RESUMO
Over the last few years, oncolytic virus therapy has been recognised as a promising approach in cancer treatment, due to the potential of these viruses to induce systemic anti-tumour immunity and selectively killing tumour cells. However, the effectiveness of these viruses depends significantly on their interactions with the host immune responses, both innate (e.g., macrophages, which accumulate in high numbers inside solid tumours) and adaptive (e.g., [Formula: see text] T cells). In this article, we consider a mathematical approach to investigate the possible outcomes of the complex interactions between two extreme types of macrophages (M1 and M2 cells), effector [Formula: see text] T cells and an oncolytic Vesicular Stomatitis Virus (VSV), on the growth/elimination of B16F10 melanoma. We discuss, in terms of VSV, [Formula: see text] and macrophages levels, two different types of immune responses which could ensure tumour control and eventual elimination. We show that both innate and adaptive anti-tumour immune responses, as well as the oncolytic virus, could be very important in delaying tumour relapse and eventually eliminating the tumour. Overall this study supports the use mathematical modelling to increase our understanding of the complex immune interaction following oncolytic virotherapies. However, the complexity of the model combined with a lack of sufficient data for model parametrisation has an impact on the possibility of making quantitative predictions.
Assuntos
Macrófagos/imunologia , Melanoma Experimental/terapia , Modelos Teóricos , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Células Cultivadas , Macrófagos/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/virologia , Camundongos , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.
Assuntos
Anticorpos Bloqueadores/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental/prevenção & controle , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular Tumoral , Feminino , Humanos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/virologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/virologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/prevenção & controle , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Melanoma Experimental/imunologia , Melanoma Experimental/virologia , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismoRESUMO
CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.
Assuntos
Vacinas Anticâncer/imunologia , Citotoxicidade Imunológica , Galactosilceramidas/administração & dosagem , Proteínas Oncogênicas Virais/imunologia , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Feminino , Galactosilceramidas/imunologia , Papillomavirus Humano 18 , Humanos , Células Matadoras Naturais/imunologia , Melanoma Experimental/prevenção & controle , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/administração & dosagem , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/terapia , Transplantes , Células Tumorais Cultivadas/imunologia , VacinaçãoRESUMO
Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.
Assuntos
Infecções por Arenaviridae/imunologia , Neoplasias do Colo/imunologia , Interferon Tipo I/farmacologia , Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/virologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interferon gama/metabolismo , Coriomeningite Linfocítica/tratamento farmacológico , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Oncolytic viruses selectively lyse tumor cells, disrupt immunosuppression within the tumor, and reactivate antitumor immunity, but they have yet to live up to their therapeutic potential. Immune checkpoint modulation has been efficacious in a variety of cancer with an immunogenic microenvironment, but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies would likely result in both effective and specific cancer therapy. To test the hypothesis, we first constructed oncolytic adenovirus Delta-24-RGDOX expressing the immune costimulator OX40 ligand (OX40L). Like its predecessor Delta-24-RGD, Delta-24-RGDOX induced immunogenic cell death and recruit lymphocytes to the tumor site. Compared with Delta-24-RGD, Delta-24-RGDOX exhibited superior tumor-specific activation of lymphocytes and proliferation of CD8+ T cells specific to tumor-associated antigens, resulting in cancer-specific immunity. Delta-24-RGDOX mediated more potent antiglioma activity in immunocompetent C57BL/6 but not immunodeficient athymic mice, leading to specific immune memory against the tumor. To further overcome the immune suppression mediated by programmed death-ligand 1 (PD-L1) expression on cancer cells accompanied with virotherapy, intratumoral injection of Delta-24-RGDOX and an anti-PD-L1 antibody showed synergistic inhibition of gliomas and significantly increased survival in mice. Our data demonstrate that combining an oncolytic virus with tumor-targeting immune checkpoint modulators elicits potent in situ autologous cancer vaccination, resulting in an efficacious, tumor-specific, and long-lasting therapeutic effect. Cancer Res; 77(14); 3894-907. ©2017 AACR.
