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Cancer Research 67, 4996, May 15, 2007. doi: 10.1158/0008-5472.CAN-06-3138
© 2007 American Association for Cancer Research
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Immunology

Scavenger Receptor-A Negatively Regulates Antitumor Immunity

Xiang-Yang Wang1,2, John Facciponte3, Xing Chen1, John R. Subjeck1 and Elizabeth A. Repasky3

Departments of 1 Cellular Stress Biology, 2 Urologic Oncology, and 3 Immunology, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Xiang-Yang Wang, Department of Cellular Stress Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-2375; E-mail: xiang-yang.wang{at}roswellpark.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The scavenger receptor-A (SR-A), originally recognized by its ability to internalize modified lipoproteins, has largely been studied in relation to atherosclerosis as well as innate immunity against pathogen infection. SR-A was recently shown to be a receptor on antigen-presenting cell for heat shock protein (HSP) and was implicated in the cross-presentation of HSP-chaperoned antigens. Here, we show that SR-A is not required for antitumor immunity generated by HSP-based (e.g., grp170) vaccine approaches in vivo. The lack of SR-A significantly enhances HSP- or lipopolysaccharide-mediated vaccine activities against poorly immunogenic tumors, indicating that SR-A is able to attenuate immunostimulatory effects of adjuvants or "danger" molecules. The improved antitumor response in SR-A knockout mice is correlated with an increased antigen-specific T-cell response. Moreover, SR-A–deficient dendritic cells are more responsive to inflammatory stimuli and display a more effective antigen-presenting capability compared with wild-type cells. This is the first report illustrating that SR-A negatively regulates antigen-specific antitumor immunity, which has important clinical implications in vaccine design for cancer immunotherapy. [Cancer Res 2007;67(10):4996–5002]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Scavenger receptor-A (SR-A), also known as CD204, is the first cloned member of an expanding family of structurally diverse receptors collectively termed scavenger receptors (1). SR-A is expressed primarily on the innate immune cells [e.g., macrophages (M{phi}) and dendritic cells] and functions as a pattern recognition receptor (PRR). SR-A is able to bind a broad range of ligands, including chemically modified or altered molecules, bacterial surface components [e.g., lipopolysaccharide (LPS)], and apoptotic cells (2). SR-A has been studied extensively in the context of atherosclerosis, where it was identified initially as a major receptor on M{phi} for internalization of oxidative or acylated low-density lipoproteins (3–5). Suzuki et al. (4) and Thomas et al. (6) have reported that SR-A–/– mice have impaired protection against pathogen infection. The nature of the protective mechanism provided by SR-A is not fully defined. However, SR-A–deficient mice are more susceptible to endotoxic mortality caused by overproduction of proinflammatory cytokines [e.g., tumor necrosis factor-{alpha} (TNF-{alpha})] by activated M{phi} (7). Recent studies from Kobzik and Platt's groups suggest that SR-A may act as an inhibitory receptor in proinflammatory responses (8, 9). Therefore, the capability of SR-A to recognize pathogen-related molecules and suppress cytokine responses to bacterial component may explain, at least in part, increased mortality in the infected SR-A knockout (SR-A–/–) mice. Although a large body of information is accumulating about the role of SR-A in atherosclerosis and pathogen recognition, the contribution of this receptor to antigen-specific antitumor immunity has not been explored.

Stress proteins, also called heat shock proteins (HSP), mainly act as molecular chaperones in a variety of cellular functions under physiologic as well as stress conditions. The immunologic roles of HSPs have been extensively studied because Srivastava et al. (10) discovered that tumor-derived HSPs are capable of eliciting a potent antitumor response. A large body of evidence supports the idea of using HSPs for vaccine design because of its unique abilities to act as both an antigen carrier and an immune modulator (11). Recently, several surface receptors on antigen-presenting cells (APC), including SR-A, have been implicated in the HSP-mediated cross-priming event (12, 13). However, the involvement of SR-A in HSP vaccine-generated antitumor immunity has not been well characterized.

