were cocultured with DC and HSP70.PC-F ({circ}), and CD8 or CD4 T cells from HSP70.PC-Tu immunized mice were cocultured with DC and HSP70.PC-Tu ({square}) at indicated concentration, respectively. After 5 days, the cultures were pulsed with 1 µCi/well [3H]thymidine and harvested on filters. Radioactivity was measured by liquid scintillation counting. The measurement of cells cultured with medium alone was used as baseline. Results were obtained in two separate experiments. E and F, In vivo activation of T cells by HSP70 complexes from DC-tumor fusion cells. LNC were freshly isolated from MUC1.Tg mice immunized twice with HSP70.PC-F, HSP70.PC-Tu, or injection with PBS. The LNC were stained with mAbs against CD4, CD8, IFN-{gamma}, CD69, CD44, and IL-15R and analyzed by flow cytometry.

 
To determine whether there are qualitative and quantitative differences in T cells primed by HSP70.PC-F and HSP70.PC-Tu in vivo, we investigated the phenotypes of CD4 and CD8 T cells isolated from immunized mice. MUC1.Tg mice were immunized with HSP70-PC obtained from FC/MUC1 or tumor cells. LNC were then purified, stained with a panel of Abs, and quantitated by FACS analysis. Immunization with HSP70.PC-F, and to a lesser extent, with HSP70.PC-Tu resulted in increased numbers of CD4 and CD8 T cells in LN compared with the numbers of T cells in naive mice (Fig. 2, E and F). A strikingly significant increase in CD8 T cell number was observed in mice immunized with HSP70.PC-F. Immunization with HSP70.PC-F resulted in 46% increase of CD8 T cells compared with mice immunized with HSP70.PC-Tu (Fig. 2, E and F). Importantly, CD4 or CD8 T cells expressing markers for activation, effector or memory T cells increased in the immunized mice (Fig. 2, E and F). The increase of CD4 and CD8 T cells expressing IFN-{gamma}, CD69, CD44, and CD44/IL-15R was greatly enhanced in T cells isolated from mice immunized with HSP70.PC-F compared with mice immunized with HSP70.PC-Tu. Thus, HSP70.PC-F complexes possesses a greatly enhanced capacity to stimulate T cells both in vivo and in vitro settings.

Enhanced maturation of DC by HSP70-PC from fusion cells

We next asked whether T cell stimulation by HSP70-PC requires the participation of APC such as DC for Ag representation. DC maturation is essential for its ability to present Ag. We asked therefore, can HSP70.PC-F stimulate DC to maturation? To answer this question, we next assessed the expression of MHC class II and costimulatory molecules on DC. We generated immature DC and then cocultured them with HSP70.PC-Tu, HSP70.PC-F, or medium. Coculture of DC with HSP70.PC-F resulted in significant up-regulation of MHC class II and costimulatory molecules in DC (Fig. 3, A and B). By contrast, up-regulation of these molecules in DC cocultured with HSP70.PC-Tu was minimal. There is statistical significance in DC maturation stimulated by HSP70.PC-F and HSP70.PC-Tu (Fig. 3B). As shown in Fig. 3, A and B, the up-regulation of MHC class II, CD86, and B7-DC was observed as early as 6 h after culture, while the up-regulation of CD40 and ICAM-1 was observed late in the culture.


Figure 3
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  		FIGURE 3. Effect on maturation of DC by HSP70 complexes derived from DC-tumor fusion cells. A and B, Phenotype of DC stimulated by HSP70.PC-F or HSP70.PC-Tu. DC were generated from bone marrow cells of WT mice by Ab/complement treatment and cultured in the presence of GM-CSF medium. On the third day, the immature DC were selected by adherent method and further cultured for ON. The immature DC were then cocultured with HSP70.PC-F or HSP70.PC-Tu (10 µg/ml) in the presence of GM-CSF medium. DC cultured in GM-CSF medium alone were used as control. A, After 6 or 24 h culture, the DC were collected, stained with indicated mAbs, and analyzed by FACS. The expression of indicated molecules on DC cocultured with HSP70.PC-F, HSP70.PC-Tu, or medium alone was indicated by red, blue, or black line, respectively. B, Quantification of DC positive for individual mAb cocultured with HSP70.PC-F, HSP70.PC-Tu, or medium is derived from three independent experiments. C, Immature DC were cocultured with LPS, medium alone, HSP70.PC-F, boiled HSP70.PC-F, HSP70.PC-Tu, HSP70-PC from tumor-tumor fusions (HSP70.PC-Tu+PEG) or mixture of DC and tumor cells (HSP70.PC-Tu+DC). After 24 h culture, the DC were collected and analyzed for the expression of CD86. D, Inguinal LN collected from mice immunized with HSP70.PC-F, HSP70.PC-Tu, or PBS were frozen and cryosectioned. The sections were stained with anti-CD86 (red color) and examined under microscopy (magnification, x100). B and C, The statistical significance of DC maturation was determined using {chi}2 analysis (* and *** indicate p < 0.05 and 0.005, respectively).

