Iranzu González

Imatinib Inhibits Proliferation of Ewing Tumor Cells Mediated by the Stem Cell Factor/KIT Receptor Pathway, and Sensitizes Cells to Vincristine and Doxorubicin-Induced Apoptosis

Purpose and Experimental Design: The stem cell factor/KIT receptor loop may represent a novel target for molecular-based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWS-FLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 μm) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 μm). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 μm) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 μm) was able to increase the antitumor in vitro effect of doxorubicin (DXR) and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.

Effects of IFN-α as a signal-3 cytokine on human naïve and antigen-experienced CD8(+) T cells.

IFN‐α/β link innate and adaptive immune responses by directly acting on naïve CD8+ T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8+CD45RO− cells were stimulated with beads coated with anti‐CD3 and anti‐CD28 mAb, mimicking Ag (type‐1) and co‐stimulatory (type‐2) signals, in the presence or absence of IFN‐α and their transcriptional profiles were defined by cDNA‐microarrays. We show that IFN‐α provides a strong third signal directly to human CD8+ T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8+ T‐cell population. Thus, whereas IFN‐α increases the proliferative capacity of naïve CD8+ T cells, it inhibits or does not affect the proliferation of Ag‐experienced cells, such as memory and effector CTL, including CMV‐specific lymphocytes. Cytolysis and IFN‐γ‐secretion of all these populations are enhanced by IFN‐α‐derived signals, which are critical in naïve CD8+ T cells for acquisition of effector functions. Our findings in human CD8+ T cells are informative to understand and improve IFN‐α‐based therapies for viral and malignant diseases.

Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN‐α

Migration of DC into lymphatic vessels ferries antigenic cargo and pro‐inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF‐α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN‐α2b or IFN‐α5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro‐adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA‐1. Furthermore, incubation of DC with IFN‐α led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN‐α also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.

EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model

Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET) are characterized by specific chromosomal translocations most often generating a chimeric EWS/FLI-1 gene. Depending on the number of juxtaposed exons assembled, several fusion types have been described with different incidences and prognoses. To assess the impact of each fusion type on the specific phenotypic, tumorigenic, and metastatic features of EWS/PNET, we developed an amenable system using a murine mesenchymal multipotent C3H10T1/2 cell line. Upon transduction of EWS/FLI-1, cells acquired dramatic morphological changes in vitro, including a smaller size and “neurite-like” membrane elongations. Chimeric fusion proteins conferred oncogenic properties in vitro, including anchorage-independent growth and an increased rate of proliferation. Furthermore, EWS/FLI-1 expression blocked mineralization, with concomitant repression of osteoblastic genes, and induced a dramatic repression of the adipocytic differentiation program. Moreover, EWS/FLI-1 promoted an aberrant neural phenotype by the de novo expression of specific neural genes. The intramuscular injection of transduced cells led to tumor development and the induction of overt osteolytic lesions. Analogously, to what was observed in human tumors, type 2 EWS/FLI-1 cells formed primary tumors in immunodeficient mice with a higher incidence and a lower latency than cells bearing types 1 and 3 fusions. By contrast, cells expressing types 2 and 3 fusions showed specific metastatic activity with a higher number of macroscopic metastases in soft tissues and osteolytic lesions in the limbs as compared to type-1-expressing cells. Therefore, the structure of each oncoprotein strongly influenced its tumorigenicity and metastagenicity. Thus, this model provides a basis for understanding the genetic determinants involved in Ewing tumor development and metastatic activity and represents a cellular system to analyze other oncoproteins involved in human sarcomagenesis.

CD8 T Cell Priming in the Presence of IFN-α Renders CTLs with Improved Responsiveness to Homeostatic Cytokines and Recall Antigens: Important Traits for Adoptive T Cell Therapy

Previous mouse and human studies have demonstrated that direct IFN-α/β signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α–primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15−/− and IL-7−/− recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2+ CD8 T cells with autologous dendritic cells loaded with MART126–35 peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.

