Source Of Dendritic Cells Research Articles

Mathematical modeling of interactions between colon cancer and immune system with a deep learning algorithm

Colon cancer is a complex disease with genetically unstable cell lines. In order to better understand the complexity of colon cancer cells and their metastatic mechanisms, we develop a mathematical model in this study. The model is based on a system of fractional-order differential equations and Fractional-Cancer-Informed Neural Networks (FCINN). The model captures a dynamic network of interactions between dendritic cells (DCs), cytotoxic T-cells (CD8+\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$8^+$$\\end{document}), helper T-cells (CD4+\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$4^+$$\\end{document}), and colon cancer cells through fractional differential equations. By varying the fractional order between 0 and 1, we can classify patients into different groups based on their immune patterns. The goal of this paper is to identify different immune patterns and cancer cell behaviors, as well as the parameters that play an important role in metastasis, control, or elimination of cancer cells in the model. However, several parameters in the model are difficult to estimate in a patient-specific manner. To address this challenge, we use FCINN as an effective deep-learning tool for parameter estimation and numerical simulation of the model. Our findings suggest that the most effective factors in controlling the progression and preventing metastasis of colon cancer are the initial number of cancer cells, the inhibiting rates of tumor cells by DCs, the source of DCs, and the activation of helper T-cells by DCs. These findings suggest that DCs can be used as an immunotherapy tool for the control and treatment of colon cancer.

Dendritic Cell-Based Immunotherapy in Multiple Myeloma: Challenges, Opportunities, and Future Directions.

Immunotherapeutic approaches, including adoptive cell therapy, revolutionized treatment in multiple myeloma (MM). As dendritic cells (DCs) are professional antigen-presenting cells and key initiators of tumor-specific immune responses, DC-based immunotherapy represents an attractive therapeutic approach in cancer. The past years, various DC-based approaches, using particularly ex-vivo-generated monocyte-derived DCs, have been tested in preclinical and clinical MM studies. However, long-term and durable responses in MM patients were limited, potentially attributed to the source of monocyte-derived DCs and the immunosuppressive bone marrow microenvironment. In this review, we briefly summarize the DC development in the bone marrow niche and the phenotypical and functional characteristics of the major DC subsets. We address the known DC deficiencies in MM and give an overview of the DC-based vaccination protocols that were tested in MM patients. Lastly, we also provide strategies to improve the efficacy of DC vaccines using new, improved DC-based approaches and combination therapies for MM patients.

Heterogeneous populations from in vitro cultures of antigen presenting cells in pigs

Dendritic cells (DCs) are the most potent antigen presenting cells (APCs). Because of the difficulty in obtaining these cells directly from tissues, different sources of DCs are frequently used for in vitro experimentation and many of their biological and functional characteristics were studied using these systems. Until recently, it was assumed that specific culture conditions polarized the differentiation of either DCs or macrophages (Macs); however, it was shown that some DC culture systems in other species generate heterogeneous cell populations that can be identified according to their CD11c and MHC class II (MHC-II) expression. Following this approach, porcine DCs were directly isolated from peripheral blood or differentiated in vitro by culturing bone marrow (BM) progenitor cells or blood monocytes treated with growth factors. Mostly homogeneous monocyte-derived DCs (MoDCs) were obtained with similar phenotype and phagocytic characteristics to that of blood DCs. On the contrary, BM-derived DC (BMDC) cultures generated two distinct heterogeneous populations identified as MHC-II+ and MHC-II++ cells. BMDCs MHC-II+ had similar phenotypic and phagocytic characteristics to those of MoDCs and blood DCs. However, BMDCs MHC-II++ population expressed a higher amount of surface markers and transcribed genes associated with Macs-lineage exhibiting a higher phagocytic capacity than all the other cells. Noteworthy, every cell system expressed different genetic signatures. These results will help interpreting and re-interpreting data obtained using in vitro systems.

Mixed cultures of allogeneic dendritic cells are phenotypically and functionally stable - a potential for primary cell-based "off the shelf" product generation.

