CCL3 and CCL20-recruited dendritic cells modified by melanoma antigen gene-1 induce anti-tumor immunity against gastric cancer ex vivo and in vivo

<p>Abstract</p> <p>Background</p> <p>To investigate whether dendritic cell (DC) precursors, recruited by injection of <b>chemokine ligand 3 (CCL3) and CCL20</b>, induce anti-tumor immunity against gastric cancer induced by a DC vaccine expressing melanoma antigen gene-1 (MAGE-1) ex vivo and in vivo.</p> <p>Methods</p> <p>B6 mice were injected with CCL3 and CCL20 via the tail vein. Freshly isolated F4/80^-B220^-CD11c⁺cells cultured with cytokines were analyzed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured F4/80^-B220^-CD11c⁺cells were incubated with Ad-MAGE-1. Vaccination of stimulated DC induced T lymphocytes. The killing effect of these T cells against gastric carcinoma cells was assayed by MTT. INF-γ production was determined with an INF-γ ELISA kit. In the solid tumor and metastases model, DC-based vaccines were used for immunization after challenge with MFC cells. <b>Tumor size, survival of mice, and number of pulmonary metastatic foci were used to assess the therapeutic effect of DC vaccines</b>.</p> <p>Results</p> <p>F4/80^-B220^-CD11c⁺cell numbers increased after <b>CCL3 and CCL20 </b>injection. Freshly isolated F4/80^-B220^-CD11c⁺cells cultured with cytokines were phenotyically identical to typical DC and gained the capacity to stimulate allogeneic T cells. These DCs were transduced with Ad-MAGE-1, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated by DCs transduced with Ad-MAGE-1 exhibited specific killing effects on gastric carcinoma cells and produced high levels of INF-γ ex vivo. In vivo, tumor sizes of the experimental group were much smaller than both the positive control group and the negative control groups (<it>P </it>< 0.05). Kaplan-Meier survival curves showed that survival of the experimental group mice was significantly longer than the control groups (<it>P </it>< 0.05). In addition, MAGE-1-transduced DCs were also a therapeutic benefit on an established metastatic tumor, resulting in a tremendous decrease in the number of pulmonary metastatic foci.</p> <p>Conclusions</p> <p><b>CCL3 and CCL20</b>-recruited DCs modified by adenovirus-trasnsduced, tumor-associated antigen, MAGE-1, can stimulate anti-tumor immunity specific to gastric cancer ex vivo and in vivo. This system may prove to be an efficient strategy for anti-tumor immunotherapy.</p

beta 2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional beta 2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8(+) T-cell response. BM-DCs expressing dysfunctional beta 2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, beta 2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting beta 2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.Peer reviewe

Doctor of Philosophy

dissertationGene expression data repositories provide large and ever increasing data for secondary use by translational informatics methods. For example, Gene Expression Omnibus (GEO) houses over 37,000 experiments with the goal of supporting further research. To use these published results in a larger meta-analysis, consolidation of the data are needed; however, the data are largely unstructured, thus hindering data integration efforts. Here, I propose the use of a novel pipeline, Ontology Based Data Integration (OBDI), which uses an ontological approach to combine the samples across multiple GEO experiments. The ODBI pipeline uses machine learning algorithms that permit researchers to consolidate and analyze data across GEO experiments. Here, I demonstrate how using an ontological approach to integrate samples across experiments can be used to explore the immune response at a molecular level. As part of this process, a Web Ontology Language (OWL) was developed for each data platform used. OWL serves as a core component in successfully processing different sample types. Immunological experiments from GEO were consolidated to evaluate this methodology. The experiments included samples analyzed on expression arrays, BeadChips, and sequencing technologies. The integration of a complex biological system and the incorporation of different biological data types will validate the potential of OBDI. iv The nature of biological data is highly dimensional. OBDI incorporates tools and techniques that can handle the analysis of various biological data. The machine learning analysis performed within the OBDI pipeline successfully evaluated the newly annotated experiments and provides insights that can be further explored. The OBDI pipeline can help researchers annotate experiments using ontologies and analyze the annotated experiments. To successfully build the pipeline, ontologies served as the backbone of integrating samples from GEO Series records into machine learning experiments using ML-Flex. By using the OBDI pipeline, researchers can access the uncurated experiments from GEO (GEO Data Series) and annotate the data using the terms in the ontologies. This mechanism allows for the organization of data sets in relationship to new experiments independent of GEO's GDS curation process. The OBDI system allows ontologies to grow organically around a cluster of experiments. These experiments are then further analyzed in ML-Flex using machine learning algorithms. The curated experiments are analyzed in silico and the computational analyses are supported by the OBDI ontological system

