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We evaluate the anti-cancer capacity of plasma-treated PBS (pPBS), by measuring the concentrations of NO2− and H2O2 in pPBS, treated with a plasma jet, for different values of gas flow rate, gap and plasma treatment time, as well as the effect of pPBS on cancer cell cytotoxicity, for three different glioblastoma cancer cell lines, at exactly the same plasma treatment conditions. Our experiments reveal that pPBS is cytotoxic for all conditions investigated. A small variation in gap between plasma jet and liquid surface (10 mm vs 15 mm) significantly affects the chemical composition of pPBS and its anti-cancer capacity, attributed to the occurrence of discharges onto the liquid. By correlating the effect of gap, gas flow rate and plasma treatment time on the chemical composition and anti-cancer capacity of pPBS, we may conclude that H2O2 is a more important species for the anti-cancer capacity of pPBS than NO2−. We also used a 0D model, developed for plasma-liquid interactions, to elucidate the most important mechanisms for the generation of H2O2 and NO2−. Finally, we found that pPBS might be more suitable for practical applications in a clinical setting than (commonly used) plasma-activated media (PAM), because of its higher stability.

Around 30% of individuals will develop herpes zoster (HZ), caused by the varicella zoster virus (VZV), during their life. While several risk factors for HZ, such as immunosuppressive therapy, are well known, the genetic and molecular components that determine the risk of otherwise healthy individuals to develop HZ are still poorly understood. We created a computational model for the Human Leukocyte Antigen (HLA-A, -B, and -C) presentation capacity of peptides derived from the VZV Immediate Early 62 (IE62) protein. This model could then be applied to a HZ cohort with known HLA molecules. We found that HLA-A molecules with poor VZV IE62 presentation capabilities were more common in a cohort of 50 individuals with a history of HZ compared to a nationwide control group, which equated to a HZ risk increase of 60%. This tendency was most pronounced for cases of HZ at a young age, where other risk factors are less prevalent. These findings provide new molecular insights into the development of HZ and reveal a genetic predisposition in those individuals most at risk to develop HZ.

Personalized dendritic cell- (DC-) based vaccination has proven to be safe and effective as second-line therapy against various cancer types. In terms of overall survival, there is still room for improvement of DC-based therapies, including the development of more immunostimulatory DC vaccines. In this context, we redesigned our currently clinically used DC vaccine generation protocol to enable transpresentation of interleukin- (IL-) 15 to IL-15Rβγ-expressing cells aiming at boosting the antitumor immune response. In this study, we demonstrate that upon electroporation with both IL-15 and IL-15Rα-encoding messenger RNA, mature DC become highly positive for surface IL-15, without influencing the expression of prototypic mature DC markers and with preservation of their cytokine-producing capacity and their migratory profile. Functionally, we show that IL-15-transpresenting DC are equal if not better inducers of T-cell proliferation and are superior in tumor antigen-specific T-cell activation compared with DC without IL-15 conditioning. In view of the clinical use of DC vaccines, we evidence with a time- and cost-effective manner that clinical grade DC can be safely engineered to transpresent IL-15, hereby gaining the ability to transfer the immune-stimulating IL-15 signal towards antitumor immune effector cells.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

Aims During recent years, immune checkpoint inhibition has proved to be effective in several solid malignancies. The aim of this study was to identify novel targets for immunotherapy in anaplastic thyroid cancer by analysis of the expression of tumour antigens for which therapeutic agents are available. Method and results By immunohistochemistry we observed tumoral expression of CD70 in 49% of cases. Expression of its receptor, CD27, was present mainly in lymphocytes surrounding and infiltrating the tumour and observed only rarely in tumour cells. CD70 expression was associated with the presence of a precursor papillary thyroid carcinoma and the presence of BRAF V600E mutations in the anaplastic thyroid cancer lesion. Furthermore, the expression of CD70 seems stable during progression of the disease. Tumoral expression of programmed cell death ligand 1 (PD-L1) was found in 28.6% of the anaplastic thyroid cancer cases. Programmed cell death 1 (PD-1), the receptor of PD-L1, was not expressed on the tumour cells. No association between CD70 expression and PD-L1 expression could be demonstrated. Conclusion These data suggest that targeted immunotherapy for CD70/CD27 and PD-L1/PD-1 might be promising in anaplastic thyroid cancer. However, as a low amount of tumour-infiltrating lymphocytes was observed in most lesions, combined therapy with agents enhancing the invasion of lymphocytes in the tumour region needs to be considered.

Contains fulltext : 177224.pdf (Publisher’s version ) (Closed access) 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 IL15Ralpha mRNA, preserved their mature DC profile and rendered the DCs superior in inducing T-cell proliferation and IFNgamma and TNFalpha production. Translated into an ex vivo hematological disease setting, DCs deprived of PD-1 ligands (PD-L), equipped with IL15/IL15Ralpha 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. (c)2017 AACR.

Re-exposure to chickenpox may boost varicella-zoster virus (VZV) immunity in the elderly. This secondary immune response is hypothesized to confer protection against herpes zoster. We longitudinally sampled 36 adults over the course of one year after re-exposure to chickenpox. The resulting 183 samples and those of 14 controls were assessed for VZV-specific T-cell immunity and antibody titres. The percentages of VZV-specific CD4+ IL-2-producing T-cells were increased in reexposed grandparents compared to control participants up to 9 months after re-exposure. Using a longitudinal mixture modelling approach, we found that 25% and 17% of re-exposed grandparents showed a boosting of VZV-specific CD4+ IL-2-producing T-cells and VZV-specific antibodies, respectively. The antibody boosting occurred exclusively in cytomegalovirus (CMV) IgG-positive participants. CMV IgG-positive participants also had higher VZV IE62-specific CD4+ IFN-.-producing T-cell percentages and VZV-specific antibody titres. The protective effect of re-exposure to chickenpox is likely limited, as boosting only occurred in 17-25% of the VZV re-exposed grandparents and for less than one year. This work was supported by grants of the Research Foundation Flanders (predoctoral fellowship to B.O.; postdoctoral fellowship to E.L.; project grants G.0409.12N and G.0903.13N); the Hercules Foundation-Belgium and the University of Antwerp (Concerted Research Action; predoctoral fellowships to J.V.D.B. and A.V.; Methusalem funding).

