PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2010 September 1.
Published in final edited form as:
PMCID: PMC2754732
NIHMSID: NIHMS139619

T cell activation by HSP70 vaccine requires TLR signaling and scavenger receptor SREC-1

Abstract

HSP70 isolated from tumor-dendritic cell fusions (HSP70.PC-F) induces potent anti-tumor immunity and prevents growth of such tumors. In the present study, we have examined mechanisms underlying such anti-tumor activity of the HSP70.PC-F vaccine. The degree of anti-tumor immunity induced by HSP70.PC-F depended on intact TLR signaling in immunized animals, and mice in which the tlr2 and tlr4 genes were both inactivated did not respond to the vaccine. The reduced responses to HSP70.PC-F vaccine in such tlr knockout mice were restored by immunization of animals with HSP70.PC-F pulsed wild-type DC, indicating a key role for this cell type in HSP70.PC-F mediated immunity. Our studies also indicate a role for the HSP70 receptor SREC-1 in anti-tumor immunity induced by HSP70.PC-F. These two receptor types appeared functionally interdependent as indicated by the finding that tlr2 and tlr4 knockout decreases HSP70 binding in double knockout DC and reduces SREC-1 expression. In addition, TLR-dependent, tumor cell killing was suppressed by SREC-1 knockdown in DC, suggesting a significant role for this receptor in HSP70.PC-F-mediated tumor immunity.

Introduction

We have recently developed a molecular chaperone based anti-cancer vaccine that reverses the immune tolerance of murine cancer cells and leads to protective immunity against tumor challenge (1). This vaccine was developed by isolation of Heat shock protein70 (HSP70) from fusion cells derived from dendritic and murine cancer cells (HSP70.PC-F). Such DC-tumor fusion possesses desirable properties as mediators of tumor immunity due to increased presentation of tumor antigens to T lymphocytes (2). We found that HSP70 plays a key role in such immunity and that HSP70 depletion from tumor-DC fusion cells leads to significant loss of ability to stimulate immunity (unpublished data). HSP70 and other molecular chaperones have been shown to have potential as anti-cancer vaccines due to their ability to capture and chaperone tumor antigens in a relatively non-selective manner (3-6). We therefore examined the potential of HSP70.PC-F in tumor immunotherapy. Indeed, HSP70.PC-F possesses superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells that have not undergone fusion with DC (1). Of most significance however, immunization of mice with HSP70.PC-F resulted in a T-cell-mediated immune response including a significant increase in CD8+ T cell proliferation and the induction of the effector and memory T cells capable of breaking T cell unresponsiveness to a non-mutated tumor antigen (MUC1) and providing protection of mice against challenge with tumor cells. By contrast, immune responses to vaccination with a conventional HSP70 based vaccine derived from tumor cells were less potent against such a non-mutated tumor antigen (1). HSP70.PC-F complexes differed from those derived from tumor cells alone in a number of key properties. Most notable among these differences was an enhanced association with immunologic peptides. HSP70.PC-F evidently chaperones an increased repertoire of antigenic peptides as indicated by co-immunoprecipitation experiments. In addition, activation of DC by HSP70.PC-F was dependent on the expression of the myd88 gene a finding that suggests a potential role for toll-like receptor (TLR) signaling in DC activation and T cell stimulation by the vaccine. These experiments indicated that HSP70-PC-F derived from DC-tumor fusion cells have increased immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine (1).

In the present study, we have examined mechanisms underlying anti-tumor immunity induced by the HSP70.PC-F vaccine. Effective vaccination was shown to depend on intact TLR signaling in immunized animals. Reduced responses to the HSP70.PC-F observed in tlr2−/− / tlr4−/− double knockout mice could however be restored by the immunization of knockout mice with wild-type DC pulsed with HSP70.PC-F. We have also demonstrated a role for the HSP70 receptor SREC-1 in tumor cell responses to HSP70.PC-F vaccine. SREC-1 was responsible for an important component of HSP70 binding to DC. The two receptor types appeared interdependent in our studies, as tlr2−/− / tlr4−/− double knockout led to depletion in HSP70 binding to DC and to reduced SREC-1 expression. In addition, TLR-dependent, HSP70.PC-F induced anti-tumor immunity was suppressed by SREC-1 knockdown, indicating a significant role for this receptor in HSP70 mediated immunity.

