Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, apoptosis-inducing gene with broad-spectrum antitumor activity, making it an ideal candidate for a novel cancer gene therapy. A systemic and sustained antitumor immune response generated at the time of initial molecular-targeted therapy would provide additional clinical benefits in cancer patients, resulting in improved prevention of tumor recurrence. In this study, we explored the therapeutic efficacy of intratumoral delivery of a nonreplicating adenoviral vectors encoding mda-7/IL-24 (Ad.mda-7) and a secretable form of endoplasmic reticulum resident chaperone grp170 (Ad.sgrp170), a potent immunostimulatory adjuvant and antigen carrier. Intratumoral administration of Ad.mda-7 in combination with Ad.sgrp170 was more effective in controlling growth of TRAMP-C2 prostate tumor as compared to either Ad.mda-7 or Ad.sgrp170 treatment. Generation of systemic antitumor immunity was demonstrated by enhanced protection against subsequent tumor challenge and improved control of distant tumors. The combined treatments enhanced antigen and tumor-specific T-cell response as indicated by increased IFN-γ production and cytolytic activity. Antibody depletion suggests that CD8+ T-cell may be involved in the antitumor effect of the dual molecule-targeted therapies. Therefore, introducing immunostimulatory chaperone grp170 in situ strongly promotes the ‘immunogenic’ cell death when delivered to the mda-7/IL-24 induced apoptotic tumor cells, indicating that an improved anti-cancer efficacy may be achieved by concurrently targeting both tumor and immune compartments. Given multiple undefined antigens present endogenously within prostate cancer, these data provide a rationale for combining sgrp170-based vaccine strategy with mda-7/IL-24-targeted cancer therapy to induce durable systemic immunity.
mda-7/IL-24; cancer gene therapy; stress protein; chaperone; grp170; immunity
Efficient cross-presentation of protein antigens to cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) is essential for the success of prophylactic and therapeutic vaccines. Here, we report a previously underappreciated pathway involving antigen entry into the endoplasmic reticulum (ER) critically needed for T-cell cross-priming induced by a DC-targeted vaccine. Directing the clinically relevant, melanoma antigen Gp100 to mouse-derived DCs by molecular adjuvant and chaperone Grp170 substantially facilitates antigen access to the ER. Grp170 also strengthens the interaction of internalized protein antigen 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 antigen in mouse bone marrow-derived DCs and splenic CD8+ DCs. This results in the blockade of antigen ubiquitination and processing, which abrogates the priming of antigen-specific CD8+ T cells in vitro and in vivo. Therefore, the improved ER entry of tumor antigen 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 antigen-specific CTLs by certain vaccines. These results also reinforce the importance of the ER-associated protein quality-control machinery and the mode of the antigen delivery in regulating DC-elicited immune outcomes.
dendritic cell; cross-presentation; endoplasmic reticulum; vaccine; melanoma antigen; molecular chaperone
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 antitumor activity of the Hsp110/Grp170-tumor protein antigen complexes, demonstrated in preclinical studies, has led to a phase I clinical trial through the National Cancer Institute's RAID Program 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 antitumor immunity induced by large HSP vaccines and other immunotherapies.
molecular chaperone; dendritic cell; Grp170; flagellin; vaccine; antitumor immunity
Converting the immunosuppressive tumor environment into one that is favorable to induction of antitumor immunity is indispensable for effective cancer immunotherapy. Here we strategically incorporate a pathogen (i.e., Flagellin)-derived, NF-κB-stimulating `danger' signal into the large stress protein or chaperone Grp170 (HYOU1/ORP150) that was previously shown to facilitate antigen cross-presentation. This engineered chimeric molecule (i.e., Flagrp170) is capable of transporting tumor antigens and concurrently inducing functional activation of dendritic cells. Intratumoral administration of adenoviruses expressing Flagrp170 induces a superior antitumor response against B16 melanoma and its distant lung metastasis compared to unmodified Grp170 and Flagellin. The enhanced tumor destruction is accompanied with significantly increased tumor infiltration by CD8+ cells as well as elevation of IFN-γ and IL-12 levels in the tumor sites. In situ Ad.Flagrp170 therapy provokes systemic activation of CTLs that recognize several antigens naturally expressing in melanoma (e.g., gp100/PMEL and TRP2/DCT). The mechanistic studies using CD11c-DTR transgenic mice and Batf3-deficient mice reveal that CD8α+ DCs are required for the improved T cell cross-priming. Antibody neutralization assays show that IL-12 and IFN-γ are essential for the Flagrp170-elicited antitumor response, which also involves CD8+ T cells and NK cells. The therapeutic efficacy of Flagrp170 and its immune stimulating activity are also confirmed in mouse prostate cancer and colon carcinoma. Together, targeting the tumor microenvironment with this chimeric chaperone is highly effective in mobilizing or restoring antitumor immunity, supporting the potential therapeutic use of this novel immune modulator in the treatment of metastatic diseases.