 
To determine the role of DC-tumor fusion in the maturation of DC by HSP70, immature DC were cultured with HSP70-PC from various sources. As expected, culture of immature DC with HSP70.PC-F resulted in up-regulation of CD86 molecules, although the level of increase is less than those stimulated by LPS (Fig. 3C). By contrast, comparable expression of CD86 was observed on control DC cultured with HSP70-PC derived from tumor cells, tumor-tumor fusion with PEG, mixture of DC and tumor cells, or medium alone (Fig. 3C). Furthermore, the maturation effect of HSP70.PC-F was abrogated by boiling, suggesting a requirement for native tertiary structure in the effects of HSP70. There is statistical significance in the up-regulation of CD86 molecules by HSP70.PC-F and HSP70-PC derived from tumor cells, tumor-tumor fusion with PEG, mixture of DC and tumor cells, or medium alone (Fig. 3C). These results indicate that physical fusion of DC and tumor cells before extraction of HSP70-PC is required for the increased maturation of DC by HSP70. In addition, it is unlikely that the maturation of DC by HSP70.PC-F is due to the contamination of endotoxin because we used the same method and condition to prepare HSP70.PC-F and HSP70.PC-Tu and we failed to observe the enhanced maturation of DC by HSP70.PC-Tu.

To assess the DC maturation in vivo by HSP70.PC-F, frozen sections were obtained from draining LN of mice immunized with HSP70-PC from tumor or fusion cells or injected with PBS, stained with mAb against CD86 and examined under microscopy. CD86-positive cells increased in mice vaccinated with HSP70.PC-F (data not shown). In contrast, few CD86-positive cells were observed in mice vaccinated with HSP70.PC-Tu or injected with PBS. Under high magnification, veiled cytoplasmic processes, a morphological characteristic of DC maturation, can be observed in CD86-positive cells from mice immunized with HSP70.PC-F (Fig. 3D). These results indicate that HSP70.PC from fusion cells possess enhanced stimulatory ability to mature DC compared with unconjugated tumor cells.

Involvement of MyD88 in the maturation of DC by HSP70-PC from fusion cells

To study the potential signaling mechanism underlying such DC maturation, we next cultured DC from mice deficient in MyD88–/– or WT mice with HSP70.PC-F and compared their phenotype in response to the HSP70.PC-F. Stimulation of TLR plays a broad role in the maturation of DC (41) and MyD88 is an adaptor molecule required for downstream signaling for the TLR. Up-regulation of MHC class II and costimulatory molecules was observed on WT-DC cocultured with HSP70.PC-F (Fig. 4). In contrast, the effect of maturation of DC by HSP70.PC-F was obliterated in DC from MyD88 mice (Fig. 4), resulting in comparable expression of MHC class II, CD86, B7-DC, CD40, or ICAM on DC from MyD88–/– mice cocultured with HSP70.PC-F to those cocultured with medium at 6 h (Fig. 4A) or 24 h (Fig. 4B). To determine the involvement of MyD88 in vivo in the induction of T cell-mediated immunity, MyD88–/–, MUC1.Tg, and WT mice were immunized with HSP70.PC-F or HSP70.PC-Tu. Seven days after the second immunization, LNC and splenocytes were isolated and assayed for proliferation and CTL activity. T cells from immunized WT and MUC1.Tg mice proliferated vigorously (Fig. 4D). By contrast, minimal cell proliferation was observed in T cells from immunized MyD88–/– mice (Fig. 4D). Similar results were obtained in CTL activity. CTLs from immunized WT and MUC1.Tg mice showed higher killings of tumor cells than those from immunized MyD88–/– mice (Fig. 4E). In addition, the CTLs from WT and MUC1.Tg mice immunized with HSP70.PC-F lysed MC38/MUC1, B16/MUC1 and, to a lesser extent, MC38 tumor cells (Fig. 4E). By contrast, there was no, if any lysis of irrelevant B16 tumor cells. These results strongly suggest that the TLR/MyD88 pathway is involved in the maturation of DC by HSP70.PC-F and that enhanced innate immune stimulation by HSP70.PC-F may be responsible, at least in part, for the augmented antitumor immunity.