Materials and Methods

Mice

C57BL/6 wild-type (WT) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). MyD88 knockout (myd88−/−) and TLR2 / TLR4 double knockout tlr2−/− / tlr4−/− mice were obtained from Dr. Stuart Levitz (Boston Medical School, Boston, MA). The mice were maintained in microisolator cages under specific pathogen-free conditions.

DC generation and stimulation

Dendritic cells were obtained from bone marrow culture of mice using the method described previously (7) 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 μg/ml; Sigma). On the third day of culture, the non-adherent cells were collected and cultured in medium containing GM-CSF overnight. Then the loosely adherent DC were collected and further cultured in the presence or absence of LPS (6 μg/ml), HSP70 immunoprecipitation from DC-tumor fusion cells by anti-HSP70 antibody (HSP70.PC-F, 6 μg/ml) or recombinant HSP70 purified by ADP-agarose column (rHSP70, 6 μg/ml) for 12, 48 or 96 hours in 12 well plates. DC cultured in medium alone was used as control. The DC were collected at indicated time for further analysis.

Hsp70.PC were prepared as described in our earlier study (1). The HSP70 preparations were routinely checked by Limulus amebocyte lysate (LAL Cambrex BioScience) assay to ensure no contamination of endotoxin. The level of endotoxin was always less than the lowest control standard (<0.1 EU/ml).

Binding Assay

DC collected on day 3–5 of culture were washed twice with serum-free RPMI medium. The DC were incubated with 10 μg/ml of Alexa 488-labeled HSP70 for 1 hour at 37°C. In some experiments, cells were incubated with Alexa 488-labeled HSP70 on ice or at 37°C. For scavenger receptor agonist / inhibition assays, the cells were pre-incubated with 2.5 mM mBSA for 30 min followed by incubation with 10 μg/ml of Alexa 488-labeled HSP70. The cells were washed three times with PBS, fixed with 2% paraformaldehyde, and then analyzed by FACScan (Becton Dickinson, Bedford, MA) with CellQuest analysis software.

Flow Cytometry

DC cultured for 3–4 days were purified. The DC were washed twice with PFNC buffer (PBS containing 0.5% FBS, 0.05% NaN3 and 1mM CaCl2), and then stained with anti-SREC1 antibody (1:50 dilution) for one hour followed by FITC anti-Rat IgG (1:100 dilution) for 30 min at 4°C. In some experiments, DC were further stained with phycoerythrin (PE)-conjugated anti-MHC class II (M5/114), CD86 (GL1) or B7-DC (TY25) (BD Pharmingen) for an additional 40 min on ice. The cells were then washed, fixed, and analyzed by FACScan (Becton Dickinson, Bedford, MA) with CellQuest analysis software.

Immunohistochemistry

The loosely adherent cells (immature DC) isolated from day 3 culture of BM cells in the presence of GM-CSF were stimulated with LPS, HSP70.PC-F or HSP70 overnight. DC cultured in GM-CSF medium and exposed to vehicle (PBS) were used as control. The cells were sedimented by cytospin on the slides, stained with anti-SREC1 mAb and then examined under microscopy.

Confocal Microscopy

DC were cultured for 4 days, purified and placed in a #1 coverslide cell chamber O.N. The DCs were labeled with Alexa 488-labeled HSP70 (green color) for 30 minutes on ice for efficient binding and later cold media was replaced with warm media and incubated for 2–5 minutes at 37°C. Cells were then fixed with 4% para-formaldehyde for 10 minutes at room temperature. Cells were permeabilized and blocked before antibody staining. Fixed DCs were stained with anti-SREC-1 antibody followed by staining with Cy3-conjugated goat anti-rabbit IgG (red color). The immunofluorescence stained cells were analyzed using a Zeiss 510 confocal microscope. Fluorophores were visualized using the following filter sets: 488 nm excitation and BP 505–530 emission filter for Alexa 488, 543 nm excitation and BP 560–615 for Cy3, 633 excitation and LP 650 for Cy5. Images were normally taken using a 63 X NA 1.4 oil immersion objective lens. Figures were created using Adobe Photoshop 7.0 with some contrast adjustments

