Phenotypic Maturation Of Dendritic Cells Research Articles

Differentially Charged Liposomes Stimulate Dendritic Cells with Varying Effects on Uptake and Processing When Used Alone or in Combination with an Adjuvant.

Liposomes carrying differential charges have been extensively studied for their role in stimulating dendritic cells (DCs), major antigen-presenting cells, known to serve as a pivotal bridge between innate and adaptive immunity. However, the impact of the differentially charged liposomes on activating DCs remains to be understood. In this study, we have investigated the impact of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based neutral, anionic, and cationic liposomes on the uptake, immunostimulation, and intracellular fate in mouse bone-marrow-derived DCs. We observed that liposomes could induce phenotypic maturation of DCs by inducing the expression of costimulatory molecules (CD40 and CD86) and production of cytokines tumor necrosis factor-α, interleukin-12,and nitric oxide. Interestingly, admixing monophosphoryl lipid A with charged liposomes further enhances the expression of the costimulatory molecules and production of cytokines, with preferential activation by positively charged liposomes. Fluorometric analysis using a pH-sensitive dye and flow-cytometry-based pathway inhibition assays revealed that cationic liposomes were taken up more efficiently by DCs through endocytosis and transported to neutral compartments for further processing, whereas anionic and neutral liposomes were inclined to accumulate in acidic compartments. These findings therefore endorse the use of cationic DSPC liposomes as a preferred option for vaccine delivery vehicles over neutral and negatively charged liposomes, particularly for the preferential activation of DCs.

The effect of cortisol on equine monocyte–derived dendritic cell phenotype and cytokine production

AbstractBackgroundIncreased risk of infection in neonates, including foals, is associated with the naivety of the immune system and the immaturity of the endocrine system. Foal dendritic cells (DCs) are phenotypically and functionally less mature than adult horse DCs. Exposure of foal DCs to foal plasma alters DC phenotypic maturation, cytokine secretion and bacterial endocytosis. Specific plasma factors that impact equine DC activity are unknown. Endocrine dysfunction, resulting in altered cortisol availability, is common in foals. Cortisol impacts DC maturation and function in other species, but the effect of cortisol on equine DCs is not described. We hypothesized that exposure to cortisol would impact both the phenotype and function of equine monocyte–derived DCs (MoDCs), perpetuating an immature phenotype and altered cytokine profile.MethodsMoDCs were generated from foals (n = 8) on 1, 7 and 30 days of age and adult horses (n = 9). MoDCs were exposed to killed bacteria in the presence or absence of cortisol. The expression of surface markers (MHC class‐II, CD86 and CD14) was measured by flow cytometry. Supernatant cytokine concentrations (IL‐4, IL‐17, IFN‐γ and IL‐10) were quantified using a validated bead‐based immunoassay.ResultsCortisol exposure reduced the percentage of equine MoDCs expressing MHC class‐II and CD86 (p ≤ 0.03). Adult horse MoDCs exposed to bacteria and cortisol produced less IL‐4, IL‐17, IFN‐γ and IL‐10 than cells not treated with cortisol, and foal MoDCs secreted less IL‐10 and IL‐4 (p ≤ 0.009).ConclusionsIn sum, cortisol exposure perpetuates an immature MoDC phenotype and alters cytokine responses, which might impact the foal's susceptibility to infection.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

The role of the 47-kDa membrane lipoprotein of Treponema pallidum in promoting maturation of peripheral blood monocyte-derived dendritic cells without enhancing C-C chemokine receptor type 7-mediated dendritic cell migration.