// Elly Marcq 1 , Jorrit De Waele 1 , Jonas Van Audenaerde 1 , Eva Lion 2 , Eva Santermans 3 , Niel Hens 3, 4 , Patrick Pauwels 1, 5 , Jan P. van Meerbeeck 1, 6, * and Evelien L.J. Smits 1, 2, * 1 Center for Oncological Research, University of Antwerp, Antwerp, Belgium 2 Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium 3 Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium 4 Center for Health Economics Research and Modelling Infectious Diseases, University of Antwerp, Antwerp, Belgium 5 Department of Pathology, Antwerp University Hospital, Antwerp, Belgium 6 Thoracic Oncology/MOCA, Antwerp University Hospital, Antwerp, Belgium * Shared senior authors Correspondence to: Elly Marcq, email: Elly.Marcq@uantwerpen.be Keywords: mesothelioma, immune checkpoints, effusions, tumor microenvironment, flow cytometry Received: April 21, 2017 Accepted: September 01, 2017 Published: September 21, 2017 ABSTRACT Malignant pleural mesothelioma (MPM) is an aggressive cancer with an increasing incidence, poor prognosis and limited effective treatment options. Hence, new treatment strategies are warranted which include immune checkpoint blockade approaches with encouraging preliminary data. Research on the immunological aspects of the easily accessible mesothelioma microenvironment could identify prognostic and/or predictive biomarkers and provide useful insights for developing effective immunotherapy. In this context, we investigated the immune cell composition of effusions (pleural and ascites fluids) from 11 different chemotherapy-treated MPM patients. We used multicolor flow cytometry to describe different subsets of immune cells and their expression of immune checkpoint molecules TIM-3, LAG-3, PD-1 and PD-L1. We demonstrate a patient-dependent inter- and intraspecific variation comparing pleural and ascites fluids in immune cell composition and immune checkpoint expression. We found CD4 + and CD8 + T cells, B cells, macrophages, natural killer cells, dendritic cells and tumor cells in the fluids. To the best of our knowledge, we are the first to report TIM-3 and LAG-3 expression and we confirm PD-1 and PD-L1 expression on different MPM effusion-resident immune cells. Moreover, we identified two MPM effusion-related factors with clinical value: CD4+ T cells were significantly correlated with better response to chemotherapy, while the percentage of PD-L1 + podoplanin (PDPN) + tumor cells is a significant prognostic factor for worse outcome. Our data provide a basis for more elaborate research on MPM effusion material in the context of treatment follow-up and prognostic biomarkers and the development of immune checkpoint-targeted immunotherapy.

Introduction Glioblastoma (GBM) still remains one of the deadliest cancer types, emphasising the urgent need for novel treatments. While immunotherapy is generating promising results, combination strategies are warranted to unlock its full potential. Here, we investigated how poly(I:C) affects immunomodulation by GBM cells as well as potential to potentiate immune checkpoint blocking therapy, in particular programmed death ligand (PD-L) blockade. Material and methods Primary human GBM cells were cultured from residual tumour tissue obtained from standard surgery of GBM patients. Their phenotype following poly(I:C) treatment was investigated by flow cytometry, immunohistochemistry and multiplex electrochemiluminescence. Mechanistic studies were performed using qRT-PCR, blocking antibodies and chloroquine (inhibitor of Toll-like receptor 3 or TLR3 signalling). Cocultures with peripheral blood mononuclear cells were studied for lymphocyte migration using a transwell assay, and immune activation using flow cytometry and ELISA. Additional PD-L1 or PD-L2 blockade was evaluated using ELISA and CFSE-labelled CD8+ T-cell proliferation. Results and discussions Poly(IC) stimulated a pro-inflammatory secretome by GBM cells, including type I interferons (IFN), interleukin-15, reduced transforming growth factor-beta, and chemokines CXCL9, CXCL10, CCL4 and CCL5. These treated GBM cells doubled the attraction of CD8+ T cells, and to a lesser extent CD4+ T cells, partly via the CXCR3 and CCR5 ligands, but NK cell migration was not significantly affected. Furthermore, lymphocytes cocultured with poly(I:C)-treated GBM cells showed increased activation (CD69, IFN-gamma) and cytotoxic capacity (CD107a, granzyme B). Poly(I:C) treatment of GBM cells also increased their PD-L1 and PD-L2 expression. Here, poly(I:C) triggered TLR3, resulting in de novo protein, partly via downstream auto-/paracrine IFN-beta. In cocultures, additional blockade of PD-L1, but not PD-L2, strongly invigorated the immune activation. Our results suggest that poly(I:C) modulates GBM cells to prepare the tumour microenvironment for immune checkpoint blocking therapy. Chemokines attract CD8+ T cells to the tumour site, a prerequisite for effective PD-1/PD-L1 blockade, while other cytokines activate the immune cells. Concomitantly, PD-L1 is stimulated on the GBM cells, providing a target for additional PD-L1 blockade, which further propagates the immune activation. Conclusion In conclusion, our data proposes poly(I:C) to unlock PD-L1 blocking therapy in GBM.

Cancer metastasis is one of the leading causes of mortality in patients with solid tumors [1]. The complex metastatic process involves cell migration and remodeling of the tumor microenvironment, among other factors. The intrinsic limitations of conventional cancer therapies for solid tumors have motivated the development and application of alternative technologies. In this context, the use of low-temperature plasmas (LTPs) has been explored in vitro and in vivo with considerable success against a variety of cancers [2,3]. However, whether LTP can inhibit or promote metastasis of solid tumors is unknown. The aim of this study is to investigate the effect of direct and indirect LTP treatments on metastatic processes of glioblastoma, adenocarcinoma and pancreatic cancers. For this purpose, we used a 3-dimensional multicellular spheroid model that mimics the pathophysiological conditions and complex architecture of solid tumors [4]. Spheroids were generated using glioblastoma U87, U251 and LN229, adenocarcinoma MDA-MB-231 and human pancreatic carcinoma MIA PaCa-2 cell lines and glioblastoma primary cells GR04, GR08 and GR15. Direct and indirect plasma treatments were administered to spheroids in PBS solution using the COST plasma jet and kINPen IND. We used live cell imaging to assess cytotoxicity and spheroid morphology, microscopy to monitor cell migration and immunohistochemistry to assess extracellular matrix remodeling in response to direct and indirect LTP treatment. Our study provides insight on the application of LTP treatment as potential cancer therapy for solid tumors. Download : Download high-res image (421KB) Download : Download full-size image LN229 spheroids in PBS: (a) untreated control, directly treated with (b) kINPen IND or (c) COST jet. Outer border: total spheroid area; dark filled area: proliferative core (live cells); grey dots: dead cells.

