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DFNA5 was first identified as a gene causing autosomal dominant hearing loss (HL). Different mutations have been found, all exerting a highly specific gain-of-function effect, in which skipping of exon 8 causes the HL. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53-dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein, which was not seen after transfection of the wild-type protein. Here, we demonstrate that DFNA5 is composed of two domains, separated by a hinge region. The first region induces apoptosis when transfected in HEK293T cells, the second region masks and probably regulates this apoptosis inducing capability. Moreover, the involvement of DFNA5 in apoptosis-related pathways in a physiological setting was demonstrated through gene expression microarray analysis using Dfna5 knockout mice. In view of its important role in carcinogenesis, this finding is expected to lead to new insights on the role of apoptosis in many types of cancer. In addition, it provides a new line of evidence supporting an important role for apoptosis in monogenic and complex forms of HL. [ABSTRACT FROM AUTHOR]

Background aims RNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients. Methods CD14+ cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients. Results In a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14+ cells (94.5±3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25±10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients. Conclusions Our method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe. [ABSTRACT FROM AUTHOR]

Following standard therapy that consists of chemotherapy with or without stem cell transplantation, both relapsed and refractory disease shorten the survival of acute myeloid leukemia (AML) patients. Therefore, additional treatment options are urgently needed, especially to fight residual AML cells. The identification of leukemia-associated antigens and the observation that administration of allogeneic T cells can mediate a graft-versus- leukemia effect paved the way to the development of active and passive immunotherapy strategies, respectively. The aim of these strategies is the eradication of AML cells by the immune system. In this review, an overview is provided of both active and passive immunotherapy strategies that are under investigation or in use for the treatment of AML. For each strategy, a critical view on the state of the art is given and future perspectives are discussed. The Oncologist 2009;14: 240--252 [ABSTRACT FROM AUTHOR]

Background: Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. Results: First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSCNT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. Conclusion: In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo. [ABSTRACT FROM AUTHOR]

Blockade of programmed cell death protein 1 (PD-1) immune checkpoint receptor signaling is an established standard treatment for many types of cancer and indications are expanding. Successful clinical trials using monoclonal antibodies targeting PD-1 signaling have boosted preclinical research, encouraging development of novel therapeutics. Standardized assays to evaluate their bioactivity, however, remain restricted. The robust bioassays available all lack antigen-specificity. Here, we developed an antigen-specific, short-term and high-throughput T cell assay with versatile readout possibilities. A genetically modified T cell receptor (TCR)-deficient T cell line was stably transduced with PD-1. Transfection with messenger RNA encoding a TCR of interest and subsequent overnight stimulation with antigen-presenting cells, results in eGFP-positive and granzyme B-producing T cells for single cell or bulk analysis. Control antigen-presenting cells induced reproducible high antigen-specific eGFP and granzyme B expression. Upon PD-1 interaction, ligand-positive antigen-presenting immune or tumor cells elicited significantly lower eGFP and granzyme B expression, which could be restored by anti-PD-(L)1 blocking antibodies. This convenient cell-based assay shows a valuable tool for translational and clinical research on antigen-specific checkpoint-targeted therapy approaches., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Although allogeneic stem cell transplantation (allo-SCT) can elicit graft-versus-tumor (GVT) immunity, patients often relapse due to residual tumor cells. As essential orchestrators of the immune system, vaccination with dendritic cells (DC) is an appealing strategy to boost the GVT response. Nevertheless, durable clinical responses after DC vaccination are still limited, stressing the need to improve current DC vaccines. Aiming to empower DC potency, we engineered monocyte-derived DCs to deprive them of ligands for the immune checkpoint regulated by programmed death 1 (PD-1). We also equipped them with interleukin (IL)-15 "transpresentation" skills. Transfection with short interfering (si)RNA targeting the PD-1 ligands PD-L1 and PD-L2, in combination with IL15 and IL15Rα mRNA, preserved their mature DC profile and rendered the DCs superior in inducing T-cell proliferation and IFNγ and TNFα production. Translated into an ex vivo hematological disease setting, DCs deprived of PD-1 ligands (PD-L), equipped with IL15/IL15Rα expression, or most effectively, both, induced superior expansion of minor histocompatibility antigen-specific CD8 + T cells from transplanted cancer patients. These data support the combinatorial approach of in situ suppression of the PD-L inhibitory checkpoints with DC-mediated IL15 transpresentation to promote antigen-specific T-cell responses and, ultimately, contribute to GVT immunity. Cancer Immunol Res; 5(8); 710-5. ©2017 AACR ., (©2017 American Association for Cancer Research.)