The 47-kDa membrane lipoprotein (Tp47) is the most representative membrane protein of Treponema pallidum (T. pallidum). Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that connect innate and acquired immunity. The regulatory role of Tp47 on DCs remains unclear. To evaluate the effects of Tp47 on DC maturation and migration, and research the changes of the main chemokine C-C chemokine receptor type 7 (CCR7) involved in DC migration. A transwell assay was applied to assess the migration of DCs. Cytokines (interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α)) in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA), and the expression of cell surface markers (CD80, CD86, CD40, and human leukocyte antigen (HLA)-DR) and CCR7 was assessed using flow cytometry. The expression of CCR7 in DCs was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The Tp47 promoted DC phenotypic maturation, such as increased CD40, CD80, CD86, and HLA-DR expression, as well as DC functional maturation, thus stimulating DCs to secrete inflammatory cytokines, including IL-6, IL-10, IL-12, and TNF-α. At the same time, Tp47 did not enhance DC migration and did not increase the expression of CCR7. The Tp47 promoted the maturation of DCs while not enhancing CCR7-mediated DC migration ability. This may be one of the mechanisms by which T. pallidum escapes host immune clearance.

HIV-1 induction of tolerogenic dendritic cells is mediated by cellular interaction with suppressive T cells.

HIV-1 infection gives rise to a multi-layered immune impairment in most infected individuals. The chronic presence of HIV-1 during the priming and activation of T cells by dendritic cells (DCs) promotes the expansion of suppressive T cells in a contact-dependent manner. The mechanism behind the T cell side of this HIV-induced impairment is well studied, whereas little is known about the reverse effects exerted on the DCs. Herein we assessed the phenotype and transcriptome profile of mature DCs that have been in contact with suppressive T cells. The HIV exposed DCs from cocultures between DCs and T cells resulted in a more tolerogenic phenotype with increased expression of e.g., PDL1, Gal-9, HVEM, and B7H3, mediated by interaction with T cells. Transcriptomic analysis of the DCs separated from the DC-T cell coculture revealed a type I IFN response profile as well as an activation of pathways involved in T cell exhaustion. Taken together, our data indicate that the prolonged and strong type I IFN signaling in DCs, induced by the presence of HIV during DC-T cell cross talk, could play an important role in the induction of tolerogenic DCs and suppressed immune responses seen in HIV-1 infected individuals.

Influence of photon, proton and carbon ion irradiation on differentiation, maturation and functionality of dendritic cells.

While the primary purpose of radiotherapy (RT) is the elimination of cancer cells by inducing DNA-damage, considerable evidence emerges that anti-neoplastic effects extend beyond mere tumor cell killing. These secondary effects are based on activation of dendritic cells (DCs) via induction of antitumoral immune reactions. However, there is an ongoing debate whether or not irradiation of the DCs themselves may negatively affect their maturation and functionality. Human monocytes were irradiated with different absorbed doses (1 × 15 Gy relative biological effectiveness (RBE), 5 × 2 Gy (RBE), 1 × 0.5 Gy (RBE)) with photons, protons and carbon ions. Differentiation and maturation of DCs were assessed by staining of corresponding cell surface molecules and functional analysis of irradiated DCs was based on in vitro analysis of phagocytosis, migration and IL-12 secretion. Irradiation of CD14-positive DCs did not alter surface phenotypes of immature DCs and mature DCs. Not only differentiation, but also functionality of immature DCs regarding phagocytosis, migration and IL-12 secretion capacity was not negatively influenced through RT with photons, protons or carbon ions as well as with different dose levels. After proton irradiation migratory capacity of immature DCs was increased. Our experiments reveal that phenotypic maturation of DCs remains unchanged after RT with different fractionations and after irradiation with particle therapy. Unaffected functionality (phagocytosis, migration and cytokine secretion) after RT of DCs indicated possible persistent potential for inducing adaptive immune response. Additional effects on the immunogenic potential of DCs will be investigated by further functional assays.

