RESUMO
Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic ß cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to ß-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic ß cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced ß-like cells are mono-hormonal, express genes essential for ß cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of ß cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.
Assuntos
Reprogramação Celular , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ductos Pancreáticos/citologia , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenoviridae/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Reprogramação Celular/genética , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/genética , Hepatócitos/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.
Assuntos
Melanoma Experimental/terapia , Fator 88 de Diferenciação Mieloide/metabolismo , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interleucina-1/deficiência , Interleucina-1/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Linfócitos T/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação Viral/genéticaRESUMO
Despite having potent oncolytic activity, in vitro, direct intratumoral injection of oncolytic vesicular stomatitis virus (VSV) into established AE17ova mesothelioma tumors in C57Bl/6 mice had no therapeutic effect. During studies to combine systemic cyclophosphamide (CPA) with VSV to suppress the innate immune reaction against VSV, we observed that CPA alone had highly significant antitumor effects in this model. However, against our expectations, the combination of CPA and VSV consistently reduced therapeutic efficacy compared to CPA alone, despite the fact that the combination increased intratumoral VSV titers. We show here that CPA-mediated therapy against AE17ova tumors was immune-mediated and dependent upon both CD4 T cells and natural killer (NK) cells. However, intratumoral VSV induced a transforming growth factor-ß (TGF-ß)-dependent suppressive activity, mediated by CD11b(+)GR-1(+) cells that significantly inhibited both antigen-specific T-cell activation, and CPA-activated, NK-dependent killing of AE17ova tumor cells. Overall, our results show that treatment with oncolytic viruses can induce a variety of immune-mediated consequences in vivo with both positive, or negative, effects on antitumor therapy. These underexplored immune consequences of treatment with oncolytic viruses may have significant, and possibly unexpected, impacts on how virotherapy interacts in combination with other agents which modulate antitumor immune effectors.
Assuntos
Ciclofosfamida/farmacologia , Terapia Genética/métodos , Mesotelioma/imunologia , Mesotelioma/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Terapia Combinada , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Mesotelioma/tratamento farmacológico , Mesotelioma/virologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação ViralRESUMO
Diabetes mellitus, caused by loss or dysfunction of the insulin-producing beta cells of the pancreas, is a promising target for recombinant adeno-associated virus (rAAV)-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell type-specific expression control element is needed. In this study, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INS × 2) and microRNA (miRNA) recognition elements (MREs). The INS × 2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2 × -MRE expression vector was highly specific to human beta cells and stem cell-derived beta cells.
Assuntos
Dependovirus , MicroRNAs , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Humanos , Insulina/metabolismo , MicroRNAs/metabolismo , TransgenesRESUMO
There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (i.v.) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.
Assuntos
Interleucina-2/uso terapêutico , Depleção Linfocítica , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Terapia Combinada , Endotélio Vascular/efeitos dos fármacos , Interleucina-2/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Vírus Oncolíticos/efeitos dos fármacos , Vírus Oncolíticos/fisiologia , Linfócitos T Reguladores/imunologia , Internalização do Vírus , Replicação Viral/efeitos dos fármacosRESUMO
There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (IV) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.
RESUMO
To protect viral particles from neutralization, sequestration, nonspecific adhesion, and mislocalization following systemic delivery, we have previously exploited the natural tumor-homing properties of antigen-specific CD8+ T cells. Thus, OT-I T cells, preloaded in vitro with the oncolytic vesicular stomatitis virus (VSV), can deliver virus to established B16ova tumors to generate significantly better therapy than that achievable with OT-I T cells, or systemically delivered VSV, alone. Here, we demonstrate that preconditioning immune-competent mice with Treg depletion and interleukin-2 (IL-2), before adoptive T-cell therapy with OT-I T cells loaded with VSV, leads to further highly significant increases in antitumor therapy. Therapy was associated with antitumor immune memory, but with no detectable toxicities associated with IL-2, Treg depletion, or systemic dissemination of the oncolytic virus. Efficacy was contributed by multiple factors, including improved persistence of T cells; enhanced delivery of VSV to tumors; increased persistence of OT-I cells in vivo resulting from tumor oncolysis; and activation of NK cells, which acquire potent antitumor and proviral activities. By controlling the levels of virus loaded onto the OT-I cells, adoptive therapy was still effective in mice preimmune to the virus, indicating that therapy with virus-loaded T cells may be useful even in virus-immune patients. Taken together, our data show that it is possible to combine adoptive T-cell therapy, with biological therapy (Treg depletion+IL-2), and VSV virotherapy, to treat established tumors under conditions where none of the individual modalities alone is successful.
