Heat shock protein (HSP) 70 isolated from tumor-dendritic cell (DC) fusions (HSP70.PC-F) induces potent antitumor immunity and prevents growth of such tumors. In the present study, we have examined mechanisms underlying such antitumor activity of the HSP70.PC-F vaccine. The degree of antitumor 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 scavenger receptor expressed by endothelial cells-1 (SREC-1) in antitumor 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.
We have recently developed a molecular chaperone-based anticancer 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 protein (HSP)3 70 from fusion cells derived from dendritic cells (DC) 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 Ags 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and that HSP70 depletion from tumor-DC fusion cells leads to significant loss of ability to stimulate immunity (our unpublished data). HSP70 and other molecular chaperones have been shown to have potential as anticancer vaccines due to their ability to capture and chaperone tumor Ags in a relatively nonselective manner (3, 4, 5, 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 nonmutated tumor Ag (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 nonmutated tumor Ag (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 coimmunoprecipitation 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 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 antitumor 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 (KO) mice could, however, be restored by the immunization of KO mice with wild-type DC pulsed with HSP70.PC-F. We have also demonstrated a role for the scavenger receptor expressed by endothelial cells-1 (SREC-1) in T 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, because tlr2−/−/tlr4−/− double KO led to depletion in HSP70 binding to DC and to reduced SREC-1 expression. In addition, TLR-dependent, HSP70.PC-F-induced antitumor immunity was suppressed by SREC-1 knockdown, indicating a significant role for this receptor in HSP70-mediated immunity.
Materials and Methods
C57BL/6 wild-type (WT) mice were obtained from The Jackson Laboratory. MyD88 KO (myd88−/−) and TLR2/TLR4 double-KO tlr2−/−/tlr4−/− mice were obtained from S. Levitz (Boston Medical School, Boston, MA). The mice were maintained in microisolator cages under specific pathogen-free conditions.
DC generation and stimulation
DC 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-Aldrich). On the third day of culture, the nonadherent 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 Ab (HSP70.PC-F, 6 μg/ml), or rHSP70 purified by ADP-agarose column (rHSP70, 6 μg/ml) for 12, 48, or 96 h 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 (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).
DC collected on day 3–5 of culture were washed twice with serum-free RPMI 1640 medium. The DC were incubated with 10 μg/ml Alexa 488-labeled HSP70 for 1 h 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 preincubated with 2.5 mM maleylated BSA (mBSA) for 30 min, followed by incubation with 10 μg/ml Alexa 488-labeled HSP70. The cells were washed three times with PBS, fixed with 2% paraformaldehyde, and then analyzed by FACScan (BD Biosciences) with CellQuest analysis software.
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 1 mM CaCl2), and then stained with anti-SREC-1 Ab (1/50 dilution) for 1 h, followed by FITC anti-rat IgG (1/100 dilution) for 30 min at 4°C. In some experiments, DC were further stained with 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 (BD Biosciences) with CellQuest analysis software.
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-SREC-1 mAb, and then examined under microscopy.
DC were cultured for 4 days, purified, and placed in a no. 1 coverslide cell chamber overnight. The DC were labeled with Alexa 488-labeled HSP70 (green color) for 30 min on ice for efficient binding, and later, cold medium was replaced with warm medium and incubated for 2–5 min at 37°C. Cells were then fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized and blocked before Ab staining. Fixed DC were stained with anti-SREC-1 Ab, 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, and 633 excitation and LP 650 for Cy5. Images were normally taken using a ×63 NA 1.4 oil immersion objective lens. Figures were created using Adobe Photoshop 7.0 with some contrast adjustments.
Lentivirus production and transduction
MISSION small hairpin RNA (shRNA) plasmids were purchased (Sigma-Aldrich), and the lentivirus generation and transduction were performed according to 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 RNA-induced silencing complex and the small interfering RNA pathway in the cells) was used as negative control. Three-day-old DC were collected, purified, and placed in 96-well round-bottom plates with 1 × 106 cell/well for overnight culture containing GM-CSF medium. On the second day, half of the medium (100 μl) was removed, and 100 μl of lentivirus supernatant (1 virus:1 DC) was added. After 20 h, 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 uninfected 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, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10−5 M 2-ME. 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 (DakoCytomation) with Summit v3.0 analysis software. The sorted CD4+ or CD8+ T cells were washed and resuspended in RPMI 1640 medium, and coincubated 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 [3H]thymidine for 12 h.