Human Embryonic Stem Cell Derivatives as Cancer Therapeutics

Ph.DDOCTOR OF PHILOSOPH

Modulation of tumoricidal activities of dendritic cells to enhance antigen uptake and cross-presentation

Abstract Dendritic cells (DCs) are professional antigen-presenting cells that are integral to the induction of primary, antigen-specific T cell responses. In the cancer setting, DCs mediate cross-priming of tumor-reactive T cells by presenting tumor antigens acquired from viable or dead cancer cells. Due to their unique functional properties, DCs have been utilized as both vectors and targets for immunological intervention in numerous diseases and are optimal candidates for vaccination protocols in cancer. In addition to their antigen presentation function(s), recent evidence suggests that DCs may also perform an innate immune effector function, with human DCs reported to mediate direct tumoricidal activity in vitro. However, the mechanism(s) by which DCs directly kill tumor cells remain unclear. The goal of this study is to further characterize the mechanism(s) associated with murine DC tumoricidal function and to determine whether and how this function may be enhanced to promote anti-tumor immune responses that translate into therapeutic effectiveness. One way we sought to enhance this DC effector function was through the genetic engineering of DCs themselves. After transduction with mIL-12 and/or mIL-18 cDNA using recombinant adenoviral vectors, DCs exhibited significantly elevated tumor killing activity. This was mediated, at least in part, by TNF ligand-receptor complexes, as demonstrated by antibody blocking assays. When injected in situ, these engineered DCs exhibited prolonged survival, in association with enhanced levels of tumor apoptosis proximal to imaged DCs and our capacity to image DC that had engulfed tumor apoptotic bodies. We also observed notable therapeutic benefits upon intratumoral delivery of these DCs in concert with an expanded in vivo repertoire of anti-tumor CD8+ T cells. In addition to DC modification, we also evaluated treatments applied to tumor cells that resulted in enhanced sensitivity to (control) DC-mediated killing. Specifically, we found that pretreatment of A20 lymphoma cells with a nitric oxide (NO) donor compound, PAPA-NO, markedly increased the sensitivity of tumor cells to consequent apoptosis mediated by DCs. This appeared to provide DCs with a preferred source of tumor antigens, with which, they were capable of activating specific T cells via a cross-presentation pathway. We have also discovered that multiple TNF family ligands participated in DC-mediated tumoricidal function and that tumor cell-expressed survivin may represent a critical downstream factor regulating the apoptotic sensitivity of tumor cells to DC-mediated apoptosis. When taken together, these studies provide novel details regarding mechanisms involved in DC anti-tumor effector function, and suggest two DC-based, combinational cancer therapies that target the effective cross-priming of therapeutic T cells. The results presented in this dissertation support an efficient model in which DCs may not only serve as the gatherers and presenters of antigens, but also the hunters as well, with tumoricidal activity mediated via TNF family ligands

Molecular analysis of pattern recognition receptors involved in sensing human rhinoviruses by dendritic cells

