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The Journal of Immunology, 2006, 177: 1543-1551.
Copyright © 2006 by The American Association of Immunologists

Extracellular Targeting of Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 170 Enhances Tumor Immunity to a Poorly Immunogenic Melanoma1

Xiang-Yang Wang*,{dagger}, Hilal Arnouk*, Xing Chen*, Latif Kazim*, Elizabeth A. Repasky{ddagger} and John R. Subjeck*

* Department of Cellular Stress Biology, {dagger} Department of Urologic Oncology, and {ddagger} Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have demonstrated previously that immunization with tumor-derived endoplasmic reticulum (ER) chaperone glucose-regulated protein 170 (grp170) elicits potent antitumor immunity. In the present study, we determine the impact of extracellular targeting grp170 by molecular engineering on tumor immunogenicity and potential use of grp170-secreting tumor cells as a cancer vaccine. grp170 depleted of ER retention sequence "KNDEL," when secreted by B16 tumor cells, maintained its highly efficient chaperoning activities and was significantly superior to both hsp70 and gp96. The continued secretion of grp170 dramatically reduced the tumorigenicity of B16 tumor cells in vivo, although the modification did not alter its transformation phenotype and cell growth rate. C57BL/6 mice that rejected grp170-secreting B16 tumor cells (B16-sgrp170) developed a strong CTL response recognizing melanocyte differentiation Ag TRP2 and were resistant to subsequent tumor challenge. B16-sgrp170 cells also stimulated the production of proinflammatory cytokines by cocultured dendritic cells. Depletion studies in vivo indicate that NK cells play a primary role in elimination of viable B16-sgrp170 tumor cells inoculated into the animals, whereas both NK cells and CD8+ T cells are required for a long-term protection against wild-type B16 tumor challenge. Both the secreted and endogenous grp170, when purified from the B16 tumor, exhibited potent tumor-protective activities. However, the B16-sgrp170 cell appears to be more effective than tumor-derived grp170. Thus, molecular engineering of tumor cell to release the largest ER chaperone grp170 is capable of eliciting innate as well as adaptive immune responses, which may provide an effective cell-based vaccination approach for cancer immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Molecular chaperones actively assist folding process of proteins and counteract their incorrect aggregation under stress conditions (1). The majority of chaperone proteins, although constitutively expressed, augment their levels of expression upon cellular stress and are therefore classified as stress proteins. The pioneering studies of Srivastava et al. (2) demonstrate that tumor-derived stress proteins elicits potent antitumor immunity, which has provided promising new immunotherapeutic agents for cancer treatment. Considerable immunological evidence now supports the notion that immunogenicity of tumor-derived stress proteins is attributed to the tumor-specific Ags (e.g., peptides) associated with these chaperones (3). Although biochemical basis and biological relevance for peptide interactions with chaperones needs to be further explored (4, 5, 6, 7, 8), it has been well-documented that certain chaperones purified from tumor can serve as effective cancer vaccines in both prophylactic and therapeutic settings (9, 10, 11). The success of the preclinical studies has led to several clinical trials using autologous tumor-derived chaperone (particularly gp96/glucose-regulated protein (grp)943 and heat shock protein (hsp)70) preparations for immunotherapy (12, 13).

Recent studies have shown that receptor recognition and receptor-mediated endocytosis of the stress protein-peptide complexes is critical for Ag presentation on MHC molecules (14, 15). Indeed, several endocytic receptors, e.g., CD91, LOX1, and SR-A, have been identified (16, 17, 18). In addition to promoting cross-priming events, stress protein can promote phenotypic and functional maturation of professional APCs such as dendritic cells (DCs), indicated by up-regulation of MHC class II, CD86 and CD83 expression, and secretion of proinflammatory cytokines and chemokines (19, 20, 21, 22). TLRs 2/4 and the downstream MyD88/NF-{kappa}B pathway have been proposed to be involved in hsp70 and gp96-mediated APC activation (23, 24). Other cell surface receptors, including CD14 (20) and CD40 (25, 26), have also been found to transduce activation signals of hsp70. Thus, stress protein as an Ag carrier and as an immunostimulatory adjuvant offer a unique opportunity to use them for cancer immunotherapy.