Lentivirus production and transduction

MISSION™ shRNA plasmids (shRNA) were purchased (Sigma-Aldrich, St. Louis, MI) and the Lentivirus generation and transduction were performed according the manual of ViralPower™ Lentiviral Expression Systems (Invitrogen). The effectiveness of shRNA for knockdown of murine SREC-1 (five clones: TRCN0000067873; TRCN0000067874; TRCN0000067875; TRCN0000067876 and TRCN0000067877) was examined by both Real-Time PCR and Western Blot analysis. The most effective construct for mSREC-1 mRNA knockdown (TRCN0000067875) was used in the experiments. The Lentivirus (insert sequence is 5'–CCGCAGGTATGCACGCGT-3' which does not target any mouse genes, but will activate RISC and the RNAi pathway in the cells) was used as negative control. Three-day-old DC were collected, purified, placed in 96 well round bottom plates with 1×106 cell per well for ON culture containing GM-CSF medium. On the second day, half of the medium (100 μl) was removed and 100 μl Lenti-virus supernatant (1 virus :1 DC) was added. After 20 hours, 150 μl of medium from each well was replaced by fresh GM-CSF medium for additional culture. DC infected with SREC-1 shRNA or control constructs or un-infected DC were collected for SREC-1 expression and T cell stimulation assay.

T cell proliferation

Splenocytes and/or lymph node cells (LNC) were isolated from mice immunized with HSP70.PC extracted from fusions of DC and tumor cells (HSP70.PC-F). 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, 100U/ml penicillin, 100μg/ml streptomycin, and 5 × 10−5 M β-mercaptoethanol. In some experiments, T cells were further dual-stained with FITC-conjugated anti-CD4 (H129, 19) mAb and PE-conjugated anti-CD8α (53–6.7) mAb (BD Pharmingen) for 30 min on ice and then sorted into CD4+ or CD8+ subsets by MoFlo (Cytomation, Fort Collins, CO) with Summit v3.0 analysis software. The sorted CD4+ or CD8+ T cells were washed and resuspended in RPMI-1640 medium, and co-incubated with indicated DC at ratio 10:1 (10 T cells: 1 DC) in the presence or absence of HSP70.PC-F in 96-well, U-bottom plates for 5 days. T-cell proliferation was measured by [3H] thymidine incorporation after the cells were pulsed with 1 μCi/well of [3H] thymidine for 12 hours.

Measurement of CTL

To assess the antigen-specific T cells, LNC were isolated from mice twice immunized with HSP70.PC-F or injected with PBS, stained with MUC1–8 iTAg (SAPDTRPA) or irrelevant iTAg (SIINFEKL OVA peptide) and analyzed by FACS. To measure the CTL activity, splenocytes were isolated from vaccinated or control mice. MC38/MUC1, B16/MUC1 and B16 tumor cells (1–2 × 106 cells) were labeled with 100–200 μCi Na251CrO4 for 60 min at 37°C, and then washed to remove unincorporated isotope. Splenocytes and tumor targets were incubated at 100:1 effector-to-target ratios (E:T) for 5 h at 37°C with 5% CO2 in 96-well U-bottom plates. 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) × 100.

Statistical analysis

Statistical significance was analyzed using χ2 and Student's t test. Percentage of positive cells in phenotype assay of DC was derived from two or three independent experiments and presented as means ± SD.

Results

Activation of immunity and T cell mediated cytotoxicity requires MyD88 and TLR expression

We first examined the role of MyD88 / TLR signaling in the induction of antigen-specific CTL by HSP70 based vaccine (HSP70.PC-F) derived from DC fusion with MC38/MUC1 cells (FC/MUC1) (Fig. 1). This vaccine has been shown to induce immunity against MC38/MUC1 tumors (1).

FIGURE 1
Inactivation of MyD88 and TLR2/TLR4 inhibits HSP70.PC-F induced antitumor immunity (A) HSP70 induced T cell proliferation. LNC and splenocytes were isolated from WT, MyD88 KO and TLR2/4 KO mice (N=3) previously immunized twice with 10μg HSP70.PC-F ...