Baoganning formula alleviates liver fibrosis in mice by inhibiting hepatic IDO1 expression and promoting phenotypic maturation of dendritic cells

To investigate the protective effect of Bao Gan Ning (BGN), a traditional Chinese medicinal formula, against CCl4-induced liver fibrosis in mice and explore the mechanism. C57BL/6 mice were randomly divided into control group, liver fibrosis model group, positive control group and the low-, middle-, and high-dose BGN groups (n=10). In all but the control group, the mice were subjected to daily intraperitoneal injection of 25% CCl4 (in olive oil) to induce liver fibrosis and intragastric gavage of corresponding drugs. After 8 weeks, serum levels of ALT and AST were detected. Pathological examination of the liver was performed using HE and Sirius Red staining and α-SMA immunohistochemistry. The expression level of indoleamine 2, 3-dioxygenase 1 (IDO1) and phenotypic changes of hepatic DCs in the liver were measured. Another 18 mice were randomly divided into AAV9-NC, AAV9-IDO1 and high-dose BGN + AAV-IDO1 groups (n=6) for corresponding treatment, and 4 weeks later the deposit of hepatic IDO1 and phenotypic changes of the hepatic DCs were analyzed. Compared with those in the model group, serum AST and ALT levels decreased significantly in BGN group (P < 0.01). Obvious liver fibrosis was observed in the model group, while the mice treated with BGN showed obviously reduced cell necrosis and collagen proliferation in the liver with significantly lowered the expression levels of hepatic α-SMA and IDO1 (P < 0.05). The percentages of CD11C + DCs, CD11C +CD80 + DCs, CD11C +CD86 + DCs, CD11C +CD40 + DCs, CD11C +MHCII + DCs, CD3 + T cells, and CD3 +CD4 + T cells all increased significantly in BGN group as compared with the model group (P < 0.05). In mice with adenovirus-mediated IDO1 overexpression in the liver, BGN treatment significantly lowered the expression level of IDO1 (P=0.000) and increased the percentages of hepatic CD11C +CD40 + DCs, CD11C +MHCII + DCs and CD3 +CD4 + T cells (P < 0.05). BGN can effectively inhibit liver fibrosis in mice possibly by lowering the expression level of IDO1 in the liver, thus improving the function of hepatic DCs and subsequently promoting proliferation of T cells.

The Collective Effect of MIP-3α and FL Promotes Dendritic Cell Function Within the Immune Microenvironment of Murine Liver Cancer.

Hepatocellular carcinoma is a highly malignant and lethal tumor. In addition to surgery, immunotherapy is currently a more effective treatment for hepatocellular carcinoma. The tumor immune microenvironment (TIME) largely determines the efficacy of cancer immunotherapy. Based on the universal targeting of TIME modulators in clinical treatment, TIME modulators are promising targets for tumor immunotherapy. We investigated the effect of a double gene expression vector (recombinant galactose-terminal glycol-poly-L-lysine coupled MIP-3α-FL) on dendritic cells (DCs) regulation within the TIME of mice with liver cancer. H22 cells were transfected with a recombinant MIP-3α-FL plasmid to induce DCs differentiation and chemotaxis. The effects of transfection were investigated by flow cytometry following the modified Boyden’s method. Cytokine-induced killer (CIK) cells co-culture revealed changes in the antigen presentation ability of DCs. Further, tumor-bearing mice were injected with the recombinant double gene vector via the tail vein. We compared the survival time, tumor volume, weight of the mice, as well as the number and phenotype of tumor-infiltrating DCs (TIDCs) between groups. The supernatant of transfected H22 cells promoted the phenotypic maturation of DCs, enhancing their chemotaxis. Further, treated DCs promoted the cytokine secretion and killing ability of CIK cells. The survival time of mice injected with the double gene vector was significantly prolonged, while their tumor weight and volume were relatively reduced. Flow cytometry revealed that the number of TIDCs (as well as CD80 and CD86 expression) in the MouseMIP-3α-FL group, were significantly higher than in the control group. The combination of MIP-3α and FL can significantly promote DCs aggregation, maturation, and enhance their antigen presentation ability. The coupling of the double gene vector with glycosylated polylysine can improve the precise targeting of the liver and inhibit tumor growth in vivo, providing a novel approach for immune therapy in liver cancer.

Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade.

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

Newcastle Disease Virus Inhibits the Proliferation of T Cells Induced by Dendritic Cells In Vitro and In Vivo.