In the present study, we report that HSP-mediated (i.e., grp170) antitumor activities not only remain intact but also are enhanced in SR-A knockout mice (SR-A–/–). Further, the absence of SR-A can restore the immunogenicity of poorly immunogenic tumors and significantly improve the antitumor efficacy of vaccines using Toll-like receptor (TLR) agonists as adjuvants. Consequently, SR-A seems capable of dampening the immunostimulatory effects derived from adjuvants of both mammalian and nonmammalian origins. Our studies suggest that SR-A plays an important role in regulating the antigen-presenting capability of APCs, thereby affecting the induction of an antigen-specific CTL response and antitumor immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines. Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory. SR-A knockout mice (4), provided by Mason W. Freeman (Harvard Medical School, Boston, MA) and Brent Berwin (Dartmouth Medical School, Hanover, NH), have been backcrossed to C57BL/6J mice for at least 10 generations before being used. The mice were maintained in a pathogen-free facility at Roswell Park Cancer Institute (Buffalo, NY). Animal care and experiments were approved by the Institutional Animal Care and Use Committee. B16 (F10) cells (H-2b), B16-OVA (provided by Ken Rock, University of Massachusetts, Worcester, MA), and D121 Lewis lung carcinoma cell line (H-2b) were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies).

Tumor studies. Tumor-derived grp170 was purified from culture medium of B16-sgrp170 cells as described previously (14). Recombinant gp100 and grp170 protein were expressed using baculovirus-insect cell system (15). gp100-grp170 chaperone complex was generated by incubating gp100 and grp170 at an equal molar ratio under heat shock conditions as described previously (15, 16). For tumor challenge study, mice were immunized s.c. with irradiated tumor cells (1 x 106), tumor-derived grp170 (30 µg), and gp100-grp170 complex (30 µg) in the left flank. Two weeks after immunization, mice were challenged by i.d. injections of 2 x 105 live tumor cells into the right flank. In some experiments, mice were immunized with recombinant gp100 (30 µg) or gp100 emulsified in complete Freund's adjuvant (CFA; Sigma-Aldrich) or mixed with 5 µg LPS (Escherichia coli serotype 026:B6; Sigma-Aldrich). For therapeutic studies, mice were inoculated with 2 x 105 D121 tumor cells on day 0 followed by treatment with irradiated D121 cells on days 2, 4, 6, and 8. The tumor volume was calculated using the following formula: V = (the shortest diameter2 x the longest diameter) / 2. Depletion of CD4+ and CD8+ T-cell subsets was accomplished by i.p. injection of GK1.5 and 2.43 monoclonal antibody, respectively, as described previously (16). For inhibition of phagocytic cells, 1 mg carrageenan (type II; Sigma-Aldrich) in 200 µL PBS was administered by i.p. (17).

Enzyme-linked immunosorbent spot and CTL assay. Splenocytes were isolated from immunized mice 2 weeks after immunization and stimulated with 1 µg/mL H-2Kb–restricted CTL epitope OVA257-264 (SIINFEKL) or irrelevant E749-57 (RAHYNIVTF) to determine antigen-specific IFN-{gamma}–secreting T cells as described previously (16). For CTL assay, splenocytes were stimulated with 1 µmol/L OVA257-264 in the presence of interleukin (IL)-2 (20 units/mL) for 5 days. CD8+ T cells were isolated by magnetic-activated cell sorting CD8 (Ly-2) microbeads (Miltenyi Biotec) and used as effector cells in a chromium release assay as described (16).

Cytokine assay. Dendritic cells were generated from mouse bone marrow in the presence of granulocyte macrophage colony-stimulating factor (20 ng/mL; BD Biosciences) and IL-4 (10 ng/mL; BD Biosciences) as described previously (18). For cytokine assay, 1x 106 day 7 dendritic cells were cultured in the presence of LPS or with irradiated tumor cells for 24 h. Irradiated tumor cells were cultured in serum-free medium for 24 h before use. The supernatants were harvested for measuring TNF-{alpha} using ELISA kits (eBioscience).