Assuntos
Adaptação Biológica/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica , Linfócitos T/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos/imunologia , Linhagem Celular Tumoral , Memória Imunológica/imunologia , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Neoplasias/genética , Linfócitos T/efeitos dos fármacos , Vesiculovirus/genéticaRESUMO
Relatively little attention has been paid to the role of virotherapy in promoting antitumor immune responses. Here, we show that CD8+ T cells are critical for the efficacy of intratumoral vesicular stomatitis virus virotherapy and are induced against both virally encoded and tumor-associated immunodominant epitopes. We tested three separate immune interventions to increase the frequency/activity of activated antitumoral T cells. Depletion of Treg had a negative therapeutic effect because it relieved suppression of the antiviral immune response, leading to early viral clearance. In contrast, increasing the circulating levels of tumor antigen-specific T cells using adoptive T cell transfer therapy, in combination with intratumoral virotherapy, generated significantly improved therapy over either adoptive therapy or virotherapy alone. Moreover, the incorporation of a tumor-associated antigen within the oncolytic vesicular stomatitis virus increased the levels of activation of naïve T cells against the antigen, which translated into increased antitumor therapy. Therefore, our results show that strategies which enhance immune activation against tumor-associated antigens can also be used to enhance the efficacy of virotherapy.
Assuntos
Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Melanoma Experimental/virologia , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/imunologiaRESUMO
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional ß cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived ß cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
Assuntos
Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Células Cultivadas , Diabetes Mellitus , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Biblioteca Gênica , Técnicas de Transferência de Genes , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
PURPOSE OF REVIEW: This report examines recent publications identifying phenotypic and functional heterogeneity among pancreatic ß cells and investigating their potential roles in normal and abnormal islet function. The development of new methods and tools for the study of individual islet cells has produced a surge of interest in this topic. RECENT FINDINGS: Studies of ß cell maturation and pregnancy-induced proliferation have identified changes in serotonin and transcription factors SIX2/3 expression as markers of temporal heterogeneity. Structural and functional heterogeneity in the form of functionally distinct 'hub' and 'follower' ß cells was found in mouse islets. Heterogeneous expression of Fltp (in mouse ß cells) and ST8SIA1 and CD9 (in human ß cells) were associated with distinct functional potential. Several impressive reports describing the transcriptomes of individual ß cells were also published in recent months. Some of these reveal previously unknown ß cell subpopulations. SUMMARY: A wealth of information on functional and phenotypic heterogeneity has been collected recently, including the transcriptomes of individual ß cells and the identities of functionally distinct ß cell subpopulations. Several studies suggest the existence of two broad categories: a more proliferative but less functional and a less proliferative but more functional ß cell type. The identification of functionally distinct subpopulations and their association with type 2 diabetes underlines the potential clinical importance of these investigations.
Assuntos
Diferenciação Celular , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologiaRESUMO
The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to ß-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic ß-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall ß-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to ß cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs-derived from multiple unrelated donors-into pancreatic ß-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.
Assuntos
Reprogramação Celular , Vesícula Biliar/citologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Transdiferenciação Celular , Transplante de Células , Células Cultivadas , Técnicas de Reprogramação Celular , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , MicroRNAs/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , TransgenesRESUMO
Direct reprogramming is a promising approach for the replacement of ß cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to ß cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional ß cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of ß cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with ß cells and had a metabolic gene expression profile resembling neonatal ß cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.
