Enhanced endoplasmic reticulum entry of tumor antigen is crucial for cross-presentation induced by dendritic cell-targeted vaccination.

Efficient cross-presentation of protein Ags to CTLs by dendritic cells (DCs) is essential for the success of prophylactic and therapeutic vaccines. In this study, we report a previously underappreciated pathway involving Ag entry into the endoplasmic reticulum (ER) critically needed for T cell cross-priming induced by a DC-targeted vaccine. Directing the clinically relevant, melanoma Ag gp100 to mouse-derived DCs by molecular adjuvant and chaperone Grp170 substantially facilitates Ag access to the ER. Grp170 also strengthens the interaction of internalized protein Ag with molecular components involved in ER-associated protein dislocation and/or degradation, which culminates in cytosolic translocation for proteasome-dependent degradation and processing. Targeted disruption of protein retrotranslocation causes exclusive ER retention of tumor Ag in mouse bone marrow-derived DCs and splenic CD8(+) DCs. This results in the blockade of Ag ubiquitination and processing, which abrogates the priming of Ag-specific CD8(+) T cells in vitro and in vivo. Therefore, the improved ER entry of tumor Ag serves as a molecular basis for the superior cross-presenting capacity of Grp170-based vaccine platform. The ER access and retrotranslocation represents a distinct pathway that operates within DCs for cross-presentation and is required for the activation of Ag-specific CTLs by certain vaccines. These results also reinforce the importance of the ER-associated protein quality control machinery and the mode of the Ag delivery in regulating DC-elicited immune outcomes.

Scavenger receptor A restrains T-cell activation and protects against concanavalin A-induced hepatic injury.

UNLABELLED: Negative feedback immune mechanisms are essential for maintenance of hepatic homeostasis and prevention of immune-mediated liver injury. We show here that scavenger receptor A (SRA/CD204), a pattern recognition molecule, is highly up-regulated in the livers of patients with autoimmune or viral hepatitis, and of mice during concanavalin A (Con A)-induced hepatitis (CIH). Strikingly, genetic SRA ablation strongly sensitizes mice to Con A-induced liver injury. SRA loss, increased mortality and liver pathology correlate with excessive production of IFN-γ and heightened activation of T cells. Increased liver expression of SRA primarily occurs in mobilized hepatic myeloid cells during CIH, including CD11b(+) Gr-1(+) cells. Mechanistic studies establish that SRA on these cells functions as a negative regulator limiting T-cell activity and cytokine production. SRA-mediated protection from CIH is further validated by adoptive transfer of SRA(+) hepatic mononuclear cells or administration of a lentivirus-expressing SRA, which effectively ameliorates Con A-induced hepatic injury. Also, CIH and clinical hepatitis are associated with increased levels of soluble SRA. This soluble SRA displays a direct T-cell inhibitory effect and is capable of mitigating Con A-induced liver pathology. CONCLUSION: Our findings demonstrate an unexpected role of SRA in attenuation of Con A-induced, T-cell-mediated hepatic injury. We propose that SRA serves as an important negative feedback mechanism in liver immune homeostasis, and may be exploited for therapeutic treatment of inflammatory liver diseases.

Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity.

Recognition of pathogen-associated molecular patterns by innate immune receptors is essential for host defense responses. Although extracellular stress proteins are considered as indicators of the stressful conditions (e.g., infection or cell injury), the exact roles of these molecules in the extracellular milieu remain less defined. We found that glucose-regulated protein 170 (Grp170), the largest stress protein and molecular chaperone, is highly efficient in binding CpG oligodeoxynucleotides (CpG-ODN), the microbial DNA mimetic sensed by toll-like receptor 9 (TLR9). Extracellular Grp170 markedly potentiates the endocytosis and internalization of CpG-ODN by mouse bone marrow-derived macrophages and directly interacts with endosomal TLR9 on cell entry. These molecular collaborations result in the synergistic activation of the MyD88-dependent signaling and enhanced production of proinflammatory cytokines and nitric oxide in mouse primary macrophages as well as human THP-1 monocyte-derived macrophages, suggesting that Grp170 released from injured cells facilitates the sensing of pathogen-associated "danger" signals by intracellular receptors. This CpG-ODN chaperone complex-promoted innate immunity confers increased resistance in mice to infection of Listeria monocytogenes compared with CpG-ODN treatment alone. Our studies reveal a previously unrecognized attribute of Grp170 as a superior DNA-binding chaperone capable of amplifying TLR9 activation on pathogen recognition, which provides a conceptual advance in understanding the dynamics of ancient chaperoning functions inside and outside the cell.

