Use Of Dendritic Cells Research Articles

Directing Dendritic Cell Immunotherapy Towards Successful Cancer Treatment

The use of dendritic cells (DCs) for tumor immunotherapy represents a powerful approach for harnessing the patient's own immune system to eliminate tumor cells. However, suboptimal conditions for generating potent immunostimulatory DCs, as well as the induction of tolerance and suppression mediated by the tumors and its microenvironment have contributed to limited success. Combining DC vaccines with new approaches that enhance immunogenicity and overcome the regulatory mechanisms underlying peripheral tolerance may be the key to achieving effective and durable anti-tumor immune responses that translate to better clinical outcomes.

Propagation and characterisation of dendritic cells from G-CSF mobilised peripheral blood monocytes and stem cells in common marmoset monkeys

The common marmoset is a small New World Primate that has been used as an immunological model for a number of human diseases. Dendritic cells (DC) have not been extensively characterised in this species and in particular protocols to derive DC from living donors without the need for animal sacrifice are presently lacking. This study establishes new protocols to generate substantial numbers of marmoset DC for use in cell therapy studies. Recombinant human G-CSF was used to mobilise peripheral blood monocytes and CD34 + stem cells in sufficient numbers for large scale in-vitro DC propagation using cytokine conditioning including IL-4, GM-CSF, FLT3-L, stem cell factor and thrombopoietin. Marmoset DC exhibited morphology similar to human DC, were capable of antigen uptake and presentation and had moderate allo-stimulatory ability. Monocyte-derived DC had a maturation-resistant immature phenotype, whereas haematopoietic precursor-derived DC were semi-mature in phenotype and function. This study confirms the feasibility of the marmoset as a unique small primate model in which to pursue DC-based immunotherapy strategies.

Use of dendritic cells for the identification and characterization of chemical allergens

Use of dendritic cells for the identification and characterization of chemical allergens

A triterpenoid methyl antcinate K isolated from Antrodia cinnamomea promotes dendritic cell activation and Th2 differentiation

Dendritic cells (DC) play a central role in the initiation and regulation of immune responses. Increasing evidence has indicated that manipulation of DC can serve as a therapeutic mechanism for immunomodulation. In this study we tested some unique compounds isolated from Antrodia cinnamomea, a medicinal fungus in Taiwan, on mouse bone marrow-derived DC activation. A triterpenoid methyl antcinate K (me-AntK) promoted DC maturation by enhancing the expression of MHC class II, CD86, and reducing the endocytosis. TNF-alpha, MCP-1, and MIP-1beta were secreted by DC after me-AntK treatment, indicating augmentation of innate immunity by me-AntK. Interestingly, the me-AntK-activated DC induced Ag-specific T-cell proliferation and facilitated Th2 differentiation. Examining signaling responses, we found that me-AntK treatment uniquely activated JNK and ERK in DC. Our results demonstrate that me-AntK is the first natural triterpenoid to promote the ability of DC to prime Th2 responses. This suggests that me-AntK can potentially be applied to enhance immune responses and modulate DC function in immunotherapy.

Improvement of a Dendritic Cell-Based Therapeutic Cancer Vaccine with Components ofToxoplasma gondii

The use of dendritic cells (DCs) as a cellular adjuvant is a promising approach to the immunotherapy of cancer. It has previously been demonstrated that DCs pulsed ex vivo with Toxoplasma gondii antigens trigger a systemic Th1-biased specific immune response and induce protective and specific antitoxoplasma immunity. In the present study, we demonstrate that tumor antigen-pulsed DCs matured in the presence of Toxoplasma gondii components induce a potent antitumor response in a mouse model of fibrosarcoma. Bone-marrow derived DCs (BMDCs) were cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. After 5 days, tumor lysates with or without the T. gondii lysate were added to the culture for another 2 days. The cytokine production in the BMDC culture and the coculture supernatants of DCs and splenic cells was evaluated. For immunization, 7 days after tumor challenge, different groups of BALB/c mice received different kinds of DCs subcutaneously around the tumor site. Tumor growth was monitored, and 2 weeks after DC immunotherapy, the cytotoxic activity and the infiltration of CD8(+) T cells were monitored in different groups. According to the findings, immunotherapy with T. gondii-matured DCs led to a significant increase in the activity of cytotoxic T cells and decreased the rate of growth of the tumor in immunized animals. Immature DCs did not cause any change in cytotoxic activity or the tumor growth rate compared to that in the healthy controls. The current study suggests that a specific antitumor immune response can be induced by DCs matured with T. gondii components and provide the basis for the use of T. gondii in DC-targeted clinical therapies.

