Evolution of the immunomodulatory role of the heat shock protein gp96.

In mammals, certain heat shock proteins (hsps) participate in specialized responses to stressors associated with pathogens or tumors, and as such, act as agents of immune surveillance interacting with both innate and adaptive immunity. We are investigating the conservation of this role throughout the class of vertebrates. We have shown that in Xenopus as in mammals, gp96, the major resident of the endoplasmic reticulum, generates MHC-restricted thymus-dependent immunity in vivo and CR in vitro against minor histocompatibility (H) antigens. By as yet unclear mechanisms that may involve classical MHC-unrestricted cytotoxic CD8+ T cells, gp96 also elicits peptide-specific responses against MHC-class I-negative tumors in adult frogs that may involve cytotoxic NK, MHC-unrestricted CD8+ T and NK/T cells. In naturally MHC class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an innate type of anti-tumor response that is independent of chaperoned peptides. Finally, in a subset of Xenopus sIgM+ B cells, a substantial fraction of gp96 is directed to the cell surface by an active process that is upregulated by bacterial stimulation. This may allow gp96 to access the extracellular compartment without necrosis. Given the dual abilities of gp96 to chaperone antigenic peptides and to modulate innate immune responses, we propose that stimulated B cells that are up-regulating surface gp96 can directly interact with antigen presenting cells (APC) and/or T helper (Th) cells to trigger or amplify immune responses.     

Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand alpha2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.     

Adjuvanticity of alpha 2-macroglobulin, an independent ligand for the heat shock protein receptor CD91

We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for alpha(2)-macroglobulin (alpha(2)M). Gp96 and alpha(2)M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8(+) T cell responses and are re-presented by APCs, we tested alpha(2)M for similar properties. Our studies show that alpha(2)M binds peptides in vitro and that the peptides, chaperoned by alpha(2)M, efficiently prime peptide-specific CD8(+) T cell responses in mice immunized with alpha(2)M-peptide complexes. Furthermore, peptides chaperoned by alpha(2)M, like those chaperoned by gp96, can be re-presented by CD91(+) APCs on their MHC I molecules. These studies demonstrate that alpha(2)M molecules, like the heat shock protein molecules, are T cell adjuvants that can channel exogenous Ags into the endogenous pathway of Ag presentaion. The remarkable similarities between an intracellular chaperone and an extracellular serum chaperone may have interesting physiological ramifications.     

Melanoma-reactive class I-restricted cytotoxic T cell clones are stimulated by dendritic cells loaded with synthetic peptides, but fail to respond to dendritic cells pulsed with melanoma-derived heat shock proteins in vitro

Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2(+) APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.     

Specific immunogenicity of heat shock protein gp96 derives from chaperoned antigenic peptides and not from contaminating proteins

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing beta-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating beta-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Heat shock protein-mediated cross-presentation of exogenous HIV antigen on HLA class I and class II

Strong CD4(+) and CD8(+) T cell responses are considered important immune components for controlling HIV infection, and their priming may be central to an effective HIV vaccine. We describe in this study an approach by which multiple CD4(+) and CD8(+) T cell epitopes are processed and presented from an exogenously added HIV-1 Gag-p24 peptide of 32 aa complexed to heat shock protein (HSP) gp96. CD8(+) T cell recognition of the HSP/peptide complex, but not the peptide alone, was inhibited by brefeldin A, suggesting an endoplasmic reticulum-dependent pathway. This is the first report to describe efficient processing and simultaneous presentation of overlapping class I- and class II-restricted epitopes from the same extracellularly added precursor peptide complexed to HSP. Given previous reports of the strong immunogenicity of HSP/peptide complexes, the present data suggest that HSP-complexed peptides containing multiple MHC class I- and class II-restricted epitopes represent potential vaccine candidates for HIV and other viral infections suitable to induce effective CTL memory by simultaneously providing CD4 T cell help.     

Efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8(+) T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.     