Assuntos
Reprogramação Celular/fisiologia , Vesícula Biliar/citologia , Células Secretoras de Insulina/citologia , Animais , Modelos Animais de Doenças , Feminino , Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica , Humanos , Insulina/biossíntese , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1(+)SOX9(+)) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.
Assuntos
Biomarcadores/metabolismo , Vesícula Biliar/metabolismo , Adenocarcinoma/patologia , Adulto , Animais , Anticorpos/imunologia , Ductos Biliares Extra-Hepáticos/metabolismo , Linhagem Celular Tumoral , Separação Celular , Ducto Cístico/metabolismo , Células Epiteliais/citologia , Feminino , Citometria de Fluxo , Imunofluorescência , Vesícula Biliar/patologia , Regulação da Expressão Gênica , Humanos , Mesoderma/citologia , Camundongos Endogâmicos BALB C , Pâncreas/metabolismo , Coloração e RotulagemRESUMO
Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable ß-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the ß-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true ß-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations.
Assuntos
Vesícula Biliar/citologia , Ilhotas Pancreáticas/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares , Células-Tronco Pluripotentes/citologia , Ratos , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Multiple intravenous injections of a cDNA library, derived from human melanoma cell lines and expressed using the highly immunogenic vector vesicular stomatitis virus (VSV), cured mice with established melanoma tumors. Successful tumor eradication was associated with the ability of mouse lymphoid cells to mount a tumor-specific CD4(+) interleukin (IL)-17 recall response in vitro. We used this characteristic IL-17 response to screen the VSV-cDNA library and identified three different VSV-cDNA virus clones that, when used in combination but not alone, achieved the same efficacy against tumors as the complete parental virus library. VSV-expressed cDNA libraries can therefore be used to identify tumor rejection antigens that can cooperate to induce anti-tumor responses. This technology should be applicable to antigen discovery for other cancers, as well as for other diseases in which immune reactivity against more than one target antigen contributes to disease pathology.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/isolamento & purificação , Interleucina-17/imunologia , Melanoma/imunologia , Melanoma/terapia , Neoplasias Experimentais/terapia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Epitopos de Linfócito T/imunologia , Biblioteca Gênica , Vetores Genéticos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Linfócitos/imunologia , Melanoma/genética , Melanoma Experimental , Camundongos , Neoplasias Experimentais/imunologia , VesiculovirusRESUMO
We have shown that the antitumor activity of vesicular stomatitis virus (VSV) against B16ova tumors in C57BL/6 mice is predominantly due to innate antiviral immune effectors. We have also shown that the innate immune-activating properties of VSV can be harnessed to prime adaptive T-cell responses against a tumor-associated antigen (TAA) if the virus is engineered to express the cDNA of the antigen. Here, we show that the combination of VSV expressing OVA as a model tumor antigen, along with adoptive T-cell therapy targeted against the same antigen, is superior to either treatment alone and induces systemic antitumor activity. In addition, we extend our findings with the OVA model to the therapeutic use of VSV expressing hgp100, a self TAA against which tolerance is well established in C57BL/6 mice. In contrast to VSV-ova, T-cell responses raised by VSV-hgp100 were insufficient to improve therapy against B16ova tumors compared with VSV-GFP alone. However, in combination with adoptive transfer of gp100-specific pmel T cells, intratumoral VSV-hgp100 cured significantly more mice than either virus or T cells alone. Even in an aggressive model of metastatic disease, antitumor therapy was generated at levels similar to those observed in the VSV-ova/OT-I model in which a potently immunogenic, nonself TAA was targeted. Therefore, individual poorly effective virotherapies and T-cell therapies that target self TAA of low immunogenicity, which reflects the situation in patients, can be combined to generate very effective antitumor therapy.
Assuntos
Melanoma Experimental/terapia , Linfócitos T/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Antígeno gp100 de Melanoma/imunologia , Transferência Adotiva , Animais , Terapia Combinada , Imunidade Inata , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia Viral Oncolítica , Linfócitos T/transplante , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral , Antígeno gp100 de Melanoma/genéticaRESUMO
Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.
