Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

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.

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells.

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.

Except for C-C chemokine receptor 7 expression, monocyte-derived dendritic cells from patients with multiple sclerosis are functionally comparable to those of healthy controls.

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.

Circulating dendritic cells of multiple sclerosis patients are proinflammatory and their frequency is correlated with MS-associated genetic risk factors.

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.

Active specific immunotherapy targeting the Wilms' tumor protein 1 (WT1) for patients with hematological malignancies and solid tumors: lessons from early clinical trials.

There is a growing body of evidence that Wilms' tumor protein 1 (WT1) is a promising tumor antigen for the development of a novel class of universal cancer vaccines. Recently, in a National Cancer Institute prioritization project, WT1 was ranked first in a list of 75 cancer antigens. In this light, we exhaustively reviewed all published cancer vaccine trials reporting on WT1-targeted active specific immunotherapy in patients with hematological malignancies and solid tumors. In all clinical trials, vaccine-induced immunological responses could be detected. Importantly, objective clinical responses (including stable disease) were observed in 46% and 64% of evaluable vaccinated patients with solid tumors and hematological malignancies, respectively. Immunogenicity of WT1-based cancer vaccines was demonstrated by the detection of a specific immunological response in 35% and 68% of evaluable patients with solid tumors and hematological malignancies, respectively. In order to become part of the armamentarium of the modern oncologist, it will be important to design WT1-based immunotherapies applicable to a large patient population, to standardize vaccination protocols enabling systematic review, and to further optimize the immunostimulatory capacity of the vaccine components. Moreover, improved immunomonitoring tools that reveal clinically relevant T-cell responses will further shape the ideal WT1 immunotherapy strategy. In conclusion, the clinical results obtained so far in WT1-targeted cancer vaccine trials reveal an untapped potential for inducing cancer immunity with minimal side effects and hold promise for a new adjuvant treatment against residual disease and against cancer relapse.

NK cells: key to success of DC-based cancer vaccines?

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.

Interleukin-12p70 expression by dendritic cells of HIV-1-infected patients fails to stimulate gag-specific immune responses.

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.

Clinical evaluation of cellular immunotherapy in acute myeloid leukaemia.

Immunotherapy is currently under active investigation as an adjuvant therapy to improve the overall survival of patients with acute myeloid leukaemia (AML) by eliminating residual leukaemic cells following standard therapy. The graft-versus-leukaemia effect observed following allogeneic haematopoietic stem cell transplantation has already demonstrated the significant role of immune cells in controlling AML, paving the way to further exploitation of this effect in optimized immunotherapy protocols. In this review, we discuss the current state of cellular immunotherapy as adjuvant therapy for AML, with a particular focus on new strategies and recently published results of preclinical and clinical studies. Therapeutic vaccines that are being tested in AML include whole tumour cells as an autologous source of multiple leukaemia-associated antigens (LAA) and autologous dendritic cells loaded with LAA as effective antigen-presenting cells. Furthermore, adoptive transfer of cytotoxic T cells or natural killer cells is under active investigation. Results from phase I and II trials are promising and support further investigation into the potential of cellular immunotherapeutic strategies to prevent or fight relapse in AML patients.

Dendritic cell vaccine therapy for acute myeloid leukemia: questions and answers.

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.

The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy.

Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.

The Toll-like receptor 7/8 agonist resiquimod greatly increases the immunostimulatory capacity of human acute myeloid leukemia cells.

Immunotherapy for leukemia is a promising targeted strategy to eradicate residual leukemic cells after standard therapy, in order to prevent relapse and to prolong the survival of leukemia patients. However, effective anti-leukemia immune responses are hampered by the weak immunogenicity of leukemic cells. Therefore, much effort is made to identify agents that could increase the immunogenicity of leukemic cells and activate the immune system. Synthetic agonists of Toll-like receptor (TLR)7 and TLR8 are already in use as anticancer treatment, because of their ability to activate several immune pathways simultaneously, resulting in effective antitumor immunity. However, for leukemic cells little is known about the expression of TLR7/8 and the direct effects of their agonists. We hypothesized that TLR7/8 agonist treatment of human acute myeloid leukemia (AML) cells would lead to an increased immunogenicity of AML cells. We observed expression of TLR7 and TLR8 in primary human AML cells and AML cell lines. Passive pulsing of primary AML cells with the TLR7/8 agonist R-848 resulted in increased expression of MHC molecules, production of proinflammatory cytokines, and enhanced allogeneic naïve T cell-stimulatory capacity. These effects were absent or suboptimal if R-848 was administered intracellularly by electroporation. Furthermore, when AML cells were cocultured with allogeneic PBMC in the presence of R-848, interferon (IFN)-gamma was produced by allogeneic NK and NKT cells and AML cells were killed. In conclusion, the immunostimulatory effect of the TLR7/8 agonist R-848 on human AML cells could prove useful for the design of TLR-based immunotherapy for leukemia.