We immunized wild type (WT), MyD88 KO and TLR2/4 KO mice with HSP70.PC-F and, seven days after the second immunization, draining lymph node cells (LNC) and splenocytes were isolated and assayed for proliferation and CTL activity. T cells from immunized WT mice proliferated vigorously (Fig. 1A). By contrast, minimal cell proliferation was observed in T cells from either immunized MyD88 KO or TLR2/4 KO mice (Fig. 1A). We chose to inactivate both TLR2 and TLR4 as earlier studies showed that HSP70 can initiate signaling through both TLR molecules (8). Our findings of reduced T lymphocyte proliferation in MyD88 or TLR knockout cells were reflected in the tumor cell killing activity of isolated T cells (Fig. 1B). CTL from immunized WT mice were effective in killing tumor cell targets MC38/MUC1 and this property was reduced in cells isolated from immunized MyD88 KO (myd88−/−) and TLR2/4 KO (tlr2−/− / tlr4−/Ȓ) mice (Fig. 1B, left panel). In addition, CTL from WT mice immunized with HSP70.PC-F were capable of antigen specific killing of MUC1 expressing malignant cells (B16/MUC1 tumor cells), but not MUC1-negative B16 tumor cells (Fig. 1B, middle and right panels), suggesting antigen-specific killing. HSP70.PC-F has been shown to carry high levels of MUC1 derived tumor antigens (1).

The reduced ability of tlr2−/− / tlr4−/− mice to support HSP70.PC-F induced immunity may be due to a number of mechanisms. One such mechanism could be reduced expression of HSP70 receptors on DC in tlr2−/− / tlr4−/− mice resulting in loss of ability of such DC to take up tumor antigens and induce T cells. Indeed, there was markedly reduced HSP70 binding to bone-marrow-derived DC isolated from myd88−/− mice, compared to wild-type controls (Fig. 1C). Furthermore, double knockout of the tlr2 and tlr4 genes led to almost quantitative loss of HSP70 binding to the tlr2 / tlr4−/− DC, a reduction which was more complete than the deficiency in HSP70 binding to myd88−/− cells (Fig. 1C). As our previous studies showed that scavenger receptors are important potential HSP70 receptors, we examined a role for these receptors in MyD88 / TLR-mediated HSP70 binding to DC. Indeed when cells were pre-incubated with scavenger receptor ligand maleylated bovine serum albumin (mBSA) we observed almost quantitative inhibition of HSP70 binding in wild-type DC, suggesting a profound role for scavenger receptors in HSP70 binding, consistent with earlier findings (Fig. 1C). In controls, unmodified BSA failed inhibit HSP70 binding, consistent with the known ability of scavenger receptors to bind chemically modified proteins such as mBSA while not interacting with the native protein (unmodified, purified BSA).

Involvement of TLR signaling in the expression of scavenger receptors

Earlier studies indicated that scavenger receptor family members including SREC-1, LOX-1 and FEEL-1 are potential HSP70 receptors (9, 10). We have concentrated on one such receptor, SREC-1 due to its potential role in tumor immunity. SREC-1 is expressed on the surface of a small proportion (<5%) of unstimulated immature WT-DC (3 day old) (Fig. 2A). These cells express relatively high levels of cell surface MHC class II (Fig. 2A). Stimulation of DC with LPS, HSP70.PC-F or rHSP70 led to an increase in cell surface expression of SREC-1 as demonstrated by FACS analysis (Fig. 2B). Levels of DC positive for SREC-1 increased to 16.84%, 10.86% and 9.66%, respectively, when cultured with LPS, HSP70.PC-F or rHSP70. The difference of SREC-1 expression between DCs with or without LPS or HSP70 stimulation was statistically significant (P<0.05) (Fig. 2C). Immunohistochemical staining of the DC populations also shows the up-regulated expression of SREC-1 on DC after treatment with LPS or HSP70.PC-F (Fig. 2D). In addition, increased expression of SREC-1 was associated with the formation of veiled cytoplasmic processes on DC stimulated by LPS, HSP70.PC-F or rHSP70, morphological characteristics of DC maturation (Fig. 2D).

FIGURE 2
Up-regulation of SREC-1 in DC exposed to LPS, HSP70.PC-F or rHSP70. (A) SREC-1 is preferentially expressed in cells abundant in MHC class II. Three-day-old DC were were stained with anti-SREC-1 antibody (1:50 dilution) at 4°C for 1 hour, followed ...