B3Z T-cell activation assay. Day 7 bone marrow dendritic cells were pulsed with OVA257-264 (SIINFEKL) at 37°C for 1 h and washed thrice with PBS. Alternatively, dendritic cells were cocultured with irradiated cells and isolated using CD11c microbeads (Miltenyi Biotec). Following stimulation with LPS, dendritic cells were coincubated overnight at 37°C with 1 x 105 B3Z T cells, which carry a lacZ (ß-galactosidase) construct driven by nuclear factor of activated T-cell elements from the IL-2 promoter and recognize OVA in the context of Kb MHC class I molecules. Cell pellets were lysed with PBS containing 0.125% NP40 and 1 mmol/L chromophenol red ß-galactoside (Calbiochem). Plate was read at 570 nm using a 96-well plate reader with 630 nm as reference wavelength. The response of LacZ-inducible B3Z T cells was measured as absorbance of the colored product generated by cleavage of the LacZ substrate chromophenol red ß-galactoside.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SR-A is not essential for grp170 vaccine-generated antitumor immunity. Our previous studies have shown that grp170, the largest endoplasmic reticulum chaperone, possesses highly potent immunostimulatory properties (15, 19). More recently, we showed that B16 tumor cell expressing a secretory form of grp170 (B16-sgrp170) was an effective vaccine generating an antitumor response in WT C57BL6 mice (14). Therefore, using SR-A knockout mice, we sought to address the question whether the absence of SR-A may negatively affect the immunogenicity of B16-sgrp170 cell vaccine. Both WT and SR-A–/– C57BL/6 mice were immunized with irradiated parental B16 cells and B16-sgrp170 cells or left untreated followed by tumor challenge. Consistent with our earlier report, WT mice without immunization and those immunized with irradiated B16 cells developed aggressively growing tumors on tumor challenge, whereas WT mice immunized with irradiated B16-sgrp170 were partially protected (Fig. 1A ). Strikingly, although tumors grew in the untreated SR-A–/– mice similarly to those in WT mice, all tumors inoculated in SR-A–/– mice vaccinated with either irradiated B16 cell or irradiated B16-sgrp170 were rejected (Fig. 1B).


Figure 1
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  		Figure 1. SR-A deficiency enhances antitumor efficacy of grp170-based vaccines. Age-matched WT mice (A) and SR-A–/– C57BL/6 mice (B) were immunized with irradiated (IR) B16 cells and B16-sgrp170 cells or left untreated. Two weeks later, mice were challenged with 2 x 105 viable B16 tumor cells (n = 8; P < 0.001, immunized SR-A–/– versus nonimmunized SR-A–/– or immunized WT mice). WT and SR-A–/– mice (n = 8) were immunized twice at a weekly interval with grp170 purified from culture medium of B16-sgrp170 (C) or gp100 protein complexed with grp170 (D). Mice were then challenged with B16 tumor. Results from three representative experiments.

 
Two other grp170-based vaccine approaches were subsequently evaluated: grp170 purified from B16-sgrp170 cells (i.e., the conventional tumor-derived HSP vaccine) and grp170 complexed with a well-characterized melanoma-associated antigen gp100 (i.e., a reconstituted chaperone vaccine recently developed by our group; Fig. 1C and D). After immunization, mice were challenged with B16 tumor cells that naturally express gp100. Both types of grp170 vaccine formulations (i.e., purified from tumor or in vitro reconstituted) exhibited significantly more potent antitumor efficacy in SR-A–/– mice compared with WT mice. Similar results were obtained in SR-A–/– mice that were vaccinated with gp100 chaperoned by hsp110 (16), a cytosolic homologue of grp170 (data not shown). Tumor specificity of the antitumor response was examined by rechallenging tumor-free SR-A–/– mice 3 months later with B16 tumor or D121 tumor (H-2b), a subline of the Lewis lung carcinoma. Tumor-free mice were resistant to a secondary B16 tumor challenge, indicating the development of immune memory. However, all tumor-free mice challenged with D121 cells developed tumors (data not shown).