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.

High molecular weight stress proteins: Identification, cloning and utilisation in cancer immunotherapy.

Although the large stress/heat shock proteins (HSPs), i.e. Hsp110 and Grp170, were identified over 30 years ago, these abundant and highly conserved molecules have received much less attention compared to other conventional HSPs. Large stress proteins act as molecular chaperones with exceptional protein-holding capability and prevent the aggregation of proteins induced by thermal stress. The chaperoning properties of Hsp110 and Grp170 are integral to the ability of these molecules to modulate immune functions and are essential for developing large chaperone complex vaccines for cancer immunotherapy. The potent anti-tumour activity of the Hsp110/Grp170-tumour protein antigen complexes demonstrated in preclinical studies has led to a phase I clinical trial through the National Cancer Institute's rapid access to intervention development (RAID) programme that is presently underway. Here we review aspects of the structure and function of these large stress proteins, their roles as molecular chaperones in the biology of cell stress, and prospects for their use in immune regulation and cancer immunotherapy. Lastly, we will discuss the recently revealed immunosuppressive activity of scavenger receptor A that binds to Hsp110 and Grp170, as well as the feasibility of targeting this receptor to promote T-cell activation and anti-tumour immunity induced by large HSP vaccines and other immunotherapies.

Pattern recognition scavenger receptor CD204 attenuates Toll-like receptor 4-induced NF-kappaB activation by directly inhibiting ubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6.

The collaboration and cross-talk between different classes of innate pattern recognition receptors are crucial for a well coordinated inflammatory response and host defense. Here we report a previously unrecognized role of scavenger receptor A (SRA; also known as CD204) as a signaling regulator in the context of Toll-like receptor 4 (TLR4) activation. We show that SRA/CD204 deficiency leads to greater sensitivity to LPS-induced endotoxic shock. SRA/CD204 down-regulates inflammatory gene expression in dendritic cells by suppressing TLR4-induced activation of the transcription factor NF-κB. For the first time, we demonstrate that SRA/CD204 executes its regulatory functions by directly interacting with the TRAF-C domain of TNF receptor-associated factor 6 (TRAF6), resulting in inhibition of TRAF6 dimerization and ubiquitination. The attenuation of NF-κB activity by SRA/CD204 is independent of its ligand-binding domain, indicating that the signaling-regulatory feature of SRA/CD204 can be uncoupled from its conventional endocytic functions. Collectively, we have identified the molecular linkage between SRA/CD204 and the TLR4 signaling pathways, and our results reveal a novel mechanism by which a non-TLR pattern recognition receptor restricts TLR4 activation and consequent inflammatory response.

ER stress and its regulator X-box-binding protein-1 enhance polyIC-induced innate immune response in dendritic cells.

Multiple physiological and pathological conditions interfere with the function of the endoplasmic reticulum (ER). However, much remains unknown regarding the impact of ER stress on inflammatory responses in dendritic cells (DCs) upon the recognition of pathogen molecules. We show that ER stress greatly potentiates the expression of inflammatory cytokines and IFN-ß in murine DCs stimulated by polyIC, a synthetic mimic of virus dsRNA. Both toll-like receptor 3 and melanoma differentiation-associated gene-5 are involved in the enhanced IFN-ß production, which is associated with increased activation of NF-κB and IRF3 signaling as well as the splicing of X-box-binding protein-1 (XBP-1), an important regulator involved in ER stress response. Surprisingly, silencing of XBP-1 reduces polyIC-stimulated IFN-ß expression in the presence or absence of ER stress, indicating that XBP-1 may be essential for polyIC signaling and ER stress-amplified IFN-ß production. Overexpression of a spliced form of XBP-1 (XBP-1s) synergistically augments polyIC-induced inflammatory response. For the first time, we show that XBP-1s overexpression-enhanced IFN-ß production in DCs markedly suppresses vesicular stomatitis virus infection, revealing a previously unrecognized role for XBP-1 in an antiviral response. Our findings suggest that evolutionarily conserved ER stress response and XBP-1 may function collaboratively with innate immunity to maintain cellular homeostasis.

