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Success of dendritic cell (DC) therapy in treating malignancies is depending on the DC capacity to attract immune effector cells, considering their reciprocal crosstalk is partially regulated by cell-contact-dependent mechanisms. Although critical for therapeutic efficacy, immune cell recruitment is a largely overlooked aspect regarding optimization of DC vaccination. In this paper we have made a head-to-head comparison of interleukin (IL)-15-cultured DCs and conventional IL-4-cultured DCs with regard to their proficiency in the recruitment of (innate) immune effector cells. Here, we demonstrate that IL-4 DCs are suboptimal in attracting effector lymphocytes, while IL15 DCs provide a favorable chemokine milieu for recruiting CD8+ T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines involved in antitumor immune effector cell attraction, while IL-4 DCs display a more immunoregulatory profile characterized by the expression of Th2 and regulatory T cell-attracting chemokines. This is confirmed by functional data indicating an enhanced recruitment of granzyme B+ effector lymphocytes by IL-15 DCs, as compared to IL-4 DCs, and subsequent superior killing of tumor cells by the migrated lymphocytes. Elevated CCL4 gene expression in IL-15 DCs and lowered CCR5 expression on both migrated γδ T cells and NK cells, led to validation of increased CCL4 secretion by IL15 DCs. Moreover, neutralization of CCR5 prior to migration resulted in an important inhibition of γδ T cell and NK cell recruitment by IL-15 DCs. These findings further underscore the strong immunotherapeutic potential of IL-15 DCs.

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.

First described in the 1970s, dendritic cells (DC) are currently subjects of intense investigation to exploit their unique antigen-presenting and immunoregulatory capacities. In cancer, DC show promise to elicit or amplify immune responses directed against cancer cells by activating natural killer (NK) cells and tumor antigen-specific T cells. Wilms' tumor 1 (WT1) protein is a tumor-associated antigen that is expressed in a majority of cancer types and has been designated as an antigen of major interest to be targeted in clinical cancer immunotherapy trials. In this chapter, we describe the generation, cryopreservation, and thawing of clinical grade autologous monocyte-derived DC vaccines that are loaded with WT1 by messenger RNA (mRNA) electroporation. This in-house-developed transfection method gives rise to presentation of multiple antigen epitopes and can be used for all patients without restriction of human leukocyte antigen (HLA) type.

In cancer immunotherapy, the use of dendritic cell (DC)-based vaccination strategies can improve overall survival, but until now durable clinical responses remain scarce. To date, DC vaccines are designed primarily to induce effective T-cell responses, ignoring the antitumor activity potential of natural killer (NK) cells. Aiming to further improve current DC vaccination outcome, we engineered monocyte-derived DC to produce interleukin (IL)-15 and/or IL-15 receptor alpha (IL-15Rα) using mRNA electroporation. The addition of IL-15Rα to the protocol, enabling IL-15 transpresentation to neighboring NK cells, resulted in significantly better NK-cell activation compared to IL-15 alone. Next to upregulation of NK-cell membrane activation markers, IL-15 transpresentation resulted in increased NK-cell secretion of IFN-γ, granzyme B and perforin. Moreover, IL-15-transpresenting DC/NK cell cocultures from both healthy donors and acute myeloid leukemia (AML) patients in remission showed markedly enhanced cytotoxic activity against NK cell sensitive and resistant tumor cells. Blocking IL-15 transpresentation abrogated NK cell-mediated cytotoxicity against tumor cells, pointing to a pivotal role of IL-15 transpresentation by IL-15Rα to exert its NK cell-activating effects. In conclusion, we report an attractive approach to improve antitumoral NK-cell activity in DC-based vaccine strategies through the use of IL-15/IL-15Rα mRNA-engineered designer DC.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.