The therapeutic efficacy of vaccination in SR-A–/– mice bearing preestablished D121 Lewis lung carcinoma was examined in light of the increased immunogenicity of the poorly immunogenic tumors in SR-A–/– mice. Administration of irradiated D121 cells to tumor-bearing SR-A–/– mice resulted in a reduced tumor growth rate and 50% of the treated mice remained tumor-free (Fig. 2 ).


Figure 2
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  		Figure 2. SR-A deficiency enhances the potency of therapeutic vaccination. SR-A–/– mice (n = 8) established with D121 tumors were treated with irradiated D121 cells on days indicated (P < 0.01, Student's t test, immunized versus nonimmunized SR-A–/– mice). Curve, tumor growth in each individual mouse. Data represent three independent experiments.

 
The impairment of apoptotic/dying cell clearance may cause the breakdown of self-tolerance (20). However, no significant difference was observed in the phagocytosis of apoptotic tumor cells by WT and SR-A–/– cells (data not shown). In addition, internalization of FITC-labeled grp170 by dendritic cells from SR-A–/– mice was also comparable with that of WT dendritic cells (data not shown), suggesting that the presence of other redundant endocytic receptors on the cell surface can compensate for the loss of SR-A (21).

Enhanced antitumor response in SR-A–/– mice immunized with non-HSP adjuvant formulated vaccines. The initial observations of highly effective HSP vaccine activities in SR-A–/– mice led to evaluation of the ability of gp100 formulated with non-HSP adjuvants to augment an antitumor response. As a nonimmunogenic self-antigen, recombinant gp100 protein alone failed to elicit measurable antitumor effects in both stains of mice. However, gp100 protein emulsified in CFA or mixed with LPS protected SR-A–/– mice, not WT mice, from B16 tumor challenge (Fig. 3 ). Interestingly, priming of animals with gp100 protein emulsified in incomplete Freund's adjuvant did not generate antitumor responses in both WT and SR-A–/– mice (data not shown), suggesting that immunostimulatory mycobacterial components are required for the highly potent vaccine activity in SR-A–/– mice.


Figure 3
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  		Figure 3. SR-A deficiency enhances antitumor immunity generated by gp100 formulated with non-HSP adjuvants. WT or SR-A–/– mice (n = 10) were immunized with gp100 protein, gp100 emulsified in CFA, or gp100 mixed with LPS followed by tumor challenge with B16 cells 2 wks later (P < 0.001, log-rank test, immunized SR-A–/– versus immunized WT mice).

 
Enhanced CD8+ T-cell response in SR-A–/– mice compared with WT mice. A clue that there may be involvement of immune effector cells in the enhanced tumor rejection in SR-A–/– mice was determined by antibody depletion studies in vivo (Fig. 4A ). Elimination of CD8+ T cells completely abrogated tumor protective immunity, whereas lack of CD4+ T cells had no effect. Carrageenan, which impairs function of phagocytic cells, also diminished the antitumor response, indicating an important role of phagocytes (e.g., M{phi} and dendritic cells) in antigen capture and immune initiation.


Figure 4
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  		Figure 4. The enhanced antitumor immunity in SR-A–/– mice is associated with a robust CTL response. A, depletion of T-cell subsets and phagocytes was done by antibody injections. SR-A–/– mice (n = 10) were immunized with irradiated D121 cells followed by D121 tumor challenge (P < 0.001, CD8+ T-cell depletion or carrageenan group versus IgG group). B, increased antigen-specific CD8+ T-cell frequency in SR-A–/– mice. Following immunization with irradiated B16-OVA cells, splenocytes were stimulated with or without OVA257-264. IFN-{gamma} production was measured using an enzyme-linked immunosorbent spot (ELISPOT) assay. *, P < 0.01, Student's t test. C, splenocytes from mice immunized with OVA-CFA were stimulated with OVA257-264 or E749-57 and subjected to ELISPOT assay. D, splenocytes from OVA-CFA–immunized mice were restimulated with OVA257-264 in vitro. The CD8+ T cells were analyzed for cytotoxic activity using 51Cr-labeled B16-OVA as targets.