Assuntos
Vacinas Anticâncer/farmacologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Células A549 , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Glioma/imunologia , Glioma/terapia , Glioma/virologia , Células HEK293 , Humanos , Imunomodulação , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/imunologia , Neoplasias/virologia , Ligante OX40/biossíntese , Ligante OX40/genética , Ligante OX40/imunologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologiaRESUMO
Cytomegalovirus (CMV) is a herpesvirus that induces an extremely robust and sustained immune response. For this reason, CMV has been proposed as a vaccine vector to promote immunity to both pathogens and cancer. However, exploration of CMV as a vaccine vector is at an early stage and there are many questions. Using a mouse melanoma model, we recently found that a CMV-based vaccine induced large populations of melanoma-specific T cells, but was not effective at slowing tumor growth unless it was injected directly into the tumor. These surprising results have led us to hypothesize that CMV may be adept at modulating the tumor micro-environment through its infection of macrophages. Importantly, injection of CMV into the growing tumor synergized with blockade of the PD-1 checkpoint to clear well-established tumors. Here, we discuss our results in the context of CMV-based vaccines for pathogens and cancer.
Assuntos
Citomegalovirus/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Melanoma Experimental/imunologia , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/administração & dosagem , Vacinas contra Citomegalovirus/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Melanoma Experimental/virologia , Camundongos , Receptor de Morte Celular Programada 1/imunologiaRESUMO
The tumor microenvironment (TME) fosters tumors by attenuating anti-tumor immunity, reinforcing tumor cell survival and increasing angiogenesis. Among the constituents of the TME, here, we focused on tumor-associated neutrophils (TANs). First, we found that the combination of poly I:C and inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) synergistically suppressed tumor growth in the B16-F10 melanoma mouse model. In this model, poly I:C contributed to the recruitment of CD11b+Ly6G+ neutrophils to the TME, and co-injection of poly I:C and HVJ-E increased CD11b+Ly6G+FAS+ TAN in the TME. Depletion of neutrophils abolished the synergistic anti-tumor effect of HVJ-E and poly I:C in B16-F10 tumors. We revealed that C-X-C motif chemokine ligand 2 (CXCL2) is produced in the TME by poly I:C, but HVJ-E enhanced neutrophil infiltration of the TME does not occur. An anti-CXCL2 antibody inhibited the tumor suppression by HVJ-E+poly I:C. HVJ-E in combination with recombinant CXCL2 protein or CXCL2 pDNA suppressed mouse melanoma by increasing cytotoxic T lymphocyte activity against B16-F10 melanoma, which was abolished by an anti-Ly6G antibody. HVJ-E directly and indirectly increased FAS and ICAM-1 expression in cultured bone marrow-derived naïve neutrophils. Thus, HVJ-E activates anti-tumor immunity via anti-tumorigenic neutrophils in the TME. An HVJ-E vector containing the CXCL2 gene may be applicable as a novel cancer gene therapy strategy.
Assuntos
Melanoma Experimental/virologia , Neutrófilos/imunologia , Neutrófilos/virologia , Linfócitos T/imunologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Quimiocina CXCL2/metabolismo , Ligantes , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Terapia Viral Oncolítica , Poli I-C/imunologia , Vírus Sendai , Transdução de Sinais , Baço/citologiaRESUMO
Targeted oncolytic vaccinia viruses are being developed as a novel strategy in cancer therapy. Arming vaccinia viruses with immunostimulatory cytokines can enhance antitumor efficacy. Such engineered oncolytic viruses, like JX-594, a Wyeth strain vaccinia virus modified with human granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown promising results and have proceeded rapidly in clinical trials. However, the oncolytic potential of the Chinese vaccine strain Tian Tan (VTT) has not been explored. In this study, we constructed a targeted oncolytic vaccinia virus of Tian Tan strain Guang9 (VG9) expressing murine GM-CSF (VG9-GMCSF) and evaluated the antitumor effect of this recombinant vaccinia virus in a murine melanoma model. In vitro, viral replication and cytotoxicity of VG9-GMCSF was as potent as VG9; in vivo, VG9-GMCSF significantly inhibited the growth of subcutaneously implanted melanoma tumors, prolonged the survival of tumor-bearing mice, and produced an antitumor cytotoxic response. Such antitumor effect may be due to the lytic nature of virus as well as the stimulation of immune activity by GM-CSF production. Our results indicate that VG9-GMCSF induces strong tumoricidal activity, providing a potential therapeutic strategy for combating cancer.