Figure 4
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  		FIGURE 4. Comparison of DC phenotype from WT and MyD88 KO mice stimulated by HSP70.PC-F. DC were generated from wild-type (WT-DC, red line) or MyD88KO (MyD88-DC, green line) mice using the same method as described in Fig. 3 and cocultured with HSP70.PC-F in the presence of GM-CSF medium for 6 (A) or 24 h (B). WT-DC or MyD88-DC cultured in medium containing GM-CSF (black line) were used as controls. The harvested DC were stained with indicated mAbs and analyzed by FACS. C, Quantification of DC positive for individual mAb is derived from two independent experiments. The statistical significance of DC maturation was determined using {chi}2 analysis (*** indicates p < 0.005). D, T cell proliferation. LNC were isolated from WT, MUC1.Tg, or MyD88 KO mice immunized with either HSP70.PC-F (red circle) or HSP70.PC-Tu (green triangle). After 5 day culture with 5 µg/ml HSP70.PC-F or HSP70.PC-Tu, respectively, the cells were pulsed with 1 µCi/well [3H]thymidine and harvested on filters. Radioactivity was measured by liquid scintillation counting. E, CTL assay. Splenocytes were isolated from WT, MUC1.Tg, or MyD88 KO mice immunized with either HSP70.PC-F or HSP70.PC-Tu as effector cells. MUC1-positive and -negative targets were labeled with 51Cr and then mixed with splenocytes isolated from mice immunized with HSP70.PC-Tu (blue bar) or HSP70.PC-F (orange bar) at a E:T ratio of 60:1. Percentage of cytotoxicity was determined by standard 51Cr release assay.

 
Enhanced MUC1 peptide association in HSP70 complexes from DC-tumor fusion cells

To determine the relative levels of HSP70 and other HSPs in DC-tumor fusion cells and assess their association with tumor Ag MUC1, we prepared lysates from heat shock-treated (HS) or untreated DC, MC38/MUC1 tumor cells, and FC/MUC1 fusion cells. Extracts were loaded in equal concentrations into 6 or 10% SDS gel, transferred to nitrocellulose and immunoblotted with mAbs against HSP25, 70, 90, and 110. Although HSP expression in DC alone was relatively low, expression of HSP25 and HSP70 in FC/MUC1 fusion cells was increased by heat shock (Fig. 5A). To assess the association of HSP70 with MUC1, lysates from FC/MUC1 and MC38/MUC1 were immunoprecipitated with anti-HSP70 mAb followed by immunoblot analysis with anti-MUC1 mAb. Immunoprecipitation with anti-HSP70 Ab led to the coprecipitation of MUC1, indicating that HSP70 forms complexes with MUC1 (Fig. 5B). The relative density of MUC1 in lanes 4 and 6 was 10.7 and 41.5%, respectively, suggesting stronger association of HSP70 with MUC1 in FC/MUC1 cells. The difference between lanes 4 and 6 is statistical significance (p < 0.005). These experiments indicate that DC-tumor fusion cells express heat shock proteins including HSP70 that are associated with tumor Ag.