Newcastle disease virus (NDV) infects poultry and antagonizes host immunity via several mechanisms. Dendritic cells (DCs) are characterized as specialized antigen presenting cells, bridging innate and adaptive immunity and regulating host resistance to viral invasion. However, there is little specific knowledge of the role of DCs in NDV infection. In this study, the representative NDV lentogenic strain LaSota was used to explore whether murine bone marrow derived DCs mature following infection. We examined surface molecule expression and cytokine release from DCs as well as proliferation and activation of T cells in vivo and in vitro in the context of NDV. The results demonstrated that infection with lentogenic strain LaSota induced a phenotypic maturation of immature DCs (imDCs), which actually led to curtailed T cell responses. Upon infection, the phenotypic maturation of DCs was reflected by markedly enhanced MHC and costimulatory molecule expression and secretion of proinflammatory cytokines. Nevertheless, NDV-infected DCs produced the anti-inflammatory cytokine IL-10 and attenuated T cell proliferation, inducing Th2-biased responses. Therefore, our study reveals a novel understanding that DCs are phenotypically mature but dysfunctional in priming T cell responses during NDV infection.

Immune-Enhancing Activity of Aqueous Extracts from Artemisia rupestris L. via MAPK and NF-kB Pathways of TLR4/TLR2 Downstream in Dendritic Cells.

Artemisia rupestris L. has long been used as a traditional herbal medicine owing to its immunomodulatory activity. Aqueous extracts of Artemisia rupestris L. (AEAR) contain the main functional component and can activate the maturation of dendritic cells (DCs) and enhance the adaptive immunity as the adjuvant against infections. To explore the underlying mechanism of immunomodulatory activities of AEAR, DCs were produced from bone-marrow cells of mice and the effects of AEAR on cell viability were assessed by the Cell Counting Kit 8 (CCK8) method and annexin V/propidium iodide staining assays. Then, the effects of AEAR on the morphology, maturation, and function of DCs were detected using a microscope, flow cytometry-based surface receptor characterization, and endocytosis assays. The secretion levels of cytokines were then analyzed with enzyme-linked immunosorbent assay (ELISA). The activation state of DCs was evaluated by the mixed lymphocyte reaction (MLR). The activity of MAPKs and NF-κB pathways, which were involved in the regulation of AEAR on DCs, was further detected by Western blot. AEAR did not have a cytotoxic effect on DCs or mouse splenocytes. AEAR remarkably enhanced the phenotypic maturation of DCs and promoted the expression of costimulatory molecules and the secretion of cytokines in DCs. AEAR also significantly decreased the phagocytic ability of DCs and augmented the abilities of DCs to present antigens and stimulate allogeneic T-cell proliferation. Simultaneously, AEAR potently activated toll-like receptor (TLR)4-/TLR2-related MAPKs and induced the degradation of IκB and the translocation of NF-κB. In short, AEAR can profoundly enhance the immune-modulating activities of DCs via TLR4-/TLR2-mediated activation of MAPKs and NF-κB signaling pathways and is a promising candidate immunopotentiator for vaccines.

Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and β-glucans.

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives.

Radiation-induced augmentation in dendritic cell function is mediated by apoptotic bodies/STAT5/Zbtb46 signaling

