Despite the potency of dendritic cells (DCs) as a vaccine carrier, they are short-lived and sensitive to CTL-mediated elimination. Thus, it is believed that the longevity of Ag presentation by peptide-pulsed DC is limited in vivo. Surprisingly, however, we found that although the majority of injected DCs disappeared from the draining lymph nodes within 7 days, Ag presentation persisted for at least 14 days following DC immunization. This prolonged Ag presentation was not mediated by the remaining injected DCs or through Ag transfer to endogenous APCs. We provide evidence that exosomes released by DCs might be responsible for the persistence of Ag presentation. Functional exosomes could be recovered from the draining lymph nodes of C57BL/6 mice following DC vaccination and, in contrast to DCs, T cell stimulation by exosomes in vivo was not affected by the presence of CTL. Our findings demonstrate that Ag presentation following delivery of DC vaccines persists for longer than expected and indicate that the exosome may play a previously unrecognized role in Ag presentation following DC vaccination. Furthermore, our study reinforces the application of exosomes as a vaccination platform and suggests that exosome-based vaccines may be advantageous for booster immunizations due to their resistance to CTL.
Oncolytic virotherapy, the selective killing of tumor cells by oncolytic viruses (OVs), has emerged as a promising avenue of anticancer research. We have previously shown that KM100, a Herpes simplex virus type-1 (HSV) deficient for infected cell protein 0 (ICP0), possesses substantial oncolytic properties in vitro and has antitumor efficacy in vivo, in part by inducing antitumor immunity. Here, we illustrate through T-cell immunodepletion studies in nontolerized tumor-associated antigen models of breast cancer that KM100 treatment promotes antiviral and antitumor CD8(+) cytotoxic T-cell responses necessary for complete tumor regression. In tolerized tumor-associated antigen models of breast cancer, antiviral CD8(+) cytotoxic T-cell responses against infected tumor cells correlated with the induction of significant tumoristasis in the absence of tumor-associated antigen-specific CD8(+) cytotoxic T-cells. To enhance oncolysis, we tested a more cytopathic ICP0-null HSV and a vesicular stomatitis virus M protein mutant and found that despite improved in vitro replication, oncolysis in vivo did not improve. These studies illustrate that the in vitro cytolytic properties of OVs are poor prognostic indicators of in vivo antitumor activity, and underscore the importance of adaptive antiviral CD8(+) cytotoxic T-cells in effective cancer virotherapy.
The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2-5-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.Viral infection of mammalian cells induces a robust antiviral response intended to restrict viral replication and propagation. A predominant characteristic of this innate immune response is the expression and secretion of interferons (IFNs), a family of immunomodulatory cytokines with antiviral and antiproliferative activities (43). Once secreted from infected cells, the type I IFNs (alpha IFN [IFN-␣] and IFN-) bind to their cognate class II cytokine receptors in both paracrine and autocrine fashions and induce the phosphorylation of Jak/Stat molecules, leading to the nuclear translocation of activated Stat1-Stat2-IRF9 heterotrimers. These complexes bind to the IFN-stimulated response elements (ISRE) in promoters of IFN-stimulated genes (ISGs), resulting in the robust induction of these antiviral effector molecules. Among the ISGs most intently studied are the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), which induces protein synthesis arrest (52), and the ubiquitous 2Ј-5Ј-oligoadenylate synthetase (OAS) family of proteins, which function to promote RNA degradation (44). Once upregulated, PKR and OAS become activated by binding to dsRNA, a by-product of viral replication.OAS catalyzes the synthesis of variable short 2Ј-5Ј-linked oligoadenylates (2Ј-5ЈA) from ATP, which in turn activate the latent endoribonuclease RNase L to cleave cellular and viral RNA (36). Infection with several viruses, including vaccinia virus and encephalomyocarditis virus, leads to RNase L-dependent rRNA degradation (44). Ultimately, rRNA cleavage results in protein synthesis inhibition and apoptosis and constitutes a significant cellular antiviral event to prevent viral propagation (7). Indeed, several viruses have evolved specific mechanisms to counteract the 2Ј-5ЈA pathway, including human immunodeficiency virus type 1, vaccinia virus, and the alphaherpesvirus herpes simplex virus type 1 (HSV-1) (8, 27, 53). HSV-1 infection of conjunctival cells induces the synthesis of 2Ј-5ЈA derivatives, which antagonize RNase L activation by competing with genuine 2Ј-5ЈA...
The oncotropic phenotypes of several viruses correlate with tumor-associated deficiencies within interferon (IFN) signaling pathways. This observation formed the conceptual basis for developing oncolytic viruses deleted for viral proteins that inhibit the host IFN-dependent antiviral response, such as herpes simplex virus type-1 infected cell protein-0 (ICP0) and vesicular stomatitis virus matrix protein. Many viruses have evolved means to disrupt promyelocytic leukemia protein (PML) nuclear bodies. For example, ICP0 promotes PML degradation to inhibit the antiviral activities of this IFN-stimulated gene. As PML is downregulated in a variety of tumors, we hypothesized ICP0-null herpes simplex type-1 viruses are selectively oncolytic in tumors with impaired PML expression. We illustrate that ICP0-null herpes simplex type-1 viruses target tumor cells that either possess impaired PML signaling or cannot upregulate PML because of impaired IFN responsiveness. Disruption of PML signaling through overexpression of the dominant-negative protein PML-retinoic acid receptor alpha in PML-positive cells renders them sensitive to oncolysis by ICP0-null herpes simplex virus type-1 and vesicular stomatitis virus M protein mutant viruses, whereas PML overexpression reverses this phenomenon. Together, these data illustrate that PML mediates an antiviral mechanism that predicts the tropism of IFN-sensitive oncolytic viruses. To our knowledge, these viruses are the first examples of anti-cancer therapeutics capable of targeting deficiencies in PML expression.
Statins are drugs that have been utilized for years to treat hyperlipidemia through inhibition of the rate-limiting enzyme of the mevalonate (MVA) pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Preclinical evidence has demonstrated statins to possess anti-cancer properties against a wide range of tumors but not normal cells. Through the use of a chemical library screen, we hypothesize that the identification of compounds which potentiate the anti-cancer effects of statins will uncover novel molecular pathways and/or targets that can be exploited in combination with the MVA pathway to maximize tumor cell death. A pilot 100-compound library, composed of off-patent pharmacologically active drugs clinically used for a wide spectrum of diseases was screened in the multiple myeloma (MM) KMS11 cell line. Dipyridamole (DP), a commonly prescribed anti-platelet agent potentiated the anti-cancer effects of atorvastatin. The DP-statin combination was synergistic and capable of inducing apoptosis in a variety of acute myelogenous leukemia (AML), MM and breast cancer cell lines. The DP-statin combination also induced apoptosis in primary AML patient samples, but was not toxic to normal PBSCs. In an in vivo AML tumor model, the DP-statin combination was found to be effective at inhibiting tumor growth. DP is known to elicit numerous effects, amongst them, phosphodiesterase (PDE) inhibition. In AML cell lines, activators of the PKA pathway including other PDE inhibitors, also induced apoptosis in combination with statins similar to DP. Interestingly, the DP-statin combination prevented the increase of HMGCR, which occurs following statin treatment as part of a classic feedback response. Further mechanistic investigations to determine how DP potentiates statin-induced apoptosis are underway. As both statins and DP are pre-approved for use in humans, off-patent, and readily available, they have the potential to directly impact patient care. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C170.
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