Tumours are complex systems formed by cellular (malignant, immune, and endothelial cells, fibroblasts) and acellular components (extracellular matrix (ECM) constituents and secreted factors). A close interplay between these factors, collectively called the tumour microenvironment, is required to respond appropriately to external cues and to determine the treatment outcome. Cold plasma (here referred as ‘plasma’) is an emerging anticancer technology that generates a unique cocktail of reactive oxygen and nitrogen species to eliminate cancerous cells via multiple mechanisms of action. While plasma is currently regarded as a local therapy, it can also modulate the mechanisms of cell-to-cell and cell-to-ECM communication, which could facilitate the propagation of its effect in tissue and distant sites. However, it is still largely unknown how the physical interactions occurring between cells and/or the ECM in the tumour microenvironment affect the plasma therapy outcome. In this review, we discuss the effect of plasma on cell-to-cell and cell-to-ECM communication in the context of the tumour microenvironment and suggest new avenues of research to advance our knowledge in the field. Furthermore, we revise the relevant state-of-the-art in three-dimensional in vitro models that could be used to analyse cell-to-cell and cell-to-ECM communication and further strengthen our understanding of the effect of plasma in solid tumours.

Dendritic cell-based and other vaccination strategies that use the patient’s own immune system for the treatment of cancer are gaining momentum. Most studies of therapeutic cancer vaccination have been performed in adults. However, since cancer is one of the leading causes of death among children past infancy in the Western world, the hope is that this form of active specific immunotherapy can play an important role in the pediatric population as well. Since children have more vigorous and adaptable immune systems than adults, therapeutic cancer vaccines are expected to have a better chance of creating protective immunity and preventing cancer recurrence in pediatric patients. Moreover, in contrast to conventional cancer treatments such as chemotherapy, therapeutic cancer vaccines are designed to specifically target tumor cells and not healthy cells or tissues. This reduces the likelihood of side effects, which is an important asset in this vulnerable patient population. In this review, we present an overview of the different therapeutic cancer vaccines that have been studied in the pediatric population, with a main focus on dendritic cell-based strategies. In addition, new approaches that are currently being investigated in clinical trials are discussed to provide guidance for further improvement and optimization of pediatric cancer vaccines.

Encouraging results from clinical trials demonstrate that dendritic cell (DC) vaccination is a valuable tool in cancer immunotherapy. Empowering DC vaccine immunogenicity to improve clinical outcome is at center stage in the rapidly evolving immunotherapeutic landscape. Developing a unique type of DC vaccine, so-called interleukin (IL)-15 DCs generated with IL-15 and strong immune stimulating maturation signals, we could demonstrate superior in vitro activities at both adaptive as well as innate immunity as compared to gold-standard IL-4 DCs. In this light, IL-15 DCs are capable of activating NK cells and gamma-delta T-cells, whereas IL-4 DCs are not. Designed to induce durable antitumor T-cell immunity, this pre-clinical research focuses on the improvement of Wilms’ Tumor protein 1 (WT1)-targeted T-cell activity. The WT1 antigen is overexpressed in a wide variety of human cancers and thus appears to fulfill the criteria of a universal tumor-associated antigen. The capacity of our novel IL-15 DCs to induce WT1-specific immunity is assessed using two in-house developed tumor antigen-specific T-cell assays. The first series of experiments demonstrate in a head-to-head comparison that WT1 mRNA-electroporated IL-15 DCs perform at least as well as IL-4 DCs in triggering a WT1 T-cell receptor (TCR)-transfected T-cell line. Using an autologous primary TCR-engineered CD8+ T-cell approach, we are now comparing their WT1-specific antitumor function. With its unique characteristics, including IL-15-transpresentation and interferon-gamma secretion, IL-15 DCs are expected to significantly promote WT1-specific T-cell-mediated tumor cell killing. Enabling its clinical application, we also evaluated the upscaled IL 15 DC vaccine production and cryopreservation processes. We were able to employ the three-day culture protocol in clinically suitable culture flasks. In addition, the use of an optimized cryopreservation medium results in a pre- to-post cryopreservation yield of 81.3 ± 7.4 % with a preserved phenotype and functionality. High-scale production and cryopreservation allows for the implementation of DC vaccination in multimodal treatment schedules. Underscoring the tumor antigen-specific T-cell-stimulating capacity among previously described superior characteristics and warranting batch production of patient-specific DC vaccines, further strengthens the impetus to bring WT1-loaded IL-15 DCs to the clinic. Citation Format: Maarten Versteven, David Damoiseaux, Diana Campillo-Davo, Heleen Van Acker, Hans De Reu, Sébastien Anguille, Zwi N Berneman, Evelien L Smits, Viggo F Van Tendeloo, Eva Lion. Preclinical evaluation of a Wilms’ tumor protein 1-targeted interleukin-15 dendritic cell vaccine: T-cell activity and batch production [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B137.