Vaccination against tumors using antigen-pulsed dendritic cell (DC) vaccines has greatly evolved over the last decade, with hundreds of active human clinical trials well on the way. The use of an autologous source for DC-based vaccine therapeutics remains the obvious choice in the majority of clinical studies; however, novel evidence suggests that an allogeneic source of DCs can yield success if administered in the right context. One of the challenges facing successful DC vaccination protocols is the generation of large enough numbers of DCs intended for vaccination and standardization of these procedures. In addition, variations in the quality of DC vaccines due to donor-to-donor variation represent an important therapeutic factor. To this day it has not been shown whether DCs from different donors can readily co-exist within the same co-culture for the extended periods required for vaccine manufacture. We demonstrate that generation of allogeneic DC co-cultures, generated from multiple unrelated donors, allows the preservation of their phenotypical and functional properties in vitro for up to 72 hours. Therefore, in the case of an allogeneic vaccination approach, one could ensure large numbers of DCs generated from a primary cell source intended for multiple vaccinations. By generating large amounts of ex vivo manufactured DCs from multiple donors, this would represent the possibility to ensure sufficient amounts of equipotent “off the shelf” product that could e.g. be used for an entire cohort of patients within a study.

A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro.

Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8+ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone-marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona-fide cross-presenting DCs. We also describe methods to assess cross-presentation by DCs using the activation of primary CD8+ T cells as a readout. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of bone marrow progenitor cells Basic Protocol 2: In vitro differentiation of dendritic cells with GM-CSF Support Protocol 1: Preparation of conditioned medium from GM-CSF producing J558L cells Basic Protocol 3: In vitro differentiation of dendritic cells with Flt3L Support Protocol 2: Preparation of Flt3L containing medium from B16-Flt3L cells Basic Protocol 4: Expansion of cDC1s in vivo for use in ex vivo experiments Basic Protocol 5: Characterizing resting and activated dendritic cells Basic Protocol 6: Dendritic cell stimulation, antigenic cargo, and fixation Support Protocol 3: Preparation of model antigen coated microbeads Support Protocol 4: Preparation of apoptotic cells Support Protocol 5: Preparation of recombinant bacteria Basic Protocol 7: Immunocytochemistry immunofluorescence (ICC/IF) Support Protocol 6: Preparation of Alcian blue-coated coverslips Basic Protocol 8: CD8+ T cell activation to assess cross-presentation Support Protocol 7: Isolation and labeling of CD8+ T cells with CFSE.

Fasciola gigantica excretory-secretory products (FgESPs) modulate the differentiation and immune functions of buffalo dendritic cells through a mechanism involving DNMT1 and TET1

BackgroundFasciola gigantica infection threatens the health of both humans and animals in the world. The excretory/secretory products (ESPs) of this fluke has been reported to impair the activation and maturation of immune cells. We have previously shown the influence of F. gigantica ESPs (FgESPs) on the maturation of buffalo dendritic cells (DCs). However, the underlying mechanisms remain unclear. The objective of this study was to investigate the potency of FgESPs in shifting the differentiation and immune functions of buffalo DCs.MethodsBuffalo DCs were incubated with FgESPs directly or further co-cultured with lymphocytes in vitro. qRT-PCR was employed to determine the gene expression profile of DCs or the mixed cells, and an ELISA was used to measure cytokine levels in the supernatants. Hoechst and Giemsa staining assays, transmission electron microscopy, caspase-3/7 activity test and histone methylation test were performed to determine DC phenotyping, apoptosis and methylation. To investigate the mechanism involved with DNA methylation, a Co-IP assay and immunofluorescent staining assay were performed to observe if there was any direct interaction between FgESPs and DNMT1/TET1 in buffalo DCs, while RNAi technology was employed to knockdown DNMT1 and TET1 in order to evaluate any different influence of FgESPs on DCs when these genes were absent.ResultsqRT-PCR and ELISA data together demonstrated the upregulation of DC2 and Th2/Treg markers in DCs alone and DCs with a mixed lymphocyte reaction (MLR), suggesting a bias of DC2 that potentially directed Th2 differentiation in vitro. DC apoptosis was also found and evidenced morphologically and biochemically, which might be a source of tolerogenic DCs that led to Treg differentiation. In addition, FgESPs induced methylation level changes of histones H3K4 and H3K9, which correlate with DNA methylation. Co-IP and immunofluorescent subcellular localization assays showed no direct interaction between the FgESPs and DNMT1/TET1 in buffalo DCs. The productions of IL-6 and IL-12 were found separately altered by the knockdown of DNMT1 and TET1 in DCs after FgESPs treatment.ConclusionsFgESPs may induce the DC2 phenotype or the apoptosis of buffalo DCs to induce the downstream Th2/Treg response of T cells, possibly through a DNMT1- or TET1-dependent manner(s).