Co-localization of SREC-1 and HSP70 in DC

The previous studies indicate that HSP70 binding and SREC-1 expression are correlated, at least in a proportion of the DC population. We therefore examined whether HSP70 and SREC-1 become co-localized in DC exposed to extracellular HSP70, using confocal microscopy (Fig. 3). When DC were incubated with HSP70 at 4°C, HSP70 associated with SREC-1 in punctate structures on the cell surface and tight association between ligand and receptor is indicated in the merged figure (Fig. 3A). When cells were warmed to 37°C after binding at 4°C, both SREC-1 and HSP70 were co-associated in intracellular vesicles that may be internalizing endosomes (Fig. 3B). SREC-1 is thus a strong candidate for a receptor that can bind and internalize HSP70-peptide complexes. Although co-localization is extensive, inspection of Figure 3 shows that it was not exclusive. There were cell surface zones in which Hsp70 was bound to regions low in SREC-1 staining (green) while some of the SREC-1 was not Hsp70 bound as indicated by maintenance of red color (Fig. 3A, B Merge). The experiment is consistent with the existence of other potential Hsp70 receptors such as LOX-1, SRA-1 and CD91 as discussed earlier. It is conceivable that the Hsp70 bound and internalized cell structures indicated in Fig 3B also contain these receptors and other molecules as well as SREC-1.

FIGURE 3
Co-localization of SREC-1 and HSP70 in DC. (A) DCs from 4 day old BMDC cultures were purified and labeled with Alexa 488-HSP70 (green) for 30 minutes at 4°C. Subsequently cells were stained for SREC-1 using anti-SREC-1 antibody (Cy3-IgG, red). ...

Inhibition of dendritic cell SREC-1 expression by small hairpin RNA infection

We next investigated whether SREC-1 plays a causal role in TLR dependent tumor immune responses stimulated by HSP70.PC-F. We used small hairpin RNA (shRNA) to deplete SREC-1 by RNA interference. Of five individual shRNA constructs tested in the RAW 231 mouse macrophage cell line each was more-or-less effective in knocking down SREC-1 mRNA levels (Fig. 4A). The most effective of these constructs (#5) was then selected, packaged into lentivirus and tested for effectiveness in inhibiting SREC-1 protein expression in the RAW264.7 macrophage-derived cell line (Fig. 4B). Infection of these cells with the shRNA-packaged lentivirus effectively inhibited SREC-1 expression (Fig. 4A and B). The expression of SREC-1 in DC was inhibited by this shRNA species. Infection with SREC-1 shRNA reduced SREC-1 levels in control cells and cells exposed to LPS, HSP70.PC-F or rHSP70, and in DC subpopulations positive for MHC class II (Fig. 4C) or B7-DC (Fig. 4D). To assess the potential role of SREC-1 on the ability of DC to stimulate T cells, we obtained T cells from mice immunized with HSP70.PC-F and sorted such T cells into CD4+ and CD8+ subsets. The CD4+ and CD8+ T cells were then co-cultured with DC infected with SREC-1 shRNA or control shRNA constructs in the presence of HSP70.PC-F. Infection of DC with SREC-1 shRNA constructs led to partial inhibition of HSP70 induced CD8+ T cell proliferation and almost complete inhibition of CD4+ T cell proliferation (Fig. 4E). In addition, SREC-1 knockdown inhibited expression of interferon gamma in both CD4+ and CD8+ T lymphocytes, suggesting inhibition of activation of T cells. These experiments indicate a significant role for SREC-1 in mediating antigen presentation of HSP70 chaperoned tumor antigen to both CD4+and CD8+ T cells by DC. These data indicate a mechanism involving HSP70.PC-F binding to SREC-1 followed by uptake of ligand-receptor complexes and facilitated transfer of antigenic peptides to MHC class I and class II molecules in DC.

FIGURE 4
Inhibition of expression of SREC-1 in DC by RNA interference blocks T cell stimulation in vitro. (A-B) The effectiveness of SREC1-shRNA (five clones) and control-shRNA (one clone) in depleting SREC-1 expression was first analyzed in RAW 264.7 mouse macrophages ...