Oncoimmunologists are in a constant search for more effective immunotherapeutic treatments, helping to cure cancer. In this respect, immune cell participation is a key issue. A particularly interesting marker to identify antitumor immune cells is the neural cell adhesion molecule (NCAM), known as CD56. Namely, hematopoietic expression of CD56 seems to be confined to activated immune cells exhibiting some level of cytotoxic properties (Van Acker et al., Frontiers in Immunology 2017). Unfortunately, the current knowledge on the expression and functional role of CD56 is very fragmented. Therefore, we sought to elucidate the role of CD56 expression on various killer immune cells. First, we identified the high motility NCAM-120 isoform to be the main subset on immune cells. Next, through neutralization of surface CD56, we were able to demonstrate for the first time a direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells such as natural killer cells, gamma delta (γδ) T-cells and interleukin (IL)-15-cultured dendritic cells (DCs). We also detected a putative crosstalk mechanism, suggesting CD56 as a co-stimulatory molecule in the interaction between IL-15 DCs and CD8 T-cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)–AKT pathway, with respectively trametinib and afuresertib, we confirmed our hypothesis that IL-15 stimulation directly leads to CD56 upregulation via the recruitment of shc, binding a phosphotyrosine residue on the IL-2/15Rβ chain. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options. Citation Format: Heleen H. Van Acker, Maarten Versteven, Hans De Reu, Peter Ponsaerts, Zwi N Berneman, Viggo F. Van Tendeloo, Evelien L. Smits. CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B192.

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.

Colorectal cancer is one of the most prevalent types of cancer worldwide and a leading cause of cancer-related mortality. The reported incidence of recurrent disease increases with the stage, and the 5-year survival rate is poor, especially for tumors with regional spread and for late-stage disease. This has prompted the search for more advanced treatment options, and therefore avenues for clinical testing of rationally designed immunotherapeutic strategies, including vaccination, as adjuvant treatment are being explored. In this review, we discuss the study design and results of the clinical trials that have been conducted using active specific immunotherapy in colorectal cancer, including autologous and allogeneic tumor cell vaccines, peptide vaccines, viral-vector-based vaccines, and dendritic-cell-based vaccines as well as some future recommendations.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value. To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required. This work was performed with the support of the Belgian Foundation Against Cancer (grant number: FA/2014/263), the Research Foundation Flanders (grant number: 1510215N), AstraZeneca and a Methusalem grant of the University of Antwerp and Hasselt awarded to Prof. Herman Goossens and Prof Geert Molenberghs. E. Marcq is research fellow of Flanders Innovation & Entrepreneurship (fellowship number: 141433), J. De Waele and J. Van Audenaerde of the Research Foundation Flanders (fellowship numbers: 1121016N and 1S32316N).

The advent of new cell-based immunotherapies for leukemia offers treatment possibilities for certain leukemia subgroups. The wider acceptability of these new technologies in clinical practice will depend on its impact on survival and costs. Due to the small patient groups who have received it, these aspects have remained understudied. This non-randomized single-center study evaluated medical costs and survival for acute myeloid leukemia between 2005 and 2010 in 50 patients: patients treated with induction and consolidation chemotherapy (ICT) alone; patients treated with ICT plus allogeneic hematopoietic stem cell transplantation (HCT), which is the current preferred post-remission therapy in patients with intermediate- and poor-risk AML with few co-morbidities, and patients treated with ICT plus immunotherapy using autologous dendritic cells (DC) engineered to express the Wilms tumor protein (WT1). Total costs including post- consolidation costs on medical care at the hematology ward and outpatient clinic, pharmaceutical prescriptions, intensive care ward, laboratory tests and medical imaging were analyzed. Survival was markedly better in HCT and DC. HCT and DC were more costly than ICT. The median total costs for HCT and DC were similar. These results need to be confirmed to enable more thorough cost-effectiveness analyses, based on observations from multicenter, randomized clinical trials and preferably using quality-adjusted life-years as an outcome measure.

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.

Dendritic cells (DCs) are a specialized family of professional antigen presenting cells that serve as a bridge linking the innate and adaptive arms of our immune system. DCs sense pathogens or interact with harmless antigens or nonpathogenic bacteria thereby tightly regulating the balance between tolerance and immunity. Despite their indispensable role in eliciting immune responses, DCs are a rather rare and heterogeneous type of immune cell, which differ in phenotype and function depending on maturation status, subsets, and age as well as their localization and microenvironment. Although scarce in numbers, cultured or naturally occurring DCs have been extensively investigated in clinical trials for both their capacity of priming antigen specific cytotoxic and helper T cells and humoral responses and their potential to induce immunological memory, which are capacities that distinct them from other, nowadays, exploited forms of immunotherapy.

Contains fulltext : 172712pub.pdf (Publisher’s version ) (Open Access) Human blood dendritic cells (DCs) hold great potential for use in anticancer immunotherapies. CD1c+ myeloid DCs and plasmacytoid DCs (pDCs) have been successfully utilized in clinical vaccination trials against melanoma. We hypothesize that combining both DC subsets in a single vaccine can further improve vaccine efficacy. Here, we have determined the potential synergy between the two subsets in vitro on the level of maturation, cytokine expression, and effector cell induction. Toll-like receptor (TLR) stimulation of CD1c+ DCs induced cross-activation of immature pDCs and vice versa. When both subsets were stimulated together using TLR agonists, CD86 expression on pDCs was increased and higher levels of interferon (IFN)-alpha were produced by DC co-cultures. Although the two subsets did not display any synergistic effect on naive CD4+ and CD8+ T cell polarization, CD1c+ DCs and pDCs were able to complement each other's induction of other immune effector cells. The mere presence of pDCs in DC co-cultures promoted plasma cell differentiation from activated autologous B cells. Similarly, CD1c+ DCs, alone or in co-cultures, induced high levels of IFN-gamma from allogeneic peripheral blood lymphocytes or activated autologous natural killer (NK) cells. Both CD1c+ DCs and pDCs could enhance NK cell cytotoxicity, and interestingly DC co-cultures further enhanced NK cell-mediated killing of an NK-resistant tumor cell line. These results indicate that co-application of human blood DC subsets could render DC-based anticancer vaccines more efficacious.