Purpose: To evaluate the effect of ionizing radiation (IR) exposure on differentiation and maturation of dendritic cells (DC).Materials and methods: Bone marrow progenitor cells irradiated in vitro or isolated from mice exposed to whole body or localized tumor irradiation were differentiated into DC. Phenotypic maturation of DC was characterized by labeling with specific antibodies and flow cytometry analysis. Cytokines were estimated by ELISA.Results: Splenic and bone marrow-derived DC (BMDC) from tumor-bearing mice exposed to localized irradiation showed abrogation of tumor-induced immunosuppression. This was not due to the effect of radiation on tumor cells as DC derived from normal mice exposed to whole-body irradiation (WBI) also showed increase in immune-activating potential of DC. This was observed in terms of increased phenotypic and functional activation of DCs. This phenomenon was also recapitulated if DC were differentiated from in vitro irradiated progenitor cells and was found to be due to STAT5/Zbtb46 signaling mediated by the irradiation-induced apoptotic bodies (ABs). When these ABs were depleted using annexin-beads, these effects were reversed confirming the involvement of this pathway. The role of ABs was further validated in DC derived from mice exposed to WBI in adaptive response experiments with 0.1 Gy priming dose prior to 2 Gy challenge dose. A corresponding reduction in DC maturation markers was observed with decrease in apoptosis in vivo. Further, these DCs derived from irradiated progenitors (IP) could resist the suppressive effects of tumor conditioned medium (TCM) and had increased immune-activating potential as seen in the tumor-bearing mice.Conclusions: Though radiation is the most commonly used therapeutic modality for cancer, its effects on dendritic cell differentiation is not completely understood. We demonstrate here for the first time that exposure to select doses of IR can increase immune-activating potential of DC through ABs. This can have implications in selection of appropriate doses of IR during radiotherapy of cancer patients.

Exosomes from 5-aminolevulinic acid photodynamic therapy-treated squamous carcinoma cells promote dendritic cell maturation.

5-aminolevulinic acid photodynamic therapy (ALA-PDT) is effective in skin tumours. Studies have demonstrated that the therapy has anti-tumour immunity in squamous cell carcinoma (SCC). Exosomes play an important role in tumour microenvironment (TME) crosstalk. Nevertheless, whether exosomes mediate the ALA-PDT anti-tumour effect is unclear. This study aims to investigate whether exosomes secreted from ALA-PDT-treated squamous carcinoma cells (SCCs) demonstrate an anti-tumour effect by inducing dendritic cell (DCs) maturation. In this study, we used electron microscopy, nanoparticle tracking analysis and western blotting to identify exosomes. Subsequently, BCA assay and fluorescence staining were used to evaluate the biological activity of exosomes. Exosomes derived from ALA-PDT-treated SCCs were incubated with SCCs, fibroblasts and immature DCs, separately. A CCK-8 kit was used to analyse the cytotoxicity of exosomes to SCCs. ELISA was utilised to analyse IL-6, VEGF, MMP-3, and TGF-β1 secreted from fibroblasts. FACS and ELISA were used to analysed DC phenotypic maturation (CD80, MHC-II) and IL-12 secretion. Herein we show that exosomes secreted from SCCs after ALA-PDT cannot exert cytotoxicity towards SCCs. However, exosomes derived from ALA-PDT-treated SCCs could induce DCs maturation and IL-12 secretion. Furthermore, exosomes secreted from SCCs after ALA-PDT promote the secretion of TGF-β1 from fibroblast. In conclusion, we found that exosomes derived from ALA-PDT-treated SCCs have the ability to stimulate DC maturation and fibroblast secretion of TGF-β1, which results in the elevation of anti-tumour immunity. These findings provide a new promising strategy of anti-tumour immune response for ALA-PDT in treating SCCs.

PPE39 of the Mycobacterium tuberculosis strain Beijing/K induces Th1-cell polarization through dendritic cell maturation.

In a previous study, we have identified MTBK_24820, the complete protein form of PPE39 in the hypervirulent Mycobacterium tuberculosis (Mtb) strain Beijing/K by using comparative genomic analysis. PPE39 exhibited vaccine potential against Mtb challenge in a murine model. Thus, in this present study, we characterize PPE39-induced immunological features by investigating the interaction of PPE39 with dendritic cells (DCs). PPE39-treated DCs display reduced dextran uptake and enhanced MHC-I, MHC-II, CD80 and CD86 expression, indicating that this PPE protein induces phenotypic DC maturation. In addition, PPE39-treated DCs produce TNF-α, IL-6 and IL-12p70 to a similar and/or greater extent than lipopolysaccharide-treated DCs in a dose-dependent manner. The activating effect of PPE39 on DCs was mediated by TLR4 through downstream MAPK and NF-κB signaling pathways. Moreover, PPE39-treated DCs promoted naïve CD4+ T-cell proliferation accompanied by remarkable increases of IFN-γ and IL-2 secretion levels, and an increase in the Th1-related transcription factor T-bet but not in Th2-associated expression of GATA-3, suggesting that PPE39 induces Th1-type T-cell responses through DC activation. Collectively, the results indicate that the complete form of PPE39 is a so-far-unknown TLR4 agonist that induces Th1-cell biased immune responses by interacting with DCs.This article has an associated First Person interview with the first author of the paper.