Sequence analysis indicates that the largest endoplasmic reticulum (ER) chaperone grp170 represents a large and highly "diverged" relative of the hsp70 family (27, 28). Initial studies observed that grp170 associates with Ig chains as well as gp96/grp94 and grp78/BiP, suggesting that grp170 may have a role in the assembly/folding of secretory proteins (29). Grp170 also interacts with TAP-translocated peptides and may be involved in polypeptide trafficking in the Ag presentation pathway (30, 31). We have demonstrated recently that grp170 purified from tumor exhibits a more potent therapeutic efficacy as a cancer vaccine than other chaperones i.e., hsp70 and hsp110 (11).

Yamazaki et al. (32) first demonstrated that immunization with gp96-Ig secreting tumors generates potent tumor-rejecting CTLs. Subsequent studies by other groups also support the concept that extracellular targeting of stress protein enhances tumor immunogenicity (33, 34). In the present study, we determine the possibility of eliciting an antitumor response when the ER chaperone grp170 is directed to extracellular milieu. We showed that B16 melanoma cells secreting grp170 exhibited an enhanced immunogenicity in vivo, possibly due to activation of innate immunity (e.g., NK cells) and Ag (e.g., TRP2)-specific CTLs. Our data suggest that tumor-derived grp170 may act as a danger signal and concurrently present tumor-associated Ags, which support the potential use of tumor cell secreting grp170 as a vaccine in the clinical cancer therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

Eight- to 12-wk-old C57BL/6 mice purchased from the National Institutes of Health animal facilities were housed under pathogen-free conditions. B16 (F10) cells (H-2b), a spontaneous murine melanoma from American Type Culture Collection, and D121 cell line (H-2b), a subline of the Lewis Lung carcinoma, were maintained in DMEM supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All of the experimental procedures were conducted according to the protocols approved by the Institutional Animal Care and Use Committee.

Generation of B16 tumor cell line secreting grp170

Using mouse grp170 cDNA as a template, the sense primer (5'-ACCATCTCGCAAATAAAATAGT-3') and the antisense primer (5'-GTACAGTCTAGATTAATGGTGATGGTGATGATGTGAAGGCCGCTTCTGTCC-3') were used to generate a KNDEL-minus grp170 cDNA. PCR product was digested with BamHI/XbaI and ligated into pcDNA3.1. The sequence of the construct was verified by DNA sequencing. B16 cells were transfected with constructs using LipofectAMINE reagent (Invitrogen Life Technologies), following the manufacturer’s instruction. Cells were washed twice with medium 24 h later and allowed to recover for another 24 h. Cells were then trypsinized and subcloned by limiting dilution in medium containing 800 µg/ml G418. To characterize tumor cell-secreting grp170, culture media and cell lysates were collected from parental B16 cells, pcDNA-neo mock, or pcDNA-grp170-secreting B16 tumor cell (sgrp170)-transfected B16 cells, followed by immunoblotting using Abs against grp170 or His-tag as described previously (35). The secreted grp170 in culture supernatants is quantitated using ELISA with grp170 Abs and His-tag Abs. Recombinant grp170 protein was serially diluted and used as a standard.

Purification of the secreted grp170

Culture medium B16-sgrp170 cells were collected and centrifuged to remove cell debris. The supernatant was passed through a Ni-NTA-agarose (Qiagen) column (36). Following the extensive wash, grp170 was eluted off the column with buffer containing 250 mM imidazole. The presence of grp170 in eluted fractions was examined using Western blotting and SDS-PAGE staining. Endogenous grp170 derived from the B16 tumor was purified as described previously (11). Recombinant mouse grp170 protein and TRP2 protein were expressed and produced using a BacPAK baculovirus expression system (BD Clontech). Protein purity was assessed using SDS-PAGE stained with Coomassie brilliant blue. Protein concentration was determined using BCA protein assay kit (Pierce).

Cell proliferation assay

Cells were plated at a density of 1 x 104/well of a 12-well culture dish. At various time points after plating, cells were incubated with MTT purchased from Sigma-Aldrich. The resulting formazan was solubilized with 0.04 M HCl in isopropanol, and OD was determined at 570 nm using a microplate absorbance reader.

Thermal aggregation assay

A total of 150 nM luciferase alone or in the presence of 1:1 molar ratio of grp170, hsp70, or gp96/grp94 was equilibrated to room temperature in 25 mM HEPES (pH 7.4), 5 mM magnesium acetate, 50 mM KCl, and 5 mM 2-ME, followed by incubation at the indicated temperatures in a thermostated cuvette. Light scattering by protein aggregation was determined by measuring the increase of OD at 320 nm with a spectrophotometer.