Contains fulltext : 108168.pdf (Publisher’s version ) (Open Access) Human plasmacytoid dendritic cells (pDCs) represent a highly specialized naturally occurring dendritic-cell subset and are the main producers of type I interferons (IFNs) in response to viral infections. We show that human pDCs activated by the preventive vaccine FSME specifically up-regulate CD56 on their surface, a marker that was thought to be specific for NK cells and associated with cytolytic effector functions. We observed that FSME-activated pDCs specifically lysed NK target cells and expressed cytotoxic molecules, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and granzyme B. Elevated levels of these molecules coincided with the expression of CD56, indicative for skewing human pDCs toward an interferon-producing killer DC subset. Detailed phenotypical and functional analysis revealed that pDCs attained a mature phenotype, secreted proinflammatory cytokines, and had the capacity to present antigens and stimulate T cells. Here, we report on the generation of CD56(+) human interferon producing killer pDCs with the capacity to present antigens. These findings aid in deciphering the role for pDCs in antitumor immunity and present a promising prospect of developing antitumor therapy using pDCs.

Learning Objectives After completing this course, the reader will be able to: Describe the current in vivo experimental and clinical dendritic cell (DC) vaccination studies encompassing the monitoring of natural killer (NK) cells.Discuss the evaluation of NK cell stimulating potency in the design of DC-based cancer vaccines in the preclinical phase and in clinical trials.Explain the added value of immune monitoring of NK cells in cancer vaccination trials. CME This article is available for continuing medical education credit at CME.TheOncologist.com 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-α (IFN-α), a type I IFN, is a well-known antitumoral agent. The investigation of its clinical properties in acute myeloid leukemia (AML) has been prompted by its pleiotropic antiproliferative and immune effects. So far, integration of IFN-α in the therapeutic arsenal against AML has been modest in view of the divergent results of clinical trials. Recent insights into the key pharmacokinetic determinants of the clinical efficacy of IFN along with advances in its pharmaceutical formulation, have sparked renewed interest in its use. This paper reviews the possible applicability of IFN-α in the treatment of AML and provides a rational basis to re-explore its efficacy in clinical trials.

Contains fulltext : 196561.pdf (Publisher’s version ) (Open Access) Dendritic cell (DC) vaccines show promising effects in cancer immunotherapy. However, their efficacy is affected by a number of factors, including (1) the quality of the DC vaccine and (2) tumor immune evasion. The recently characterized BDCA1+CD14+ immunosuppressive cells combine both aspects; their presence in DC vaccines may directly hamper vaccine efficacy, whereas, in patients, BDCA1+CD14+ cells may suppress the induced immune response in an antigen-specific manner systemically and at the tumor site. We hypothesize that BDCA1+CD14+ cells are present in a broad spectrum of cancers and demand further investigation to reveal treatment opportunities and/or improvement for DC vaccines. In this review, we summarize the findings on BDCA1+CD14+ cells in solid cancers. In addition, we evaluate the presence of BDCA1+CD14+ cells in leukemic cancers. Preliminary results suggest that the presence of BDCA1+CD14+ cells correlates with clinical features of acute and chronic myeloid leukemia. Future research focusing on the differentiation from monocytes towards BDCA1+CD14+ cells could reveal more about their cell biology and clinical significance. Targeting these cells in cancer patients may improve the outcome of cancer immunotherapy.

Introduction Till today, human malignant pleural mesothelioma (MPM) remains an aggressive cancer with a poor prognosis due to the limited impact on overall survival of the current treatments. Data from us and others about the presence of the immune checkpoint-related molecules PD-1, PD-L1, TIM-3 and LAG-3 in MPM lay the basis to evaluate their suitability as immunotherapeutic targets. Two clinical trials that investigated PD-1 and PD-L1 inhibition in mesothelioma (KEYNOTE-28, JAVELIN trial) have shown promising results with room for improvement. It is of great interest to investigate the effect of combined treatments and compare them to stand-alone treatment to select the best therapeutic strategy for MPM. Material and methods Human cell lines representative for the epithelioid (NCI-H2818 and NCI-H2795) and sarcomatoid (NCI-H2731) subtypes of MPM were placed in allogeneic co-cultures with healthy donor peripheral blood mononuclear cells. The co-cultures were treated with the following immune checkpoint blocking antibodies: anti PD-1 (Nivolumab, BMS) or anti PD-L1 (Durvalumab, AstraZeneca) in combination with anti TIM-3 or anti LAG-3. Supernatant was collected and enzyme-linked immunosorbent assays and multiplex electrochemo-luminescence were used to look at the secretion of 7 cytokines, being IFNg, IL-2/5/6/10, IL-1b and TNF-a, as well as the enzyme granzyme B. Statistical analysis was done to investigate the differences between the treatment conditions. Results and discussions Treatment with immune checkpoint blockers as monotherapy or in combination resulted in a significant increase in the secretion of granzyme B and the cytokines IFNg, IL-2, IL-5 and IL-10. Although the increased secretion was not always statistically significant for all 3 MPM cell lines of the two subtypes, the same trends were observed among them. Interestingly, highest concentrations of granzyme B and these 4 cytokines were noticed for monotherapy treatment with anti PD-1, anti PD-L1 or either of these antibodies with anti TIM-3. In vivo investigation of PD-1 or PD-L1 blockade in combination with TIM-3 or LAG-3 blockade is currently ongoing to validate our in vitro results. Conclusion Our data show that treatment with anti PD-1, anti PD-L1 or their respective combination with anti TIM-3 resulted in the highest secretion of cytokines and granzyme B, suggesting that these treatments stimulate the antitumor response the most. In vivo experiments are currently ongoing for validation.