 
Given that CD8+ T cells were involved in the tumor rejection in SR-A–/– mice, we next determined the effect of SR-A deficiency on vaccination-elicited antigen-specific CTL response. Splenocytes were isolated from mice immunized with irradiated B16 cell transfected with a model antigen OVA. On stimulation with the MHC I–restricted OVA257-264 (SIINFEKL) peptide, splenocytes derived from SR-A–/– mice displayed a robust production of IFN-{gamma} in comparison with those from immunized WT mice (Fig. 4B). Moreover, splenocytes from OVA-CFA–immunized SR-A–/– mice produced higher levels of IFN-{gamma} than did WT cells after stimulation with OVA257-264 peptide, not irrelevant E7 peptide, indicating an antigen specificity of the primed CTLs (Fig. 4C). Effector CD8+-T-cell function (i.e., cytolytic activity), as assessed by chromium release assay, was also enhanced in the immunized SR-A–/– mice (Fig. 4D).

Using standard surface markers, fluorescence-activated cell sorting (FACS) analyses were carried out to assess the potential effect of SR-A deficiency on cellular composition of the immune compartments. Naive SR-A–/– mice showed normal lymphocyte composition (e.g., B-cell, T-cell, and T-cell subtype) in spleen and lymph nodes, with numbers and ratios of CD4+ and CD8+ T cells in the periphery that were not different from those of WT mice (data not shown). In addition, there were no substantial differences between mitogen-stimulated proliferation response of naive T cells derived from WT and SR-A–/– mice (data not shown).

SR-A–deficient APCs are more responsive to inflammatory stimuli. Examination of SR-A expression in various cell types by immunoblotting showed that SR-A was present primarily in professional phagocytes or APCs (e.g., dendritic cell and M{phi}), whereas SR-A is absent or undetectable in B16 tumor cells and Miltenyi microbeads isolated T and B cells (Fig. 5A ). Further, it was seen that SR-A expression increased in LPS-treated WT bone marrow dendritic cells (Fig. 5B).


Figure 5
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  		Figure 5. Dendritic cells derived from SR-A–/– mice are more sensitive to inflammatory stimuli. A, cell lysates prepared from DC1.2 line (lane 1), fresh M{phi} (lane 2), CD3+ T cells (lane 3), B220+ B cells (lane 4), and B16 tumor line (lane 5) were separated on a 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Expression of SR-A was detected using SR-A–specific rabbit polyclonal antibodies (1:10,000). B, dendritic cells from WT or SR-A–/– mice were stimulated with or without LPS and subjected to immunoblotting. C and D, day 7 bone marrow dendritic cells (DC) were cultured in 24-well plate with 10 ng/mL LPS (C) or irradiated D121 cells (D) at a 1:5 ratio for 24 h. NS, no stimulation. The supernatants were measured for secretion of TNF-{alpha} using ELISA. *, P < 0.01, Student's t test.

 
When stimulated with LPS, bone marrow dendritic cells from SR-A–/– mice produced more TNF-{alpha} and IL-6 (data not shown) than did WT counterparts (Fig. 5C). SR-A–/– dendritic cells also exhibited a more mature phenotype on LPS stimulation, indicated by surface staining with MHC II and CD86 antibodies (data not shown). In addition, the enhanced production of TNF-{alpha} (Fig. 5D) was observed when bone marrow dendritic cells from SR-A–/– mice were coincubated with irradiated dying tumor cells.

Bone marrow dendritic cells were used to examine whether SR-A deficiency may have influenced expression of TLR4 signaling receptors [e.g., TLR4-myeloid differentiation protein-2 (MD-2) complex and CD14], which are critical for LPS response. FACS analysis of CD11c+ cells costained with antibodies specific for either TLR4/MD-2 or CD14 revealed similar expression levels in SR-A–/– and WT cells (data not shown).