We formerly demonstrated that vaccination with Wilms' tumor 1 (WT1)-loaded autologous monocyte-derived dendritic cells (mo-DCs) can be a well-tolerated effective treatment in acute myeloid leukemia (AML) patients. Here, we investigated whether we could introduce the receptor for hyaluronic acid-mediated motility (RHAMM/HMMR/CD168), another clinically relevant tumor-associated antigen, into these mo-DCs through mRNA electroporation and elicit RHAMM-specific immune responses. While RHAMM mRNA electroporation significantly increased RHAMM protein expression by mo-DCs, our data indicate that classical mo-DCs already express and present RHAMM at sufficient levels to activate RHAMM-specific T cells, regardless of electroporation. Moreover, we found that RHAMM-specific T cells are present at vaccination sites in AML patients. Our findings implicate that we and others who are using classical mo-DCs for cancer immunotherapy are already vaccinating against RHAMM.

Interleukin (IL)-15 is one of the most promising molecules to be used in antitumor immune therapy, as it is able to stimulate the main killer cells of both the innate and adaptive immune system. Although this cytokine can be used as a stand-alone immunotherapeutic agent, IL-15 will probably be most efficient in combination with other strategies to overcome high tumor burden, immune suppression of the tumor microenvironment and/or the short half-life of IL-15. In this review, we will discuss the combination strategies with IL-15 that have been tested to date in different animal tumor models, which include chemotherapy, other immunostimulatory cytokines, targeted therapy, adoptive cell transfer and gene therapy. In addition, we give an overview of IL-15 combination therapies that are currently tested in clinical studies to treat patients with hematological or advanced solid tumors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Infection with human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in solid organ and hematopoietic stem cell transplant (HSCT) recipients., Methods: The present study explored the safety, feasibility, and immunogenicity of CMV pp65 messenger RNA-loaded autologous monocyte-derived dendritic cells (DC) as a cellular vaccine for active immunization in healthy volunteers and allogeneic HSCT recipients. Four CMV-seronegative healthy volunteers and three allogeneic HSCT recipients were included in the study. Four clinical-grade autologous monocyte-derived DC vaccines were prepared after a single leukapheresis procedure and administered intradermally at a weekly interval., Results: De novo induction of CMV-specific T-cell responses was detected in three of four healthy volunteers without serious adverse events. Of the HSCT recipients, none developed CMV disease and one of two patients displayed a remarkable threefold increase in CMV pp65-specific T cells on completion of the DC vaccination trial., Conclusion: In conclusion, our DC vaccination strategy induced or expanded a CMV-specific cellular response in four of six efficacy-evaluable study subjects, providing a base for its further exploration in larger cohorts.

Background Aims: Dendritic cell (DC)-based immunotherapy has shown potential to counteract autoimmunity in multiple sclerosis (MS)., Methods: We compared the phenotype and T-cell stimulatory capacity of in vitro generated monocyte-derived DC from MS patients with those from healthy controls., Results: Except for an increase in the number of C-C chemokine receptor 7-expressing DC from MS patients, no major differences were found between groups in the expression of maturation-associated membrane markers or in the in vitro capacity to stimulate autologous T cells., Conclusions: Our observations may pave the way for the development of patient-tailored DC-based vaccination strategies to treat MS., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: The role of the adaptive immune system and more specifically T cells in the pathogenesis of multiple sclerosis (MS) has been studied extensively. Emerging evidence suggests that dendritic cells (DCs), which are innate immune cells, also contribute to MS., Objectives: This study aimed to characterize circulating DC populations in MS and to investigate the contribution of MS-associated genetic risk factors to DCs., Methods: Ex vivo analysis of conventional (cDCs) and plasmacytoid DCs (pDCs) was carried out on peripheral blood of MS patients (n = 110) and age- and gender-matched healthy controls (n = 112)., Results: Circulating pDCs were significantly decreased in patients with chronic progressive MS compared to relapsing-remitting MS and healthy controls. While no differences in cDCs frequency were found between the different study groups, HLA-DRB1*1501(+) MS patients and patients not carrying the protective IL-7Rα haplotype 2 have reduced frequencies of circulating cDCs and pDCs, respectively. MS-derived DCs showed enhanced IL-12p70 production upon TLR ligation and had an increased expression of the migratory molecules CCR5 and CCR7 as well as an enhanced in vitro chemotaxis., Conclusion: DCs in MS are in a pro-inflammatory state, have a migratory phenotype and are affected by genetic risk factors, thereby contributing to pathogenic responses.