IPS cell serves as a source of dendritic cells for in vitro dengue virus infection model.

The lack of an appropriate model has been a serious concern in dengue research pertinent to immune response and vaccine development. It remains a matter of impediment in dengue virus (DENV) studies when it comes to an in vitro model, which requires adequate quantity of dendritic cells (DC) with uniform characters. Other sources of DC, mostly monocyte derived DC (moDC), have been used despite their limitations such as quantity, proliferation, and donor dependent characters. Recent development of human iPS cells with consistent proliferation for long, stable, functional characteristics and desired HLA background has certainly offered added advantages. Therefore, we hypothesised that iPS derived cells would be a reliable alternative to the traditional DCs to be used with an in vitro DENV system. To develop a DENV infection and T cell activation model, we utilised iPS cells (HLA-A*24) as the source of DC. iPS-ML-DC was prepared and DENV infectivity was assessed apart from the major surface markers expression and cytokine production potential. Our iPS-ML-DC had major DC markers expression, DENV infection efficiency and cytokine production properties similar to that of moDC. Moreover, DENV infected iPS-ML-DC demonstrated the ability to activate HLA-matched T cell (but not mismatched) in vitro as evidenced by significantly higher proportion of IFN-γ+ CD69+ T cells compared to non-infected iPS-ML-DC. This affirmed the antigen-specific T cell activation by iPS-ML-DC as a function of antigen presenting cells. To conclude, maturation potential, DENV infection efficiency and T cell activation ability collectively suggest that iPS-ML-DC serves as an attractive option of DC for use in DENV studies in vitro.

Compromised functionality of monocyte-derived dendritic cells in multiple myeloma patients may limit their use in cancer immunotherapy

Dendritic cells (DCs) have the potential to elicit long-lasting anti-tumour immune responses. Most of the clinical trials of anti-cancer DC vaccines are based on monocyte-derived DCs (Mo-DCs). However, their outcomes have shown limited promise especially in multiple myeloma (MM) patients. Here, we investigated whether in vitro generated Mo-DCs from MM patients (MM-DCs) possess impaired functionality, thus contributing to the limited success of DC vaccines. We generated MM-DCs and compared them with DCs from healthy donors (HD-DCs). The yield of DCs in MM was 3.5 fold lower than in HD sets. However morphology, phenotype, antigen uptake and allo-T cell stimulation were comparable. Migration and secretion of IL12p70 and IFN-γ (in DC-T cell co-cultures) were significantly reduced in MM-DCs. Thus, MM-DCs were compromised in functionality. This impairment could be attributed to autocrine secretion of IL6 by MM-monocytes and activation of their P38 MAPK pathway. This indicates a need to look for alternative sources of DCs.

The Effects of T4 and A3/R Bacteriophages on Differentiation of Human Myeloid Dendritic Cells.

Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3/R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3/R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs.

Analysis of peripheral blood dendritic cells as a non-invasive tool in the follow-up of patients with chronic hepatitis C.

Hepatitis C virus (HCV) has a high propensity to establish chronic infections. Failure of HCV-infected individuals to activate effective antiviral immune responses is at least in part related to HCV-induced impairment of dendritic cells (DCs) that play a central role in activating T cell responses. Although the impact of HCV on DC phenotype and function is likely to be more prominent in the liver, major HCV-induced alterations are detectable in peripheral blood DCs (pbDCs) that represent the most accessible source of DCs. These alterations include numerical reduction, impaired production of inflammatory cytokines and increased production of immunosuppressive IL10. These changes in DCs are relevant to our understanding the immune mechanisms underlying the propensity of HCV to establish persistent infection. Importantly, the non-invasive accessibility of pbDCs renders the analysis of these cells a convenient procedure that can be serially repeated in patient follow-up. Accordingly, the study of pbDCs in HCV-infected patients during conventional treatment with pegylated interferon and ribavirin indicated that restoration of normal plasmacytoid DC count may represent an additional mechanism contributing to the efficacy of the dual therapy. It also identified the pre-treatment levels of plasmacytoid DCs and IL10 as putative predictors of response to therapy. Treatment of chronic HCV infection is changing, as new generation direct-acting antiviral agents will soon be available for use in interferon-free therapeutic strategies. The phenotypic and functional analysis of pbDCs in this novel therapeutic setting will provide a valuable tool for investigating mechanisms underlying treatment efficacy and for identifying predictors of treatment response.

