Dormant tumors circumvent tumor-specific adaptive immunity by establishing a Treg-dominated niche via DKK3.

Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.

Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines.

CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Replication-attenuated Human Adenoviral Type 4 vectors elicit capsid dependent enhanced innate immune responses that are partially dependent upon interactions with the complement system.

Human Adenovirus Type 4 (HAdV-4) is responsible for epidemic outbreaks of Acute Respiratory Disease (especially in military recruits), and is known to cause significant morbidity with several reported cases of mortality. However, we do not understand why this serotype causes such high morbidity, and have little insight into the immunobiology of HAdV-4 infections. We have now developed a replication attenuated HAdV-4 vector system, and through it, demonstrate that HAdV-4 virions have enhanced infectivity of certain cell types and reveal aspects of the serotype-specific heightened innate immunogenicity of infectious HAdV-4 capsids both in vitro and in vivo. We further found that elements of this serotype-specific immunogenicity were dependent upon interactions with the complement system. These findings provide insights into the mechanisms possibly underlying the known morbidity accompanying wild-type HAdV-4 infections as well as highlight important considerations when considering development of alternative serotype vectors.

Adenovirus infection triggers a rapid, MyD88-regulated transcriptome response critical to acute-phase and adaptive immune responses in vivo.

Nearly 50 years ago, the discovery of interferon prompted the notion that host cells innately respond to viral invasion. Since that time, technological advances have allowed this response to be extensively characterized and dissected in vitro. However, these advances have only recently been applied to highly complex, in vivo biological systems. To this end, we exploited high-titer adenovirus (Ad) vectors to globally investigate the innate immune response to nonenveloped viral infection in vivo. Our results indicated a potent cellular transcriptome response shortly after infection, with global assessments revealing significant dysregulation in approximately 15% of the measured transcripts derived from Ad vector-transduced tissue. Bioinformatics-based transcriptome analysis revealed a complex innate response to Ad infection, with induction of proinflammatory responses (and suppression of metabolism and mitochondrial genes) akin to those observed when mice are challenged with lipopolysaccharide. Despite this commonality, there were many unique aspects of the Ad-dependent transcriptome response, including the upregulation of several RNA regulatory mechanisms and apoptosis-related pathways, accompanied by the suppression of lysosomal and endocytic genes. Our results also implicated the Toll-like receptors (TLRs) in these responses, prompting specific investigations into this pathway. By using MyD88KO mice, our results confirmed that Ad-induced dysregulation of five functionally related gene clusters are significantly dependent on this TLR adaptor gene. MyD88 deficiency also resulted in significantly diminished, although not abolished, adaptive and acute-phase immune responses to Ad, confirming the transcriptome data, as well as specifically identifying MyD88 as a significant Ad immunity amplifier and regulator in vivo.

Applications of adenoviral vector-mediated gene transfer in cardiovascular research.

Cardiovascular disease is a leading cause of morbidity and mortality worldwide. New studies are needed to explore novel therapeutic options for patients that are refractory to existing therapies. Gene transfer using adenoviral vectors has shown promising results in animal studies, and is now being tested in many clinical trials. In this chapter, the advantages of adenoviral vector-mediated gene transfer for cardiovascular disease applications, and the methods on how to construct, propagate, and evaluate adenoviral vectors, are discussed.

Multiple innate inflammatory responses induced after systemic adenovirus vector delivery depend on a functional complement system.

Excessive complement activation can result in extreme tissue damage and systemic inflammatory responses, similar to innate immune responses rapidly elicited after systemic adenovirus (Ad) injections. To determine if Ad interactions with the complement system impact upon Ad-induced innate immune responses, we injected Ad into complement-deficient, C3-knockout mice (C3-KO) or wild-type mice (WT) and quantitatively compared multiple anti-Ad innate immune responses in both strains of mice. In Ad-treated WT mice, we noted rapid increases in plasma KC levels (1 h post injection), followed by increases in IL-6, IFN-gamma, RANTES, IL-12(p40), IL-5, G-CSF, and GM-CSF and subsequently thrombocytopenia. Conversely, in Ad-treated C3-KO mice, many of these inflammatory responses were significantly blunted, including the avoidance of Ad-induced thrombocytopenia. Global liver transcriptome responses in Ad-treated WT mice were assessed by RT-PCR-validated gene array analysis and were found to be also significantly affected by the lack of complement activity in Ad-treated C3-KO mice. Finally, our results confirmed the ability of high dose Ads to transduce hepatocytes despite a lack of complement activity. In summary, Ad interactions with the mammalian complement system are significant and likely initiate and/or exacerbate many of the inflammatory responses noted after systemic Ad injections.