In this chapter, we describe the technique of electroporation as an efficient method to load primary leukemic cells with the double-stranded RNA (dsRNA) analogue, polyriboinosinic polyribocytidylic acid (poly(I:C)), and detail on the delicate freezing and thawing procedure of primary leukemic cells.Electroporation is a non-viral gene transfer method by which short-term pores in the membrane of cells are generated by an electrical pulse, allowing molecules to enter the cell. RNA electroporation, a technique developed in our laboratory, is a widely used and versatile transfection method for efficient introduction of both coding RNA (messenger RNA) and non-coding RNA, e.g., dsRNA and small interfering (siRNA), into mammalian cells. Accurate cell processing and storage of patient material is essential for optimal recovery and quality of the cell product for downstream applications.

Background: In an effort to raise protective antiviral immunity, dendritic cell immunotherapy was evaluated in six adults infected with human immunodeficiency virus (HIV)-1 and stable under highly active antiretroviral therapy (HAART)., Design and Methods: Autologous monocyte-derived dendritic cells electroporated with mRNA encoding Gag and a chimeric Tat-Rev-Nef protein were administered, whereas patients remained on HAART. Feasibility, safety, immunogenicity and antiviral responses were investigated., Results: Dendritic cell vaccine preparation and administration were successful in all patients and only mild adverse events were seen. There was a significant increase post-dendritic cell as compared to pre-dendritic cell vaccination in magnitude and breadth of HIV-1-specific interferon (IFN)-γ response, in particular to Gag, and in T-cell proliferation. Breadth of IFN-γ response and T-cell proliferation were both correlated with CD4(+) and CD8(+) polyfunctional T-cell responses. Importantly, dendritic cell vaccination induced or increased the capacity of autologous CD8(+) T cells to inhibit superinfection of CD4(+) T cells with the vaccine-related IIIB virus and some but not all other HIV-1 strains tested. This HIV-1-inhibitory activity, indicative of improved antiviral response, was correlated with magnitude and breadth of Gag-specific IFN-γ response., Conclusions: Therapeutic immunization with dendritic cells was safe and successful in raising antiviral cellular immune responses, including effector CD8(+) T cells with virus inhibitory activity. The stimulation of those potent immunological and antiviral effects, which have been associated with control of HIV-1, underscores the potential of dendritic cell vaccination in the treatment of HIV-1. The incomplete nature of the response in some patients helped to identify potential targets for future improvement, that is increasing antigenic spectrum and enhancing T-cell response.

A variety of immune-based therapies has been developed in order to boost or induce protective CD8(+) T cell responses in order to control HIV replication. Since dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique capability to stimulate naïve T cells into effector T cells, their use for the induction of HIV-specific immune responses has been studied intensively. In the present study we investigated whether modulation of the activation state of DCs electroporated with consensus codon-optimized HxB2 gag mRNA enhances their capacity to induce HIV gag-specific T cell responses. To this end, mature DCs were (i) co-electroporated with mRNA encoding interleukin (IL)-12p70 mRNA, or (ii) activated with a cytokine cocktail consisting of R848 and interferon (IFN)-γ. Our results confirm the ability of HxB2 gag-expressing DCs to expand functional HIV-specific CD8(+) T cells. However, although most of the patients had detectable gag-specific CD8(+) T cell responses, no significant differences in the level of expansion of functional CD8(+) T cells could be demonstrated when comparing conventional or immune-modulated DCs expressing IL-12p70. This result which goes against expectation may lead to a re-evaluation of the need for IL-12 expression by DCs in order to improve T-cell responses in HIV-1-infected individuals.

The cytotoxic and regulatory antitumor functions of natural killer (NK) cells have become attractive targets for immunotherapy. Manipulation of specific NK cell functions and their reciprocal interactions with dendritic cells (DCs) might hold therapeutic promise. In this review, we focus on the engagement of NK cells in DC-based cancer vaccination strategies, providing a comprehensive overview of current in vivo experimental and clinical DC vaccination studies encompassing the monitoring of NK cells. From these studies, it is clear that NK cells play a key regulatory role in the generation of DC-induced antitumor immunity, favoring the concept that targeting both innate and adaptive immune mechanisms may synergistically promote clinical outcome. However, to date, DC vaccination trials are only infrequently accompanied by NK cell monitoring. Here, we discuss different strategies to improve DC vaccine preparations via exploitation of NK cells and provide a summary of relevant NK cell parameters for immune monitoring. We underscore that the design of DC-based cancer vaccines should include the evaluation of their NK cell stimulating potency both in the preclinical phase and in clinical trials.