Expression of SREC-1 on DC with induction of antigen-specific CTL in mice immunized with HSP70 peptide complexes

The previous experiment showed a significant role for SREC-1 in the stimulation of T cells by DC in the in vitro setting (Fig. 4). We next asked whether SREC-1 plays a similar role in vivo. We therefore immunized WT mice twice with the HSP70.PC-F at the base of the tail. Our earlier published studies showed that HSP70.PC-F derived from MUC1-positive fusion cells contains immunogenic MUC1 peptides (1). MyD88 and TLR2/4 KO mice were similarly immunized as controls since the previous experiments indicate that expression of SREC-1 is dependent on TLR signaling (Fig. 2). Seven days after the second immunization, the draining inguinal lymph nodes (LN) were collected and processed into frozen sections. The sections were then stained with anti-SREC1 and anti-CD3 antibodies. Immunization with HSP70.PC-F resulted in the expression within lymph nodes of SREC-1 positive cells with DC morphology in the midst of CD3 stained T cells in WT mice, and to a lesser extent, in MyD88 KO mice (Fig. 5A). By contrast, SREC-1-positive cells in tlr2−/− / tlr4−/− mice were at a similar low level compared to those in WT control mice injected with PBS instead of vaccine (Fig. 5A). We did not observe a similar distribution of DC expressing other potential HSP70 binding scavenger receptors such as LOX-1 or FEEL-1 (data not shown). These experiments therefore suggest that SREC-1 expressing DC can migrate to draining lymph nodes and potentially interact with T cells, but only in mice with intact TLR signaling.

FIGURE 5
Expression of SREC-1 on DC and induction of antigen-specific CTL induction in vivo. (A) Inguinal LN were collected from WT, MyD88 KO and TLR2/4 KO mice immunized with HSP70.PC-F or PBS injection, and then frozen and cryosectioned. The sections were stained ...

We next addressed the question of whether SREC-1 plays a role in HSP70.PC-F induced tumor antigen-specific CTL in vivo. To address this issue, we first examined whether the depleted immunity in tlr2−/− / tlr4−/− mice (Fig 1) could be reversed by immunization with HSP70.PC-F pulsed wild-type DC (WT-DC). We had shown previously that immunization with DC-tumor (MC38/MUC1) fusions cells could overcome the effects of tlr 2/4 knockout and mediate anti-tumor immunity and T cell killing of tumor cells (data not shown). This suggested that the major effects of TLR inactivation are mediated through a block in DC function and that this can be reversed by immunization with DC that possess wild-type TLR function. As shown above, immunization of WT mice with HSP70.PC-F-pulsed DC induces tumor specific CTL (Fig 5B, lane 2) compared to naïve DC alone that did not induce CTL (Fig. 5B, lane 1). Groups of tlr2−/− / tlr4−/− mice were next immunized with DC from WT mice infected in vitro with SREC-1 shRNA or control shRNA encoding lentivirus and then pulsed with HSP70.PC-F. Under these conditions, control DC infected with lentivirus containing an irrelevant small hairpin RNA sequence were able to activate CTL in TLR KO mice essentially as effectively as in wild type mice (compare Fig 5B lanes 3 and 2). However, SREC-1 knockdown inhibited the ability of WT-DC to compensate for TLR knockout (Fig. 5B, lane 4). Thus SREC-1 appears to be a significant component of the HSP70.PC-F-mediated, TLR-dependent tumor cell killing. Next, in order to assess the frequency of antigen-specific T cells induced, we assembled an MHC-class-I/peptide tetramer (iTAg). The 8-mer peptide (SAPDTRPA) is a dominant epitope from MUC1 that binds to C57BL/6 MHC class I, H-2Kb (11). The MUC1–8 iTAg was used to identify and assess the tetramer-positive T cells. As expected, the numbers of MUC1–8 iTAg-positive T cells were increased after immunization with WT-DC pulsed with HSP70.PC-F compared to naïve DC (Fig 5C, compare lanes 1 and 2). Tetramer positive T cells in tlr2−/− / tlr4−/− mice immunized with SREC-1 knockdown WT-DC were significantly decreased compared with those from mice immunized with WT-DC infected with control virus (Fig. 5C). Immunization of tlr2−/− / tlr4−/− mice either with HSP70.PC-F pulsed WT-DC after SREC-1 knockdown or after infection with control virus resulted 1.45% and 3.51% CD8 T cells positive for MUC1–8 (Fig. 5C) respectively. These experiments provide direct evidence that SREC-1 participates in the induction of antigen-specific T cells by HSP70.PC-F.

Discussion

Our previous studies showed that HSP70 complexes extracted from tumor-DC fusion heterokaryons (HSP70.PC-F) function as effective vaccines and provide protective antitumor immunity (1). Here we show that HSP70.PC-F induced anti-tumor immunity requires intact TLR signaling in the tumor bearing host in order to induce CTL activity (Figs. 1 and and5).5). Immunity was restored to tlr2−/− / tlr4−/− animals by vaccination with wild-type DC pulsed with HSP70 peptide complexes, indicating that DC are an essential population of immune cells that require TLR function for HSP70.PC-F induced immunity (Fig. 5).