molecular chaperone; Grp170; Flagellin; immune modulator; antitumor immunity
Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation.
This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics.
Strategies to specifically identify and inhibit critical determinants that promote therapy-resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying small molecule inhibitors with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies.
BH3 domain; apoptosis; Mcl-1; radiation resistance; chemotherapy resistance
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 upregulated 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. We also report for the first time that CIH and clinical hepatitis are associated with the 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. 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.
Liver injury; CD204; T cell; Interferon-γ; myeloid cells
Therapeutic vaccines represent a viable option for active immunotherapy of cancers that aim to treat late stage disease by using a patient's own immune system. The promising results from clinical trials recently led to the approval of the first therapeutic cancer vaccine by the U.S. Food and Drug Administration. This major breakthrough not only provides a new treatment modality for cancer management, but also paves the way for rationally designing and optimizing future vaccines with improved anticancer efficacy. Numerous vaccine strategies are currently being evaluated both pre-clinically and clinically. This review discusses therapeutic cancer vaccines of diverse platforms or targets as well as the preclinical and clinical studies employing these therapeutic vaccines. We will also consider tumor-induced immune suppression that hinders the potency of therapeutic vaccines, and potential strategies to counteract these mechanisms for generating more robust and durable antitumor immune responses.
cancer vaccine; immunotherapy; tumor-associated antigen; immune modulator; immunosuppression; tumor microenvironment
Glucose-regulated protein 170 (GRP170) is the largest member of glucose-regulated protein family that resides in the endoplasmic reticulum (ER). As a component of the ER chaperone network, GRP170 assists in protein folding, assembly, and transportation of secretory or transmembrane proteins. The well documented cytoprotective activity of intracellular GRP170 due to its intrinsic chaperoning property has been shown to provide a survival benefit in cancer cells during tumor progression or metastasis. Accumulating evidence shows that extracellular GRP170 displays a superior capacity in delivering tumor antigens to specialized antigen-presenting cells for cross-presentation, resulting in generation of an anti-tumor immune response dependent on cytotoxic CD8+ T cells. This unique feature of GRP170 provides a molecular basis for using GRP170 as an immunostimulatory adjuvant to develop a recombinant vaccine for therapeutic immunization against cancers. This review summarizes the latest findings in understanding the biological effects of GRP170 on cell functions and tumor progression. The immunomodulating activities of GRP170 during interactions with the innate and adaptive arms of the immune system as well as its therapeutic applications in cancer immunotherapy will be discussed.