Absence of scavenger receptor A promotes dendritic cell-mediated cross-presentation of cell-associated antigen and antitumor immune response.

Given the primary expression of scavenger receptor A (SRA) or CD204 on antigen-presenting cells, we investigate the immunoregulatory activities of SRA/CD204 in the context of cross-presentation of cell-associated antigen and the immunogenicity of dying tumor cells. Immunization with dying prostate cancer cells results in profoundly increased control of subsequently inoculated tumors in SRA/CD204 knockout mice. Using OVA-expressing RM1 prostate tumor line (RM1-OVA), we show for the first time that SRA absence greatly enhances dendritic cells (DCs)-mediated cross-presentation of OVA antigen derived from dying RM1 cells. While the phagocytic ability of DCs is not significantly impacted by the lack of SRA/CD204, DCs deficient in SRA/CD204 display increased expression of inflammatory cytokines and chemokines, as well as co-stimulatory molecules upon interaction with dying RM1 cells, implicating a suppressive regulation of the functional activation of DCs by SRA/CD204. Further, SRA/CD204-deficient DCs pulsed with dying RM1-OVA cells are more effective than wild-type counterparts in priming antigen-specific T-cell responses, resulting in improved control of RM1 tumor growth in both prophylactic and therapeutic settings. Our findings suggest that the increased immunogenicity of dying tumor cells in SRA/CD204 knockout mice is attributed to the altered functions of DCs in the absence of SRA/CD204, which underscores the important role of SRA/CD204 in host immune homeostasis. Selective downregulation or blockade of this immunoregulatory molecule may lead to enhanced potency of DC-based vaccines capable of breaking immune tolerance against cancer.

Superior antitumor response induced by large stress protein chaperoned protein antigen compared with peptide antigen.

Our previous studies have demonstrated that the natural chaperone complexes of full-length tumor protein Ags (e.g., gp100) and large stress proteins (e.g., hsp110 and grp170) with exceptional Ag-holding capabilities augment potent tumor protective immunity. In this study, we assess the peptide-interacting property of these large chaperones and, for the first time, compare the immunogenicity of the recombinant chaperone vaccines targeting two forms of Ags (protein versus peptide). Both hsp110 and grp170 readily formed complexes with antigenic peptides under physiologic conditions, and the peptide association could be further stimulated by heat shock. The large chaperones displayed similar but distinct peptide-binding features compared with hsp70 and grp94/gp96. Immunization with hsp110- or grp170-tyrosinase-related protein 2 (TRP2(175-192)) peptide complexes effectively primed CD8(+) T cells reactive with TRP2-derived, MHC class I-restricted epitope. However, the tumor protective effect elicited by the TRP2(175-192) peptide vaccine was much weaker than that achieved by full-length TRP2 protein Ag chaperoned by grp170. Furthermore, immunization with combined chaperone vaccines directed against two melanoma protein Ags (i.e., gp100 and TRP2) significantly improved overall anti-tumor efficacy when compared with either of the single Ag vaccine. Lastly, treatment of tumor-bearing mice with these dual Ag-targeted chaperone complexes resulted in an immune activation involving epitope spreading, which was associated with a strong growth inhibition of the established tumors. Our results suggest that high m.w. chaperones are superior to conventional chaperones as a vaccine platform to deliver large protein Ags, and provide a rationale for translating this recombinant chaperoning-based vaccine to future clinical investigation.

Recombinant human Hsp110-gp100 chaperone complex vaccine is nontoxic and induces response in advanced stage melanoma patients.