The immunostimulatory effects and pro-apoptotic activity of rhCNB against Lewis lung cancer is mediated by Toll-like receptor 4.

BackgroundRecombinant human calcineurin B subunit (rhCNB) has been shown to be an immune‐stimulatory protein promoting cytokine production and inducing phenotypic maturation of Dendritic cells (DCs). In vivo, it has good antitumor efficacy, and has potential as an antitumor drug. Exogenous rhCNB was found to be internalized into tumor cells via the Toll‐like receptor 4 (TLR4) complex, but it was not known whether its immuno‐modulatory and antitumor functions involved entry by this same route.MethodsThe production and secretion of the cytokines and chemokines in innate immune cells induced by rhCNB were determined by ELISA, and the expression of CD40, CD80, CD86, and MHCII was analyzed by FACs. Experimental Lewis lung cancer (LLC) model was prepared in C57 BL/6 wild‐type (WT) mice, TLR4−/− mice or their littermates by the inoculation of LLCs in their right armpit, and then administrated daily intraperitoneal injections (0.2 mL) of normal saline, rhCNB 20 mg/kg, and rhCNB 40 mg/kg, respectively.ResultsRecombinant human calcineurin B subunit promoted the production of antitumor cytokines by innate immune cells, and culture supernatants of rhCNB‐stimulated immune cells induced apoptosis of LLCs. In addition, rhCNB up‐regulated CD40, CD80, CD86, and MHCII expression in macrophages and DCs in TLR4+ cells but failed to do so in TLR4 deficient cells. rhCNB also induced the formation of CD4+ and CD8+T cells in splenocytes from WT mice, but not from TLR4‐deficient littermates. Intraperitoneal administration of WT C57BL/6 mice with rhCNB resulted in a 50% reduction in LLC tumor growth, but failed to inhibit tumor growth in TLR4−/− littermates.ConclusionsThe in vivo antitumor and immunomodulatory effects of rhCNB are mediated by the TLR4. This conclusion is important for the further understanding and development of rhCNB as an antitumor drug.

PB1982 AN IN VITRO STUDY OF THE INTERACTIONS BETWEEN THE CELL BASED CANCER VACCINE DCP‐001 AND ANTIGEN PRESENTING CELLS

Background:DCP‐001 is a whole cell‐based vaccine derived from the DCOne® human myelomonocytic leukemic cell line. DCOne cells endogenously express known and unknown tumour‐associated antigens. In the DCP‐001 manufacturing process, DCOne cells are differentiated and matured into cells with a mature dendritic cell phenotype. A phase I clinical trial demonstrated that the vaccine is safe and induces local and systemic cellular and humoral immune responses. DCP‐001 is currently studied in an international Phase II trial in AML patients ineligible for HSCT.Aims:To study the role of the interaction of whole cell‐based vaccine DCP‐001 with host immune cells.Methods:DCP‐001 and antigen presenting cell interaction studies were performed using flow cytometry and confocal microscopy. Cytokine/chemokine analyses were performed using luminex technology.Results:The strong immunogenicity of DCP‐001 is demonstrated by the broad range of chemokines and pro‐inflammatory cytokines released upon co‐culture of DCP‐001 with human peripheral blood mononuclear cells. Using different methods, we further demonstrate that antigen presenting cells efficiently process DCP‐001 through phagocytosis.Summary/Conclusion:These in vitro data suggest that in vivo, upon intradermal injection, DCP‐001 induces a strong inflammatory response, and is ingested by both resident as well as attracted host's APC, which subsequently prime tumour‐reactive T cells.