Figure 5
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  		FIGURE 5. Chaperoning of MUC1 by HSP70 derived from FC/MUC1 fusion cells. A, Immunoblotting analysis of HSP SDS contents in cell lysates from DC, MC38/MUC1 tumor cells, and FC/MUC1 fusion cells treated with or without HS at 43°C for 1 h and 37°C for 6 h. Lysates were analyzed by SDS-PAGE followed by immunoblotting with Abs against HSP-25, -70, -90, or -110. Equal loading of the protein was determined by SDS-PAGE. B, Association of HSP70 with MUC1. Lysates from HS-treated FC/MUC1 fusion cells, MC38/MUC1 tumor cells or DC alone were immunoprecipitated with anti-HSP70 or anti-MHC class II Abs followed by immunoblotting with anti-MUC1 mAb DF3. The relative density of MUC1 in lanes 4 and 6 was determined by densitometric analysis of x-ray films in the chemiluminescence-based protein detection. C, Detection of MUC1 peptide from GST-HSP70 complexes by anti-MUC1 peptide Ab. GST-HSP70 complexes were pulled down from GST-HSP70-transfected MC38/MUC1 tumor cells ({triangleup}), DC fused with GST-HSP70-transfected MC38/MUC1 cells (FC/MUC1/GST-HSP70, {circ}) or vector-transfected tumor cells ({square}) and used to coat the ELISA plate at indicated concentrations ON at 4°C. After washing five times, the anti-MUC1 peptide Abs BCP8 (left panel) or BCP9 (right panel) were added into the plate for 2 h at RT. Following washing and blocking, the second Ab HRP-anti-mouse lgG at 1/5000 dilution was added and the color was developed by o-phenylenediamine tetrahydrochloride buffer with H2O2. The absorbance was measured by an autoreader at 492 nm. D, Comparison of MUC1 peptide in HSP70-PC obtained by GST pull-down or ADP affinity chromatography. The GST-HSP70-complexes pulled down from fusion cells ({circ}) or tumor cells ({triangleup}) (left panel), and HSP70-PC purified by ADP affinity chromatography from fusion cells ({circ}) or tumor cells ({triangleup}) (right panel) was assayed with BCP8 mAb using the same method in C. C and D, The statistical significance was determined using Student’s t test (***, p < 0.005).

 
To assess whether the isolated HSP70-PC carries immunogenic peptides, we next measured the ability of HSP70-PC in association with antigenic peptides. We obtained HSP70-PC using GST-HSP70 "pull-down" to optimize recovery of peptides. MC38/MUC1 carcinoma cells were transfected with a plasmid vector encoding the GST-HSP70 fusion proteins or GST vector alone, and then fused to DC to create FC/MUC1/GST-HSP70 fusion cells or FC/MUC1/GST fusion cells, respectively. The WT GST and GST-HSP70 proteins were then isolated from those cells using GST pull-down. We then assayed for two individual MUC1 peptide DTRPAPGST and GSTAPPAHG using ELISA with specific anti-peptide mAbs BCP8 and BCP9. MUC1 peptide DTRPAPGST and GSTAPPAHG were found to be restricted by H-2b (35). Efficient association with BCP8 was observed in HSP70.PC-F while recovery was greatly reduced from MC38/MUC1-HSP70 tumor cells (Fig. 5C, left panel). In contrast, there was no reaction in complexes pulled down from vector-transfected tumor cells that express WT GST. The difference in MUC1 peptide content between HSP70.PC-F and HSP70.PC-Tu is statistical significant. Minimal association with BCP9 was observed in complexes pulled down from either cell type (Fig. 5C, right panel). To compare the level of peptide in HSP70-PC obtained by GST pull-down or ADP affinity chromatography, HSP70-PC from GST pull-down or ADP purification was assayed by BCP8 mAb. The recovery of MUC1 peptide was significantly reduced in HSP70.PC-F obtained by ADP affinity chromatography (Fig. 5D). Thus HSP70-associated complexes carry tumor antigenic peptides and antigenic peptide association is increased in complexes from FC/MUC1. This may constitute a portion of the increased effectiveness of HSP.PC-F as a tumor vaccine. In addition, the recovery of these peptides is affected by the method of extraction.

Promotion of association with HSP90 in HSP70 complexes from DC-tumor fusion cells

To determine whether HSP70 is associated with other proteins, lysates were next obtained from MC38/MUC1 or FC/MUC1 and immunoprecipitated with anti-HSP70 mAb. The immunoprecipitates were analyzed by immunoblotting with a panel of Abs. Fig. 6A shows that HSP70 was associated with HSP90 and such association was strongly enhanced in HSP70.PC-F, whereas the association with HSP110 or HSP40 was comparable between HSP70.PC-F (lane 3) and HSP70.PC-Tu (lane 2). In addition, the enhanced association with HSP90 was not observed in the precipitates from the mixture of unfused DC and tumor cells (Fig. 6A, lane 1), suggesting that physical fusion is essential for promotion of such association. To confirm the enhanced association with HSP90 in the precipitates from fusion cells, lysates were obtained from tumor cells, or FC/MUC1 treated with or without drug geldanamycin (GA) that is known to inhibit the HSP70-HSP90 interaction (42, 43). Fig. 6B shows that treatment of FC/MUC1 with GA abolished the association between HSP70 and HSP90 (lane 3). Interestingly, a novel HSP90-interacting band appeared in the HSP70 precipitates from FC/MUC1 (Fig. 6B, lane 2). This band is not observed in precipitates from FC/MUC1 treated with GA (lane 3) or tumor cells alone (lane 1), suggesting that fusion of DC and tumor cells promotes the interaction with a novel HSP90 isoform. To assess the immunologic effect of HSP70 association with HSP90, naive LNC, and splenocytes were isolated and cocultured with HSP70.PC-Tu, or HSP70.PC-F treated with or without GA. A higher rate of proliferation of LNC and splenocytes was observed in the culture stimulated by HSP70.PC-F (Fig. 6C). However, the stimulatory ability of HSP70.PC-F was diminished after pretreatment of FC/MUC1 with GA before HSP70 extraction (Fig. 6C). These results suggest that DC-tumor fusion promotes the association of HSP70 with HSP90 and that this HSP70-HSP90 complex enhances the immunogenicity of HSP70.PC-F.