Heat shock proteins (hsp) are intracellular chaperones that possess extracellular immunostimulatory properties when complexed with antigens. A recombinant Hsp110-gp100 chaperone complex vaccine showed an antitumor response and prolonged survival in murine melanoma. A phase Ib dose-escalation study of a recombinant human Hsp110-gp100 vaccine in advanced-stage melanoma patients was performed to evaluate toxicity, immunostimulatory potential and clinical response. Patients with pretreated, unresectable stage IIIB/C/IV melanoma received the chaperone complex vaccine in a dose-escalation protocol; three vaccinations over a 43-day-period. Tumor response, clinical toxicity and immune response were measured. Ten patients (eight female, median age 70 years) were enrolled and two patients had grade 1 adverse events; minor skin rash, hyperhidrosis and fever (no grade 2 or higher adverse events). Median progression-free survival was longer for lower vaccine doses as compared to the maximum dose of 180 mcg (4.5 vs. 2.9 months; P = 0.018). The lowest dose patients (30 and 60 mcg) had clinical tumor responses (one partial response, one stable disease). CD8+ T cell interferon-γ responses to gp100 were greater in the clinically responding patients. A pattern of B cell responses to vaccination was not observed. Regulatory T cell populations and co-stimulatory molecules including cytotoxic T-lymphocyte-associated protein 4 and PD-1 appeared to differ in responders versus nonresponders. A fully recombinant human Hsp110-gp100 chaperone complex vaccine had minimal toxicity, measurable tumor responses at lower doses and produced peripheral CD8+ T cell activation in patients with advanced, pretreated melanoma. Combination with currently available immunotherapies may augment clinical responses.

Pattern recognition scavenger receptor SRA/CD204 down-regulates Toll-like receptor 4 signaling-dependent CD8 T-cell activation.

Class A scavenger receptor (SRA), also known as CD204, has been shown to participate in the pathogenesis of atherosclerosis and the pattern recognition of pathogen infection. However, its role in adaptive immune responses has not been well defined. In this study, we report that the lack of SRA/CD204 promotes Toll-like receptor (TLR)4 agonist-augmented tumor-protective immunity, which is associated with enhanced activation of CD8(+) effector T cell and improved inhibition of tumor growth. Dendritic cells (DCs) deficient in SRA/CD204 display more effective immunostimulatory activities upon TLR4 engagement than those from wild-type counterparts. Silencing of SRA/CD204 by RNA interference improves the ability of DCs to prime antigen-specific CD8(+) T cells, suggesting that antigen-presenting cells, for example, DCs, play a major role in SRA/CD204-mediated immune modulation. Our findings reveal a previously unrecognized role for SRA/CD204, a non-TLR pattern recognition receptor, as a physiologic negative regulator of TLR4-mediated immune consequences, which has important clinical implications for development of TLR-targeted immunotherapeutic intervention.

Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity.

BACKGROUND: The efficacy of cancer immunotherapy can be limited by the poor immunogenicity of cancer and the immunosuppressive tumor microenvironment (TME). Immunologically programming the TME and creating an immune-inflamed tumor phenotype is critical for improving the immune-responsiveness of cancers. Here, we interrogate the immune modulator Flagrp170, engineered via incorporation of a pathogen-associated molecular pattern (ie, flagellin) into an immunostimulatory chaperone molecule, in transforming poorly immunogenic tumors and establishing a highly immunostimulatory milieu for immune augmentation. METHODS: Multiple murine cancer models were used to evaluate the immunostimulatory activity, antitumor potency, and potential side effects of Flagrp170 on administration into the tumors using a replication impaired adenovirus. Antibody neutralization and mice deficient in pattern recognition receptors, that is, toll-like receptor 5 (TLR5) and NOD like receptor (NLR) family caspase activation and recruitment domain (CARD) domain-containing protein 4 (NLRC4), both of which can recognize flagellin, were employed to understand the immunological mechanism of action of the Flagrp170. RESULTS: Intratumoral delivery of mouse or human version of Flagrp170 resulted in robust inhibition of multiple malignancies including head and neck squamous cell carcinoma and breast cancer, without tissue toxicities. This in situ Flagrp170 treatment induced a set of cytokines in the TME known to support Th1/Tc1-dominant antitumor immunity. Additionally, granulocyte macrophage colony-stimulating factor derived from mobilized CD8+ T cells was involved in the therapeutic activity of Flagrp170. We also made a striking finding that NLRC4, not TLR5, is required for Flagrp170-mediated antitumor immune responses. CONCLUSION: Our results elucidate a novel immune-potentiating activity of Flagrp170 via engaging the innate pattern recognition receptor NLRC4, and support its potential clinical use to reshape cancer immune phenotype for overcoming therapeutic resistance.

Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo.

PURPOSE: We employed a grp170-secreting tumour cell system to determine whether tumour cells engineered to secrete grp170 generate an antitumour-specific immune response. Further, we examine the possibility that secreted grp170 can bind to and co-transport out of tumour cells full-length tumour antigens that may play a role in the anti-tumour immune response. MATERIALS AND METHODS: Wild type Colon-26 and Colon-26 engineered to secrete grp170 were subcutaneously inoculated into BALB/c mice. Tumour growth was monitored, and variations in immunoregulatory mechanisms were evaluated using immunohistochemistry, lymphocyte depletion, ELISpot assays, and Western blot analysis. RESULTS: Immunisation of animals with grp170-secreting tumour cells results in rejection of the tumour by induction of antigen-specific, CD8-dependent immune responses. The secreted grp170 is able to deliver full-length tumour antigens to the tumour microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate tumour-specific immune responses. CONCLUSIONS: These data parallel our studies showing that hsp110 or grp170 are able to chaperone full-length proteins, and when complexed with protein antigens and used as vaccines, these complexes elicit immune responses in vivo against the protein antigens. This cell-based approach has the potential to be utilised as a tumour-specific vaccine in tumours of various histological origins.

Adrenergic stress constrains the development of anti-tumor immunity and abscopal responses following local radiation.

The abscopal effect following ionizing radiation therapy (RT) is considered to be a rare event. This effect does occur more frequently when combined with other therapies, including immunotherapy. Here we demonstrate that the frequency of abscopal events following RT alone is highly dependent upon the degree of adrenergic stress in the tumor-bearing host. Using a combination of physiologic, pharmacologic and genetic strategies, we observe improvements in the control of both irradiated and non-irradiated distant tumors, including metastatic tumors, when adrenergic stress or signaling through ß-adrenergic receptor is reduced. Further, we observe cellular and molecular evidence of improved, antigen-specific, anti-tumor immune responses which also depend upon T cell egress from draining lymph nodes. These data suggest that blockade of ß2 adrenergic stress signaling could be a useful, safe, and feasible strategy to improve efficacy in cancer patients undergoing radiation therapy.

Scavenger receptor-A negatively regulates antitumor immunity.

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.

Carbonic anhydrase IX has chaperone-like functions and is an immunoadjuvant.

Carbonic anhydrase IX (CA9) is a hypoxia-regulated, transmembrane protein associated with neoplastic growth in a large spectrum of human tumors. CA9 is expressed in nearly all clear-cell renal tumors; levels of CA9 expression predict prognosis and response to interleukin-2 therapy. These observations may be explained by a novel chaperone-like function of CA9, which allows it to serve as an immunoadjuvant and stimulate an adaptive immune response against tumor antigens. Classic heat shock proteins (HSP) such as HSP110 and HSP70 are up-regulated in response to cellular stress and function to protect intracellular proteins from aggregation. Similarly, CA9 formed complexes with client proteins and inhibited heat-induced aggregation and enabled refolding of denatured client protein. HSP released from injured cells activate an immune response. CA9 bound dendritic cells in a receptor-specific manner. Bound CA9 was internalized by dendritic cells and processed primarily through the proteosomal pathway. In a murine melanoma model, a complex of CA9 and gp100 generated a gp100-specific antitumor response. A soluble form of CA9 shed from tumor cells had the same chaperone-like functions, providing renal tumors and hypoxic cells with a mechanism for stimulating an immune response against extracellular antigens. Interleukin-2 treatment of patient renal tumors in short-term culture increased CA9 shedding, suggesting a strategy for augmenting the immunogenicity of renal tumors. CA9 has chaperone-like functions and CA9 shed from tumors may play a direct role in stimulating an adaptive immune response.

