Generation Of Dendritic Cell Vaccines Research Articles

A single-cell surgery microfluidic device for transplanting tumor cytoplasm into dendritic cells without nuclei mixing.

This study aimed to demonstrate the feasibility of generating tumor cell vaccine models by single-cell surgery in a microfluidic device that integrates one-to-one electrofusion, shear flow reseparation, and on-device culture. The device was microfabricated from polydimethylsiloxane (PDMS) and consisted of microorifices (aperture size: ∼3μm) for one-to-one fusion, and microcages for on-device culture. Using the device, we could achieve one-to-one electrofusion of leukemic plasmacytoid dendritic cells (DC-like cells) and Jurkat cells with a fusion efficiency of ∼ 80%. Fusion via the narrow microorifices allowed DC-like cells to acquire cytoplasmic contents of the Jurkat cells while preventing nuclei mixing. After fusion, the DC-like cells were selectively reseparated from the Jurkat cells by shear flow application to generate tumor nuclei-free antigen-recipient DC-like (tarDC-like) cells. When cultured as single cells on the device, these cells could survive under gentle medium perfusion with a median survival time of 11.5h, although a few cells could survive longer than 36h. Overall, this study demonstrates single-cell surgery in a microfluidic device for potential generation of dendritic cell vaccines which are uncontaminated with tumor nucleic materials. We believe that this study will inspire the generation of safer tumor cell vaccines for cancer immunotherapy.

Improving Dendritic Cell Cancer Vaccine Potency Using RNA Interference.

Dendritic cell cancer vaccines have already become a treatment modality for patients with various cancer types. However, the curative potential of this immunotherapy is limited by the existence of negative feedback mechanismsthat control dendritic cells (DCs) and T-cell function. By inhibiting the expression of inhibitory factors using RNA interference technology, a new generation of DC vaccines was developed. Vaccine-stimulated T cells showed antitumor effects both in vitro and in cancer patients. Here, we describe the development and validation of a fully GMP-compliant production process of ex vivo DC cancer vaccines combined with the blockade of immunosuppressive pathways using small interfering RNAs. The protocol can be used for DC-based therapy forall cancer types.

Impaired circulating myeloid CD1c+ dendritic cell function in human glioblastoma is restored by p38 inhibition – implications for the next generation of DC vaccines

ABSTRACTCurrent treatments for glioblastoma (GBM) have limited efficacy and significant morbidity and therefore new strategies are urgently needed. Dendritic cells have the power to create anti-tumor immune responses. The greater potency of circulating dendritic cells (DC) over laboratory-generated monocyte-derived DC makes them exciting new immunotherapeutic candidates. To determine the immune status of GBM patients we initially investigated the frequency and function of circulating DC subsets. Furthermore, we tested the therapeutic potential of inhibiting the p38 mitogen-activated protein kinase pathway (p38i) in circulating DC to overcome DC dysfunction.GBM patients (n = 16) had significantly reduced numbers of the major myeloid circulating dendritic cell (cDC2) and plasmacytoid DC vs healthy controls; 1736 vs 4975 (p = 0.028) and 893 vs 2287 cells/mL (P = <0.001) respectively. This inversely correlated with dexamethasone (Dex) dose in a log-linear model, and disease status. Patients’ cDC2 were immature with impaired interleukin (IL)-12 secretion, reduced IL-12:IL-10 ratio, and low HLA-DR and CD86 expression. Exposure of healthy donor cDC2 to Dex or GBM cell lysate resulted in a similar low IL-12:IL-10 ratio. Inhibition of p38 restored the IL-12:IL-10 balance in Dex or tumor lysate-conditioned healthy cDC2 and enhanced T-cell proliferation and interferon-gamma (IFNγ) production. Importantly, patient-derived cDC2 showed a similar reversal of DC dysfunction with p38i. This study demonstrates the therapeutic potential of developing the next generation of DC vaccines using enhanced p38i-conditioned cDC2. We will therefore shortly embark on a clinical trial of adoptively transferred, p38 MAPK-inhibited cDC2 in adults with GBM.

Static Image Analysis as New Approach for the Characterization of Tumor Cell Lysate Used in Dendritic Cell Vaccine Preparation

Background: Safety is an important consideration for the clinical application of dendritic cells (DC) loaded with autologous tumor lysate (TL). Thus, avitalization of TL from living autologous tumor tissue has to be guaranteed. Methods: Composition of TL was investigated by static image analysis (SIA) with the Morphologi G3 device, which simultaneously measures size and shape of up to 100,000 particles within one sample run. This approach was compared with sample characterization by high-resolution automated cell counting, trypan blue staining, and ATP quantification. Results: Using SIA, we only detected fragmented, non-cellular structures in completely avitalized TL, indicating complete destruction of living cells. Analysis of particle size distribution by SIA as well as CASY cell counter showed that 95% of particles had a diameter of <10 µm as a sign of cell fragmentation. Complete avitalization of TL was confirmed with trypan blue staining and ATP analysis. Conclusion: Regarding generation of DC vaccines, the proof of avitality of TL from living tumor tissue can clearly be achieved by SIA alone or in combination with standard assays. Our data show that SIA is a highly precise method for TL characterization. The SIA device complies with FDA regulation and, therefore, might be suitable for characterization of cellular therapy medicinal products.