A requirement for TLR signaling in immunity may involve a number of potential mechanisms. For instance, TLR signaling has been linked to DC maturation and increased expression of co-activator molecules required for efficient activation of T cells (12). Indeed some studies suggest that exposure of DC to purified HSP70 can directly induce co-activator expression (8). In addition, TLR 4 is thought to be involved in cross-presentation of phagocytosed antigens due to its ability to alter intralysosomal pH and slow the rate of antigen proteolysis (13, 14). The studies presented here indicate a third potential role for TLR signaling in the responsiveness of DC to exogenous antigens: the induction of expression of HSP70 receptors belonging to the scavenger receptor family (Figs. 2 and and5).5). Our studies imply that the capacity of DC to respond to HSP70 requires significant expression of scavenger receptor SREC-1 (Fig. 5). SREC-1 expression in DC was shown to be dependent on signaling through the TLR / myD88 pathways and indeed tlr2−/− / tlr4−/− cells were almost entirely deficient in SREC-1 expression. Confluent findings have also been reported for another HSP70 binding DC receptor - EWI/CD316 (15). This molecule was also induced by exposure of DC to LPS and was shown to bind the product of the HSP70 family gene Hspa8 that encodes Hsc70 (15). The TLR pathway may therefore play a complex role in the interactions of HSP70 with APC. For instance, some investigators report direct stimulation of TLR signaling and NFκB activation by extracellular HSP70 (16-19). The present study indicates that TLR signaling also indirectly affects DC responses through control of scavenger receptor expression and cell surface display of such receptors (Figs 2 and and5).5). Furthermore, additional reports show that TLR2 can signal through a pathway downstream of scavenger receptors SREC-1 and LOX-1 and induce NFκB-dependent transcription in an SR-dependent manner (20). However, other reports indicate that scavenger receptor A failed to activate antigen cross priming by heat shock proteins, findings which imply that not all HSP-SR interactions lead to enhanced immunity (21, 22). The finding of a requirement for TLR pathway intermediates in HSP70.PC-F induced tumor immunity suggests that HSP signaling and immune stimulation may be dependent on a complex relationship between the environment in which the APC is located (more or less inflammatory), local HSP-peptide complex concentrations, and the phenotype of the APC that encounters such extracellular HSP (23). Furthermore, receptors in addition to scavenger receptors and TLR family proteins play significant roles in the immune effects of HSPs. These receptors include CD40, CD91 and CCR5 (10). Further studies will therefore be required to construct an inclusive model for network signaling and immune stimulation triggered by HSP70.PC.

In conclusion therefore, SREC-1 appears to be an important receptor for HSP70 in the immune response. SREC-1 is expressed at relatively low levels in murine bone marrow-derived DC, although its expression is expanded on encountering the HSP70-based vaccine. Such HSP70-induced SREC-1 induction requires TLR expression in dendritic cells. The anti-cancer immune response to HSP70.PC-F vaccination therefore depends on positive cross-talk between at least two classes of pattern recognition receptors and mutual interactions among these molecules may finely tune the outcome of HSP70.PC-DC encounters.

Acknowledgments

Support: This work was supported by NIH research grants R01CA047407 (to S.K.C.); R01CA094397 (to S.K.C.) and R01CA119045 (to S.K.C.); by the US Department of Defense Breast Cancer Research programs (to J.G.); by the Ellison Foundation (to J.G.).

Abbreviations

Hsp
Heat shock protein
APC
Antigen presenting cells
CTL
cytotoxic T lymphocytes
SR
scavenger receptor
LOX-1
Lectin-like oxidized low-density lipoprotein receptor 1
FEEL-1
fasciclin, EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1
SREC-1
Scavenger receptor expressed by endothelial cells-1
SR-PSOX
Scavenger receptor that binds phosphatidylserine and oxidized lipoprotein
CXCL16
CXC chemokine ligand (CXCL)16, TLR, Toll Like Receptor