Introduction CD70 has emerged as a promising immunotherapeutic target in various haematological and solid malignancies. Its overexpression on tumour cells is associated with immunosuppression in the tumour microenvironment. We have previously demonstrated constitutive CD70 overexpression on tumour cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. More importantly, preliminary findings from our group demonstrated the ability of cisplatin to induce CD70 overexpression on NSCLC cells, which broadens the therapeutic window of anti-CD70 immunotherapy. On the other hand, certain chemotherapeutic agents can elicit immunostimulatory changes in the tumour microenvironment by triggering immunogenic cell death and enabling tumor-specific cytotoxic T cell responses. In this regard, strategically combining such a chemotherapeutic agent with anti-CD70 immunotherapy has the potential to achieve enhanced anti-tumour effects. Material and methods The immunostimulatory potential of different chemotherapeutics (cisplatin, oxaliplatin, cyclophosphamide, docetaxel and carboplatin) was evaluated on several NSCLC cell lines, varying in genetic aberrations, by assessing the most important hallmarks of immunogenic cell death: IFNα and HMGB1 production (ELISA), membrane calreticulin expression (flow cytometry) and ATP secretion (bioluminescence). Furthermore, the potential of these different chemotherapeutics to induce CD70 overexpression on NSCLC cells was explored (flow cytometry). Results and discussions Cytotoxicity experiments demonstrated different mechanisms of action for all chemotherapeutics with divergent sensitivity to NSCLC cell lines. At least three out of five chemotherapeutic agents demonstrated ATP secretion and ecto-calreticulin induction on various NSCLC cell lines. In addition, initial experiments showed the highest induction of CD70 expression on NSCLC cells upon low doses of cisplatin and carboplatin. In vitro data on the immunogenic effects of clinically relevant combination strategies in different cell lines are currently being analysed. Conclusion These in vitro results demonstrate the immunostimulatory effects of clinically relevant chemotherapeutics, making it worthwhile to further investigate the potential of a novel combination strategy of chemotherapy and anti-CD70 immunotherapy in NSCLC.

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a tumour with a fibroblastic stroma compartment that consists of pancreatic stellate cells (PSC) which play a complex role in supporting carcinogenesis, immunosuppression and therapy resistance. Therefore, the 5 year survival rate for PDAC patients remains below a disappointing 8%, stressing the need for new and more effective treatments. Recently, cold atmospheric plasma (CAP) has emerged as a potent treatment option for cancer. Although, CAP is being investigated for several years, the involvement of the immune system after CAP treatment remains poorly understood. The immunogenic cell death (ICD) concept describes that the killing of cancer cells leads to direct activation of the immune system through release of so-called ‘danger-associated molecular patterns’. ICD can be elicited by several physical means such as irradiation and photodynamic therapy, providing a rationale for the induction of ICD after CAP treatment. The aim of this study is to investigate the induction of a specific antitumoral immune response after CAP treatment in PDAC, in vitro . Material and methods Phosphate-buffered saline (PBS) was treated with CAP, generated by the kINPenIND, and subsequently added to monocultures of both pancreatic cancer cell (PCC) lines and PSC lines. To evaluate the four most important hallmarks of ICD, being membrane exposure of calreticulin, secretion of ATP and release of HMGB1 and type I interferon, the treatment parameters were optimised (i.e. treatment time, gas flow and gap distance) to obtain 50% cell death. The cellular difference in sensitivity for CAP treatment was assessed through cytotoxic analysis. After attaining the optimal treatment parameters, we investigated the translocation of calreticulin onto the cell surface with flow cytometry. ATP secretion was investigated with a bioluminescence assay, while ELISA was used to monitor the release of HMGB1 and interferon type I in the supernatants. Results and discussions Our data report a cytotoxic effect of CAP treatment in vitro on both PCC and PSC. In both PCC and PSC our results show a significant expression of calreticulin after CAP treatment, together with a significant release of ATP in PCC, but not in PSC. The evaluation of other ICD hallmarks after CAP treatment is currently ongoing. Conclusion We strongly believe that CAP therapy can be a good alternative for the treatment of PDAC. However, the results warrant further in vivo validation to refine the involvement of the immune system after CAP treatment.

The use of cold atmospheric plasma, and more specifically plasma-treated liquids (PTL), for cancer treatment is a very promising topic within the field of plasma medicine. [1,2] Simple solutions to be used as PTL (e.g. plasma-treated PBS (pPBS)) are more generally applicable than the plasma-treated media (PTM) that are now mostly reported, because they have a higher stability for storage and are not cell line dependent. [3,4] However, the underlying mechanisms of the plasma-liquid interactions as well as of the selectivity mechanisms of plasma cancer treatment are not yet fully understood. [5,6] In this study, we want to address these two major questions in plasma oncology. Firstly, we investigated the selectivity of plasma treatment for cancer cells vs normal cells. For this purpose, we studied the effect of plasma-treated PBS (pPBS) on the cell cytotoxicity of both non-small cell lung cancer (NSCLC) and normal lung cells. The effects on the cells were monitored by live cell imaging with the IncuCyte ZOOM®. To gain insight in which species are crucial for this selectivity, we measured the concentrations of several reactive oxygen and nitrogen species (RONS, e.g. H2O2, NO2-, NO3-) by UV-VIS spectrophotometry in the pPBS. Secondly, based on the knowledge on which species are important for the killing of and the selectivity towards cancer cells, we aim to promote the formation of these species in the liquid. Therefore, fundamental insight in the plasma-liquid chemistry is needed. In order to gain this insight, we performed both experimental and computational studies on the effect of the composition of the liquid on the formation of RONS in the liquid. For the computational work, we used 0D and 2D models to reveal the most important reactions and species in the gas and liquid phase. Experimentally, concentrations of RONS in the liquid were measured by both UV-VIS spectrophotometry (e.g. H2O2, NO2-, NO3-) and electron paramagnetic resonance (EPR, e.g. OH, O3, O, 1O2). The effect of chlorine concentration, buffer capacity and pH of the liquid will be presented.

Development of non-thermal plasma for cancer immunotherapeutic applications has received growing attention, namely for induction of immunogenic cancer cell death (ICD). Cancer cells undergoing ICD emit signals known as damage-associated molecular patterns (DAMPs), that can attract and stimulate local immune cells [1]. Of these, membrane-bound calreticulin is a key DAMP signal that facilitates engulfment of cancer cells by dendritic cells, a critical process for the development of a specific, anti-tumor immune response [2]. Several studies have shown that plasma treatment of cancer cell lines increased the exposure of CRT on the cell surface [3, 4]. However, the mechanism by which plasma elicits ICD is not fully elucidated. We therefore studied the interaction of plasma with melanoma cells, at ICD-inducing regimes. The B16-F10 murine melanoma cell line and the A375 human melanoma cell line were treated with a microsecond-pulsed dielectric barrier discharge system over a range of energies and evaluated for CRT emission. We also studied changes to liquid chemistry following plasma treatment, as cells were not treated under dry conditions. Using electron paramagnetic resonance spectroscopy and colorimetric assays, we identified the reactive oxygen and nitrogen species (RONS) present in the liquid after treatment with plasma. By comparing the trends between the exposure of CRT on the cell membrane and the concentration of RONS in the liquid following increasing plasma treatment energies, we can gain insight into which species are most crucial for plasma-induction of ICD. Knowing this will facilitate the optimization of plasma systems for immunotherapy of cancers. Ongoing work includes evaluation of ICD induction of melanoma cells in an in vivo mouse model.