Lentiviral calnexin‐modified dendritic cells promote expansion of high‐avidity effector T cells with central memory phenotype

Dendritic cells (DCs) are key immune mediators for the education and activation of effector cytotoxic T lymphocytes (CTLs). Ex vivo manipulation of DCs is an attractive strategy in immunotherapy. The chaperone proteins are known to hold the keys to proper protein folding and antigen processing. However, little is known of the role of molecular chaperones in DC and T-cell functions. We report that DCs expressing supraphysiological levels of calnexin, a chaperone protein, via lentiviral gene transfer stimulated the expansion of high-avidity CTLs with increased central memory phenotype. Microarray RNA profiling and analyses of protein expression with flow cytometry and multiplex enzyme-linked immunosorbent assay indicated that calnexin had a global effect on DCs with up-regulation of immune modulatory signals including costimulatory molecules, cytokines, chemokines and adhesion molecules. Compared with unmodified DCs, calnexin-DCs were capable of activating T cells to exhibit increased functional avidity associated with up-regulation of CCR7 and costimulatory tumour necrosis factor receptor superfamily molecules. These findings demonstrate a prominent role of calnexin in optimizing DC immunity with potential for improving immunotherapy.

Induction of protective cytotoxic T-cell responses by a B-cell-based cellular vaccine requires stable expression of antigen

2. SummaryB cell-based cellular vaccines represent a promising approach to active immunotherapy of cancer complementing the use of dendritic cells, especially in pediatric patients and patients with low bone marrow reserves. B cells can be easily prepared in large numbers and readily home to secondary lymphoid organs, the primary site of induction of cytotoxic T lymphocyte (CTL) responses. However, most B cell-based vaccines tested so far failed to induce functional and protective CTLs in in vivo models. Here we demonstrate that B cells activated via the Toll like receptor-9 (TLR-9) and CD40 up-regulate surface expression of MHC and costimulatory molecules, produce IL-12, and exhibit potent antigen-presenting properties in vitro. Importantly, while administration of peptide-coated or transiently transfected B cells fails to induce immune responses, therapeutic immunization with low numbers of genetically modified B cells stably expressing antigen results in an induction of functional CTLs and protection against the growth of tumor in an animal model. Following activation, B cells partially loose their ability to home to organized lymphoid tissue due to the shedding of CD62L; however, this property can be restored by expression of protease-resistant mutant of CD62L. In summary, the data presented in this report suggest that genetically modified activated B cells represent a promising candidate for a cancer vaccine eliciting functional systemic CTLs.

Effect of Lentiviral Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Gene Therapy

Dendritic cells (DCs) play a pivotal role in orchestrating and bridging innate, adaptive, and memory immunity. For this reason, genetic modification of DCs functions is an attractive approach to treat disease, both using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with viral vectors on the phenotype and function of DCs. Adenovirus (Ad5), and Lentivirus (LV) are able to up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as inducing production of Th1 and proinflammatory cytokines. Moreover, the function of virally infected DCs is altered. In fact, iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Similar results were observed when the capability of transduced-DCs to present recall- antigen was investigated. Of interest, in the context of vaccine design, these vectors were able to induce an antigen- independent T lymphocytes proliferative response. These data are relevant not only in the context of genetic manipulation of DCs for vaccine development and active immunotherapy, but also in our understanding of the response of DCs to viral infection.

Costimulatory ligand CD70 allows induction of CD8+ T-cell immunity by immature dendritic cells in a vaccination setting

AbstractThe use of dendritic cells (DCs) as anticancer vaccines holds promise for therapy but requires optimization. We have explored the potential of costimulatory ligand CD70 to boost the capacity of DCs to evoke effective CD8+ T-cell immunity. We show that immature conventional DCs, when endowed with CD70 expression by transgenesis, are converted from a tolerogenic state into an immunogenic state. Adoptively transferred CD70-expressing immature DCs could prime CD8+ T cells, by CD27, to become tumor-eradicating cytolytic effectors and memory cells with a capacity for robust secondary expansion. The CD8+ T-cell response, including memory programming, was independent of CD4+ T-cell help, because the transferred immature DCs were loaded with major histocompatibility complex class I–restricted peptide only. Without CD70 expression, the DCs generated abortive clonal expansion, dysfunctional antitumor responses, and no CD8+ T-cell memory. CD70-expressing CD8+ DCs were the primary subset responsible for CD8+ T-cell priming and performed comparably to fully matured DCs. These data highlight the importance of CD27/CD70 interactions at the T-cell/DC interface and indicate that CD70 should be considered in the design of DC vaccination strategies.