endoplasmic reticulum; glucose-regulated protein 170; molecular chaperone; anti-tumor immunity; cancer vaccine
Myeloid-derived suppressive cells (MDSCs) have been a focus of recent study on tumor-mediated immune suppression. However, its role in type 17 helper T (Th17) cell differentiation and the pathogenesis of autoimmune diseases (e.g., multiple sclerosis) has not been determined. We show here that development of experimental autoimmune encephalomyelitis (EAE) in mice is associated with a profound expansion of CD11b+Gr-1+ MDSCs, which display efficient T cell inhibitory functions in vitro. Unexpectedly, these MDSCs enhance the differentiation of naïve CD4+ T cell precursors into Th17 cells in a highly efficient manner under Th17 polarizing conditions, as indicated by significantly increased number of Th17 cell, elevation of IL-17A production, and upregulation of the orphan nuclear receptor RORA and RORC. Mechanistic studies show that IL-1β represents a major mediator of MDSC-facilitated Th17 differentiation, which depends on the IL-1 receptor on CD4+ T cells but not MDSCs. Selective depletion of MDSCs using gemcitabine results in a marked reduction in the severity of EAE (e.g., decreased clinical scores and myelin injury), which correlates with reduced Th17 cells and inflammatory cytokines (IL-17A and IL-1β) in the lymphoid tissues and spinal cords. Adoptively transfer of MDSCs after gemcitabine treatment restores EAE disease progression. Together, we demonstrate for the first time that excessive and prolonged presence of MDSCs can drive a Th17 response and consequently contributes to the pathogenesis of EAE. These new findings provide unique insights into the pleiotropic functions of MDSCs, and may help explain the failure of immunosuppressive MDSCs to control Th17/IL-17-dependent autoimmune disorders.
T helper 17 cell; Myeloid-derived suppressor cells; Interleukin-1; multiple sclerosis
Given the complexity of prostate cancer progression and metastasis, multimodalities that target different aspects of tumor biology, e.g., radiotherapy (RT) in conjunction with immunotherapy, may provide the best opportunities for promoting clinical benefits in patients with high risk localized prostate cancer. Here we show that intratumoral administration of unmodified dendritic cells (DCs) failed to synergize with fractionated RT. However, ionizing radiation combined with in situ vaccination with DCs, in which the immunosuppressive scavenger receptor A (SRA/CD204) has been downregulated by lentivirus-mediated gene silencing, profoundly suppressed the growth of two mouse prostate cancers (e.g., RM1 and TRAMP-C2), and prolonged the lifespan of tumor-bearing animals. Treatment of subcutaneous tumors with this novel combinatorial radio-immunotherapeutic regimen resulted in a significant reduction in distant experimental metastases. SRA/CD204-silenced DCs were highly efficient in generating antigen or tumor-specific T cells with increased effector functions (e.g., cytokine production and tumoricidal activity). SRA/CD204 silencing-enhanced tumor cell death was associated with elevated IFN-γ levels in tumor tissue and increased tumor-infiltrating CD8+ cells. IFN-γ neutralization or depletion of CD8+ cells abrogated the SRA/CD204 downregulation-promoted antitumor efficacy, indicating a critical role of IFN-γ-producing CD8+ T cells. Therefore, blocking SRA/CD204 activity significantly enhances the therapeutic potency of local RT combined with in situ DC vaccination by promoting a robust systemic antitumor immunity. Further studies are warranted to test this novel combinatorial approach for translating into improved clinical outcomes in prostate cancer patients.
Prostate cancer; radiotherapy; dendritic cell; T cell; tumor immunity; IFN-γ; CD204; scavenger receptor A
To study the anatomic parameters related to clival screw and establish reference data concerning the craniovertebral fixation technique.
Morphometric measurement of the clivus and the surrounding anatomic structures were obtained on 41 dry bone specimens. Then, 2-D CT reconstruction of the craniovertebral region of 30 patients (19 men and 11 women, ranging in age from 20–64 years with an average age of 38.8 years) were performed to measure the safety range for a 3.5-mm screw placement. Nine entry points were evaluated. Finally, one male fresh cadaver specimen (age 46 years) was dissected to observe the craniovertebral region.
The clivus faces the basilar artery, the V ~ XII cranial nerves, the pons, and ventral medulla oblongata at its intracranial surface. The longitudinal diameter of extracranial clivus was 25.87 ± 2.64 mm. The narrowest diameter of the clivus was 12.84 ± 1.08 mm, the distance between the left and right hypoglossal canal was 32.70 ± 2.09 mm at its widest part. The distance between the left and right structures, the maximum value was 49.31 ± 4.16 mm at carotid canal, the minimum value was 16.54 ± 2.04 mm at the occipital condyle. The measurement of clival screws placement simulation via 2-D CT reconstruction images shows the maximum upper insertion angle of three components the optimal entry points, the candidate points, the limit entry points was 130.19°, 125.23° and 85.72°, and the total mean screw length was 7.57, 10.13 and 15.6 mm at the vertical entry angle, respectively.