Secretion of cytokines by APCs

Syngeneic bone marrow-derived DCs were prepared after 6-day culture in the presence of GM-CSF (20 ng/ml; BD Pharmingen) as described previously (11). A total of 1 x 106 bone marrow-derived DCs or thioglycolate-elicited macrophages was cultured in a 0.4-µm Transwell filter (Millipore) with medium alone, 5 x 105 B16-neo cells, B16-sgrp170 cells, LPS (100 ng/ml), or B16-sgrp170 cells in the presence of polymyxin B (20 µg/ml; Sigma-Aldrich) for 24 h. The supernatants were measured for the secretion of TNF-{alpha} and IL-1beta using Luminex multiplex bead immunoassays.

Tumor studies

For tumorigenicity assay, mice were injected s.c. with different doses of live B16 cells, B16-neo cells, and B16-sgrp170 cells in 100 µl of PBS into the left flank of mice. Tumor free mice were rechallenged with parental B16 tumor cells. Splenocytes were isolated from tumor-free mice to determine Ag-specific IFN-{gamma}-secreting T cells using ELISPOT assay as described previously (35). For tumor challenge study, mice (five mice per group) were immunized s.c. with 1 x 106 irradiated tumor cells in the right flank. Two immunizations at 2-wk intervals were given. Two weeks later, mice were challenged by s.c. injections of the indicated number of live parental B16 tumor cells into the left flank. Tumor growth was monitored every 2 days by measuring perpendicular tumor diameters using an electronic digital caliper. The tumor volume is calculated using the formula V = (the shortest diameter2 x the longest diameter)/2.

In vivo Ab depletion

Depletion of NK cell and CD4+ and CD8+ T cell subsets was accomplished by i.p. injection of 200 µg of PK136, GK1.5, and 2.43 mAb, respectively, given every other day for 6 days. Effective depletion of cell subsets was confirmed by FACS analysis of splenocytes 1 day before inoculation of tumor cells and maintained by the Ab injections once a week for the duration of experiment. Isotype-matched Abs were also used as control.

Statistical analysis

Tumor size data and cell proliferation data were analyzed using Student’s t test. Survival time was compared by the Kaplan-Meier method, and tumor incidence was analyzed using the log-rank tests. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Extracellular targeting of grp170 does not change the growth rate of the B16 tumor in vitro

Grp170 resides normally in the lumen of ER due to the presence of an ER retention signal, KNDEL, at its carboxyl terminus. To study impact of extracellular grp170 on tumor immunogenicity, we targeted grp170 to the extracellular environment by molecular engineering. Construction of the expression vector encoding a secreted form of grp170 was achieved by deletion of the KNDEL sequence followed by sequential fusion in frame to a 6x His tag, which is used to distinguish the secreted grp170 from the endogenous grp170. The well-characterized B16 melanoma tumor was chosen as a model in the present study because B16 cells are nonimmunogenic or poorly immunogenic (37, 38). Extracellular secretion of grp170 was confirmed by immunoblotting analysis of culture medium using grp170-specific Abs (Fig. 1A) and His-tag-specific Abs (data not shown). Tumor cells transfected with KNDEL-depleted grp170 cDNA (sgrp170) consistently secreted grp170 to the supernatant, whereas parental B16 cells and mock-transfected cells did not. The level of secreted grp170 in the culture supernatant was quantitated using ELISA. B16-sgrp170 tumor produced 320 ± 58 ng of grp170 per 106 cells within a period of 24 h. Expression of other stress proteins, such as gp96 and hsp70, was not affected (data not shown).


Figure 1
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  		FIGURE 1. Extracellular targeting of grp170 does not alter the growth rate of B16 tumor cells in vitro. A, Characterization of B16 tumor cells expressing a secretory form of grp170. Media samples and cell lysates were collected from parental B16 cells and pcDNA-neo mock- or pcDNA-sgrp170-transfected B16 cells, followed by immunoblotting analysis using anti-grp170 Abs. beta-actin Ab was also used for a control. B, In vitro proliferation rate of parental, mock-, or sgrp170-transfected B16 cells. Cells were plated at a density of 1 x 104/well in a 12-well plate. Cells proliferation was determined by a MTT assay at various time points. The cell number is the mean of quadruplicate samples.

 
We next compared the growth kinetics of B16, B16-neo, and B16-sgrp170 cell in vitro. MTT assay showed that B16-sgrp170 cell exhibited a similar proliferation rate as parental or mock-transfected cell (p > 0.05, B16-sgrp170 vs B16-neo), suggesting that grp170 secretion did not affect the growth of B16 tumor cells under culture condition (Fig. 1B). In addition, these cells displayed a similar anchorage-independent growth in soft agar, indicating that transformation phenotype of B16 tumor cells remains unchanged following transfections (data not shown).