SR-A deficiency promotes antigen-presenting function of dendritic cells. To determine whether SR-A deficiency promotes dendritic cell stimulation of antigen-specific T cells, bone marrow dendritic cells from WT or SR-A–/– mice were pulsed with OVA257-264 peptide (Fig. 6A ). After stimulation with LPS, dendritic cells were cocultured with H-2Kb–restricted, SIINFEKL-specific responder B3Z T cells. Dendritic cells from SR-A–/– mice stimulated OVA-specific CD8 T cells more effectively than WT dendritic cells. After antigen pulsing and LPS stimulation, a similar expression level of H-2/Kb molecules was seen on both WT and SR-A–/– dendritic cells indicated by FACS analysis (data not shown), suggesting that SR-A deficiency does not alter the expression levels of class I MHC molecules. In addition, SR-A–/– dendritic cells pulsed with irradiated OVA-expressing B16 cells were also seen more effective in stimulating B3Z cells compared with WT cells (Fig. 6B).


Figure 6
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  		Figure 6. Dendritic cells derived from SR-A–/– mice exhibit enhanced antigen presentation capability. A, bone marrow dendritic cells were pulsed with 100 ng/mL H2-Kb–restricted OVA257-264 peptide and washed thrice with PBS. After LPS (10 ng/mL) stimulation, cells were coincubated with B3Z cells for 20 h. A colorimetric assay was used to detect ß-galactosidase activity in B3Z cells. *, P < 0.01. B, bone marrow dendritic cells were pulsed with irradiated B16-OVA cells and subjected to B3Z assay. Columns, mean of one of three experiments; bars, SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The recent report of SR-A as a binding receptor for grp94/gp96, an extensively studied immunoadjuvant (13), prompted investigation of whether SR-A is involved in HSP-mediated antitumor effects in vivo. We made an unexpected observation that the antitumor response elicited by various grp170-based vaccines was enhanced in SR-A genetically deficient mice. Although more studies are required to determine the involvement of SR-A in other HSP-mediated immune activities, these findings suggest that SR-A may not be essential for in vivo presentation of HSP-associated antigens or subsequent antitumor immunity. It is conceivable that the presence of other redundant endocytic receptors or HSP receptors (e.g., SREC, LOX-1, and CD91) on APCs may sufficiently compensate for the loss of SR-A in vivo. More importantly, we have shown that vaccines formulated with either HSP or non-HSP (e.g., CFA and LPS) adjuvants exhibit enhanced antitumor potency in SR-A–/– mice, suggesting that SR-A is capable of dampening the immunostimulatory activities derived from adjuvants of both mammalian and nonmammalian origins. Although LPS is a well-recognized TLR4 ligand, the immunostimulatory properties of HSPs (22, 23) and CFA (24) have been attributed to the TLR2/TLR4 signaling pathways. Thus, SR-A seems to attenuate the adjuvant effects provided by the activation of TLR signaling.

The significance of TLR signaling in enhancing antigen presentation and activating adaptive or acquired immune responses has been well established (25). The engagement of TLR signaling pathways is a promising mechanism for boosting vaccine responses against infectious diseases as well as cancer (26, 27). The action mechanism of the exogenous adjuvants or the nonmammalian microbial pattern molecules (e.g., LPS) can be depicted by the "stranger hypothesis" (28). In addition, the "danger hypothesis" proposes that the immune system can discriminate endogenous "danger" molecules released from injured host cells, which are distinct from the exogenous adjuvants of microbes (29). Indeed, these endogenous damage-associated molecular pattern molecules, including HSPs, signal through the same TLRs and the downstream MyD88/nuclear factor-{kappa}B pathway as those used by nonmammalian microbial pattern molecules (30–34). Therefore, TLRs are important in recognition of danger molecules generated from self or infectious materials and are representative adjuvant receptors. The inhibition of adjuvant effects of TLR agonists as shown by both in vivo and in vitro studies here further underscores the important role of SR-A in immune modulation.