Dendritic Cells Use Endocytic Pathway for Cross-Priming Class Ib MHC-Restricted CD8αα+TCRαβ+ T Cells with Regulatory Properties

Understanding the mechanisms leading to effective priming of lymphocytes with regulatory properties is crucial for the manipulation of immune responses. CD8alphaalpha(+)TCRalphabeta(+) T cells are a special subset of innate-like lymphocytes that have been shown to be involved in immune regulation. These cells can recognize self-peptides in the context of a class Ib molecule, Qa-1. How self-Ags are processed in the Qa-1 pathway and presented to CD8alphaalpha(+)TCRalphabeta(+) T cells is not understood. In this study we demonstrate a cross-presentation pathway by which bone marrow-derived dendritic cells (DCs) capture apoptotic CD4(+) T cells and process and present TCR-derived peptides in the context of Qa-1 to prime CD8alphaalpha(+)TCRalphabeta(+) T cells. The priming ability of the DCs is enhanced following TLR signaling using TLR3, TLR4, and TLR9 agonists. DC-mediated cross-presentation is inhibited in the presence of endosomal and proteasomal Ag-processing antagonists. Importantly, DCs loaded with apoptotic T cells prime CD8alphaalpha(+)TCRalphabeta(+) T cells in vivo, which in turn provides protection from CD4(+) T cell-mediated autoimmune disease. These data provide a key insight related to processing and presentation of self-Ags in the Qa-1 pathway for priming of CD8alphaalpha(+)TCRalphabeta(+) T cells and have implications for a DC-based immunotherapeutic approach to inflammatory diseases.

Tumour antigen-loaded mouse dendritic cells maturing in the presence of inflammatory cytokines are potent activators of immune response in vitro but not in vivo

The use of dendritic cells (DCs) loaded with tumour antigen is one of the most promising approaches to induce tumour-specific immune response. However, methods of the vaccine preparation have not yet been standardized. The purpose of the study was to analyse the anti-tumour efficacy of tumour antigen-loaded mouse bone marrow-derived dendritic cells (BM-DC/TAg) at different maturation stages. BM-DCs were loaded with MC38 colon carcinoma cell lysate (TAg) alone, to become partially differentiated, or were additionally stimulated with inflammatory cytokines such as TNF-alpha, IFN-gamma, or IL-12 to reach complete maturity. BM-DCs simultaneously stimulated with TAg and cytokines (especially IL-12 or IFN-gamma+IL-12) were in vitro more effective immune response activators than BM-DC/TAg cells. However, the highest anti-tumour effect in vivo was noted when mice were treated just with BM-DC/TAg. In a further study, the ability of IL-12 gene transduced BM-DCs (BM-DC/IL-12) to augment the immune response induced by BM-DC/TAg cells at different stages of maturation was examined. The highest anti-tumour effect was observed when partially differentiated BM-DC/TAg cells were injected simultaneously with BM-DC/IL-12 cells. The results suggest that partially differentiated BM-DC/TAg cells are more potent in evoking a strong anti-tumour response in vivo than mature BM-DCs. Moreover, the capacity of BM-DC/TAg cells for further differentiation and their sensitivity to factors secreted in vivo by the host or cells engineered to cytokine production seem to be of great importance.

Phenotypic and functional markers for 1α,25-dihydroxyvitamin D3-modified regulatory dendritic cells

The clinical use of dendritic cells (DCs) to induce antigen-specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non-existence of reliable markers. Thus, we set out to find reliable markers that can be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1alpha,25-dihydroxyvitamin D(3) (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high CD14 and reduced CD1a on the cell surface. These VD3-treated DCs exert a long-lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3-treated DCs were readily distinguishable from untreated DCs by low levels of interleukin-23 secretion and low expression of miR-155 upon exposure to maturation stimuli. Furthermore, VD3-treated DCs showed over-expression of miR-378. All these features can be used as robust markers for quality control of VD3-treated regulatory DCs in future clinical studies.

Dendritic Cell-Based Therapeutic Cancer Vaccines: What We Have and What We Need

Therapeutic cancer vaccines rely on the immune system to eliminate tumor cells. In contrast to chemotherapy or passive (adoptive) immunotherapies with antibodies or ex vivo-expanded T cells, therapeutic vaccines do not have a direct anti-tumor activity, but aim to reset patients' immune systems to achieve this goal. Recent identification of effective ways of enhancing immunogenicity of tumor-associated antigens, including the use of dendritic cells and other potent vectors of cancer vaccines, provide effective tools to induce high numbers of circulating tumor-specific T cells. However, despite indications that some of the new cancer vaccines may be able to delay tumor recurrence or prolong the survival of cancer patients, their ability to induce cancer regression remains low. Recent reports help to identify and prospectively remove the remaining obstacles towards effective therapeutic vaccination of cancer patients. They indicate that the successful induction of tumor-specific T cells by cancer vaccines is not necessarily associated with the induction of functional cytotoxic T lymphocytes, and that current cancer vaccines may promote undesirable expansion of Treg cells. Furthermore, recent studies also identify the tools to counteract such phenomena, in order to assure the desirable induction of Th1-cytotoxic T lymphocytes, NK-mediated type-1 immunity and appropriate homing of effector cells to tumors.