Assuntos
Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Imunoterapia/métodos , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/patogenicidade , Vaccinia virus/patogenicidade , Animais , Sobrevivência Celular , Chlorocebus aethiops , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Células HEK293 , Humanos , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Vírus Oncolíticos/metabolismo , Fatores de Tempo , Carga Tumoral , Vaccinia virus/genética , Vaccinia virus/imunologia , Vaccinia virus/metabolismo , Células Vero , Replicação ViralRESUMO
Malignant melanoma requires precise resection in order to avoid metastatic recurrence. We report here that the telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401 could label malignant melanoma with GFP in situ in orthotopic mouse models. OBP-401-based fluorescence-guided surgery (FGS) resulted in the complete resection of malignant melanoma in the orthotopic models, where conventional bright-light surgery (BLS) could not. High-dose administration of OBP-401 enabled FGS without residual cancer cells or recurrence, due to its dual effect of cancer-cell labeling with GFP and killing.
Assuntos
Adenoviridae/metabolismo , Melanoma Experimental/cirurgia , Imagem Óptica/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fluorescência , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/virologia , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/prevenção & controleRESUMO
BACKGROUND: Using immune checkpoint modulators in the clinic to increase the number and activity of cytotoxic T lymphocytes that recognize tumor antigens can prolong survival for metastatic melanoma. Yet, only a fraction of the patient population receives clinical benefit. In short, these clinical trials demonstrate proof-of-principle but optimizing the specific therapeutic strategies remains a challenge. In many fields, CAD (computer-aided design) is a tool used to optimize integrated system behavior using a mechanistic model that is based upon knowledge of constitutive elements. The objective of this study was to develop a predictive simulation platform for optimizing anti-tumor immunity using different treatment strategies. METHODS: To better understand the therapeutic role that cytotoxic CD8(+) T cells can play in controlling tumor growth, we developed a multi-scale mechanistic model of the biology using impulsive differential equations and calibrated it to a self-consistent data set. RESULTS: The multi-scale model captures the activation and differentiation of naïve CD8(+) T cells into effector cytotoxic T cells in the lymph node following adenovirus-mediated vaccination against a tumor antigen, the trafficking of the resulting cytotoxic T cells into blood and tumor microenvironment, the production of cytokines within the tumor microenvironment, and the interactions between tumor cells, T cells and cytokines that control tumor growth. The calibrated model captures the modest suppression of tumor cell growth observed in the B16F10 model, a transplantable mouse model for metastatic melanoma, and was used to explore the impact of multiple vaccinations on controlling tumor growth. CONCLUSIONS: Using the calibrated mechanistic model, we found that the cytotoxic CD8(+) T cell response was prolonged by multiple adenovirus vaccinations. However, the strength of the immune response cannot be improved enough by multiple adenovirus vaccinations to reduce tumor burden if the cytotoxic activity or local proliferation of cytotoxic T cells in response to tumor antigens is not greatly enhanced. Overall, this study illustrates how mechanistic models can be used for in silico screening of the optimal therapeutic dosage and timing in cancer treatment.