Infektionen durch humane Rhinoviren (HRV) sind die häufigste Ursache für Erkältungen. Sie bedingen aber auch eine Prädisposition für bakterielle Infektionen sowie Verschlimmerungen von Asthmaanfällen. Die pathogenen Mechanismen von HRV Infektionen, die jene Komplikationen verursachen, werden hingegen schlecht verstanden. Dendritische Zellen (DC) sind die wichtigsten Antigen-präsentierenden Zellen (APC) des Immunsystems. Mit Hilfe von viral pattern recognition Rezeptoren (VPRR) können DC evolutionär hoch konservierte molekulare Muster von Viren erkennen. Die nach solcher Aktivierung resultierenden reifen DC besitzen die Fähigkeit, eine adaptive Immunantwort einzuleiten. HRV sind kleine, ikosahedrische, nicht-Membran-umhüllte (+)-Strang Einzelstrang RNS (ssRNA) Viren. Wir haben kürzlich gezeigt, dass HRV14, ein Mitglied der major group HRV, die akzessorische Funktion von DC herunterreguliert. HRV14 behandelte DC reifen nicht und weisen durch die Expression inhibitorischer Oberflächenmoleküle eine stark herabgesetzte Kapazität zur T-Zell Proliferationsstimulation auf. HRV replizieren in DC nicht. DC sind aber mit unterschiedlichen VPRR ausgestattet, die virale ssRNA (vssRNA) erkennen. Aus diesem Grund war unsere Fragestellung, ob die genomische RNS von HRV alleine - als potentielles danger signal - die Kapazität hat, DC Reifung zu induzieren. Diese Studie zeigt, dass HRV ssvRNA durch DC erkannt wird, was zu einer starken Typ 1 Interferon (IFN) Antwort führt. Diese IFN Induktion wurde auch in einem Luciferase Reporter Assay unter Verwendung von RIG-I transfizierten HEK Zellen gefunden. Dieses Ergebnis identifiziert RIG-I als potentiellen VPRR für HRV RNA. Dennoch konnten wir in DC, im Gegensatz zu HEK Zellen, keine NFκB Aktivierung, die für die DC Reifung wesentlich ist, durch ssvRNA feststellen. Weiters wurden typische Reifungsmarker wie CD83, sowie die Freisetzung der proinflammatorischen Cytokine TNFα, IL-12 und IL6 nicht induziert. Letzlich konnte ssvRNA in einer allo-mixed leukocyte reaction keine erhöhte T-Zell stimulatorische Kapazität von DC bewirken. HRV ssvRNA ist also nur ein semi-danger Signal für DC: Trotz Erkennung, induziert ssvRNA keine DC Reifung.Human rhinovirus (HRV) infections are the most prevalent cause of the common cold but may also induce a predisposition for bacterial infections as well as exacerbations of asthma. The pathogenic mechanisms of HRV infections leading to such complications are still poorly understood. Dendritic cells (DC) are the main professional antigen presenting cells of the immune system. With the help of viral pattern recognition receptors (VPRR) DC can recognize evolutionary highly conserved molecular patterns of viruses. Once activated, the resulting mature DC are capable of inducing adaptive immune responses. HRV, are small, icosahedral, nonenveloped (+)-strand single-stranded RNA (ssRNA) viruses. We have previously shown that HRV14, a member of the major group rhinoviruses, specifically down-modulates the accessory function of monocyte-derived-DC (DC). HRV14-treated DC do not mature and show a strongly diminished T-cell stimulatory capacity due to the upregulation of inhibitory surface molecules. HRV do not replicate in DC. However, DC are well equipped with different single-stranded viral RNA (ssvRNA) detecting VPRR. Therefore, we wondered whether the genomic RNA of HRV alone, as a potential danger signal, has the capacity to induce DC maturation. This study shows that HRV ssvRNA is recognized by DC, causing a strong Type I interferon (IFN) response. A profound IFN induction was also observed in a luciferase reporter assay using RIG-I-transfected HEK cells, identifying RIG-I as a potential VPRR for HRV RNA. However, in DC, in contrast to HEK cells, ssvRNA failed to induce NFκB activation, which is required for proper maturation induction. Furthermore, markers of maturation like the surface molecule CD83 were absent, and the proinflammatory cytokines TNFα, IL-12 and IL-6 were not produced. In addition, HRV ssvRNA failed to induce an increased T-cell stimulatory capacity in an allo-mixed leukocyte reaction. Therefore, ssvRNA of HRV is only a semi-danger signal for DC: Despite recognition, ssvRNA fails to induce proper DC maturation

Dendritic Cell as Potential Immunotherapy for Nasopharyngeal Cancer: A Review

BACKGROUND: Dendritic cell (DC)-based cancer therapy is a promising adjuvant therapy for nasopharyngeal cancer (NPC) after chemoradiation. Owing to low immunity after chemoradiation, DC therapy activates immune responses. Moreover, DC-based cancer therapy can decrease tumor progression, prolong lifespan, and increase the quality of life of patients. Various studies regarding the use of DC therapy for NPC have been reported, however there are limited reviews on the implementation and foundation of DC immunotherapy to expand this technology.METHODS: A literature search was performed on EMBASE, ScienceDirect, PubMed (MEDLINE), and Cochrane Library, with the term dendritic cells therapy for nasopharyngeal cancer, dendritic cell immunotherapy in nasopharyngeal cancer patients, and DC therapy in NPC, as the search keywords.RESULTS: A total of 199 literatures were reviewed, and four clinical trials were identified as relevant for this review. DC vaccines can be processed with various maturation and activation processes. Selected literatures reported antigens used when incubating the DC are latent membrane protein (LMP) 1, LMP2, and Epstein–Barr virus nuclear antigen 1 (EBNA1). Although DC therapy was produced from different pathways, it has been reported that there are increases of cluster of differentiation (CD)8+ T cells, CD4+ T cells, and the progression free survival (PFS) rate in DC immunotherapy patients than the radiochemotherapy patients.CONCLUSION: It can be concluded that DC could be used as an adjuvant therapy alongside the standard therapy of NPC, which prolongs NPC patient survival.KEYWORDS: adjuvant cell therapy, nasopharyngeal cancer therapy, dendritic cell