The role of 2-arachidonoylglycerol in the regulation of the tumor-immune microenvironment in murine models of pancreatic cancer

The pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a critical role in the antitumor immune response. 2-arachidonoylglycerol (2-AG) exhibits a direct antitumor effect in various tumor models. However, the immunomodulatory effect of 2-AG on PDAC remains obscure. The aim of this study was to explore the tumor microenvironment response to 2-AG in pancreatic cancer. A PDAC orthotopic tumor model was used to investigate tumor proliferation and the population of immune cells in vivo, including dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), CD8+ T cells and CD4+ T cells. The effect of 2-AG on panc02 cell proliferation and DC2.4 cell maturation in vitro by mediating activation the typical cannabinoid receptors (CB1, CB2) was evaluated by flow cytometry and CCK8. The protein levels of P-STAT6, STAT6 and GADPH were measured by Western blotting.2-AG inhibited pancreatic cancer cell proliferation in tumor bearing mice and panc02 cell. Inhibition of proliferation was blocked by the CB1receptor antagonist (AM251) but not the CB2 receptor antagonist (AM630). In addition, 2-AG promoted DC phenotypic maturation and the production of proinflammatory cytokines by up-regulating p-STAT6. These effects were also blocked by AM251 but not AM630. Moreover, we also provide evidence that 2-AG administration induced the expansion of MDSCs in tumor bearing mice. However, no effect on the population of CD8+ T cells and CD4+ T cells was observed. Our findings support 2-AG exhibited direct antitumor effects via inhibiting pancreatic cancer proliferation and inducing DC phenotypic maturation, but also significantly promoted an immunosuppressive microenvironment via increasing the suppressive immune cell population of MDSCs.

The interactions of dendritic cells with osteoblasts on titanium surfaces: an in vitro investigation.

Osteoimmune interactions possess a critical part in the integration of materials and hosts. Dendritic cells (DCs) are the most common members of osteoimmune cells family. The titanium surfaces of dental implants tend to promote a mature dendritic cell phenotype with increased proinflammatory secretion. However, very little is known to the effects of this microenvironment on the behaviors of cells around implants, especially osteoblasts, and how the tissue integrations take place on such biomaterial surfaces. Therefore, the present study was to investigate the interactions of DCs with osteoblasts on titanium surfaces. DCs seeded on PT and SLA titanium surfaces were compared by assays for the proliferations, surface markers, and inflammatory genes expressions. DCs seeded on PT and SLA titanium surfaces were compared by assays for the proliferations, surface markers, and inflammatory genes expressions. Next, we harvested the dendritic cell-conditioned medium (CM) and investigated the effects of CM on MC3T3-E1. The results showed an increase in CD86 and proinflammatory expressions of DCs seeded on PT and SLA surfaces, as well as a decrease in anti-inflammatory cytokines. The CM from titanium surfaces inhibited the osteoblast differentiation with reduced expression of osteogenic genes RUNX2, COL1, ALP, and OCN and decreased ALP activity as well as Alizarin red staining. These findings suggested that titanium surfaces switch DCs toward maturation phenotypes and thus inhibit the differentiation and mineralization of osteoblasts. Knowing the significant impact of immune cells on osteogenesis behaviors, some efforts to decrease the immune reaction might be of clinical significance. Favorable immune environments can increase the dental implants survival rate in patients.

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma.

Glioblastoma (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen-presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in modifying DC function. We found that VEGF blockade results in a more mature DC phenotype in the brain, as demonstrated by an increase in the expression of the co-stimulatory molecules B7-1, B7-2, and MHC II. Furthermore, we observed reduced levels of the exhaustion markers PD-1 and Tim-3 on brain-infiltrating CD8 T cells, indicating improved functionality. Thus, anti-angiogenic therapy has the potential to be used in conjunction with and enhance immunotherapy for GBM.