The contribution of natural killer (NK) cells to the treatment efficacy of dendritic cell (DC)-based cancer vaccines is being increasingly recognized. Much current efforts to optimize this form of immunotherapy are therefore geared towards harnessing the NK cell-stimulatory ability of DCs. In this study, we investigated whether generation of human monocyte-derived DCs with interleukin (IL)-15 followed by activation with a Toll-like receptor stimulus endows these DCs, commonly referred to as "IL-15 DCs", with the capacity to stimulate NK cells. In a head-to-head comparison with "IL-4 DCs" used routinely for clinical studies, IL-15 DCs were found to induce a more activated, cytotoxic effector phenotype in NK cells, in particular in the CD56bright NK cell subset. With the exception of GM-CSF, no significant enhancement of cytokine/chemokine secretion was observed following co-culture of NK cells with IL-15 DCs. IL-15 DCs, but not IL-4 DCs, promoted NK cell tumoricidal activity towards both NK-sensitive and NK-resistant targets. This effect was found to require cell-to-cell contact and to be mediated by DC surface-bound IL-15. This study shows that DCs can express a membrane-bound form of IL-15 through which they enhance NK cell cytotoxic function. The observed lack of membrane-bound IL-15 on "gold-standard" IL-4 DCs and their consequent inability to effectively promote NK cell cytotoxicity may have important implications for the future design of DC-based cancer vaccine studies.

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Owing to their professional antigen-presenting capacity and unique potential to induce tumor antigen-specific T cell immunity, dendritic cells (DCs) have attracted much interest over the past decades for therapeutic vaccination against cancer. Clinical trials have shown that the use of tumor antigen-loaded DCs in cancer patients is safe and that it has the potential to induce anti-tumor immunity which, in some cases, culminates in striking clinical responses. Unfortunately, in a considerable number of patients, DC vaccination is unable to mount effective anti-tumor immune responses and, if it does so, the resultant immunity is often insufficient to translate into tangible clinical benefit. This underscores the necessity to re-design and optimize the current procedures for DC vaccine manufacturing. A new generation of DC vaccines with improved potency has now become available for clinical use as a result of extensive pre-clinical research. One of the promising next-generation DC vaccine candidates are interleukin (IL)-15-differentiated DCs. In this commentary, we will compile the research data that have been obtained by our group and other groups with these so-called IL-15 DCs and summarize the evidence supporting the implementation of IL-15 DCs in DC-based cancer vaccination regimens.

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.

Dendritic cells (DC) have the capacity to induce potent tumor antigen-specific T-cell immunity. We have completed vaccination in the adjuvant setting in 77 cancer patients (acute myeloid leukemia (AML, n=30), metastatic breast cancer (MBC, n=12), glioblastoma multiforme (GBM, n=13), malignant pleural mesothelioma (MPM, n=10) and other solid tumors (n=12)) with autologous DC electroporated with mRNA encoding the nearly universal tumor-associated antigen Wilms' tumor 1 protein (WT1). WT1-targeted DC vaccination was feasible and safe in all patients. The majority of the patients showed a positive delayed type hypersensitivity (DTH) response to the vaccine. Objective clinical responses were observed among all tumor types, including complete (CR) and/or molecular remissions or stable disease (SD) in the AML group, partial responses (PR) and SD in the GBM group and SD in the MBC and MPM groups. In AML patients in first CR, median overall survival (OS) calculated from time of diagnosis was 56.1 months (mo). Median OS from time of diagnosis of metastasis was 41.9 mo in MBC, 43.7 mo from diagnosis in GBM and 35.7 mo from start of therapy in MPM; this compares favorably to numbers reported in the literature, respectively 24.8 mo, 14.7 mo and 22 mo. In AML, long-term OS was correlated with WT1-specific polyfunctional CD8+ T-cells in the DTH reaction sites and long-term CR with polyepitope WT1-specific tetramer+ CD8+ T-lymphocytes. In solid tumors, PR or SD was correlated with interferon (IFN)-gamma+ and/or tumor necrosis factor (TNF)-alpha+ WT1-specific CD4+ and/or CD8+ T-cells; increased OS in the GBM+MPM cohorts was correlated with IFN-gamma+ WT1-specific CD4+ T-lymphocytes. In conclusion, WT1-targeted DC vaccination is feasible, safe and immunogenic, and displays relevant anti-tumor activity in patients with hematological and solid malignancies. Most importantly, this treatment modality can confer a significant survival benefit to the patients. [ABSTRACT FROM AUTHOR]