Induction of CD4(+) Regulatory and Polarized Effector/helper T Cells by Dendritic Cells.

Dendritic cells (DCs) are considered to play major roles during the induction of T cell immune responses as well as the maintenance of T cell tolerance. Naive CD4+ T cells have been shown to respond with high plasticity to signals inducing their polarization into effector/helper or regulatory T cells. Data obtained from in vitro generated bone-marrow (BM)-derived DCs as well as genetic mouse models revealed an important but not exclusive role of DCs in shaping CD4+ T cell responses. Besides the specialization of some conventional DC subsets for the induction of polarized immunity, also the maturation stage, activation of specialized transcription factors and the cytokine production of DCs have major impact on CD4+ T cells. Since in vitro generated BM-DCs show a high diversity to shape CD4+ T cells and their high similarity to monocyte-derived DCs in vivo, this review reports data mainly on BM-DCs in this process and only touches the roles of transcription factors or of DC subsets, which have been discussed elsewhere. Here, recent findings on 1) the conversion of naive into anergic and further into Foxp3− regulatory T cells (Treg) by immature DCs, 2) the role of RelB in steady state migratory DCs (ssmDCs) for conversion of naive T cells into Foxp3+ Treg, 3) the DC maturation signature for polarized Th2 cell induction and 4) the DC source of IL-12 for Th1 induction are discussed.

Efficient isolation of monocytes from PBMCs utilizing a newly developed material

Meeting abstracts Recent years immunotherapy has been used to treat various diseases especially cancer. The dendritic cell therapy has been one of the most widely practiced therapies in clinics and usually monocytes are isolated as the source of dendritic cells. However, although dendritic cell

Can the KG1 cell line be used as a model of dendritic cells and discriminate the sensitising potential of chemicals?

The KG1 myeloid leukaemia was used as source of dendritic cells (DC) to discriminate between respiratory and contact sensitising chemicals. A cocktail of cytokines was used to differentiate KG1 to dendritic like cells (termed dKG1) and the effects of nine chemicals (respiratory and contact sensitisers) and an irritant control on surface marker expression, 'antigen presenting' function and cytokine expression investigated. The stability of these chemicals when dissolved was characterised using MALDI ToF MS. A Hill plot model was used with the cellular viability data to quantify the lethal dose 50% (LD50) and a maximum sub toxic concentration of each chemical defined. Cytokine expression by the treated dKG1 was quantified using multiplex immunobead analysis. Whilst dKG1 cells were morphologically similar to DCs, expression of specific surface markers was not typical for DCs derived from healthy precursor cells. When the chemicals were applied at defined sub toxic doses no effects on dKG1 phenotype, function, or cytokine expression, attributable to the sensitisation properties were discriminated. However, dKG1 cells were much more sensitive to the toxic effects of these chemicals compared to the parent KG1 cells. Only 4 of the 9 chemicals tested were stable when dissolved indicating that the effect of sensitising chemicals on antigen presenting cells may be related to species other than the parent compound.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

Dendritic cells generated from naïve and tumor-bearing mice uniquely restores different leukocyte subpopulations in chemotherapy-treated tumor-bearing mice

Background: Dendritic cell (DC)-based vaccination has shown promising application in tumor immunotherapy. However, it is not clear whether the presence of tumor impacts the efficacy of generation and functionality of DCs. Aim : To compare the phenotype of DCs generated from naive or tumor bearing mice and their capability to restore leukopenia-associated chemotherapy. Materials and Methods: DCs were generated from bone marrow (BM) of naive or Ehrlich ascites carcinoma (EAC) bearing mice. EAC is an undifferentiated breast cancer cell line with the high transplantable capability and rapid proliferation. BM cells were cultured in vitro for 7 days with granulocyte macrophage colony-stimulating factor and interleukin-4 (20 ng/ml each), loaded with different concentrations of EAC cell lysate (0.5, 1, 3 and 5 mg/10 6 ) DCs followed by activation with the toll-like receptor 3 ligand poly(I:C). For DC-based vaccination, CD1 mice ( n = 5/group) were inoculated with an intraperitoneal (i.p.) injection of 0.25 × 10 6 EAC cells to form ascites, treated on day 14 with an i.p. injection of cyclophosphamide (4 mg/mouse) and on day 15 with subcutaneous injection of 2 × 10 6 DCs from control or EAC bearing mice. Injected DCs were loaded with or without EAC lysate followed by i.p. injection of 50 μg/mouse poly(I:C). On day 21, mice were bled and sacrificed for peripheral blood count and spleen and BM cellularity. Results : Yield of DCs generated from naIve or EAC bearing mice, as well as their phenotype (CD11c + CD11b + ) and activation (CD40 and CD80) with poly(I:C) were similar. Loading DCs with 1 mg EAC lysate induced better viability and activation phenotype as compared with the other concentrations. Regardless the source of DCs, DCs vaccination restored the total numbers of leukocytes in blood but not in the spleen and BM. The effect on peripheral blood leukocytes was coincided with the restoration of the relative numbers of lymphocytes, monocytes, and granulocytes. Conclusion: These data support the use of allogenic DCs from healthy donors in anticancer DC-based vaccination.