Tumor-produced secreted form of binding of immunoglobulin protein elicits antigen-specific tumor immunity

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8(+) T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8(+) T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag-BiP complex.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8(+) T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8(+) T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4(+) T cell effector function, but did not directly prime CD8(+) T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8(+) T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4(+) Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8(+) DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8(+) T cells will ultimately dictate the success of immunotherapy.     

Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules

The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing.     

Heat shock proteins and the antitumor T cell response

Heat shock proteins (HSP) have been shown to participate in the antitumor T cell response. First, HSP play a crucial role in the intracellular pathway for antigen processing where HSP can make complexes with a broad spectrum of cellular proteins and peptides through their chaperone functions. In this pathway, macrophages are required for processing the chaperoned peptides to make stable molecules with the major histocompatibility complex (MHC) class I molecules, even when HSP-peptide complexes are exogenously administered. Through this pathway, vaccination with HSP-peptide complexes is thus able to elicit the response of CD8⁺ T cells specific for the chaperoned peptides. These findings suggest an essential role of HSP in ‘cross-priming’ and their usefulness for antitumor vaccination with tumor peptides. Second, HSP have been suggested to be expressed on the cell surface by transformation and, in addition, to function as antigen-presenting molecules for double negative T cells. Third, HSP derived from tumor cells have reportedly been recognized by T cells with either T cell receptor (TCR)-αβ or TCR-γδ. These lines of evidence therefore indicate that HSP may be potentially promising target molecules for antitumor T cell immunotherapy.     

Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CD8 cell repertoire induced by a minor transplantation antigen

T cell immunity is often focused on one peptide segment of a complex protein Ag, with other epitopes inducing weaker, low frequency responses or no responses at all. Such determinant hierarchy has been well characterized for MHC class II-restricted CD4 cell immunity, but is less well understood for class I-restricted CD8 cell responses. We studied class I determinant recognition in a skin transplant model with beta-galactosidase (beta-gal) as a minor transplantation Ag. CD8 T cells from C57BL/6 mice that rejected congenic C57BL/6 beta-gal transgenic skin were tested in enzyme-linked immunospot assays for recall responses to single-step, overlapping, 9-mer peptides that spanned a 94-aa region of the beta-gal sequence. This approach provided every possible class I-restricted peptide for CD8 cell recognition, allowing us to define the in vivo frequency of CD8 cells specific for each of the 86 individual peptides. While four peptides were predicted to bind to the Kb or Db molecules, only one (beta-gal96-103) actually induced an immune response. No peptides outside of the motifs were recognized. Tolerization to beta-gal96-103 significantly prolonged beta-gal transgenic skin graft survival, confirming its immune dominance. Therefore, single-determinant dominance characterized this CD8 cell response. The data demonstrate the feasibility of large-scale, comprehensive, class I determinant mapping, an approach that should be indispensable in measuring CD8 cell immunity in humans.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

Glycoprotein 96 can chaperone both MHC class I- and class II-restricted epitopes for in vivo presentation, but selectively primes CD8+ T cell effector function

The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the dual function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8(+) T cell effector responses. Although the ability of HSPs to elicit effector CD8 cell responses has been well established, their potential to prime CD4 cell effector responses has been relatively unexplored. In the current study we compared the ability of the endoplasmic reticulum-resident HSP gp96 to prime CD4 vs CD8 cells using TCR transgenic adoptive transfer systems and soluble gp96-peptide complexes. As expected, gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide; however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.     

Variable immunodominance hierarchies for H2-M3-restricted N-formyl peptides following bacterial infection

H2-M3-restricted presentation of N-formyl methionine (f-Met) peptides to CD8(+) T cells provides a mechanism for selective recognition of bacterial infection. In this report we demonstrate that Listeria monocytogenes infection induces distinct CD8(+) T cell populations specific for each of the known Listeria-derived formyl methionine peptides presented by M3. The sum H2-M3-restricted, Listeria-specific T cell response constitutes a major fraction of the total CD8(+) T cell response to primary infection. H2-M3-restricted T cell populations expand synchronously in vivo and achieve peak frequencies approximately 2 days earlier than MHC class Ia-restricted T cell populations. Although cross-recognition of different f-Met peptides by M3-restricted T cells was previously described, costaining of CD8(+) T cells ex vivo with H2-M3 tetramers complexed with different f-Met peptides shows that the majority of Listeria-specific, M3-restricted CD8(+) T cells are peptide specific. In contrast to the highly predictable size and immunodominance hierarchies of MHC class Ia-restricted T cell responses, the magnitudes of T cell responses specific for H2-M3-restricted peptides are remarkably variable between genetically identical mice. Our findings demonstrate that H2-M3-restricted T cell responses are distinct from classically restricted T cell responses to bacterial infection.     