MP29-11 TRANSCRIPTION FACTOR PROFILE OF MONOCYTES AND DENDRITIC CELLS (DC) IN RENAL CELL CARCINOMA (RCC) PATIENTS MAY DETERMINE RESPONSE TO IMMUNOTHERAPY

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-11 TRANSCRIPTION FACTOR PROFILE OF MONOCYTES AND DENDRITIC CELLS (DC) IN RENAL CELL CARCINOMA (RCC) PATIENTS MAY DETERMINE RESPONSE TO IMMUNOTHERAPY Alexander Wald, Jan Hoffmeyer, Mohammad Habiby Kermany, Lilia Heit, Marc Ernstoff, and Thomas Schwaab Alexander WaldAlexander Wald More articles by this author , Jan HoffmeyerJan Hoffmeyer More articles by this author , Mohammad Habiby KermanyMohammad Habiby Kermany More articles by this author , Lilia HeitLilia Heit More articles by this author , Marc ErnstoffMarc Ernstoff More articles by this author , and Thomas SchwaabThomas Schwaab More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.753AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Dendritic Cell (DC)-based, immunotherapy has shown great promise in some cancer vaccines. Clinical and immunologic results vary. We have recently shown that genomic profiling of peripheral blood mononuclear cells (PBMC) may predict response. Transcription factors play a key role in the myelopoesis of monocytes and DC. We hypothesized that the profile of key transcription factors plays a key role in the efficacy of DC vaccines. METHODS Monocytes from RCC patients and healthy donors were isolated via cold agglutination or ELUTRA. For some experiments, DC were matured with GM-CSF/ IL-4 and a variety of maturation protocols (TNF-alpha, cytokine cocktail, Flt-3).RNA was extracted using QIAGEN RNeasy Mini Kit. It's concentration and quality was assessed using Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent Technologies). Only high quality RNA (RIN > 7.2) was used for reverse transcription (Bio-Rad iScript cDNA synthesis kit) and real time qPCR (in triplicates using ABI SYBR-Select Master Mix with target specific primers and the ABI 7900HT Thermocycler). The expression of key transcription factors IRF-4, IRF-8, IL-10, IL-12 were analyzed using previously reported primer pairs. To determine relative concentrations, cycle threshold (Ct) values were compared to the reference gene GAP-DH. The statistical analysis was conducted in JMP 11 from SAS. RESULTS Healthy donors demonstrated a significant variation of expression levels of all transcription factors examined. When DC were matured with a variety of maturation protocols, these resulted in significant differences in expression of all transcription factors. The greatest variations were observed in the expression levels of IRF-4, IL-10 and IL-12. The greatest induction of IRF-4 and IL-12 and lowest induction of IL-10 were observed with the cytokine cocktail. In an IFN-gamma release assay, these cells also demonstrated the greatest T cell stimulatory capacity. RCC patient monocytes showed showed significantly lower expression of IRF-4 and IRF-8 than healthy age-adjusted donor monocytes (p=0.004 and 0.0214, respectively). Immunotherapy resulted in upregulation of IL-10 when compared to pretreatment mRNA levels (p=0.007). CONCLUSIONS In summary, this is the first study to assess the profile of key transcription factors as predictors for DC vaccine quality. We demonstrate significant inter-individual variability in monocytes prior to DC vaccine and throughout the generation of DC vaccines. These data may lead to improved cellular anti-cancer vaccines. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e308-e309 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Alexander Wald More articles by this author Jan Hoffmeyer More articles by this author Mohammad Habiby Kermany More articles by this author Lilia Heit More articles by this author Marc Ernstoff More articles by this author Thomas Schwaab More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antitumor efficacy of a photodynamic therapy-generated dendritic cell glioma vaccine