Function and dysfunction of dendritic cells in autoimmune rheumatic diseases

Dendritic cells (DC) have been implicated both in initiation of immunity and in immune tolerance. The mechanisms whereby tolerogenic DC may induce and maintain peripheral tolerance include the generation or expansion of regulatory T cells (Treg) and the promotion of T-cell anergy or deletion. A wide spectrum of hematopoietic growth factors and cytokines are endowed with the ability to differentiate tolerogenic DC both in vitro and in vivo. Based on this knowledge, therapeutic vaccination with cytokine-modulated tolerogenic DC has been applied to animal models of autoimmune disorders. This article will review the current experimental evidence underpinning DC dysfunction in rheumatic autoimmune diseases and will discuss how the manipulation of DC and Treg number and function may control undesired T-cell responses.

Browsing issues

Browsing issues

Phenotype of dendritic cells generated in the presence of non-small cell lung cancer antigens - preliminary report.

Therapeutic outcomes of definitively treated non-small-cell lung cancer (NSCLC) are unacceptably poor. It has been proposed that the manipulation of dendritic cells (DCs) as a "natural" vaccine adjuvant may prove to be a particularly effective way to stimulate antitumor immunity. Presently, there is no standardized methodology for preparing vaccines and many questions concerning the optimal source and type of antigens as well as maturation state and activity of DCs are still unsolved. The study population comprised of ten patients with histologically confirmed NSCLC (mean age: 67.63 +/- 6.15 years). Resected small tumor pieces were placed in tissue culture dishes containing different growth factors in order to obtain pure cancer cells. Seven days after the operation, the PBMC were collected and monocytes were purified by the adherence to culture dishes. Monocytes were cultured in RPMI 1640 medium supplemented with 10% of autologous plasma in the presence of rhIL-4 and rhGM-CSF to generate immature autologous (DCs). TNF-alpha with or without tumor cells' lysate were added to maturation of DCs. After 7 days of culture, DCs were harvested and the expression of CD1a, CD83, CD80, CD86 and HLA-DR antigens were analyzed by flow cytometry. We discovered higher (p=0.07) percentage of semimature DCs in tumor cell lysate culture in comparison with TNF-alpha culture (21.22 +/- 16.82% versus 11.27 +/- 11.64%). The expression of co-stimulatory and maturation markers (CD86, CD83 and HLA-DR) was higher on DCs from the culture with tumor cell lysate compared with TNF-alpha culture as a control. Specimen of NSCLC's culture prepared in this way could generate differences in DCs phenotype, which may have an influence on the therapeutic and protective antitumor immunity of the vaccine. Our research seems to be the next step in the development of DC-based vaccine. We are going to continue the investigation to start the preparation of a pattern of immunological vaccine against lung cancer.

Dendritic Compounds as Immune Response Modulators. New Approaches for Vaccine Development

Despite several infectious diseases which have been eradicated or controlled worldwide nowadays, there are still some infectious diseases such as AIDS, caused by HIV, which are supposed to be a serious social health problem and demand the search for new vaccines. A classical approach for generation of vaccines against infection was initially based on the use of killed or attenuated pathogens. With the increase of information about mechanisms governing immune responses after microorganism infection, new approaches have been investigated. The use of dendritic cells (DCs) pulsed with whole pathogen organisms has been considered and explored; however, this approach still presents serious concerns about safety issues. This fact has conducted to a “non-classical” approach where well defined antigens with complete control of molecular design are used. Two strategies are generally envisaged. One is based on the direct stimulation of T and B-cells using immunogenic peptides for producing antibodies and the other one is based on the use of antigen-pulsed dendritic cells producing both cellular and humoral response. This review will be focused on the state of the art of these new approaches for vaccine development to infectious diseases. Synthetic strategies, technical problems, and applications of multivalent dendritic systems will be described and commented. This is a topic of enormous interest which could open interesting alternatives and expectancy in infectious diseases for the development of effective therapies. Keywords: Dendritic Compounds, Vaccine Development, Immune Response, Modulators, infectious diseases, AIDS, HIV, T and B-cells, immunogenic peptides

Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, and seven transmembrane prostate specific G-protein coupled receptor (PSGR). Mice were immunized with these DCs and followed by repeated booster immunization with plasmids expressing each protein. The immunized mice produced significant amounts of antibodies against these proteins. Our results suggest that DC immunization is an effective method to produce antibodies reactive to extracellular regions of plasma membrane proteins with multiple-transmembrane domains, and may be useful to develop antibody mediated antitumor therapies.

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFN gamma and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8 + T cell-mediated cell death

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8 + cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8 + T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8 + T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.