Glucose-regulated protein 94/glycoprotein 96 elicits bystander activation of CD4+ T cell Th1 cytokine production in vivo

Glucose-regulated protein 94 (GRP94/gp96), the endoplasmic reticulum heat shock protein 90 paralog, elicits both innate and adaptive immune responses. Regarding the former, GRP94/gp96 stimulates APC cytokine expression and dendritic cell maturation. The adaptive component of GRP94/gp96 function reflects a proposed peptide-binding activity and, consequently, a role for native GRP94/gp96-peptide complexes in cross-presentation. It is by this mechanism that tumor-derived GRP94/gp96 is thought to suppress tumor growth and metastasis. Recent data have demonstrated that GRP94/gp96-elicited innate immune responses can be sufficient to suppress tumor growth and metastasis. However, the immunological processes activated in response to tumor Ag-negative sources of GRP94/gp96 are currently unknown. We have examined the in vivo immunological response to nontumor sources of GRP94/gp96 and report that administration of syngeneic GRP94/gp96- or GRP94/gp96-N-terminal domain-secreting KBALB fibroblasts to BALB/c mice stimulates CD11b(+) and CD11c(+) APC function and promotes bystander activation of CD4(+) T cell Th1 cytokine production. Only modest activation of CD8(+) T cell or NK cell cytolytic function was observed. The GRP94/gp96-dependent induction of CD4(+) T cell cytokine production was markedly inhibited by carrageenan, indicating an essential role for APC in this response. These results identify the bystander activation of CD4(+) T lymphocytes as a previously unappreciated immunological consequence of GRP94/gp96 administration and demonstrate that GRP94/gp96-elicited alterations in the in vivo cytokine environment influence the development of CD4(+) T cell effector functions, independently of its proposed function as a peptide chaperone.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.     

CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

We previously reported that scavenger receptor A (SRA/CD204), a binding structure on dendritic cells (DCs) for large stress/heat shock proteins (HSPs; e.g., hsp110 and grp170), attenuated an antitumor response elicited by large HSP-based vaccines. In this study, we show that SRA/CD204 interacts directly with exogenous hsp110, and lack of SRA/CD204 results in a reduction in the hsp110 binding and internalization by DCs. However, SRA(-/-) DCs pulsed with hsp110 or grp170-reconstituted gp100 chaperone complexes exhibit a profoundly increased capability of stimulating melanoma Ag gp100-specific naive T cells compared with wild-type (WT) DCs. Similar results were obtained when SRA/CD204 was silenced in DCs using short hairpin RNA-encoding lentiviruses. In addition, hsp110-stimulated SRA(-/-) DCs produced more inflammatory cytokines associated with increased NF-κB activation, implicating an immunosuppressive role for SRA/CD204. Immunization with the hsp110-gp100 vaccine resulted in a more robust gp100-specific CD8(+) T cell response in SRA(-/-) mice than in WT mice. Lastly, SRA/CD204 absence markedly improved the therapeutic efficacy of the hsp110-gp100 vaccine in mice established with B16 melanoma, which was accompanied by enhanced activation and tumor infiltration of CD8(+) T cells. Given the presence of multiple HSP-binding scavenger receptors on APCs, we propose that selective scavenger receptor interactions with HSPs may lead to highly distinct immunological consequences. Our findings provide new insights into the immune regulatory functions of SRA/CD204 and have important implications in the rational design of protein Ag-targeted recombinant chaperone vaccines for the treatment of cancer.