The role of CD40 expression in dendritic cells in cancer biology; a systematic review.

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor superfamily and is essential in activation of dendritic cells. Dendritic cells (DCs) are antigen-presenting cells capable of initiating cytotoxic T-lymphocyte immune response against cancer cells. However, there are few studies on the characterization of DCs in cancer, specifically their expression of CD40, despite its implication in cancer immunotherapy. We reviewed available data on the expression of CD40 on DCs in various cancers, and its implications for cancer immunotherapy. A systematic review on CD40 expression on DCs in cancer was performed with reference to preferred reporting items for systematic reviews and meta-analyses (PRISMA). Studies that satisfied the inclusion and exclusion criteria were 21 out of 927. Variations in type and status of the cancers, source of DCs and methodology for detecting CD40 expression amongst the studies resulted in contrasting results. DCs generally expressed low CD40 in tumor infiltrating DCs (tiDCs), in DCs derived by in vitro culture from blood monocytes using cytokine stimulation (MoDCs) and in DCs exposed in vitro to tumor cells lines; the studies suggested that CD40 expression in DCs is impaired in cancer particularly in metastatic disease. However, DCs identified in fresh peripheral blood mononuclear cells (PBMC) expressed higher numbers of CD40 positive cells in some cancer patients, which could be due to tumor-derived factors leading to partially-stimulated DCs. The results provide evidence that some cancer patients may show partial systemic DC activation and expression of increased CD40 in response to the presence of tumor but that such activity may become abortive in the presence of factors produced by the tumor. This review has thus identified key papers on CD40 expression on DCs in various cancers and discusses the limitations and contrasting results of these studies in relation to variations in methodology. The results highlight the need for further studies on the role of CD40-CD40 ligand pathway to inform cancer treatment.

Allergic airway inflammation induces the migration of dendritic cells into airway sensory ganglia.

BackgroundA neuroimmune crosstalk between dendritic cells (DCs) and airway nerves in the lung has recently been reported. However, the presence of DCs in airway sensory ganglia under normal and allergic conditions has not been explored so far. Therefore, this study aims to investigate the localisation, distribution and proliferation of DCs in airway sensory ganglia under allergic airway inflammation.MethodsUsing the house dust mite (HDM) model for allergic airway inflammation BALB/c mice were exposed to HDM extract intranasally (25 μg/50 μl) for 5 consecutive days a week over 7 weeks. With the help of the immunohistochemistry, vagal jugular-nodose ganglia complex (JNC) sections were analysed regarding their expression of DC-markers (MHC II, CD11c, CD103), the neuronal marker PGP 9.5 and the neuropeptide calcitonin gene-related peptide (CGRP) and glutamine synthetase (GS) as a marker for satellite glia cells (SGCs). To address the original source of DCs in sensory ganglia, a proliferation experiment was also carried in this study.ResultsImmune cells with characteristic DC-phenotype were found to be closely located to SGCs and vagal sensory neurons under physiological conditions. The percentage of DCs in relation to neurons was significantly increased by allergic airway inflammation in comparison to the controls (HDM 51.38 ± 2.38% vs. control 28.16 ± 2.86%, p < 0.001). The present study also demonstrated that DCs were shown to proliferate in jugular-nodose ganglia, however, the proliferation rate of DCs is not significantly changed in the two treated animal groups (proliferating DCs/ total DCs: HDM 0.89 ± 0.38%, vs. control 1.19 ± 0.54%, p = 0.68). Also, increased number of CGRP-positive neurons was found in JNC after allergic sensitisation and challenge (HDM 31.16 ± 5.41% vs. control 7.16 ± 1.53%, p < 0.001).ConclusionThe present findings suggest that DCs may migrate from outside into the ganglia to interact with sensory neurons enhancing or protecting the allergic airway inflammation. The increase of DCs as well as CGRP-positive neurons in airway ganglia by allergic airway inflammation indicate that intraganglionic DCs and neurons expressing CGRP may contribute to the pathogenesis of bronchial asthma. To understand this neuroimmune interaction in allergic airway inflammation further functional experiments should be carried out in future studies.