Assuntos
Adenoviridae/fisiologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Modelos Biológicos , Vacinação , Adenoviridae/genética , Algoritmos , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Humanos , Interferon gama/metabolismo , Melanoma Experimental/prevenção & controle , Melanoma Experimental/virologia , Camundongos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cancer therapy using oncolytic viruses has gained interest in the last decade. Vesicular stomatitis virus is an attractive candidate for this alternative treatment approach. The importance of the immune response against tumor antigens in virotherapy efficacy is now well recognized, however, its relative contribution versus the intrinsic oncolytic capacity of viruses has been difficult to evaluate. To start addressing this question, we compared glycoprotein and matrix mutants of vesicular stomatitis virus (VSV), showing different oncolytic potentials for B16/B16gp33 melanoma tumor cells in vitro, with the wild-type virus in their ability to induce tumor-specific CD8(+) T cell responses and control tumor progression in vivo. Despite the fact that wild-type and G mutants induced a stronger gp33-specific immune response compared to the MM51R mutant, all VSV strains showed a similar capacity to slow down tumor progression. The effectiveness of the matrix mutant treatment proved to be CD8(+) dependent and directed against tumor antigens other than gp33 since adoptive transfer of isolated CD8(+) T lymphocytes from treated B16gp33-bearing mice resulted in significant protection of naive mice against challenge with the parental tumor. Remarkably, the VSV matrix mutant induced the upregulation of major histocompatibility class-I antigen at the tumor cell surface thus favoring recognition by CD8(+) T cells. These results demonstrate that VSV mutants induce an antitumor immune response using several mechanisms. A better understanding of these mechanisms will prove useful for the rational design of viruses with improved therapeutic efficacy.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/genética , Vírus Oncolíticos/genética , Vesiculovirus/imunologia , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Genes MHC Classe I , Células Hep G2 , Humanos , Imunoterapia Adotiva , Melanoma Experimental/virologia , Antígenos Específicos de Melanoma/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Células Vero , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
Cytolytic activity by CD8(+) cytotoxic T lymphocytes (CTLs) is a powerful strategy for the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTLs is progressively dampened during chronic infection, yet the environmental cues and molecular pathways that influence immunological 'exhaustion' remain unclear. Here we found that CTL immunity was regulated by the central transcriptional response to hypoxia, which is controlled in part by hypoxia-inducible factors (HIFs) and the von Hippel-Lindau tumor suppressor VHL. Loss of VHL, the main negative regulator of HIFs, led to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTLs displayed enhanced control of persistent viral infection and neoplastic growth. We found that HIFs and oxygen influenced the expression of pivotal transcription, effector and costimulatory-inhibitory molecules of CTLs, which was relevant to strategies that promote the clearance of viruses and tumors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Proteína Supressora de Tumor Von Hippel-Lindau/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Melanoma Experimental/virologia , Camundongos , Camundongos Knockout , Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Análise de Sobrevida , Linfócitos T Citotóxicos/patologia , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
MicroRNAs (miRNAs) are important posttranscriptional regulators in immune cells, but how viral infection regulates miRNA expression to shape dendritic cell (DC) responses has not been well characterized. We identified 20 miRNAs that were differentially expressed in primary murine DCs in response to the dsRNA agonist polyinosinic-polycytidylic acid, a subset of which were modestly regulated by influenza infection. miR-451 was unique because it was induced more strongly in primary splenic and lung DCs by live viral infection than by purified agonists of pattern recognition receptors. We determined that miR-451 regulates a subset of proinflammatory cytokine responses. Three types of primary DCs treated with antisense RNA antagomirs directed against miR-451 secreted elevated levels of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α, and these results were confirmed using miR-451(null) cells. miR-451 negatively regulates YWHAZ/14-3-3ζ protein levels in various cell types, and we measured a similar inhibition of YWHAZ levels in DCs. It is known that YWHAZ can control the activity of two negative regulators of cytokine production: FOXO3, which is an inhibitory transcription factor, and ZFP36/Tristetraprolin, which binds to AU-rich elements within 3'-untranslated regions to destabilize cytokine mRNAs. Inhibition of miR-451 expression correlated with increased YWHAZ protein expression and decreased ZFP36 expression, providing a possible mechanism for the elevated secretion of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α. miR-451 levels are themselves increased by IL-6 and type I IFN, potentially forming a regulatory loop. These data suggest that viral infection specifically induces a miRNA that directs a negative regulatory cascade to tune DC cytokine production.
Assuntos
Citocinas/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/virologia , MicroRNAs/fisiologia , Animais , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Células Dendríticas/metabolismo , Regulação para Baixo/imunologia , Regulação Viral da Expressão Gênica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Cultura Primária de Células , Regulação para Cima/imunologiaRESUMO
OBJECTIVE: The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF. METHODS: oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. RESULTS: Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree. CONCLUSION: The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.
