Transfection Of Dendritic Cells Research Articles

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy.

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

Expression of Long Interspersed Nuclear Element 1 Retroelements and Induction of Type I Interferon in Patients With Systemic Autoimmune Disease.

Increased expression of type I interferon (IFN) and a broad signature of type I IFN-induced gene transcripts are observed in patients with systemic lupus erythematosus (SLE) and other systemic autoimmune diseases. To identify disease-relevant triggers of the type I IFN pathway, this study sought to investigate whether endogenous virus-like genomic repeat elements, normally silent, are expressed in patients with systemic autoimmune disease, and whether these retroelements could activate an innate immune response and induce type I IFN. Expression of type I IFN and long interspersed nuclear element 1 (LINE-1; L1) was studied by polymerase chain reaction, Western blotting, and immunohistochemistry in samples of kidney tissue from patients with lupus nephritis and minor salivary gland (MSG) tissue from patients with primary Sjögren's syndrome (SS). Induction of type I IFN by L1 was investigated by transfection of plasmacytoid dendritic cells (PDCs) or monocytes with an L1-encoding plasmid or L1 RNA. Involvement of innate immune pathways and altered L1 methylation were assessed. Levels of L1 messenger RNA transcripts were increased in lupus nephritis kidneys and in MSG tissue from patients with SS. Transcript expression correlated with the expression of type I IFN and L1 DNA demethylation. L1 open-reading frame 1/p40 protein and IFNβ were expressed in MSG ductal epithelial cells and in lupus nephritis kidneys, and IFNα was detected in infiltrating PDCs. Transfection of PDCs or monocytes with L1-encoding DNA or RNA induced type I IFN. Inhibition of Toll-like receptor 7 (TLR-7)/TLR-8 reduced the induction of IFNα by L1 in PDCs, and an inhibitor of IKKε/TANK-binding kinase 1 abrogated the induction of type I IFN by L1 RNA in monocytes. L1 genomic repeat elements represent endogenous nucleic acid triggers of the type I IFN pathway in SLE and SS and may contribute to initiation or amplification of autoimmune disease.

Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems.

Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75% with survival rates of >90%. This chapter describes the instrumentation and methods needed for the efficient transfection by electroporation of millions of dendritic cells in one continuous flow process.

P70: Construction and studying expression in eukaryotic cells of allel-specific HLA-A∗0201 DNA-vaccine against breast cancer

Background Breast cancer ranks the first among female oncological diseases and ranks the second after lung cancer among causes of cancer related deaths. Use of contemporary anticancer drugs significantly enhanced possibilities of cancer therapy. However, most of medicines against cancer have such disadvantages as toxicity, narrowness of therapeutic action, and emergence of drug resistance. Recently, an approach based on potential of dendritic cells (DCs) to induce formation of cytotoxic T-cell immune response against cancer cells is widely used. Priming of dendritic cells by DNA-constructs encoding immunogenic epitopes of tumor antigens is regarded as a rather promising approach to design cancer vaccines. The aim of this study is to construct, obtain and study expression in eukaryotic cells of recombinant plasmid – candidate DNA-vaccines inducing T-cell immune response against breast cancer. Materials and methods Using the PolyCTLDesigner software, DNA-vaccine construct was made encoding polyepitope T-cell immunogen BC-A ∗ 0201. Gene encoding target immunogen was synthesized and cloned in the composition of plasmid pcDNA3.1 that is eukaryotic expression vector. Accuracy of gene insertion encoding polyepitopeimmunogen was verified using restriction analysis. Obtained target plasmid pBC-A ∗ 0201 – candidate DNA-vaccine was used for evaluation of gene expression in transfected eukaryotic cells 293T and dendritic cells by means of flow cytofluorometry and immunohistochemistry using MAT to p24 epitope-marker inserted into the structure of target plasmid. Dendritic cells were obtained from peripheral blood precursors by 2-h plastic adhesion. The nonadherent cells were removed 2 h later and the adherent monolayer was cultivated in the medium containing growth factors (GM-CSF and IL-4) according to (Naik, 2010; Ganul and Khranovskaya, 2012). Transfection of immature DCs (iDC) was performed by using the reagents purchased from Promokine. Further cultivation of iDCs was carried out in the medium containing TNF- α to obtain fully mature DCs (mDC). Analysis of mDCsurface markers expression was made using MAO (HLA-DR, CD83, CD86) and flow cytofluorometer (BD FACSCalibur). Results Plasmid pBC-A ∗ 0201 encoding epitopes of breast cancer antigens was constructed and produced in preparative amount. Using the method of intracellular staining of product of expression by specific MAO 29F2 to epitope p24, we showed expression of gene BC-A ∗ 0201 in eukaryotic cells 293T and dendritic cells transfected by plasmid pBC-A ∗ 0201. Maturity of dendritic cells was also proved. Conclusion Thus, obtained candidate DNA-vaccine encoding target polyepitopeimmunogen provides synthesis of relevant protein in the eukaryotic cells culture. In future, we plan to study biological activity of DNA-vaccine to evaluate level of induction of cytotoxic response against SKBR3 breast cancer cells.