Oncostatin M Enhances the Antiviral Effects of Type I Interferon and Activates Immunostimulatory Functions in Liver Epithelial Cells

ABSTRACT Oncostatin M (OSM) is released together with type I interferon (IFN) by activated dendritic cells, suggesting a concerted action of these cytokines in the biological response against infection. We found that OSM increases the antiviral effect of IFN-α in Huh7 hepatoma cells infected with hepatitis A or hepatitis C virus and synergizes with IFN-α in the induction of antiviral genes. The combination of OSM and IFN-α led to upregulation of both STAT1 and STAT3 together with intense and prolonged activation of STAT1, STAT3, and Jak1. OSM with or without IFN-α also activated p38 mitogen-activated protein kinase, which is known to enhance transcription of IFN-α-inducible genes. Interestingly, OSM combined with IFN-α strongly induced immunoproteasome genes and other genes involved in antigen processing and presentation. Moreover, OSM, alone or in combination with IFN-α, upregulated relevant innate immunity molecules and increased the expression of intracellular adhesion molecule 1 and interleukin-15 receptor alpha (IL-15Rα) in liver cells. Hepatoma cells transfected with a plasmid encoding a viral antigen were able to activate effector T cells when pretreated with IFN-α plus OSM but not with each cytokine separately. Also, OSM, more than IFN-α, augmented the ability of Huh7 cells to transpresent IL-15 to responding lymphocytes and increased the immunostimulatory activity of liver epithelial cells by presenting a short viral peptide to sensitized cytotoxic T cells. In conclusion, OSM enhances the antiviral effects of type I interferon and cooperates with it in the induction of adaptive immune responses to pathogens. These findings may have therapeutic implications.

Cardiotrophin-1 Promotes a High Survival Rate in Rabbits with Lethal Fulminant Hepatitis of Viral Origin

ABSTRACT Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 104 hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 μg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort (n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1β, Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor β, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.

Anchoring interferon alpha to apolipoprotein A-I reduces hematological toxicity while enhancing immunostimulatory properties†‡§

Interferon alpha (IFNα) is widely used for the treatment of viral hepatitis but substantial toxicity hampers its clinical use. In this work, we aimed at improving the efficacy of IFNα therapy by increasing the IFNα half‐life and providing liver tropism. We selected apolipoprotein A‐I (ApoA‐I) as the stabilizing and targeting moiety. We generated plasmids encoding IFNα, albumin bound to IFNα (ALF), or IFNα linked to ApoA‐I (IA) and mice were treated either by hydrodynamic administration of the plasmids or by injection of the corresponding recombinant proteins or high‐density lipoproteins containing IA. The plasma half‐life of IA was intermediate between IFNα and ALF. IA was targeted to the liver and induced higher hepatic expression of interferon‐stimulated genes than IFNα or even ALF. IA exhibits stronger in vivo antiviral activity than IFNα and the hematologic cytopenic effects of IA are milder than those observed when using IFNα or ALF. In contrast to IFNα, IA does not cause activation‐dependent cell death of lymphocytes in vitro. Accordingly, in vivo studies showed that IA boosts T‐cell immune responses more efficiently than IFNα or ALF. The difference in immunostimulatory activity between IFNα and IA disappears in scavenger receptor class B type I (SR‐BI) knockout mice, suggesting that crosstalk between SR‐BI and IFNα receptor is essential for enhanced induction of cytotoxic T cells by IA. Conclusion: Anchoring IFNα to ApoA‐I prolongs the half‐life of IFNα and promotes targeting to the liver. Importantly, the fusion protein shows increased immunostimulatory properties and lower hematological toxicity. (HEPATOLOGY 2011;)

Validation of an antiviral assay method for quantifying IFN-α5 activity in macaque and human serum

BACKGROUND IFN-α5 has been demonstrated to induce stronger signaling and higher expression of antiviral genes than IFN-α2, which is the current treatment in chronic viral hepatitis. However, there is no specific and validated quantification method in order to conduct kinetic studies as part of the preclinical and clinical evaluation for regulatory purposes. RESULTS A novel integration of an antiviral assay against the cytopathic effect of the encephalomyocarditis virus in HeLa cells with a very sensitive method for assay processing - the Vialight(®) Plus assay - is presented for IFN-α5 activity quantification. The bioassay has been validated in macaque and human serum and it has been demonstrated to be selective, precise and accurate. CONCLUSION The validated bioassay meets suitable acceptance criteria for these types of biological assays.