Clival screw placement is a viable option for craniovertebral fixation. There is a safe scope for the screw length and angle of the screw placement. And, these parameters obtained in the present study will be helpful for anyone contemplating the use of clival screw fixation.
Clivus; Craniovertebral fusion; Screw placement; Anatomical study
The incidence of melanoma continues to rise and prognosis in patients with metastatic melanoma remains poor. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) serves as one of the primary immune checkpoints and downregulates T cell activation pathways. Enhancing T cell activation by antibody blockade of the CTLA-4 provides a novel approach to overcome tumor-induced immune tolerance. Recently, anti-CTLA-4 therapy demonstrated significant clinical benefit in patients with metastatic melanoma, which led to the approval of ipilimumab by the Food and Drug Administration in early 2011.
The fundamental concepts underlying CTLA-4 blockade-potentiated immune activation, the scientific rationale for and the preclinical evidence supporting CTLA-4-targeted cancer immunotherapy are presented. We also provide an update on clinical trials with anti-CTLA-4 inhibitors and discuss the associated autoimmune toxicity.
Given that overall survival is the only validated endpoint for the anti-CTLA-4 therapy, the clinical implications of the antigen or tumor-specific immunity in patients remain to be clarified. Additional research is necessary to elucidate the prognostic significance of immune-related side effects and significantly optimize the treatment regimens. An improved understanding of the mechanisms of action of CTLA-4 antibodies may also culminate in wide-ranging clinical applications of this novel therapy for other tumor types.
cytotoxic T-lymphocyte-associated antigen; CTL-A blockade; T cell activation; tumor immunity; overall survival
We have recently demonstrated that glucose-regulated protein 170 (Grp170), a stress-responsive molecular chaperone of the endoplasmic reticulum, can be exploited to stimulate anticancer immunity due to its superior antigen chaperoning and delivering capacity. The immune remodeling of the tumor microenvironment induced by a Grp170-based chaperone leads to immune responses that effectively control the progression of both primary neoplasms and their metastases. Our findings support the development of Grp170-based immunomodulating strategies to potentiate antitumor immune responses.
dendritic cells; glucose-regulated protein 170; immune modulator molecular chaperone; tumor microenvironment; vaccine
Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, endotoxic shock, host defense and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here we for the first time report that SRA/CD204 knockout (SRA−/−) mice developed a more robust CD4+ T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA−/− mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA−/− DCs with or without lipopolysaccharide treatment exhibited increased T cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus IFN-γ stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, MAPK p38 and NF-κB signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA−/− OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC II-restricted OVA323-339 peptide compared with cells from the SRA+/+ OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4+ T cell activation, but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule.
dendritic cells; CD4 T cell; immunity; CD204
Although dendritic cell (DC) vaccines offer promise as cancer immunotherapy, further improvements are needed to amplify their clinical therapeutic efficacy. The pattern recognition scavenger receptor SRA/CD204 attenuates the ability of DCs to activate CD8+ T cell responses. Therefore, we examined the impact of SRA/CD204 on antitumor responses generated by DC vaccines and we also evaluated the feasibility of enhancing DC vaccine potency by SRA/CD204 blockade. DCs from SRA/CD204 deficient mice were more immunogenic in generating antitumor responses to B16 melanoma, compared to DCs from wild-type mice. Similarly, siRNA-mediated knockdown of SRA/CD204 by lentiviral vectors improved the ability of wild-type DCs to stimulate the expansion and activation of CD8+ T cells specific for idealized or established melanoma antigens in mice. Using SRA/CD204-silenced DCs to generate antigen-targeted vaccines, we documented a marked increase in the level of antitumor immunity achieved against established B16 tumors and metastases. This increase was associated with enhanced activation of antigen-specific CTLs, greater tumor infiltration by CD8+ T cells and NK cells, and increased intratumoral ratios of both CD4+ and CD8+ T effector cells to CD4+CD25+ T regulatory cells. Our studies establish that downregulating SRA/CD204 strongly enhances DC-mediated antitumor immunity. Additionally, they provide a rationale to enhance DC vaccine potency through SRA/CD204-targeting approaches that can improve clinical outcomes in cancer treatment.