Measurement of CTL
To assess the Ag-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 of Na251CrO4 for 60 min at 37°C, and then washed to remove unincorporated isotope. Splenocytes and tumor targets were incubated at 100:1 E:T ratios 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 was 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 of specific release = (experimental – spontaneous)/(maximum – spontaneous) × 100.
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.
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 Ag-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).
We immunized WT, MyD88 KO (myd88−/−), and TLR2/4 double-KO (tlr2−/−/tlr4−/−) mice with HSP70.PC-F and, 7 days after the second immunization, draining LNC and splenocytes were isolated and assayed for proliferation and CTL activity. T cells from immunized WT mice proliferated vigorously (Fig. 1,A). By contrast, minimal cell proliferation was observed in T cells from either immunized myd88−/− or tlr2−/−/tlr4−/− mice (Fig. 1,A). We chose to inactivate both TLR2 and TLR4 because earlier studies showed that HSP70 can initiate signaling through both TLR molecules (8). Our findings of reduced T lymphocyte proliferation in MyD88 or TLR KO cells were reflected in the tumor cell-killing activity of isolated T cells (Fig. 1,B). 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−/− and tlr2−/−/tlr4−/− mice (Fig. 1,B, left panel). In addition, CTL from WT mice immunized with HSP70.PC-F were capable of Ag-specific killing of MUC1-expressing malignant cells (B16/MUC1 tumor cells), but not MUC1-negative B16 tumor cells (Fig. 1 B, middle and right panels), suggesting Ag-specific killing. HSP70.PC-F has been shown to carry high levels of MUC1-derived tumor Ags (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 Ags and induce T cells. Indeed, there was markedly reduced HSP70 binding to bone marrow-derived DC isolated from myd88−/− mice, compared with wild-type controls (Fig. 1,C). Furthermore, double KO of the tlr2 and tlr4 genes led to almost quantitative loss of HSP70 binding to the tlr2/tlr4−/− DC, a reduction that was more complete than the deficiency in HSP70 binding to myd88−/− cells (Fig. 1,C). Because 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 preincubated with scavenger receptor ligand 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. 1 C). In controls, unmodified BSA failed to 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, lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and fasciclin, 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. 2,A). These cells express relatively high levels of cell surface MHC class II (Fig. 2,A). 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. 2,B). 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 DC with or without LPS or HSP70 stimulation was statistically significant (p < 0.05) (Fig. 2,C). 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. 2,D). 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. 2 D).
Colocalization 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 colocalized 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. 3,A). When cells were warmed to 37°C after binding at 4°C, both SREC-1 and HSP70 were coassociated in intracellular vesicles that may be internalizing endosomes (Fig. 3,B). SREC-1 is thus a strong candidate for a receptor that can bind and internalize HSP70-peptide complexes. Although colocalization is extensive, inspection of Fig. 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), whereas some of the SREC-1 was not HSP70 bound, as indicated by maintenance of red color (Fig. 3, A and B, Merge). The experiment is consistent with the existence of other potential HSP70 receptors, such as LOX-1, scavenge receptor class A member 1, and CD91, as discussed earlier. It is conceivable that the HSP70-bound and internalized cell structures indicated in Fig. 3 B also contain these receptors and other molecules as well as SREC-1.
Inhibition of DC SREC-1 expression by shRNA infection
We next investigated whether SREC-1 plays a causal role in TLR-dependent tumor immune responses stimulated by HSP70.PC-F. We used shRNA to deplete SREC-1 by RNA interference. Of five individual shRNA constructs tested in the RAW231 mouse macrophage cell line, each was more or less effective in knocking down SREC-1 mRNA levels (Fig. 4,A). The most effective of these constructs (no. 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. 4,B). Infection of these cells with the shRNA-packaged lentivirus effectively inhibited SREC-1 expression (Fig. 4, A 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. 4,C) or B7-DC (Fig. 4,D). 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 cocultured 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. 4 E). In addition, SREC-1 knockdown inhibited expression of IFN-γ 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 Ag presentation of HSP70-chaperoned tumor Ag 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.