The knowledge that our immune system can be exploited for control or even eradication of acute myeloid leukemia (AML) has sparked a strong interest in therapeutic vaccine strategies to mount effective anti-leukemic immunity in AML patients. One of the most tantalizing approaches in this regard involves the use of dendritic cell-based vaccines. Dendritic cells (DCs) are antigen-presenting cells, capable of inducing anti-leukemic immune responses directed against leukemia-associated antigens. They can be obtained in high numbers following in vitro differentiation of peripheral blood monocytes. Research efforts are now focused on optimizing in vitro culture conditions and antigen loading strategies of DCs in order to maximize their potential to induce anti-leukemic immunity. Here, we will highlight some important aspects in the design of a potent DC vaccine for AML. We also discuss the importance of natural killer cells and combination strategies to further improve the outcome of DC-based vaccination in AML patients.

Interferon (IFN)-gamma ELISPOT can be used to monitor the magnitude of virus-specific cellular immune responses in vaccine trials. Often, IFN-gamma ELISPOT is performed with cryopreserved peripheral blood mononuclear cells (PBMC). However, it has not been well defined yet to what extent diminished cell viability of PBMC following cryopreservation affects IFN-gamma responses in ELISPOT. Therefore, we assessed the influence of apoptotic cells on the number of spot-forming cells (SFC) in IFN-gamma ELISPOT using a gradient of UV-irradiated apoptotic PBMC and viral antigens derived from varicella zoster virus (VZV) and cytomegalovirus (CMV). No SFC were observed when UV-irradiated apoptotic cells were stimulated with VZV or CMV antigens. Moreover, presence of apoptotic cells among viable T cells hampered the detection of SFC following stimulation with VZV or CMV cell lysates, but not with CMVpp65 peptide pool. Statistical analysis showed that mainly late apoptotic cells, staining both Annexin V and 7-amino-actinomycin D (7-AAD), were associated with a decreased number of SFC. In conclusion, it is recommended to use highly viable thawed PBMC for the detection of virus-specific cellular immune responses by IFN-gamma ELISPOT, since the detection of CMV- and VZV-specific T cell responses stimulated by cell lysates was significantly impeded by the presence of apoptotic cells.

In view of their potent antigen-presenting capacity and ability to induce effective immune responses, dendritic cells (DCs) have become an attractive target for therapeutic manipulation of the immune system. The application of tumor-associated antigen (TAA)-expressing DCs for cancer therapy has been the subject of intensive translational investigation. Previous clinical trials demonstrated tumor-specific immune responses without any significant toxicity. However, the clinical success has been modest, because only a limited number of immunized patients demonstrated cancer regression. Considerable progress has been made in the knowledge of DC biology, which opens new avenues for the development of optimized clinical protocols. One such promising approach that might carve its place in the future of DC-based therapy is the use of gene-modified DCs. DCs engineered to express TAAs allow multiepitope presentation by both major histocompatibility complex (MHC) class I and II molecules of full-length TAAs independent of the patient's HLA constitution, as opposed to peptide vaccination strategies. Besides transgene TAA expression, DCs can be genetically modified (1) to express a variety of immune-potentiating molecules (e.g., costimulatory molecules, cytokines, and chemokines) or (2) to downregulate negative modulators of DC functioning, all allowing an enhancement of their immunogenic potential. In the present review, gene delivery systems for DCs are discussed, as well as the various transgenes used for genetic modification of DCs. Moreover, a detailed review of the already published trials using gene-modified DCs is presented and future DC-based strategies targeting multiple layers of the complex cellular immune response are highlighted.