Assuntos
Herpesvirus Humano 2/genética , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Animais , Linhagem Celular Tumoral , Feminino , Deleção de Genes , Engenharia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Herpesvirus Humano 2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/fisiologia , Distribuição Aleatória , Carga Tumoral , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (T(H)1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)-mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the T(H)1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8(+) T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of T(H)1 cytokines.
Assuntos
Ligante de CD40/genética , Ligante de CD40/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Animais , Apoptose/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Humanos , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/genética , Neoplasias/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor necrosis factor superfamily members, including Fas ligand and TRAIL, have been studied extensively for cancer therapy, including as components of gene therapy. We examined the use of FasL expression to achieve tumor-selective replication of an oncolytic poxvirus (vFasL), and explored its biology and therapeutic efficacy for FasR- and FasR+ cancers. Infection of FasR+ normal and MC38 cancer cells by vFasL led to abortive viral replication owing to acute apoptosis and subsequently showed both reduced pathogenicity in non-tumor-bearing mice and reduced efficacy in FasR+ tumor-bearing mice. Infection of FasR- B16 cancer cells by vFasL led to efficient viral replication, followed by late induction of FasR and subsequent apoptosis. Treatment with vFasL as compared with its parental virus (vJS6) led to increased tumor regression and prolonged survival of mice with FasR- cancer (B16) but not with FasR+ cancer (MC38). The delayed induction of FasR by viral infection in FasR- cells provides for potential increased efficacy beyond the limit of the direct oncolytic effect. FasR induction provides one mechanism for tumor-selective replication of oncolytic poxviruses in FasR- cancers with enhanced safety. The overall result is both a safer and more effective oncolytic virus for FasR- cancer.
Assuntos
Proteína Ligante Fas/genética , Terapia Viral Oncolítica/métodos , Poxviridae/fisiologia , Receptor fas/biossíntese , Animais , Apoptose/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Neoplasias da Mama/virologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Neoplasias Colorretais/virologia , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/metabolismo , Feminino , Terapia Genética , Humanos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Poxviridae/genética , Replicação Viral , Receptor fas/genéticaRESUMO
Oncolytic virotherapy is a promising biological approach to cancer treatment that contributes to tumor eradication via immune- and non-immune-mediated mechanisms. One of the remaining challenges for these experimental therapies is the necessity to develop a durable adaptive immune response against the tumor. Vesicular stomatitis virus (VSV) is a prototypical oncolytic virus (OV) that exemplifies the multiple mechanisms of oncolysis, including direct cell lysis, cellular hypoxia resulting from the shutdown of tumor vasculature, and inflammatory cytokine release. Despite these properties, the generation of sustained antitumor immunity is observed only when VSV is engineered to express a tumor antigen directly. In the present study, we sought to increase the number of tumor-associated dendritic cells (DC) in vivo and tumor antigen presentation by combining VSV treatment with recombinant Fms-like tyrosine kinase 3 ligand (rFlt3L), a growth factor promoting the differentiation and proliferation of DC. The combination of VSV oncolysis and rFLt3L improved animal survival in two different tumor models, i.e., VSV-resistant B16 melanoma and VSV-sensitive E.G7 T lymphoma; however, increased survival was independent of the adaptive CD8 T cell response. Tumor-associated DC were actively infected by VSV in vivo, which reduced their viability and prevented their migration to the draining lymph nodes to prime a tumor-specific CD8 T cell response. These results demonstrate that VSV interferes with tumor DC functions and blocks tumor antigen presentation.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Linfoma de Células T/prevenção & controle , Melanoma Experimental/prevenção & controle , Terapia Viral Oncolítica , Vírus da Estomatite Vesicular Indiana/fisiologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Movimento Celular , Proliferação de Células , Terapia Combinada , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Feminino , Citometria de Fluxo , Terapia Genética , Linfoma de Células T/imunologia , Linfoma de Células T/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.