Figure 6
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  		FIGURE 6. Increased association of HSP70 from fusion cells with HSP90. A, Detection of HSP70-PC associated with other HSPs using Weston blot. DC mixed with MC38/MUC1 (lane 1), MC38/MUC1 tumor cells (lane 2), or FC/MUC1 fusion cells (lane 3) were lysed by lysis buffer. The lysates were subjected to immunoprecipitation with anti-HSP70 mAb (Ab46). The precipitates were analyzed by immunoblotting with indicated Abs in 10% SDS gel. B, Lysates from MC38/MUC1 tumor cells (lane 1), FC/MUC1 (lane 2), FC/MUC1 treated with GA (1 µg/ml) (lane 3) were immunoprecipitated with anti-HSP70 Ab (Ab 46). The precipitates were analyzed by immunoblotting with anti-HSP90 Ab in 6% SDS gel. Lane 4, Lysates from MC38/MUC1 tumor cells. C, LNC and splenocytes were isolated from naive mice and cocultured with HSP70.PC-Tu, or HSP70.PC-F treated with or without GA and then measured for T cell proliferation with standard [3H]thymidine incorporation assay. The data are representative of three separate experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The data presented here show a clear advantage for the molecular chaperone-based vaccine extracted from DC-tumor fusion cells compared with comparable extracts from tumor cells alone (Figs. 1 and 2). HSP70.PC-F is able to break T cell tolerance to a defined tumor Ag, kill MC38/MUC1 tumor cells, and prevent tumor growth, as opposed to HSP70 preparations from tumor alone which were ineffective in breaking tolerance under the conditions used here. These enhanced effects of HSP70.PC-F vaccine may be related to the ability of the vaccine to initiate the program of host DC maturation and lead to expression of costimulatory molecules required for T cell activation (Fig. 3). Host DC maturation may in turn underlie the increases in CD4 and CD8 T cells expressing markers for activation, effector, and memory T cells seen in mice vaccinated with HSP70.PC-F vaccine.

The mechanisms underlying the effect of extracellular HSP70 on immune cells are only beginning to be determined (44). HSP70 has been shown to bind effectively to LOX-1, a member of the C-type lectin receptors which is found on the DC cell surface (45). Mechanism of signaling through LOX-1 have recently been shown to include the activation of TLR2, a potent activator of innate immunity (46). Indeed, we show here that the increased expression of markers of host DC maturation is abrogated by inactivation of the MyD88 gene, a key downstream component of TLR signaling (Fig. 4). A role for TLR signaling in stimulation of host APC by HSP70.PC-F is thus indicated (Fig. 4). The molecular mechanisms underlying the superiority of HSP70.PC-F compared with HSP70 from tumor cells alone is complex. However, HSP70.PC-F vaccine contains an elevated concentration of MUC1 antigenic peptides which may be cross-presented to T cells and lead to some of the changes in T cell activation observed (Figs. 2 and 5). Antigenic peptides in addition to BCP8 shown here may also be chaperoned by the HSP70.PC-F vaccine. Other differences may include the particular species of HSP70 in the DC-tumor fusion cells. Human cells contain at least 12 HSP70 genes and we have found that significant levels of 8 different HSP70 family gene products HSP70–1a, HSP701b, HSP70-HOM, HSP70-4, HSP70-5, HSP70-8, HSP70-9b, HSP75, and HSC70 are expressed in tumor cells (47, 48). Three of these (HSP70–1a, HSP701b, HSP70-HOM) are encoded by genes within the MHC locus suggesting immune function (49).