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 graftversus-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

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (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. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

A growing body of evidence points toward an important anti-cancer effect of bisphosphonates, a group of inexpensive, safe, potent, and long-term stable pharmacologicals that are widely used as osteoporosis drugs. To date, they are already used in the prevention of complications of bone metastases. Because the bisphosphonates can also reduce mortality in among other multiple myeloma, breast, and prostate cancer patients, they are now thoroughly studied in oncology. In particular, the more potent nitrogen-containing bisphosphonates have the potential to improve prognosis. The first part of this review will elaborate on the direct and indirect anti-tumoral effects of bisphosphonates, including induction of tumor cell apoptosis, inhibition of tumor cell adhesion and invasion, anti-angiogenesis, synergism with anti-neoplastic drugs, and enhancement of immune surveillance (e.g., through activation of gamma delta T cells and targeting macrophages). In the second part, we shed light on the current clinical position of bisphosphonates in the treatment of hematological and solid malignancies, as well as on ongoing and completed clinical trials investigating the therapeutic effect of bisphosphonates in cancer. Based on these recent data, the role of bisphosphonates is expected to further expand in the near future outside the field of osteoporosis and to open up new avenues in the treatment of malignancies. (C) 2015 Elsevier Inc. All rights reserved.

The poor survival rates of refractory/relapsed acute myeloid leukemia (AML) patients after haematopoietic cell transplantation (HCT) requires the development of additional immune therapeutic strategies. As the elicitation of tumor-antigen specific cytotoxic T lymphocytes (CTLs) is associated with reduced relapses and enhanced survival, enhanced priming of these CTLs using an anti-AML vaccine may result in long-term immunity against AML. Cord blood (CB), as allogeneic HCT source, may provide a unique setting for such post-HCT vaccination, considering its enhanced graft-versus-leukemia (GvL) effects and population of highly responsive naïve T cells. It is our goal to develop a powerful and safe immune therapeutic strategy composed of CB-HCT followed by vaccination with CB CD34+-derived dendritic cells (DCs) presenting the oncoprotein Wilms Tumor-1 (WT1), which is expressed in AML-blasts in the majority of patients. Here, we describe the optimization of a clinically applicable DC culture protocol. This two-step protocol consisting of an expansion phase followed by the differentiation toward DCs, enables us to generate sufficient cord blood-derived DCs (CBDCs) in the clinical setting. At the end of the culture, the CBDCs exhibit a mature surface phenotype, are able to migrate, express tumor antigen (WT1) after electroporation with mRNA encoding the full-length WT1 protein, and stimulate WT1-specific T cells.

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., Supplemental digital content is available in the text.

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.

Malignant pleural mesothelioma is an aggressive cancer linked to asbestos exposure in most patients. Due to the long latency between exposure and presentation, incidence is expected to further increase in the next decade, despite the ban on asbestos import which occurred at the end of last century in industrialized countries. Platinum-based palliative chemotherapy is the only treatment with proven benefit on outcome, resulting in selected patients in a median overall survival of about 1 year. Therefore, there is room for therapeutic improvement using a new strategy to prolong survival. Dealing with cancer cell induced immunosuppression is a promising approach. Reactivating immune responses that are silenced by immune checkpoints recently gained a lot of interest. Checkpoint blockade has already shown promising preclinical and clinical results in several cancer types and is currently also being investigated in mesothelioma. Here, we discuss the expression patterns and mechanisms of action of CTLA-4 and PD-1 as the two most studied and of TIM-3 and LAG-3 as two interesting upcoming immune checkpoints. Furthermore, we review the clinical results of molecules blocking these immune checkpoints and point out their future opportunities with a special focus on mesothelioma.

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.81.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

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-15Ra) using mRNA electroporation. The addition of IL-15Ra 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-gamma, 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-15Ra 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-15Ra mRNA-engineered designer DC.

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.

Although the earliestrudimentaryattempts at exploiting the immune system for cancer therapy can be traced back to the late 18th Century, it was not until the past decade that cancer immunotherapeutics have truly entered mainstream clinical practice. Given their potential to stimulate both adaptive and innate antitumor immune responses, dendritic cells (DCs) have come under intense scrutiny in recent years as pharmacological tools for cancer immunotherapy. Conceptually, the clinical effectiveness of this form of active immunotherapy relies on the completion of three critical steps: 1) the DCs used as immunotherapeutic vehicles must properly activate the antitumor immune effector cells of the host, 2) these immune effector cells must be receptive to stimulation by the DCs and be competent to mediate their antitumor effects, which 3) requires overcoming the various immune-inhibitory mechanisms used by the tumor cells. In this review, following a brief overview of the pivotal milestones in the history of cancer immunotherapy, we will introduce the reader to the basic immunobiological and pharmacological principles of active cancer immunotherapy using DCs. We will then discuss how current research is trying to define the optimal parameters for each of the above steps to realize the full clinical potential of DC therapeutics. Given its high suitability for immune interventions, acute myeloid leukemia was chosen here to showcase the latest research trends driving the field of DC-based cancer immunotherapy.

Item does not contain fulltext Human plasmacytoid and myeloid dendritic cells (DCs), when appropriately stimulated, can express the archetypal natural killer (NK)-cell surface marker CD56. In addition to classical DC functions, CD56+ DCs are endowed with an unconventional cytotoxic capacity.

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.