Immunotherapy of acute myeloid leukemia: current approaches.

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.

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial.

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.

Human C-reactive protein activates monocyte-derived dendritic cells and induces dendritic cell-mediated T-cell activation.

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.

Chimeric Antigen Receptor-Modified T Cell Therapy in Multiple Myeloma: Beyond B Cell Maturation Antigen.

Chimeric antigen receptor (CAR)-modified T cell therapy is a rapidly emerging immunotherapeutic approach that is revolutionizing cancer treatment. The impressive clinical results obtained with CAR-T cell therapy in patients with acute lymphoblastic leukemia and lymphoma have fueled the development of CAR-T cells targeting other malignancies, including multiple myeloma (MM). The field of CAR-T cell therapy for MM is still in its infancy, but remains promising. To date, most studies have been performed with B cell maturation antigen (BCMA)-targeted CARs, for which high response rates have been obtained in early-phase clinical trials. However, responses are usually temporary, and relapses have frequently been observed. One of the major reasons for relapse is the loss or downregulation of BCMA expression following CAR-T therapy. This has fostered a search for alternative target antigens that are expressed on the MM cell surface. In this review, we provide an overview of myeloma target antigens other than BCMA that are currently being evaluated in pre-clinical and clinical studies.

The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy.

The importance of Toll-like receptors (TLRs) in stimulating innate and adaptive immunity is now well established. In view of this, TLR ligands have become interesting targets to use as stand-alone immunotherapeutics or vaccine adjuvants for cancer treatment. TLR7 and TLR8 were found to be closely related, sharing their intracellular endosomal location, as well as their ligands. In this review, we describe the agonists of TLR7 and TLR8 that are known so far, as well as their contribution to antitumor responses by affecting immune cells, tumor cells, and the tumor microenvironment. The major benefit of TLR7/8 agonists as immune response enhancers is their simultaneous stimulation of several cell types, resulting in a mix of activated immune cells, cytokines and chemokines at the tumor site. We discuss the studies that used TLR7/8 agonists as stand-alone immunotherapeutics or cancer vaccine adjuvants, as well as the potential of TLR7/8 ligands to enhance antitumor responses in passive immunotherapy approaches.

A maximal exercise bout increases the number of circulating CD34+/KDR+ endothelial progenitor cells in healthy subjects. Relation with lipid profile.

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally, we investigated possible mediators of this effect, namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1, n = 11; group 2, n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis, either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise, EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise, with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile.

Balancing between immunity and tolerance: an interplay between dendritic cells, regulatory T cells, and effector T cells.

Dendritic cells (DC), professional antigen-presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the main function of immature DC (iDC) in their in vivo steady-state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell-based therapies.

Dendritic Cells and Programmed Death-1 Blockade: A Joint Venture to Combat Cancer.

Two decades of clinical cancer research with dendritic cell (DC)-based vaccination have proved that this type of personalized medicine is safe and has the capacity to improve survival, but monotherapy is unlikely to cure the cancer. Designed to empower the patient's antitumor immunity, huge research efforts are set to improve the efficacy of next-generation DC vaccines and to find synergistic combinations with existing cancer therapies. Immune checkpoint approaches, aiming to breach immune suppression and evasion to reinforce antitumor immunity, have been a revelation in the immunotherapy field. Early success of therapeutic antibodies blocking the programmed death-1 (PD-1) pathway has sparked the development of novel inhibitors and combination therapies. Hence, merging immunoregulatory tumor-specific DC strategies with PD-1-targeted approaches is a promising path to explore. In this review, we focus on the role of PD-1-signaling in DC-mediated antitumor immunity. In the quest of exploiting the full potential of DC therapy, different strategies to leverage DC immunopotency by impeding PD-1-mediated immune regulation are discussed, including the most advanced research on targeted therapeutic antibodies, lessons learned from chemotherapy-induced immune activation, and more recent developments with soluble molecules and gene-silencing techniques. An overview of DC/PD-1 immunotherapy combinations that are currently under preclinical and clinical investigation substantiates the clinical potential of such combination strategies.

A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches.

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.