dendritic cell vaccine; CD204; CTL; antitumor immunity
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. Here 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 profoundly increased capability of stimulating melanoma antigen gp100-specific naïve T-cells compared to wild-type (WT) DCs. Similar results were obtained when SRA/CD204 was silenced in DCs using shRNAs-encoding lentiviruses. In addition, hsp110-stimulated SRA−/− DCs produced more inflammatory cytokines associated with increased NF-κB activation, implicating an immunosuppressive role of 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 antigen-presenting cells, we propose that selective scavenger receptor interactions with HSPs may lead to highly distinct immunological consequences. Our findings provide new insights to the immune regulatory functions of SRA/CD204 and have important implications in the rational design of protein antigen-targeted recombinant chaperone vaccines for the treatment of cancer.
large heat shock protein; chaperone; grp170; hsp110; melanoma antigen; CD204; vaccination
Extensive studies have established a role of scavenger receptor CD204 in pattern recognition and ligand uptake. Strikingly, we recently revealed a previously unrecognized feature of CD204 action in attenuating T-cell activation and antitumor immunity. Blocking its activity in dendritic cells represents a promising approach to the improvement of cancer immunotherapy.
innate immune receptor; dendritic cells; CD204; vaccine; T-cell response; tumor immunity
Given the primary expression of scavenger receptor A (SRA) or CD204 on antigen presenting cells, we investigate the immune-regulatory 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.
dendritic cells; antigen presentation; immune response; CD204; scavenger receptor
Large heat shock proteins (HSPs), including hsp110 and grp170, are unique immunochaperones capable of carrying and introducing antigens into professional antigen presenting cells (APCs) for efficient cross-presentation. Therefore, reconstituted chaperone complexes of large HSPs and protein antigen may be exploited for augmentation of an antigen-specific immune response. The methods for the preparation of the recombinant protein antigen chaperone complex and characterization of its T-cell priming capability in both in vitro and in vivo settings are described.
large heat shock protein; hsp110; grp170; chaperone vaccine; antigen presentation; T-cell priming
Multiple physiological and pathological conditions interfere with the function of 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-β 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-β 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-β expression in the presence or absence of ER stress, indicating that XBP-1 may be essential for polyIC signaling and ER stress-amplified IFN-β 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-β production in DCs markedly suppresses vesicular stomatitis virus infection, revealing a previously unrecognized role of XBP-1 in an antiviral response. Our findings suggest that evolutionarily conserved ER stress response and XBP-1 may function collaboratively with the innate immunity in maintaining cellular homeostasis.
endoplasmic reticulum; X-box binding protein-1; polyIC; dendritic cells; inflammatory response; VSV
Our previous studies have demonstrated that the natural chaperone complexes of full-length tumor protein antigens (e.g., gp100) and large stress proteins (e.g., hsp110 and grp170) with exceptional antigen holding capabilities augment potent tumor protective immunity. Here 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 antigens (protein vs peptide). Both hsp110 and grp170 readily formed complexes with antigenic peptides under physiological conditions, and the peptide association could be further stimulated by heat shock. The large chaperones displayed similar, but distinct peptide-binding features compared to hsp70 and grp94/gp96. Immunization with hsp110- or grp170-tyrosinase-related protein 2 (TRP2175–192) peptide complexes effectively primed CD8+ T-cells reactive with TRP2-derived, MHC I-restricted epitope. However, the tumor protective effect elicited by the TRP2175–192 peptide vaccine was much weaker than that achieved by full-length TRP2 protein antigen chaperoned by grp170. Furthermore, immunization with combined chaperone vaccines directed against two melanoma protein antigens (i.e., gp100 and TRP2) significantly improved overall antitumor efficacy when compared to either of the single antigen vaccine. Lastly, treatment of tumor-bearing mice with these dual antigen-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 molecular weight chaperones are superior to conventional chaperones as a vaccine platform to deliver large protein antigens, and provide a rationale for translating this recombinant chaperoning-based vaccine to future clinical investigation.