Natural killer (NK) cells are key players of innate immunity. Besides their major cytotoxic function, NK cells can also produce inflammatory cytokines such as interferon (IFN)-gamma. In this way, NK cells can shape adaptive immune responses through activation of dendritic cells (DC), thereby promoting the bidirectional cross-talk between NK cells and DC. Including this helper function of NK cells in cancer vaccination might be important for the induction of effective T cell responses. Here, we explored the capacity of purified human NK cells to produce IFN-gamma upon two-signal stimulation using different types of acute myeloid leukemia (AML) cells and type I IFN. Based on our previous findings that AML cells produce IFN-alpha upon electroporation with the synthetic double-stranded (ds)RNA polyriboinosinic polyribocytidylic acid (poly(I:C)), we hypothesized that dsRNA-loaded tumor cells provide both signals to elicit an NK cell-driven IFN-gamma production. Our results show that in vitro, NK cells become strong IFN-gamma-secreting cells upon stimulation with specific AML cells and IFN-alpha, with a variable responsiveness against different AML cell lines. Importantly, loading of AML cells with poly(I:C) is an elegant method to provide NK cells with both signals, a feature that could have important clinical implications because it obviates the side effects of systemic cytokine administration. Moreover, in addition to our previous findings that DC become activated upon phagocytosis of poly(I:C)-electroporated AML cells, these data strongly encourage future research on the potential of dsRNA-transfected AML cells and their effect to favor NK-DC cross-talk for the design of leukemia vaccines.

Objective: Recent studies proposed a pathogenic role for C-reactive protein (CRP), an independent predictor of cardiovascular disease (CVD), in atherosclerosis. Therefore, we tested whether CRP may modulate dendritic cell (DC) function, because these professional antigen-presenting cells have been implicated in atherogenesis., Methods and Results: Human monocyte-derived immature DCs were cultured with human CRP (0 to 60 microg/mL) for 24 hours. Thereafter, activation markers were measured by flow-cytometry and DCs were cocultured with CFSE-labeled lymphocytes to measure T-cell proliferation and interferon (IFN)-gamma secretion after 8 days. Exposure to 60 microg/mL CRP (n=5) induced an activated cell morphology and significant (CD40 increase MFI 5.23+/-0.28, P<0.01 paired t test; CD80 6.18+/-0.51, P<0.01) to modest (CD83 1.38+/-0.17, P<0.05, CCR7 1.60+/-0.29, P=0.05) upregulation of DC activation markers. The expression of CD86 and HLA-DR was high, but not affected. T-lymphocytes incubated with CRP-pulsed DCs displayed increased IFN-gamma secretion and proliferation (P<0.001). DC activation was concentration-dependent and detected from 2 mug/mL CRP; the maximum effect was equivalent to that seen with 0.1 microg/mL lipopolysaccharide (LPS). Polymyxin B abolished the LPS response, without influencing CRP effects. Finally, immunohistochemistry could demonstrate DC/CRP colocalization in human atherosclerotic lesions., Conclusions: These findings suggest that CRP in plaques or found circulating in CVD patients can influence DC function during atherogenesis.

The arsenal of therapeutic weapons against hematological malignancies is constantly growing. Unravelling the secrets of tumor immunobiology has allowed researchers to manipulate the immune system in order to stimulate tumor immunity or to bypass tumor-induced immunosuppression. An area of great interest is active specific immunotherapy where dendritic cell (DC)-based therapeutic vaccines for cancer have definitely grabbed the spotlight. DC are intensively investigated as cellular adjuvants to harness the immune system to fight off cancer by augmenting the number and effector functions of tumor-specific CD8+ cytotoxic T lymphocytes. In the present review we present a comprehensive synopsis and an update of the use of DC in hematological malignancies. In the future, more basic research as well as more clinical trials are warranted to fully establish the value of DC vaccination as an adjuvant therapy for modern hematological oncology.

In the field of nonviral gene therapy, mRNA-based gene transfer has generated much interest over the last decade. The combination of RNA as a versatile protein delivery molecule and the dendritic cell as the most potent antigen-presenting cell is an attractive approach to induce cellular and potentially therapeutic immune responses in patients with cancer. The success of mRNA transfection stems from its superior cytoplasmic expression efficiency, simplicity over viral transduction protocols, and clinical safety profile (due to a strictly transient expression and inability to integrate into the host genome). Most researchers have exploited low-voltage electrical pulses (electroporation) as a means to introduce RNA into cells, but other methods with an even higher degree of in vivo applicability are under development. Recently, more insights into the immunological properties of RNA and optimized strategies to produce highly translatable mRNA have increased its efficacy in cellular vaccination. In addition, application of RNA gene transfer into areas other than immunotherapy are slowly emerging and underscore the potential of RNA transfection as a versatile gene therapy tool.

Background: Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity., Results: In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711-20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-gamma ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-gamma-secreting CD8+ T cells in 5/5 healthy donors., Conclusion: We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.