The Wilms tumor 1 (WT1) protein is a tumor-associated antigen that is overexpressed in a wide range of solid and hematological malignancies.1 WT1 is also known to be frequently overexpressed in acute myeloid leukemia (AML) and is thus suitable for antigen-targeted immunotherapy of AML.2 Previous clinical trials, including both peptide and WT1-targeted dendritic cell (DC) vaccine approaches, have already provided tangible evidence of the clinical anti-leukemic activity of WT1-based immunotherapy in AML patients.1, 3, 4, 5, 6, 7 Several characteristics make WT1 an ideal immunotherapeutic target in AML, including its overexpression in bulk AML cells as well as in the leukemic stem cell compartment, its established role in leukemogenesis and its potent immunogenicity.2 The fact that WT1 is one of the few leukemia-associated antigens from which different CD4+ helper T-cell epitopes have been identified undoubtedly contributes to its superior immunogenicity profile,2 as CD4+ T-cell help is considered critical for the induction of effective anti-tumor immunity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis for which new therapeutic strategies are available. Data derived from a small number of mesothelioma patients suggest that blocking immune checkpoints could offer new treatment opportunities. Gaining more insight in the immunological aspect of the tumor microenvironment (TME) in MPM is essential to develop an effective immunotherapy. In this context, we investigated expression of the immune checkpoint molecules TIM-3, LAG-3, PD-1 and its ligand PD-L1 on cells in human pleural (collected via thoracocentesis, n=6) and ascites (collected via paracentesis, n=5) MPM fluids and identified different subsets of immune cells in the TME using multicolor flow cytometry. Different types of immune cells in the fluids could be detected, with predominant occurrence of CD3+CD4+ T cells, CD64+ macrophages and CD11c+CD303+ dendritic cells (DCs). CD3+CD8+ T cells, CD3-CD56+ natural killer (NK) cells and CD19+ B cells were also present to a lesser extent. While CD4+ and CD8+ T cells and CD64+ macrophages could be detected in all samples, this was not the case for the other cell types. Ascites fluids contained more podoplanin+ (PDPN) tumor cells compared to pleural fluids (average 3.0% vs 0.3%). PD-1+ T cells and NK cells could be detected in all pleural and 80% of the ascites samples. 64% of all samples contained LAG-3-expressing cells, most frequently in pleural fluids. In all samples CD4+ T-cells and NK cells showed TIM-3 expression and in 82% of the samples TIM-3 was also expressed on CD8+ T-cells. In all ascites samples PD-L1 was expressed on DCs, B cells, macrophages and PDPN+ MPM tumor cells. PD-L1 was also expressed on DCs in all pleural samples while in 67%, 83% and 83% of those samples CD19+, CD64+ and PDPN+ cells were PD-L1+, respectively. Based on the mean fluorescence intensity PDPN+ tumor cells showed the highest PD-L1 expression in both fluids. In conclusion, we provide a detailed analysis of a diversity of immune cells present in MPM fluid samples. We are the first to demonstrate TIM-3 and LAG-3 expression in mesothelioma effusions. Statistical analysis is ongoing to investigate whether there are differences between both fluid types and whether there are associations between the cellular composition of the fluids and survival of the patients. The finding of expression of PD-1, PD-L1, TIM-3 and LAG-3 on immune cells in MPM fluids warrants further investigation of the effect of immune checkpoint blockade in MPM, with TIM-3 and LAG-3 as interesting new targets. Citation Format: Elly Marcq, Jorrit De Waele, Jonas van Audenaerde, Eva Lion, Eva Santermans, Niel Hens, Patrick Pauwels, Jan P. van Meerbeeck, Evelien L. Smits. Effusions of mesothelioma patients: What's in it for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3715A. doi:10.1158/1538-7445.AM2017-3715A

Introduction: Numerous studies have reported that tumor progression and invasiveness are determined not only by the malignant cancer cells themselves but also by the surrounding tumor microenvironment, including cancer-associated fibroblasts (CAFs). Although CAFs are implicated in tumor progression, their total depletion of CAFs has been demonstrated to induce more aggressive tumors, indicating that different CAF subpopulations have opposing tumor-promoting or tumor-inhibitory roles. Unfortunately, specific markers to target these subsets of CAFs are lacking. Expression of the immune checkpoint CD70 is normally tightly regulated and limited to cells of the lymphoid lineage only. Instead, tumors hijack CD70 to facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Recently, a lot of clinical successes have been generated by the blockade of immune checkpoints. However, in colorectal cancer (CRC) the efficacy remains limited to a small subset of patients with mismatch repair-deficient (MSI) tumors which might be caused by the intense dialogue between stroma and malignant cells. Therefore, we have explored the expression patterns of the immune checkpoint molecule CD70 in CRC, with a particular focus on CAFs. Methods: The prognostic value of CD70 was analyzed by immunohistochemistry on 51 CRC specimens. In addition, the relationship with Tregs and microsatellite instability was explored. Furthermore, primary CAF cell lines were successfully cultured from 20 different primary resection specimens. These cell lines were used to study the effect of CD70 on the tumor microenvironment in vitro. Results: We revealed expression of CD70, not just on the malignant cells but on the majority of CAFs in invasive CRC specimens. Thereby, CD70-expression was significantly correlated with negative clinicopathological parameters such as metastasis (P=0.007), differentiation (P=0.053) and advanced stage (P=0.001). Moreover, CD70-positive CAFs proved to be a poor prognostic marker by univariate as well as multivariate analysis. We have also detected a significant association between elevated Treg amounts and CD70-expressing CAFs (P=0.012). In vitro data on the effects of CD70 on CRC behavior and immune escape are currently being analyzed. Conclusion: We have identified a new targetable CAF subpopulation, marked by the expression of CD70 and equipped with strong tumor-promoting properties. Thereby, we have found evidence of a potential cross talk between CD70+ CAFs and Treg, paving the way towards immune escape. The lack of association of CD70 expression and MSI-status, which highlights the potential of this target in CRC subsets that do not benefit from immune checkpoint blockade. We believe that targeting CD70 holds great potential in CRC, especially in light of the limited immunotherapeutic options available. Citation Format: Julie Jacobs, Vanessa Deschoolmeester, Karen Zwaenepoel, Christophe Hermans, Christian Rolfo, Marc Peeters, Filip Lardon, Vasiliki Siozopoulou, Evelien Smits, Patrick Pauwels. Blocking CD70+ cancer associated fibroblasts: Are we paving the way towards immunotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2017-958