Grp170 secreted by the B16 tumor exhibits a potent chaperoning activity

Given the fact that chaperoning property is important for stress protein mediated immunological functions (39, 40, 41), we examined the protein-"holding" ability of the secreted grp170 (i.e., ability to bind/chaperone a denatured protein substrate and inhibit heat-induced substrate aggregation). Taking advantage of a His-tag added to the C terminus of grp170, grp170 protein secreted into the medium was purified using a Ni-NTA column and examined for its chaperoning properties (Fig. 2, A and B). Using luciferase as a reporter protein, the ability of the secreted, KNDEL-minus grp170 to prevent heat-induced luciferase aggregation was compared with that of hsp70 family members (e.g., hsp70) and hsp90 family members (e.g., gp96/grp94). A light scattering assay demonstrated that both KNDEL-depleted grp170 (Fig. 2C) and full-length recombinant grp170 (data not shown) are equally effective in holding luciferase in a soluble state. The secreted grp170 is highly efficient in inhibiting luciferase aggregation at a 1:1 substrate to chaperone molar ratio, significantly more so than hsp70 and gp96/grp94.


Figure 2
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  		FIGURE 2. Extracellular targeting of grp170 reduces tumorigenicity. A, grp170 was purified from the culture medium of B16-sgrp170 cells using a Ni-NTA column. The presence of grp170 in different fractions was examined by immunoblotting using anti-grp170 Abs. B, Gel staining of the secreted grp170 purified from the culture medium. sgrp170 from eluted fraction 1 and eluted fraction 2 was dissolved in SDS-PAGE, followed by GelCode blue staining. C, grp170 secreted by B16 tumor cells exhibits highly efficient chaperoning activity. Luciferase was incubated alone or in the presence of purified grp170, gp96/grp94, or hsp70 (1:1 molar ratio) at 50°C for 30 min. Absorbance changes due to the luciferase aggregation were measured at 320 nm using a spectrophotometer. D, C57BL/6 mice (n = 5) were inoculated s.c. with different doses of B16, B16-neo, or B16-sgrp170 tumor cells. Mice were followed for 2 mo to determine tumor incidence. The experiments were performed independently three times with the similar results.

 
Extracellular targeting grp170 reduces tumor tumorigenicity and enhances tumor immunogenicity

To determine whether grp170 secretion affects tumorigenicity in vivo, syngeneic C57BL6 mice were injected in the left flank with different doses of live B16, B16-neo, or B16-sgrp170 tumor cell. Mice efficiently rejected inoculation of as many as 1 x 105 B16-sgrp170 cells, whereas mice injected with 1 x 104 B16 or B16-neo developed aggressive tumors (Fig. 2D). To investigate whether those mice rejecting B16-sgrp170 tumor have developed a protective immunity against wild-type B16 tumors, tumor-free animals were rechallenged with 1 x 105 B16 cells in the right flank. All of these animals were resistant to the subsequent tumor challenge (data not shown).

To evaluate the potential of using grp170-secreting tumor cells as a cancer vaccine, we immunized mice with irradiated B16, B16-neo, or B16-sgrp170 cells, followed by tumor challenge with wild-type B16 cells. It was observed that mice immunized with B16-sgrp170 cells effectively rejected tumors when challenged with up to 1 x 105 live B16 cells, whereas immunization with parental B16 or B16-neo cells offered no protection (Fig. 3A). Gamma irradiation was used because tumor cell vaccines are routinely irradiated before injections to avoid tumor cell proliferation in vivo. We found that the irradiation did not compromise the ability of B16-sgrp170 cells to synthesize and secrete grp170 (Fig. 3B). Flow cytometric analysis using annexin V and 7-aminoactinomycin D staining indicated that cells after irradiation still maintain the membrane intactness (data not shown). When mice were challenged with 5 x 105 live B16 cells, all mice developed tumors, probably due to the fast growth rate of this aggressive tumor (Fig. 3C). However, all the B16-sgrp170 cell-immunized mice displayed a significantly longer survival than untreated mice or those vaccinated with B16-neo cells (p < 0.05, B16-sgrp170 vs B16-neo).