Previously described HSP70-PC-based vaccines, though eliciting immunological response, have been proven insufficient to provide protection against tumor growth (10, 13, 17). It was thus evident that the efficacy of HSP70-PC, as a therapeutic tumor vaccine, requires improvement. The present study has made some progress toward this goal. We have extended previous findings and made new discoveries by demonstration: 1) HSP70.PC-F possess superior adjuvant effect that matures the APC by up-regulation of MHC class II and costimulatory molecules over its counterpart from tumor cells (Fig. 3); 2) HSP70.PC-F possess enhanced ability to induce effector and memory T cells (Fig. 2); 3) vaccination of mice with HSP70.PC-F confers sufficient protection against the challenge with tumor cells (Fig. 1); 4) HSP70.PC-F can break T cell tolerance to a predominant tumor Ag MUC1 (Fig. 1); 5) HSP70-PC derived from DC-tumor fusion vaccine are strongly associated with tumor Ag (Fig. 5) and contains enriched antigenic peptides (Fig. 5); 6) HSP70 from fusion cells associates with HSP90 (Fig. 6), which will increase its ability to carry antigenic peptides (50). Therefore, the present study has significant implication for the advancement of cancer immunotherapy.

The molecular events that promote the formation of immunogenic complexes of HSP with tumor antigenic peptides in fusion cell are unclear. Previous studies show that fusion of DC and tumor cell creates an immunogenic cell with integration of cytoplasm of these two cells (51). Such integration makes it possible to exchange cytoplasmic contents and share subcellular compartments between DC and tumor cell. Indeed, we have observed the expression of tumor Ag on the surface of the DC side and DC marker on the surface of tumor cell side of the fusion cells (51). The combination of DC and tumor cells makes the fusion cells potentially good candidates from which to obtain the HSP-based vaccine. It has been recognized that DC are the most potent APC in the body possessing efficient Ag processing (endosome/lysosome) and presentation machinery, whereas tumor cells express abundant tumor Ags. In contrast, the fusion cells inherit the Ag-processing and presentation machinery from DC and possess the full set of accessory molecules necessary for Ag processing and presentation. These molecules probably facilitate or participate in the Ag processing and presentation of tumor Ags, making them enriched in a wider repertoire of immunogenic peptides. In addition, the fusion cells inherit from tumor cells the ability to synthesize tumor Ag de novo, thus making these synthesized tumor Ags accessible to the endogenous processing pathway. It is likely that the Ag-processing machinery from DC can sort or select the immunogenic peptides to be processed and presented and work much more efficiently than that from tumor cells, thus increasing the quality and quantity of the HSP-associated complexes.

Induction of a robust CD4 and CD8 T cell response by HSP70-PC derived from fusion cells is probably another mechanism underlying the efficacy of HSP70.PC-F. Immunization with HSP70.PC-F and, to a lesser extent, with HSP70.PC-Tu resulted in the significant proliferation of CD4 and CD8 T cells and increased the T cell populations expressing effector and memory markers. The induction of these T cells by HSP70.PC-F probably constitutes the enhanced antitumor immunity that provide protection of the immunized mice. It should be noted that the populations expressing CD69, CD44, CD44/IL-15R, and IFN-{gamma} also increased in CD4 T cells from HSP70.PC-F-immunized mice. There is increasing evidence that CD4 T cells activated by MHC class II-restricted epitope play a critical role in the antitumor immunity (52, 53). The optimal effects of vaccination will likely involve both arms of cellular-mediated immunity.

In the present study, we have largely concentrated on HSP70 because these chaperone proteins are associated with the immunogenicity of DC-tumor fusion cells (our unpublished data). It is likely that other stress proteins, with markedly different peptide-binding domains, bind to different spectra of cellular peptide Ags and may thus play additive roles in antitumor immunity (54, 55).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Cancer Institute Grant R01 CA87057, the Susan G. Komen Breast Cancer Foundation, and by funding from Boston University School of Medicine. Back

2 Y.E. and A.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jianlin Gong, Department of Medicine, Boston University School of Medicine, Room 309, 650 Albany Street, Boston, MA 02118. E-mail address: jgong{at}bu.edu Back

4 Abbreviations used in this paper: HSP, heat shock protein; DC, dendritic cell; WT, wild type; Tg, transgenic; KO, knockout; LN, lymph node; LNC, LN cell; ON, overnight; PEG, polyethylene glycol; RT, room temperature; PC, peptide complex; GA, geldanamycin; HS, heat shock treated. Back

Received for publication April 5, 2006. Accepted for publication August 16, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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