FOXO1 regulates dendritic cell activity through ICAM-1 and CCR7.

The transcription factor FOXO1 regulates cell function and is expressed in dendritic cells (DCs). We investigated the role of FOXO1 in activating DCs to stimulate a lymphocyte response to bacteria. We show that bacteria induce FOXO1 nuclear localization through the MAPK pathway and demonstrate that FOXO1 is needed for DC activation of lymphocytes in vivo. This occurs through FOXO1 regulation of DC phagocytosis, chemotaxis, and DC-lymphocyte binding. FOXO1 induces DC activity by regulating ICAM-1 and CCR7. FOXO1 binds to the CCR7 and ICAM-1 promoters, stimulates CCR7 and ICAM-1 transcriptional activity, and regulates their expression. This is functionally important because transfection of DCs from FOXO1-deleted CD11c.Cre(+)FOXO1(L/L) mice with an ICAM-1-expressing plasmid rescues the negative effect of FOXO1 deletion on DC bacterial phagocytosis and chemotaxis. Rescue with both CCR7 and ICAM-1 reverses impaired DC homing to lymph nodes in vivo when FOXO1 is deleted. Moreover, Ab production following injection of bacteria is significantly reduced with lineage-specific FOXO1 ablation. Thus, FOXO1 coordinates upregulation of DC activity through key downstream target genes that are needed for DCs to stimulate T and B lymphocytes and generate an Ab defense to bacteria.

The inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader RNA.

Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA. Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.

Direct transfection of dendritic cells in the epidermis after plasmid delivery enhanced by surface electroporation.

The skin is rich in antigen-presenting cells and as such is an excellent target tissue for vaccination strategies. Electroporation is a physical delivery method that potentiates the uptake of DNA vaccines into target cells. Intradermal electroporation offers a minimally invasive solution to DNA delivery in the clinic. Here we describe the direct transfection of dendritic cells in the epidermis, using a surface dermal electroporation device, and specifically show a dendritic cell transfected with plasmid expressing green fluorescent protein. The dendritic cell has used its motile capabilities after transfection to move from the epidermis into the dermis, making its way to the lymphatic system.

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy.

Dendritic cell (DC)-based cancer vaccines, where the patient's own immune system is harnessed to target and destroy tumor tissue, have emerged as a potent therapeutic strategy. In the development of such DC vaccines, it is crucial to load the DCs with tumor antigens, and to simultaneously activate them to become more potent antigen-presenting cells. For this, we report on microbubbles, loaded with both antigen mRNA as well as immunomodulating TriMix mRNA, which can be used for the ultrasound-triggered transfection of DCs. In vivo experiments with in vitro sonoporated DCs show the effective induction of antigen-specific T cells, resulting in specific lysis of antigen-expressing cells. Especially in a therapeutic setting, sonoporation with TriMix has an important added value, resulting in a significant reduction of tumor outgrowth and a marked increase in overall survival. What is more, complete tumor regression was observed in 30% of the antigen+TriMix DC vaccinated animals, which also displayed long-term antigen-specific immunological memory. As a result, DC sonoporation using microbubbles loaded with a combination of antigen and TriMix mRNA can elicit powerful immune responses in vivo, and might serve as a potential tool for further in vivo DC vaccination applications.

Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production.

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1β (IL-1β) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1β. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1β, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Attenuation of allograft rejection by intragraft inhibition of class II transcativator in high responder rat liver transplantation.

Major histocompatibility complex-II (MHC-II) plays an important role in graft rejection and class II transactivator (CIITA) is the key regulator for MHC-II expression. The aim of this study was to determine the efficacy of intragraft inhibition of CIITA in attenuating liver transplant rejection. Three plasmids containing small hairpin RNA (shRNA) against rat CIITA (pCIITA-shRNA) and one control plasmid of pHK-shRNA were constructed. In vitro dendritic cell (DC) transfection and liver transfection via portal vein in donor rats (n = 8) by shRNA plasmids were performed to confirm the inhibitory effect of pCIITA-shRNA on CIITA expression. It showed that expressions of CIITA and MHC-II were significantly inhibited by pCIITA-shRNA in both DC in vitro and liver of donor rats in vivo (p < 0.05 vs. control pHK-shRNA treatment). pCIITA1-shRNA was proved to be the best inhibitor among three pCIITA-shRNAs and then used in high-responder rat liver transplantation model (DA donors-to-Lewis recipients). Transplant groups (n = 16/group) include untreated recipients transplanted with donor liver graft pretreated with either saline, or pHK-shRNA, or pCIITA1-shRNA. Cyclosporine-treated (10 mg/kg, im, day 0-7) recipients transplanted with unmodified liver grafts were used as no rejection control. The results showed that the recipient rats survived significantly longer in pCIITA1-shRNA-treated group with markedly attenuated liver graft rejection (p < 0.05 vs. saline and pHK-shRNA-treated groups). Furthermore, significantly decreased intragraft expressions of CIITA, MHC-II, IL-2, and IFN-γ were found in pCIITA1-shRNA-treated group (p < 0.05 vs. saline and pHK-shRNA-treated groups). This study suggests that intragraft inhibition of CIITA could be a novel strategy for attenuating graft rejection in liver transplantation.

In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes

Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

Dendritic Cell-Based Approaches in the Fight Against Diseases

DENDRITIC CELL ROLE IN THE IMMUNE SYSTEM AND ITS MANIPULATION The immune system works to contain infections through activation of different molecules and cell types. Correct presentation of antigens by antigen-presenting cells (APCs) is a critical step necessary to initiate an immune response. APCs have the ability to take-up and process antigens, and express high levels of co-stimulatory and major histocompatibility complex (MHC) molecules bound to antigens (1). Dendritic cells (DCs) are innate immune cells first characterized and reported by Ralph Steinman in 1973 (2). For their unique properties and features, DCs are the most important APCs acting at the interface of innate and adaptive immunity, which results in the activation of immune responses in the body. Distinct subsets of DCs are associated with lineage and receptor expression patterns (3). Dendritic cells have different roles in the immune system, such as activation and regulation of adaptive immune responses, and other opposing functions in the induction of tolerance and anergy (4). During immune responses, DCs are crucial decision makers toward the development of naive T cells to T helper type 1 (Th1) or type (Th2) profile (5). Among the different families of molecules expressed by DC to aid in their function, one of them is the family of toll-like receptors (TLR). TLR which are expressed by different types of DCs, and bind to common molecules associated with pathogens. Once bound, molecules such as bacterial lipopolysaccharide and hypomethylated CpG DNA, can induce activation of biochemical cell pathways, resulting in overexpression of MHC, co-stimulatory molecules (CD80, CD86), and cytokines (6). In this context, a number of methods have been available to manipulate DCs from diverse sites in the body resulting in activated cells for therapy. These methods include reinfusion of unloaded DCs; reinfusion of DCs co-cultured with peptides or proteins of interest; in vivo DC loading; DC transfection with antigen-encoding viruses or nucleic acids; and DC-derived exosomes (7, 8). After this, DCs might be ready to promote protection or treat specific diseases. In this context, the availability of methods to manipulate DCs in laboratory, arise as an important tool for immunointerventions in different diseases. In this opinion article, we focused on the basis of DC approaches already available in the field of cancer currently in test for infectious diseases, and future interventions that are needed.

MRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery

Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (<10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1-19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems.

Numerical solution of an optimal control model of dendritic cell treatment of a growing tumour

A new optimal control model of the interactions between a growing tumour and the host immune system, along with an immunotherapy treatment strategy, is presented. The model is based on an ordinary differential equation model of interactions between the growing tumour and the natural killer, cytotoxic T lymphocyte and dendritic cells of the host immune system, extended through the addition of a control function representing the application of a dendritic cell treatment to the system. The numerical solution of this model, obtained from a multi species Runge–Kutta forward-backward sweep scheme, is described. We investigate the effects of varying the maximum allowed amount of dendritic cell vaccine administered to the system and find that control of the tumour cell population is best effected via a high initial vaccine level, followed by reduced treatment and finally cessation of treatment. 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Human dendritic cells transfected with a human papilloma virus-18 construct display decreased mobility and upregulated cytokine production

The marked depletion of dendritic cells (DCs) in skin cancers, as well as preneoplastic and neoplastic cervical epithelium, suggests a central role for DCs in productive human papillomavirus (HPV) infection and cancer promotion. It has been suggested that HPV may facilitate tumor development by reducing DC density, contributing to a decrease in local immune surveillance. In this study, we have examined the response of human DCs transfected with a construct containing the HPV18 genome and their subsequent expression of papilloma virus proteins. Transfected cells expressed the L1 major capsid protein and upregulated E6 and E7 oncoprotein transcripts as detected by RT-PCR. Transfection of DCs also resulted in a significant increase in cytokine production. Finally, we observed that HPV18 transfection decreased the migratory activity of DCs. Our data indicate that HPV transfection of DCs leads to changes in migratory activity and cytokine production, which potentially can suppress or delay immune responses to viral antigens. Additionally, changes in cytokine production by HPV-transformed human fibroblasts and human cervical epithelial cells revealed that the migratory and antigen-presenting functions of DCs may be impaired by the suppressive effects of cytokines produced by HPV-infected epithelial and stromal cells.

Tumor RNA-transfected dendritic cells and combined therapy with low dose 5-FU induce regression of murine colon cancer

Background We have recently observed that treatment of colon tumor cells with low concentration of paclitaxel increased the expression of several genes associated with antigen-presenting machinery. Since 5-fluoruracil (5-FU) is the main antineoplastic agent for colon cancer, in this study we aimed to evaluate: a) whether transfection of dendritic cells (DC) with drug-treated tumor cells RNA, enhances the effectiveness of DC-based vaccine; b) if the modulatory effects of vaccine can be observed in vivo, and c) if the combination of DC with low dose chemotherapy schedule improves the antitumor responsiveness.

Abstract B14: Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA.

Abstract The central role of dendritic cells (DCs) in antigen presentation and induction of antitumor immunity supported the development of antitumor vaccines based on these cells. In previous studies, we have observed that treatment with ultra-low doses of paclitaxel (PAC) modulated human DCs to express the phenotype of mature and activated cells, and stimulated their antigen presentation activity. At the same time, tumor cells treated with low concentrations of drugs became more immunogenic and more susceptible to the lytic activity of cytotoxic T lymphocytes in vitro. Therefore, we aimed to verify if the transfection of DCs with drug-treated tumor cells RNA enhances the effectiveness of DC-based vaccines and if the in vitro effects are reproducible in vivo. Murine colon tumor cells (MC-38) were subcutaneously inoculated in C57Bl/6 mice. At the 10th and 17th days after inoculation, they received a DC vaccine transfected with RNA of MC-38 cells pre-treated with minimum effective concentration of PAC. These DCs expressed 12% higher levels of CD40 and were able to delay the tumor growth when compared with non-treated animals. Although the differences were not significant, our results suggest that the treatment of tumor cells with low concentration of PAC can enhance the immunogenicity of tumor cells, which is transfered for DCs by RNA transfection, and delay tumor growth. Financial support: FAPESP 2009/18331-8; FAPESP 2010/06013-9. Citation Format: Marcela Rodrigues Camargo, Cecília Pessoa Rodrigues, Carolina Mendonça Gorgulho, Fabiana Albani Zambuzi, Victória Elizabeth Galvão, Juliana Cristina Longo Frederico, Marcimara Penitenti, Ramon Kaneno. Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B14.