large stress protein; chaperone; grp170; hsp110; antigen; immuogenicity
In this study, we report a novel treatment strategy that could potentially be used to improve efficacy of adoptive cell therapy for patients with prostate cancer. We show that female C57BL/6 mice are able to effectively reject two syngeneic prostate tumors (TRAMP-C2 and RM1) in a T cell-dependent manner. The protective antitumor immunity appears to primarily involve T cell responses reactive against general prostate tumor/tissue antigens, rather than simply to male-specific H-Y antigen. For the first time we show that adoptive transfer of lymphocytes from TRAMP-C2-primed or naive female mice effectively control prostate tumor growth in male mice, when combined with host pre-conditioning (i.e., non-myeloablative lymphodepletion) and IL-2 administration. No pathological autoimmune response was observed in the treated tumor-bearing male mice. Our studies provide new insights regarding the immune-mediated recognition of male-specific tissue, such as the prostate, and may offer new immunotherapy treatment strategies for advanced prostate cancer.
prostate cancer; female lymphocyte; adoptive cell therapy; immunotherapy
It is well established that certain stress proteins or molecular chaperones are highly efficient in cross-presenting tumor-derived antigens, resulting in a potent antitumor immune response. In this study we demonstrate that genetic modification of weakly immunogenic murine prostate tumor cells (TRAMP-C2) by stable transfection with a secretable form of endoplasmic reticulum resident chaperone grp170 significantly enhances its immunogenicity in vivo. Generation of systemic antitumor immunity is indicated by the growth suppression of distant parental tumors, which is associated with increased tumor infiltration, elevated effector functions of CD8+ T-cells. Immunization with inactivated grp170-secreting C2 cells augments a CD8+ T-cell dependent, tumor-protective effect. Furthermore, infection of C2 tumor cells with a nonreplicating adenoviral vectors encoding secretable grp170 promotes tumor immunogenicity more effectively than plasmid transduction, as shown by the increased production of pro-inflammatory cytokine TNF-α by dendritice cells and enhanced therapeutic efficacy in treating pre-established tumors. Given a repertoire of undefined antigens in prostate tumor, manipulation of cellular compartmentalization of immuno-stimulatory chaperone grp170 to elicit systemic tumor immunity may be used to improve treatment outcomes for prostate cancer when combined with other treatment modalities.
Prostate cancer; Stress protein; Chaperone; grp170; Immuogenicity
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis and modulation of antitumor immune responses. In our study, we elucidated the role of MDA-7/IL-24 in inhibiting growth of breast cancer-initiating/stem cells. Ad.mda-7 infection decreased proliferation of breast cancer-initiating/stem cells without affecting normal breast stem cells. Ad.mda-7 induced apoptosis and endoplasmic reticulum stress in breast cancer-initiating/stem cells similar to unsorted breast cancer cells and inhibited the self-renewal property of breast cancer-initiating/stem cells by suppressing Wnt/β-catenin signaling. Prevention of inhibition of Wnt signaling by LiCl increased cell survival upon Ad.mda-7 treatment, suggesting that Wnt signaling inhibition might play a key role in MDA-7/IL-24-mediated death of breast cancer-initiating/stem cells. In a nude mouse subcutaneous xenograft model, Ad.mda-7 injection profoundly inhibited growth of tumors generated from breast cancer-initiating/stem cells and also exerted a potent “bystander” activity inhibiting growth of distant uninjected tumors. Further studies revealed that tumor growth inhibition by Ad.mda-7 was associated with a decrease in proliferation and angiogenesis, two intrinsic features of MDA-7/IL-24, and a reduction in vivo in the percentage of breast cancer-initiating/stem cells. Our findings demonstrate that MDA-7/IL-24 is not only nontoxic to normal cells and normal stem cells but also can kill both unsorted cancer cells and enriched populations of cancer-initiating/stem cells, providing further documentation that MDA-7/IL-24 might be a safe and effective way to eradicate cancers and also potentially establish disease-free survival.
MDA-7/IL-24; apoptosis; Wnt signaling; cancer-initiating/stem cells; breast cancer