Expression of SREC-1 on DC with induction of Ag-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 because 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-SREC-1 and anti-CD3 Abs. Immunization with HSP70.PC-F resulted in the expression within LN 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. 5,A). By contrast, SREC-1-positive cells in tlr2−/−/tlr4−/− mice were at a similar low level compared with those in WT control mice injected with PBS instead of vaccine (Fig. 5 A). We did not observe a similar distribution of DC expressing other potential HSP70-binding scavenger receptors such as LOX-1 or fasciclin (data not shown). These experiments therefore suggest that SREC-1-expressing DC can migrate to draining LN and potentially interact with T cells, but only in mice with intact TLR signaling.
We next addressed the question of whether SREC-1 plays a role in HSP70.PC-F-induced tumor Ag-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) fusion cells could overcome the effects of tlr2/4 KO and mediate antitumor 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. 5,B, lane 2) compared with naive DC alone that did not induce CTL (Fig. 5,B, 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 shRNA sequence were able to activate CTL in TLR KO mice essentially as effectively as in wild-type mice (Fig. 5,B, compare lanes 3 and 2). However, SREC-1 knockdown inhibited the ability of WT-DC to compensate for TLR KO (Fig. 5,B, lane 4). Thus, SREC-1 appears to be a significant component of the HSP70.PC-F-mediated, TLR-dependent tumor cell killing. Next, to assess the frequency of Ag-specific T cells induced, we assembled a 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 with naive DC (Fig. 5,C, 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. 5,C). Immunization of tlr2−/−/tlr4−/− mice either with HSP70.PC-F-pulsed WT-DC after SREC-1 knockdown or after infection with control virus resulted in 1.45 and 3.51% CD8 T cells positive for MUC1–8 (Fig. 5 C), respectively. These experiments provide direct evidence that SREC-1 participates in the induction of Ag-specific T cells by HSP70.PC-F.
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). In this study, we show that HSP70.PC-F-induced antitumor immunity requires intact TLR signaling in the tumor-bearing host to induce CTL activity (Figs. 1 and 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 coactivator molecules required for efficient activation of T cells (12). Indeed, some studies suggest that exposure of DC to purified HSP70 can directly induce coactivator expression (8). In addition, TLR4 is thought to be involved in cross-presentation of phagocytosed Ags due to its ability to alter intralysosomal pH and slow the rate of Ag proteolysis (13, 14). The studies presented in this work indicate a third potential role for TLR signaling in the responsiveness of DC to exogenous Ags: the induction of expression of HSP70 receptors belonging to the scavenger receptor family (Figs. 2 and 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 NF-κB activation by extracellular HSP70 (16, 17, 18, 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 5). Furthermore, additional reports show that TLR2 can signal through a pathway downstream of scavenger receptors SREC-1 and LOX-1 and induce NF-κB-dependent transcription in an scavenger receptor-dependent manner (20). However, other reports indicate that scavenger receptor class A member 1 failed to activate Ag cross-priming by heat shock proteins, findings that imply that not all HSP-scavenger receptor 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 DC. The anticancer 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.
The authors have no financial conflict of interest.
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.
This work was supported by National Institutes of Health Research Grants R01CA047407 (to S.K.C.), R01CA094397 (to S.K.C.), and R01CA119045 (to S.K.C.); the U.S. Department of Defense Breast Cancer Research programs (to J.G.); and the Ellison Foundation (to J.G.).
Abbreviations used in this paper: HSP, heat shock protein; DC, dendritic cell; KO, knockout; LN, lymph node; LNC, LN cell; LOX-1, lectin-like oxidized low-density lipoprotein receptor 1; mBSA, maleylated BSA; shRNA, small hairpin RNA; SREC-1, scavenger receptor expressed by endothelial cells-1; WT, wild type.