Gene‐carried chitosan‐linked polyethylenimine induced high gene transfection efficiency on dendritic cells

A dendritic cell (DC) networking system has become an attractive approach in cancer immunotherapy. Successful DC gene engineering depends on the development of transgene vectors. A cationic polymer, chitosan-linked polyethylenimine (PEI) (CP), possessing the advantages of both PEI and chitosan, has been applied in nonviral transfection of DCs. Physicochemical evaluation showed that CP/DNA complexes could form cationic nanoparticles. Compared with DCs transfected with commercial reagent, Lipofectamine2000, it showed higher transfection efficiency and lower cytotoxicity when DCs were transfected with CP/DNA complexes. A nuclear trafficking observation of CP/DNA complexes by a confocal laser scanning microscope further revealed that the CP could help DNA enter into the cytoplasm and finally into the nucleus of a DC. Finally, vaccination of DCs transfected with CP/DNA encoding gp100 slightly improved resistance to the B16BL6 melanoma challenge. This is the first report that CP polymer is used as a nonviral vector for DC gene delivery and DC vaccine. Essentially, these results might be helpful to design a promising nonviral vector for DC gene delivery.

Abstract 1585: Primary and metastatic uveal melanoma cell lines express MelanA that can be used as a target antigen for transfection of mature dendritic cells

Abstract The most common intra-ocular malignancy is uveal melanoma (UM). Metastatic disease occurs in approximately 50% of UM patients and presents most frequently in the liver. Novel therapies that prevent the development of metastases are needed and immunotherapy is a potential option. Because of its origin in the immune-privileged eye, UM may be particularly responsive to T cell-based immunotherapy. MelanA is frequently over-expressed by human cutaneous melanomas. MelanA expression has also been detected in UM primary and metastatic tumor-tissue sections and could serve as a target antigen for immunotherapy of UM. Here we report that primary and metastatic UM cell lines express MelanA and that this protein can be used as a target antigen for transfection of mature dendritic cells (mDC). MelanA expression was confirmed in primary and metastatic UM cell lines using intracellular flow cytometry and quantitative PCR (qPCR). In addition, sequencing of UM-derived MelanA demonstrated that MelanA in primary and metastatic UM cells has an identical sequence as compared to the published MelanA consensus sequence. As an initial step towards activating UM-specific T cells by UM-derived mRNA transfected DC we are generating MelanA mRNA transfected mDC. To facilitate both MHC class I and class II presentation of MelanA epitopes after mRNA electroporation into mDC, we included the endosomal targeting sequence DCLamp linked to the MelanA protein. Monocytes were isolated from leukapheresis products of healthy donors by density centrifugation and plastic adherence. Monocytes were differentiated to monocyte-derived DC by culturing with IL-4 and GM-CSF for 6 days to obtain immature DC (iDC). iDCs were matured with a cytokine mixture containing IL-4, GM-CSF, IL-1α, IL-6, TNFα, and PGE2 for 48 hours. The resulting mDC expressed cell surface maturation markers CD25, CD83, and CD86 as confirmed by flow cytometry. mDC were transfected with either MelanA-mRNA of MelanA-DCLamp-mRNA, using an established electroporation protocol. To assess transfection efficiency and monitor MelanA expression in transfected mDC, MelanA-mRNA and MelanA-DCLamp-mRNA expression was demonstrated by qPCR and MelanA-protein expression was shown by intracellular flow cytometry at different time points post-transfection using variable mRNA concentrations. The detected amount of MelanA-mRNA and protein was dependent on the amount of transfected mRNA and presented transiently. Subsequently, total RNA was isolated from MelanA+ primary and metastatic UM cell lines, then reversely transcribed into cDNA, amplified by PCR and in vitro transcribed to mRNA. As confirmed by qPCR, MelanA-mRNA was present in the pool of UM-derived mRNA and can be used for transfection of mDC. Collectively, these data demonstrate that mDC can be transfected with MelanA-mRNA and suggest MelanA could serve as a target for immunotherapy of UM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1585. doi:1538-7445.AM2012-1585