Figure 3
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  		FIGURE 3. B16-sgrp170 tumor exhibits enhanced immunogenicity. A, C57BL/6 mice (n = 5) were immunized twice with 1 x 106 irradiated (12,000 rad) B16 cells, B16-neo cells, B16-sgrp170 cells, or left untreated. Two weeks after the second immunization, mice were challenged with different dose of wild-type B16 tumor cells. B, Tumor cells were gamma-irradiated using a Cs137 source and changed into fresh medium. Twenty-four hours after irradiation, culture media were collected from B16-neo or B16-sgrp170 cells for immunoblotting analysis. C, Two weeks after the vaccinations, mice (n = 10) were challenged with 5 x 105 wild-type B16 cells in the right flank and followed for survival. All of the experiments were repeated three times, and representative data are shown.

 
Grp170-secreting B16 tumor stimulates DCs to produce proinflammatory cytokines

To examine whether the secreted grp170 is a potent activator of APCs, bone marrow-derived DCs were cocultured for 24 h with B16 cells with or without extracellular expression of grp170. Tumor cells and DCs were separated by a 0.4-µm Transwell filter during coculturing. The B16-sgrp170 cells, but not mock transfectants, effectively stimulated DCs to produce proinflammatory cytokines TNF-{alpha} (Fig. 4A) and IL-1beta (Fig. 4B), indicating that direct contact between DCs and B16-sgrp170 cells is not required. Studies using thioglycollate-elicited macrophages as responder cells or medium derived from B16-sgrp170 cells showed similar results (data not shown). Furthermore, all culture media were endotoxin tested to be free of LPS, and DC activation by B16-sgrp170 cells was not blocked by adding LPS inhibitor polymyxin B (Fig. 4). Radiation treatment of the grp170-secreting tumor cells did not affect the cytokine production by DCs (data not shown), which was consistent with the pervious observation (42).


Figure 4
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  		FIGURE 4. B16-sgrp170 tumor cells stimulate DCs to produce proinflammatory cytokines. A total of 1 x 106 bone-barrow-derived DCs was cultured in a 0.4-µm Transwell with medium alone, 5 x 105 B16-neo cells, B16-sgrp170 cells, LPS (100 ng/ml), or B16-sgrp170 cells in the presence of polymyxin B (10 µg/ml) for 24 h. The supernatants were harvested and measured for the production of TNF-{alpha} (A) and IL-1beta (B).

 
Immunization with B16-sgrp170 elicits a TRP2-specific CTL response

By far the largest category of peptide Ags recognized by patient-derived melanoma reactive CD8+-T cells originate from melanocyte differentiation Ag (MDA) proteins (43). B16 melanoma is a more relevant model for evaluating immune responses specific for the melanoma Ags since it expresses multiple MDAs, and the sequences of murine MDAs, including tyrosinase, gp100, and TRP2, are all highly homologous to their human counterparts. Moreover, the sequences corresponding to several identified HLA-A2-restricted peptide epitopes are highly homologous or identical (44). Using the synthetic peptide epitopes derived from MDAs, we attempted to identify the potential antigenic targets recognized by B16-sgrp170-elicited CTLs. ELISPOT results indicated that immunization with B16-sgrp170 tumor cells elicited a potent cellular response specific for TRP2180–188 (Fig. 5A). A group of mice were immunized with grp170 protein derived from B16 tumors. Splenocytes from the immunized animals displayed Ag-specific IFN-{gamma} production when stimulated with TRP2180–188 epitope in vitro. The secreted grp170 protein and culture medium of B16-sgrp170 cells were subjected subsequently to immunoblotting analysis using TRP2-specific Abs (1/4000). Although the Abs were able to detect 1 ng of recombinant TRP2 protein, no TRP2 protein or its degradation products were detected in the sgrp170 preparation and B16-sgrp170 culture medium (Fig. 5B).


Figure 5
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  		FIGURE 5. Immunization with B16-sgrp170 cells generates CTLs recognizing TRP2180–188 epitope. A, C57BL/6 mice were immunized with irradiated 1 x 106 B16 cells, B16-neo cells, and B16-sgrp170 cells. A group of mice were immunized with 30 µg of B16 tumor-derived grp170. Splenocytes were isolated and stimulated overnight with 1 µg/ml Kb-restricted CTL epitopes MART127–34, TRP2180–188, gp10025–33, and tyrosinase269–377 derived from MDAs in the presence of 20 U/ml IL-2. IFN-{gamma} production was measured using ELISPOT assay. Representative data from three independent experiments are shown. B, Immunoblotting analysis of the secreted grp170 using TRP2-specific Abs. Thirty micrograms of purified grp170 (lane 6) and 20 µl of day 3 culture medium (lane 7) from B16-sgrp170 cells were dissolved by SDS-PAGE, followed by immunoblotting with anti-TRP2 Abs (1/4000 dilution). Recombinant TRP2 protein served as a positive control. Different amount of TRP2 protein was loaded to determine Ab sensitivities (lane 1, 2 µg; lane 2, 0.5 µg; lane 3, 20 ng; lane 4, 1 ng; and lane 5, 0.05 ng).