In the present study, we have shown that SR-A is preferentially expressed in professional APCs, and dendritic cells derived from SR-A–deficient mice are highly responsive to inflammatory stimuli, such as LPS and ionizing radiation–damaged cells. These results agree with the previously reported roles of SR-A in inhibiting production of proinflammatory cytokines by LPS-activated M{phi} (7, 8), supporting the idea that SR-A may act as an inhibitory receptor to limit proinflammatory responses. The Platt group recently showed that SR-A–/– dendritic cells displayed enhanced expression of TNF-{alpha} and costimulatory molecule CD40 on LPS stimulation, suggesting that SR-A might be a limiting factor in dendritic cell maturation (9). Interestingly, the enhanced production of IL-6 and TNF-{alpha} by SR-A–/– dendritic cells was also observed when cocultured with dying tumor cells. Identities of the molecules in dying cells that stimulate dendritic cells to release inflammatory mediators are not clear. However, endogenous danger molecules, such as stress/HSPs, are likely to be the candidates.

APCs, particularly dendritic cells, can sense pathogens or "dangers" through PRRs and direct the immune system to initiate appropriate and effective responses. The default action of dendritic cells in antigen cross-presentation is likely to promote tolerance, or partial activation, in the absence of inflammatory signals or costimulation (35). Given that the decision between T-cell immunity and tolerance is decided in part by the activation state of dendritic cells, the lack of SR-A may enhance the maturation and activation of dendritic cells by endogenous or exogenous inflammatory stimuli or danger signals, which leads to improved antigen presentation and tumor immunity. Furthermore, the enhanced IL-6 production by SR-A–/– dendritic cells may block the suppressive effect of CD4+CD25+ regulatory T cells, facilitating the development of type 1 immunity in vivo (26).

The present study also raises the question of how SR-A may regulate TLR signaling at the molecular levels. We have shown no significant difference between WT and SR-A–/– dendritic cells in the uptake of dying cells or grp170 (data not shown). Similar observations have been made in the case for LPS clearance in SR-A–/– mice (7, 36). Thus, the contribution of SR-A to the removal of the inflammatory molecules may not play a major role in SR-A–mediated immune-suppressive effects. The similar expression of CD14, TLR2/TLR4, and MD-2 on SR-A–/– dendritic cells and WT counterparts also indicates that SR-A deficiency does not result in changes in TLR signaling receptors (9). Earlier studies suggested that SR-A is linked directly to signal transduction pathways (37–39). Kobzik's group recently showed that engagement of SR-A triggered production of H2O2, a signaling messenger, and inhibited release of IL-12 by M{phi} (40). The physical or functional interactions between SR-A and the TLR signaling pathway, therefore, are likely to play a key role in the immunoregulation by SR-A.

In summary, the abilities of SR-A to attenuate adjuvant-generated (e.g., TLR agonist) immunostimulatory effects suggest that SR-A represents a previously unrecognized immune-suppressive receptor, participating in maintenance of host immune homeostasis. To improve vaccine efficacy and achieve ultimate success in cancer immunotherapy, novel strategies must be developed to overcome the various inherent negative regulatory mechanisms. Selective removal or blockade of SR-A (e.g., using a RNA silencing technology or soluble decoy receptors) may be a promising approach to enhance antigen-specific immune responses. Further dissection of the global contribution of SR-A to the host immunity or tolerance and unraveling the details of inflammatory pathways modulated by the innate PRR will facilitate the design of SR-A–targeted approaches for treatment of cancer.


   Acknowledgments
 
Grant support: National Cancer Institute grant R21 CA121848, Roswell Park Alliance Foundation, and National Cancer Institute Cancer Center Support Grant CA016156 (Roswell Park Cancer Institute).

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.

We thank Mason W. Freeman and Brent Berwin who provided us with SR-A–/– mice, Ken Rock who provided B16-OVA cell, Nicholas Restifo who provided gp100 cDNA, and Motohiro Takeya who provided SR-A antibodies.

Received 8/24/06. Revised 1/23/07. Accepted 2/27/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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