References

1. Enomoto Y, Bharti A, Khaleque AA, Song B, Liu C, Apostolopoulos V, Xing PX, Calderwood SK, Gong J. Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell-tumor fusion cells. J Immunol. 2006;177:5946–5955. [PubMed]
2. Gong J, Koido S, Calderwood SK. Cell fusion: from hybridoma to dendritic cell-based vaccine. Expert Rev Vaccines. 2008;7:1055–1068. [PubMed]
3. Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol. 2002;20:395–425. [PubMed]
4. Srivastava PK. Heat shock protein-based novel immunotherapies. Drug News Perspect. 2000;13:517–522. [PubMed]
5. Murshid A, Gong J, Calderwood SK. Heat-shock proteins in cancer vaccines: agents of antigen cross-presentation. Expert Rev Vaccines. 2008;7:1019–1030. [PubMed]
6. Calderwood SK, Gong J, Theriault JR, Mambula SS, Gray PJ., Jr. Cell stress proteins: novel immunotherapeutics. Novartis Found Symp. 2008;291:115–131. discussion 131-140. [PubMed]
7. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176:1693–1702. [PMC free article] [PubMed]
8. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem. 2002;277:15028–15034. [PubMed]
9. Theriault JR, Adachi H, Calderwood SK. Role of scavenger receptors in the binding and internalization of heat shock protein 70. J Immunol. 2006;177:8604–8611. [PubMed]
10. Calderwood SK, Theriault J, Gray PJ, Gong J. Cell surface receptors for molecular chaperones. Methods. 2007;43:199–206. [PubMed]
11. Apostolopoulos V, Yu M, Corper AL, Teyton L, Pietersz GA, McKenzie IF, Wilson IA. Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design. J Mol Biol. 2002;318:1293–1305. [PubMed]
12. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335–376. [PubMed]
13. Kinchen JM, Ravichandran KS. Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol. 2008;9:781–795. [PMC free article] [PubMed]
14. Husebye H, Halaas O, Stenmark H, Tunheim G, Sandanger O, Bogen B, Brech A, Latz E, Espevik T. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. Embo J. 2006;25:683–692. [PubMed]
15. Kettner S, Kalthoff F, Graf P, Priller E, Kricek F, Lindley I, Schweighoffer T. EWI-2/CD316 Is an Inducible Receptor of HSPA8 on Human Dendritic Cells. Mol Cell Biol. 2007;27:7718–7726. [PMC free article] [PubMed]
16. Asea A, Kabingu E, Stevenson MA, Calderwood SK. HSP70 peptidembearing and peptide-negative preparations act as chaperokines. Cell Stress Chaperones. 2000;5:425–431. [PMC free article] [PubMed]
17. Quintana FJ, Cohen IR. Heat shock proteins regulate inflammation by both molecular and network cross-reactivity. Cambridge University Press; Cambridge: 2005.
18. Vabulas RM, Ahmad-Nejad P, da Costa C, Miethke T, Kirschning CJ, Hacker H, Wagner H. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J Biol Chem. 2001;276:31332–31339. [PubMed]
19. Vabulas RM, Ahmad-Nejad P, Ghose S, Kirschning CJ, Issels RD, Wagner H. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J Biol Chem. 2002;277:15107–15112. [PubMed]
20. Jeannin P, Bottazzi B, Sironi M, Doni A, Rusnati M, Presta M, Maina V, Magistrelli G, Haeuw JF, Hoeffel G, Thieblemont N, Corvaia N, Garlanda C, Delneste Y, Mantovani A. Complexity and complementarity of outer membrane protein A recognition by cellular and humoral innate immunity receptors. Immunity. 2005;22:551–560. [PubMed]
21. Wang XY, Facciponte J, Chen X, Subjeck JR, Repasky EA. Scavenger receptor-A negatively regulates antitumor immunity. Cancer Res. 2007;67:4996–5002. [PubMed]
22. Tewalt EF, Maynard JC, Walters JJ, Schell AM, Berwin BL, Nicchitta CV, Norbury CC. Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo. Immunology. 2008;125:480–491. [PubMed]
23. Kottke T, Sanchez-Perez L, Diaz RM, Thompson J, Chong H, Harrington K, Calderwood SK, Pulido J, Georgopoulos N, Selby P, Melcher A, Vile R. Induction of hsp70-mediated Th17 autoimmunity can be exploited as immunotherapy for metastatic prostate cancer. Cancer Res. 2007;67:11970–11979. [PubMed]