 
Involvement of immune cells in the reduced tumorigenicity and the enhanced immunogenicity

We first examined the immune effector cells involved in rejection of live B16 tumor cells secreting grp170. Mice were depleted of NK cell and CD4+ or CD8+ T cell subsets by Ab injections. Mice were then injected s.c with 1 x 105 B16-sgrp170 tumor cells (Fig. 6A). It was found that mice depleted of NK cells developed aggressively growing tumors, whereas depletion of CD8+ T cells only had a slight effect on rejection of B16-sgrp170 tumor cells.


Figure 6
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  		FIGURE 6. Immune cells involved in the elimination of B16-sgrp170 tumor and antitumor immunity against parental B16 tumor. A, Effect of immune cell depletion on the rejection of B16-sgrp170 tumor. Mice (n = 10) were depleted of CD4+- and CD8+-T cells or NK cells on days –6, –4, and –2. Mice were then inoculated on day 0 with live B16-sgrp170 (1 x 105 cells/mouse) and followed for tumor development. B, Effect of immune cell depletion on the rejection of wild-type B16 tumor. Mice (n = 10) were immunized with B16-sgrp170 tumor cells on days –28 and –14. Immune cell subsets were depleted on days –6, –4, and –2, followed by tumor challenge with parental B16 cells on day 0.

 
To further examine the role of these immune effector cells in memory response against challenge with the parental B16 tumor, Ab depletion of the subsets was conducted during the challenge phase. Mice were injected with B16-sgrp170 cells, followed by depletion of NK cells and CD4+ or CD8+ T cells (Fig. 6B). Mice were then challenged with wild-type B16 tumor cells. Depletion of either NK cells or CD8+ T cells abrogated the tumor-protective effect. However, antitumor immunity remained intact in mice depleted of CD4+ T cell.

The secreted grp170 protein purified from B16-sgrp170 culture medium exhibits antitumor activities

In view of the highly potent chaperoning and immunostimulatory activities of the secreted grp170, we determined ability of the secreted grp170 to provide a tumor protection. Mice were immunized with the secreted grp170 or endogenous grp170, which was purified from B16-sgrp170 culture medium and the B16 tumor, respectively. Tumor challenge studies demonstrated that the secreted grp170 is as effective as the endogenous grp170 in generating a potent tumor inhibitory effect in vivo (p < 0.05) (Fig. 7A). However, both proteins failed to significantly suppress the growth of D121 Lewis lung tumor (p > 0.05) (Fig. 7B), indicating a tumor specificity of the vaccine activity.


Figure 7
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  		FIGURE 7. The secreted grp170 exhibits tumor-specific vaccine activity. Mice (n = 5) were immunized twice at 2-wk intervals with the secreted grp170 (30 µg), the endogenous grp170 (30 µg), or left untreated. Two weeks after the second immunization, mice were challenged with B16 tumor (A) or D121 lung tumor (B) of C57BL/6 origin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Based on our previous observations that tumor-derived grp170 is highly immunostimulatory (11, 45), we reason that extracellular targeting of grp170 will provide a danger signal, as well as tumor-specific antigenic repertoire to the host immune system, which will result in a robust antitumor response. In the present study, we show that modification of tumor cells by transfection with KNDEL-depleted murine grp170 significantly enhances the immunogenicity of poorly immunogenic B16 melanoma.