Fully deleted adenovirus persistently expressing GAA accomplishes long-term skeletal muscle glycogen correction in tolerant and nontolerant GSD-II mice.

Glycogen storage disease type II (GSD-II) patients manifest symptoms of muscular dystrophy secondary to abnormal glycogen storage in cardiac and skeletal muscles. For GSD-II, we hypothesized that a fully deleted adenovirus (FDAd) vector expressing hGAA via nonviral regulatory elements (PEPCK promoter/ApoE enhancer) would facilitate long-term efficacy and decrease propensity to generate anti-hGAA antibody responses against hepatically secreted hGAA. Intravenous delivery of FDAdhGAA into GAA-tolerant or nontolerant GAA-KO mice resulted in long-term hepatic secretion of hGAA. Specifically, nontolerant mice achieved complete reversal of cardiac glycogen storage and near-complete skeletal glycogen correction for at least 180 days and tolerant mice for minimally 300 days coupled with the preservation of muscle strength. Anti-hGAA antibody levels in both mouse strains were significantly less relative to those previously generated by CMV-driven hGAA expression in nontolerant GAA-KO mice. However, plasma GAA levels decreased in nontolerant GAA-KO mice despite long-term intrahepatic GAA expression from the persistent vector. This intriguing result is discussed in light of other examples of "tolerance" induction by gene-transfer-based approaches.

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA.

BACKGROUND: Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. METHODS: An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. RESULTS: After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. CONCLUSIONS: Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.

Regulation of insulin-like growth factor (IGF)-I action by matrix metalloproteinase-3 involves selective disruption of IGF-I/IGF-binding protein-3 complexes.

IGF-I and IGF-II play important roles in growth and development via interactions with cell-surface receptors; however, in nature, IGFs are sequestered by at least six soluble, high-affinity IGF-binding proteins (IGFBPs), namely IGFBPs 1-6. Herein, we demonstrate that the stromal cell-derived extracellular matrix-degrading metalloproteinase stromelysin 1 (matrix metalloproteinase 3) disrupts IGF/IGFBP-3 complexes and liberates free, intact IGFs, leading to phosphorylation of cell surface type 1 IGF receptors and cellular proliferation. Tissue inhibitor of metalloproteinases (TIMP-1) or an antibody to the type 1 IGF receptor mitigates IGF-mediated cellular proliferation. Thus, these studies suggest that matrix metalloproteinases, beyond their effects on extracellular matrix turnover, regulate cellular proliferation by modulating the bioavailability of IGFs, an event critical for such diverse phenomena as embryo development, morphogenesis, angiogenesis, and tumorigenesis.

Efficacy of gene therapy for a prototypical lysosomal storage disease (GSD-II) is critically dependent on vector dose, transgene promoter, and the tissues targeted for vector transduction.

Lysosomal storage diseases are an intriguing target for gene therapy approaches, as transduction of a "depot" organ with a transgene encoding a lysosomal enzyme can be followed by secretion, systemic distribution, downstream uptake, and lysosomal targeting of the enzyme into non-transduced tissues. These benefits are of utmost importance when considering gene therapy approaches for glycogen storage disease type-II (GSD-II). GSD-II is a prototypical lysosomal storage disorder caused by lack of intralysosomal acid alpha-glucosidase (GAA) activity. Lack of GAA can result in a proximal limb myopathy and respiratory and cardiac failure, each due to abnormal glycogen accumulation in the skeletal muscles or cardiac tissues, respectively. After converting the liver into a "depot" organ, we found that intravenous injection of the [E1-,polymerase-]AdGAA vector allowed for hepatic secretion of GAA over an at least 20-fold dosage range. We noted that very low plasma GAA levels (derived from hepatic secretion of GAA) can allow for GAA uptake by muscle tissues (skeletal or cardiac), but significantly higher plasma GAA levels are required before glycogen "cross-correction" can occur in these same tissues. We also demonstrated that liver-specific enhancer/promoters prolonged GAA transgene expression from persistent [E1-,polymerase-] adenovirus based vector genomes for at least 180 days, and significantly diminished the amounts of neutralizing anti-GAA antibodies elicited in this animal model. Finally, we demonstrated that skeletal muscles can also serve as a "depot" organ for GAA secretion, allowing for secretion of GAA and its uptake by noninfected distal tissues, although glycogen reductions in non-injected muscles were not achieved by the latter approach.