Human Dendritic Cells Derived From Embryonic Stem Cells Stably Modified With CD1d Efficiently Stimulate Antitumor Invariant Natural Killer T Cell Response

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However, the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study, we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette, we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly, more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification, and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional, as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore, hESCs stably modified with the CD1d gene may serve as a convenient, unlimited, and competent DC source for iNKT cell-based cancer immunotherapy.

Identification of a novel Th1 promoting vaccine adjuvant from natural products (P4451)

Abstract One of the great obstacles to vaccine development is the lack of adjuvants that can safely and appropriately stimulate human immune cells. There are a limited number of adjuvants that have been approved for use in human vaccines. In the USA, all but one of the adjuvanted vaccines uses aluminum salts (alum) and these promote T helper (TH) 2 but not TH1 immunity. For this screen we have utilized Eskitis's Nature Bank library as a source of compounds. This library consists of over 200,000 natural product fractions derived from a collection of over 45,000 samples of plants and marine invertebrates from Australia, China, and Papua New Guinea. Mice on a C57BL/6 background that express the IL-12p40 promoter linked to yellow fluorescent protein (YFP) have been utilized as a source of dendritic cells (DCs). Splenic DCs were cultured in the presence of Nature Bank fractions or with toll like receptor-9 (TLR9) agonist CpG 1826. Cultures were assayed for IL-12 production by measuring YFP using a Perkin Elmer Operetta high content screening system and analyzed with Harmony software. Only a single compound was identified in our screen of over 20,000 fractions and it robustly activated DCs (86% compared to 11% for CpG positive controls) to become IL-12 positive. We have preliminary structural data on the compound and are subsequently testing “compound A” for immunogenicity and adjuvant activity utilizing in vitro and in vivo assays with mice, as well as human monocyte derived DC cultures.

Dendritic Cell-Based Immunotherapy for Myeloid Leukemias

Acute and chronic myeloid leukemia (AML, CML) are hematologic malignancies arising from oncogene-transformed hematopoietic stem/progenitor cells known as leukemia stem cells (LSCs). LSCs are selectively resistant to various forms of therapy including irradiation or cytotoxic drugs. The introduction of tyrosine kinase inhibitors has dramatically improved disease outcome in patients with CML. For AML, however, prognosis is still quite dismal. Standard treatments have been established more than 20 years ago with only limited advances ever since. Durable remission is achieved in less than 30% of patients. Minimal residual disease (MRD), reflected by the persistence of LSCs below the detection limit by conventional methods, causes a high rate of disease relapses. Therefore, the ultimate goal in the treatment of myeloid leukemia must be the eradication of LSCs. Active immunotherapy, aiming at the generation of leukemia-specific cytotoxic T cells (CTLs), may represent a powerful approach to target LSCs in the MRD situation. To fully activate CTLs, leukemia antigens have to be successfully captured, processed, and presented by mature dendritic cells (DCs). Myeloid progenitors are a prominent source of DCs under homeostatic conditions, and it is now well established that LSCs and leukemic blasts can give rise to “malignant” DCs. These leukemia-derived DCs can express leukemia antigens and may either induce anti-leukemic T cell responses or favor tolerance to the leukemia, depending on co-stimulatory or -inhibitory molecules and cytokines. This review will concentrate on the role of DCs in myeloid leukemia immunotherapy with a special focus on their generation, application, and function and how they could be improved in order to generate highly effective and specific anti-leukemic CTL responses. In addition, we discuss how DC-based immunotherapy may be successfully integrated into current treatment strategies to promote remission and potentially cure myeloid leukemias.