Depletion of ER retention signal "KNDEL" of grp170 results in constitutive release of the protein as expected. However, grp170 in the sera of immunized animals is not detectable by ELISA (data not shown). Molecular characterization reveals that retargeting of grp170 from the ER to extracellular milieu did not change biochemical properties of the protein. In vitro analysis indicate that, similar to full-length grp170, the KNDEL-depleted grp170 secreted by the B16 tumor maintains its highly efficient chaperoning ability to hold protein substrate or antigenic peptide (X.-Y. Wang and J. R. Subjeck, unpublished data). Moreover, grp170 is significantly more effective in binding to and stabilizing denatured protein substrates when compared with hsc70/hsp70 and gp96/grp94. Data obtained from our recent chaperoning studies demonstrate that grp170 contains two essential substrate-binding regions, i.e., the beta-sheet domain and the C-terminal helix domain (36). We speculate that the enhanced immunogenicity by the secretable grp170 may be attributed to its highly efficient chaperoning capability because several reports support the idea that ability of chaperone-Ag complexes to generate a CTL response correlates with affinity with which the chaperone binds Ag (39, 40, 41). Our studies of various structural depletion mutants of grp170 also suggest that the chaperoning activity of grp170 is essential for its immunologic properties, e.g., receptor binding on APCs and Ag cross-presentation (46). In addition, grp170 secreted by B16 melanoma appears to maintain its potent immunostimulatory activities, as indicated by inflammatory cytokine secretion from APCs in vitro and efficient T cell priming and tumor rejection in vivo. The secreted grp170 is at least as effective as the endogenous grp170 in stimulating an antitumor response, implicating that both proteins are able to obtain access to the Ag pool inside B16 tumor cells. Compared with the multiple steps for the purification of the endogenous grp170, a simple purification procedure using a Ni-NTA column for isolating the secreted grp170 from culture media may help circumvent the loss of Ag(s) carried by the chaperone.

TRP2 has been shown to be recognized by CTLs of patients with melanoma and has also been defined as a potential tumor-rejection Ag in murine B16 melanoma. CTL peptide from mTRP2 presented by Kb is identical to HLA-A2-restricted T cell epitope identified from hTRP2, including the immediate flanking amino acid residues (47, 48). Our studies using defined CTL epitopes derived from murine melanoma Ags indicate that immunization with B16 tumor cells secreting grp170 primed CTLs specific for TRP2180–188. Given the fact that TRP2 protein or its degradation products was not detectable by the highly sensitive TRP2-specific Abs in both purified grp170 preparation and culture medium from grp170-secreting B16 cells, we believe that the grp170 from B16 tumor cells chaperones TRP2180–188 peptide or its precursors. Because chaperoning is a promiscuous function, it is conceivable that, in addition to TRP2, a spectrum of antigenic peptides is associated with the grp170.

In agreement with other reports showing that purified chaperones stimulate APCs (20, 21), grp170 also displays an adjuvant, peptide-independent activity as indicated by production of TNF-{alpha} and IL-1beta by APCs incubated with B16-sgrp170 cell or its culture medium. It strongly suggests that grp170 itself may serve as an endogenous danger signal. A confounding factor in studies to determine innate stimulatory activities of purified chaperone proteins includes the potential contamination of endotoxin. Indeed, removal of LPS from purified chaperone proteins reduces DC activation (49, 50). Thus, genetic modification of chaperone genes to provide either a secreted or membrane-anchored protein allows study of the innate stimulating properties of chaperones in a setting devoid of possible endotoxin contamination (51, 52). However, more studies are required to exclude the possibility that other molecules, as a result of extracellular expression of grp170, are involved in APC activation.

Ab depletion studies demonstrate that NK cells play a dominant role in elimination of the grp170-secreting B16 tumor cells in vivo. It is not surprising that modification of the B16 tumor by transfection with secretable grp170 leads to activation of NK cells. Srivastava’s group (9) made the first observation that NK cells are required for heat shock protein-based cancer immunotherapy. Multhoff et al. (53, 54) have reported that membrane-associated hsp70 enhanced NK cell recognition of tumor cells. In addition, tumor cells secreting hsp70 exhibit increased susceptibility to NK cell lysis (34). Immunization with gp96-secreting tumor cells (55) or tumor-derived autologous gp96 proteins (56) has been shown to elicit a strong expansion of NK cell. In any case, the capability of activating another arm of immune system, i.e., NK cells, by grp170-secreting cells indicates the strength of chaperone-based cancer immunotherapy. More recently, Baker-LePain et al. (33) demonstrated that murine fibroblasts transfected with secretable gp96 or a truncated gp96 lacking the presumptive peptide-binding domain was able to significantly delay 4T1 mammary tumor growth, when used as prophylactic vaccines. Those cells effectively induced Th1 polarization of CD4+ T cells, with production of large amount of IFN-{gamma} (52). Tumor Ag-independent effects observed in this study highlight the role of stress protein-mediated innate immunity in tumor control. However, Argon’s group (7, 8) recently demonstrated that the peptide binding activity of the chaperone gp96 resides within the N-terminal fragment, which may account for the T cell stimulatory activity of gp96. Thus, the structure/immunological function relationship of stress protein, as well as its role in innate (Ag independent) and adaptive (Ag dependent) immunity, needs additional investigation.