Chaperoning function of stress protein grp170, a member of the hsp70 superfamily, is responsible for its immunoadjuvant activity.

When used as vaccines, tumor-derived stress proteins can elicit antitumor immune responses. For members of the hsp70 superfamily, like grp170, this seems to be due to (a) the chaperoning of antigenic peptide by the stress protein and (b) the binding of the stress protein to receptor(s) on antigen-presenting cells (APC) and subsequent antigen presentation. This suggests that domains exist on the stress protein for each function. In this study, we determine the ability of grp170 and its structural domains to (a) bind to and present melanoma-associated antigen gp100 to the immune system and (b) to bind to receptors on APCs. A direct correlation between chaperone function, binding to APCs in a receptor-like manner, and antitumor immunity was observed. Two mutants that share no common sequence, yet are both effective in their antitumor activities, compete with one another for APC binding. Studies of other members of the hsp70 superfamily, hsp110 and hsp70, or their domain deletion mutants, further confirmed that APC binding segregates with chaperoning function and not sequence. Therefore, these studies suggest that molecular chaperoning is involved in stress protein interactions with APCs, antigen binding, and in eliciting antitumor immunity, thus bridging this ancient function of stress proteins in prokaryotes to their ability to elicit immunity in higher organisms.

Creation of Recombinant Chaperone Vaccine Using Large Heat Shock Protein for Antigen-Targeted Cancer Immunotherapy.

Large heat shock proteins (HSPs) or stress proteins, including Hsp110 and Grp170, are unique molecular chaperones with superior capability of shuttling tumor protein antigens into professional antigen-presenting cells, such as dendritic cells, for highly efficient cross-presentation and T cell priming. Reconstituted chaperone complexes of large HSP and tumor protein antigen have been demonstrated to generate a robust antigen-specific T lymphocyte response with therapeutic potency against multiple cancer types in preclinical models. Here, we describe the methods for preparing this recombinant chaperone complex vaccine and analyzing the vaccine-induced activation of antigen-specific T cells using in vitro and in vivo systems.

Intestinal heat shock protein 110 regulates expression of CD1d on intestinal epithelial cells.

CD1d is expressed on the surface of professional and nonprofessional APCs, including intestinal epithelial cells (IECs), for a role in the presentation of glycolipid-based antigens to subsets of T cells. The mechanisms that regulate CD1d expression in any cell type are unknown. To investigate the possibility that expression of CD1d is influenced by exogenous factors present within the intestinal lumen, CD1d expression was analyzed in several IEC lines after culturing in the presence of lumenal contents (LC) of the normal human intestine. Exposure of the colon-derived cell lines T84, HT-29, and Caco-2 to soluble LC resulted in a marked induction of CD1d expression as determined by RT-PCR, confocal microscopy, cell surface ELISA, and Western blot analysis. Similarly, exposure of human IECs to LC isolated from mice bred in both specific pathogen-free and germfree conditions also resulted in the induction of CD1d expression, with the maximum CD1d-inducing activity observed in the small intestine. Biochemical and biophysical characterization of the human CD1d-inducing activity identified heat shock protein 110 (Hsp110) as a major functional component of the LC that contributes to CD1d surface regulation, and immunolocalization studies revealed Hsp110 expression in subsets of human IECs in vivo. These data support the presence of a novel autocrine pathway of CD1d regulation by Hsp110.

Heat shock proteins as vaccine adjuvants in infections and cancer.

In addition to maintaining cell homeostasis under physiological and stress conditions, some heat shock proteins (HSPs) are potent inducers of immunity and have been harnessed as vaccine adjuvants targeted to cancers and infections. HSPs are a group of ubiquitous intracellular molecules that function as molecular chaperones in numerous processes, such as protein folding and transport, and are induced under stress conditions, such as fever and radiation. Certain HSPs are potent inducers of innate and antigen-specific immunity. They activate dendritic cells partly through toll-like receptors, activate natural killer cells, increase presentation of antigens to effector cells and augment T-cell and humoral immune responses against their associated antigens. Their roles in priming multiple host defense pathways are being exploited in vaccine development for cancer and infectious diseases.