The objective of this study is to generate dendritic cell (DC) vaccines by exposing DCs to C6 glioma cancer cell antigenic (tumor) peptides following the exposure of C6 cells to photodynamic therapy (PDT) and acid elution. Effects of these DCs on host immunity were assessed by measuring cytokine induction (following adaptive transfer into rats) and assessing DC-induced cytotoxic T lymphocyte (CTL)-mediated lysis of C6 target cells. Precursor dendritic cells were purified from rat bone marrow and matured in vitro. C6 cells were stimulated with PDT, and adherent cells were acid-eluted to obtain cell surface antigens, whole cell antigens were also isolated from supernatants. C6 cells not stimulated with PDT were also used to isolate antigens by acid elution or freeze-thaw methods for comparison purposes. The isolated antigens from the respective purification methods were used to sensitize DCs for the generation of DC vaccines subsequently transferred into SD rats. Following adoptive transfer, the changes in interleukin (IL)-12, IL-10, and TNF-α expression were measured in rat serum by ELISA. CTL-mediated lysis was assessed using the MTT assay. PDT-generated antigens further purified by acid elution had the greatest stimulatory effect on DCs based on the elevated serum IL-12 and TNF-α levels and decreased serum IL-10 levels. CTL activity in this group was also highest (percent lysis 95.5% ± 0.016) compared with that elicited by PDT-supernatants, acid elution, and freeze-thawing (or the control group), which had 90.2% ± 0.024, 73.3% ± 0.027, 63.6% ± 0.049, or 0.4% ± 0.063 lysis, respectively. PDT significantly enhanced tumor cell immunogenicity. These data suggested that DC vaccines prepared by treating tumor cells with PDT to generate antigen-specific CTL responses can be developed as novel cancer immunotherapeutic strategies.

Dendritic cells: a critical player in cancer therapy?

Cancer immunotherapy seeks to mobilize a patient's immune system for therapeutic benefit. It can be passive, that is, transfer of immune effector cells (T cells) or proteins (antibodies), or active, that is, vaccination. Early clinical trials testing vaccination with ex vivo generated dendritic cells (DCs) pulsed with tumor antigens provide a proof-of-principle that therapeutic immunity can be elicited. Yet, the clinical benefit measured by regression of established tumors in patients with stage IV cancer has been observed in a fraction of patients only. The next generation of DC vaccines is expected to generate large numbers of high avidity effector CD8 T cells and to overcome regulatory T cells and suppressive environment established by tumors, a major obstacle in metastatic disease. Therapeutic vaccination protocols will combine improved DC vaccines with chemotherapy to exploit immunogenic chemotherapy regimens. We foresee adjuvant vaccination in patients with resected tumors but at high risk of relapse to be based on in vivo targeting of DCs with fusion proteins containing anti-DCs antibodies, antigens from tumor stem/propagating cells, and DC activators.

Taming cancer by inducing immunity via dendritic cells

Immunotherapy seeks to mobilize a patient's immune system for therapeutic benefit. It can be passive, i.e. transfer of immune effector cells (T cells) or proteins (antibodies), or active, i.e. vaccination. In cancer, passive immunotherapy can lead to some objective clinical responses, thus demonstrating that the immune system can reject tumors. However, passive immunotherapy is not expected to yield long-lived memory T cells that might control tumor outgrowth. Active immunotherapy with dendritic cell (DC)-based vaccines has the potential to induce both tumor-specific effector and memory T cells. Early clinical trials testing vaccination with ex vivo-generated DCs pulsed with tumor antigens provide a proof-of-principle that therapeutic immunity can be elicited. Yet, there is a need to improve their efficacy. The next generation of DC vaccines is expected to generate large numbers of high-avidity effector CD8(+) T cells and to overcome regulatory T cells. Therapeutic vaccination protocols will combine improved ex vivo DC vaccines with therapies that offset the suppressive environment established by tumors.

The potential of CpG oligodeoxynucleotides in the development of dendritic cell vaccines

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) capable of initiating a primary immune response, leading to the development of CD8 + cytotoxic T-cells (CTLs) and CD4 + T helper (Th) cells. These characteristics make them promising as vaccine carriers. However, despite the great potential demonstrated in murine models, DC-based vaccines have only been moderately effective in human clinical trials. The efficacy of DC-based vaccination or therapy depends on a number of variables, including antigen characteristics, DC lineage and level of maturation, vaccination route, frequency and interval, and host status. Many of these parameters need to be further optimized and/or understood before DC vaccines reach their full potential. Toll-like receptor (TLR) agonists are capable of activating DCs in response to pathogens and have potential as activation and maturation factors for DCs. The immunostimulatory properties of CpG oligodeoxynucleotides (ODNs), which signal through TLR9, have been extensively studied. The concept of using CpG ODNs stimulation of murine bone marrow-derived DCs (BM-DCs) has been proven mostly in tumor challenge models. According to several published reports, TLR9 is present on human plasmacytoid DCs and B-cells, but not on monocytes, which are the most common source of DC vaccines. However, a recent study demonstrated expression of TLR9 on human monocyte-derived DCs, which suggests that CpG ODNs might be useful as activation and maturation factors. Furthermore, TLR7 and TLR8 activation was found to have a similar mode of action to TLR9. TLR8 is expressed on human monocytes and monocyte-derived DCs, and TLR8 agonists may therefore also have potential in the generation of DC vaccines.