To further determine the involvement of immune effector cells in tumor immunity observed, following immunization with grp170-secreting B16 tumor cells, we performed Ab depletion before tumor challenge. Both NK cell and CD8+-T cell are required for rejection of the subsequently inoculated B16 tumor. It is likely that initial activation of NK cell promotes the killing and elimination of the grp170-secreting B16 tumor cells, which helps additionally provide Ag source or cytokines to APCs, leading to an Ag (e.g., TRP2)-specific CTL response. In light of recent reports (57, 58) on the cross-talk between DCs and NK cells, whether NK cells contribute to DC maturation and subsequent CTL priming remains to be elucidated.

In our experimental setting, tumor-secreted grp170 acts as a danger signal and concurrently chaperones B16 tumor-associated Ags (e.g., TRP2) to APCs. Collaboration of innate components (e.g., NK cells) and adaptive components (e.g., T cells) contributes to the long-term protective immunity against the wild-type B16 tumor. Supporting evidence also came from the finding that NK cell depletion significantly reduced T cell expansion in response to immunization with cells secreting gp96-Ig fusion protein (55). Regardless of the immunological mechanisms involved, these studies suggest that certain chaperones are associated with tumor immunogenicity (59, 60, 61) and that manipulation of compartmentalization of chaperones may provide a novel approach to boost the immune response and help break tolerance to tumor Ags that otherwise remain immunologically silent in the progressively growing tumor. Indeed, tumor cells with chaperones genetically engineered to be expressed on the cell surface (51, 62) or secreted into the extracellular environment (32, 34) display potent vaccine activities.

Extracellular targeting of grp170 through gene therapy approaches may eliminate the need to isolate chaperone proteins from tumors while maintaining the polyvalent vaccine activities of purified stress proteins (i.e., the broad spectrum of antigenic repertoire). Moreover, chaperones and whole tumor cells have been shown to require different immune effector cells for their immunologic activity (63). Tumor cell-secreting grp170 may be able to combine the potential of chaperone-Ag complexes and cell-based vaccines, which is also supported by the present study that the grp170-secreting B16 cell is more potent than the tumor-derived grp170 in eliciting a tumor-protective effect. However, a significant disadvantage with the use of genetically modified autologous tumor cells is the substantial efforts of tumor cell harvest, propagation, genetic modification, and validation. Thus, employment of effective vector, e.g., adenovirus to introduce the gene for secretable grp170 directly into primary tumors, may offer more benefits in the clinic. In addition, there is evidence that stress protein can also induce a regulatory phenotype in T cells with immunosuppressive functions (64, 65). Thus, any stress protein-based vaccine strategies for cancer treatment should be designed cautiously and evaluated rigorously. Nonetheless, exposing tumor-associated chaperones to host immune system by extracellular targeting in the present study appears to be an effective means of mounting an antitumor immune response and may provide a new therapeutic approach for cancer immunotherapy.


   Acknowledgments
 
We thank Dr. Drew Pardoll (Johns Hopkins University, Baltimore, MD) for providing the GK1.5 and 2.43 hybridomas. We also thank Dr. Christopher Nicchitta (Duke University, Durham, NC) for providing recombinant gp96/grp94 protein and Dr. Vincent Hearing (National Cancer Institute, Bethesda, MD) for providing TRP2 cDNA and Abs.


   Disclosures
Top
 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 CA121848 (to X-Y.W.) and National Institutes of Health Grants RO1 CA 099326 and PO1 CA094045 (to J.R.S.). This research used core facilities supported in part by Roswell Park Cancer Institute’s National Cancer Institute-funded Cancer Center Support Grant CA 16056. Back

2 Address correspondence and reprint requests to Dr. Xiang-Yang Wang, Department of Cellular Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263. E-mail address: xiang-yang.wang{at}roswellpark.org Back

3 Abbreviations used in this paper: grp, glucose-regulated protein; hsp, heat shock protein; DC, dendritic cell; ER, endoplasmic reticulum; B16-sgrp170, grp170-secreting B16 tumor cell; MDA, melanocyte differentiation Ag. Back

Received for publication November 17, 2005. Accepted for publication May 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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