Assuntos
Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Biblioteca Gênica , Neoplasias/terapia , Animais , Vacinas Anticâncer/imunologia , DNA Complementar/genética , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/imunologia , Reação em Cadeia da Polimerase , Neoplasias da Próstata/terapia , Vesiculovirus/genéticaRESUMO
Innate immune effector mechanisms triggered by oncolytic viruses may contribute to the clearance of both infected and uninfected tumor cells in immunocompetent murine hosts. Here, we developed an in vitro tumor cell/bone marrow coculture assay and used it to dissect innate immune sensor and effector responses to intratumoral vesicular stomatitis virus (VSV). We found that the type III IFN interleukin-28 (IL-28) was induced by viral activation of innate immune-sensing cells, acting as a key mediator of VSV-mediated virotherapy of B16ova melanomas. Using tumor variants which differentially express the IL-28 receptor, we showed that IL-28 induced by VSV within the tumor microenvironment sensitizes tumor cells to natural killer cell recognition and activation. These results revealed new insights into the immunovirological mechanisms associated with oncolytic virotherapy in immune-competent hosts. Moreover, they defined a new class of tumor-associated mutation, such as acquired loss of responsiveness to IL-28 signaling, which confers insensitivity to oncolytic virotherapy through a mechanism independent of viral replication in vitro. Lastly, the findings suggested new strategies to manipulate immune signals that may enhance viral replication, along with antitumor immune activation, and improve the efficacy of oncolytic virotherapies.
Assuntos
Citocinas/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Citocinas/biossíntese , Citotoxicidade Imunológica , Modelos Animais de Doenças , Imunidade Inata/imunologia , Hospedeiro Imunocomprometido , Injeções Intralesionais , Interferon Tipo I/imunologia , Células Matadoras Naturais/imunologia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Citocinas/biossíntese , Receptores de Citocinas/imunologia , Replicação Viral/imunologiaRESUMO
Oncolytic virotherapy can be achieved in two ways: (1) by exploiting an innate ability of certain viruses to selectively replicate in tumor tissues, and (2) by using viruses to deliver toxic or immunostimulatory genes to tumors. Vesicular stomatitis virus (VSV) selectively replicates in tumors lacking adequate type I interferon response. The efficacy of oncolytic virotherapy using VSV against B16 melanomas in C57BL/6 mice is dependent on CD8(+) T and natural killer cells. Because immunotherapies that prime specific CD8(+) T cells against melanocyte/melanoma antigens can generate significant therapeutic responses, we hypothesized that engineering VSV to express the potent T cell costimulatory molecule CD40 ligand (VSV-CD40L) would enhance virotherapy with concomitant priming of melanoma-specific T cells. However, we observed no difference in antitumor efficacy between the parental VSV-GFP and VSV-CD40L. In contrast, intratumoral injection of a replication-defective adenovirus expressing CD40L (Ad-CD40L) consistently produced significantly greater therapy than either replication-competent VSV-GFP or VSV-CD40L. The Ad-CD40L-mediated tumor regressions were associated with specific T cell responses against tumor-associated antigens (TAAs), which took several days to develop, whereas VSV-CD40L rapidly induced high levels of T cell activation without specificity for TAAs. These data suggest that the high levels of VSV-associated immunogenicity distracted immune responses away from priming of tumor-specific T cells, even in the presence of potent costimulatory signals. In contrast, a replication-defective Ad-CD40L allowed significant priming of T cells directed against TAAs. These observations suggest that an efficiently primed antitumor T cell response can produce similar, if not better, therapy against an established melanoma compared with intratumoral injection of a replication-competent oncolytic virus.
Assuntos
Ligante de CD40 , Terapia Genética/métodos , Imunoterapia/métodos , Melanoma Experimental/terapia , Vírus Oncolíticos , Vírus da Estomatite Vesicular Indiana , Adenoviridae/genética , Adenoviridae/imunologia , Adenoviridae/metabolismo , Adenoviridae/fisiologia , Animais , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cricetinae , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Vírus Oncolíticos/metabolismo , Vírus Oncolíticos/fisiologia , Transgenes , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
PURPOSE: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. EXPERIMENTAL DESIGN: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. RESULTS: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. CONCLUSION: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.