Burn injury suppresses human dermal dendritic cell and Langerhans cell function

Human skin contains epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) that are key players in induction of adaptive immunity upon infection. After major burn injury, suppressed adaptive immunity has been observed in patients. Here we demonstrate that burn injury affects adaptive immunity by altering both epidermal LC and dermal DC functions. We developed a human ex vivo burn injury model to study the function of DCs in thermally injured skin. No differences were observed in the capacity of both LCs and dermal DCs to migrate out of burned skin compared to unburned skin. Similarly, expression levels of co-stimulatory molecules were unaltered. Notably, we observed a strong reduction of T cell activation induced by antigen presenting cell (APC) subsets that migrated from burned skin through soluble burn factors. Further analyses demonstrated that both epidermal LCs and dermal DCs have a decreased T cell stimulatory capacity after burn injury. Restoring the T cell stimulatory capacity of DC subsets might improve tissue regeneration in patients with burn wounds.     

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4⁺FOXP3⁺ Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4⁺FOXP3⁺ cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4⁺FOXP3⁺ T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4⁺ that express FOXP3 was also seen when CD4⁺CD25^- purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3⁺ T cells showed suppressive activity in vitro.These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.     

The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs’ antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and Dcbased immunotherapy may be adopted in the treatment of RB.     

Carboxymethytl pachymaram up-regulates dendritic cell function in hepatitis B virus transgenic mice

Carboxymethytl pachymaram (CMP) was administered to HBV transgenic mice through abdominal injection. Lymphocytes were extracted from the spleens. MTT method was used to detect cytotoxicity of CMP. Dendritic cells (DCs) were separated from lymphocytes and incubated with granulocyte-macrophage colony-stimu-lating factor (GM-CSF) and interleukin-4 (IL-4). Phenotypes of DC's were assayed by flow cytometry (FCM). IL-12 released by DCs and IL-10 and IFN-γ pro-duced by T cells in mixed lymphocyte reaction (MLR) were measured using ELISA. Results showed that CMP within the concentration of 0-500μg/mL did not produce cyto-toxicity to lymphocytes and could even increase DC phe-notypes, and IL-12 level in HBV transgenic mice. It could also increase the secretion of IFN-γ, and inhibit the secre-tion of IL-10 in MLR. Thus it can up-regulate DC function.     

HIV-1 Nef induces dendritic cell differentiation: a possible mechanism of uninfected CD4(+) T cell activation

Human immunodeficiency virus (HIV)-1 Nef protein is an essential modulator of AIDS pathogenesis and we have previously demonstrated that rNef enters uninfected human monocytes and induces T cells bystander activation, up-regulating IL-15 production. Since dendritic cells (DCs) play a central role in HIV-1 primary infection we investigated whether rNef affects DCs phenotypic and functional maturation in order to define its role in the immunopathogenesis of AIDS. We found that rNef up-regulates the expression on immature DCs of surface molecules known to be critical for their APC function. These molecules include CD1a, HLA-DR, CD40, CD83, CXCR4, and to a lower extent CD80 and CD86. On the other hand, rNef down-regulates surface expression of HLA-ABC and mannose receptor. The functional consequence of rNef treatment of immature DCs is a decrease in their endocytic and phagocytic activities and an increase in cytokine (IL-1beta, IL-12, IL-15, TNF-alpha) and chemokine (MIP-1alpha, MIP-1beta, IL-8) production as well as in their stimulatory capacity. These results indicate that rNef induces a coordinate series of phenotypic and functional changes promoting DC differentiation and making them more competent APCs. Indeed, Nef induces CD4(+) T cell bystander activation by a novel mechanism involving DCs, thus promoting virus dissemination.     

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

Background

Damage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.

Methods

In this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.

Results

The three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8⁺ and NK cells, but not CD4⁺ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4⁺ and CD8⁺ T-cell proliferation when co-cultured with DCs.

Conclusions

Specific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.     

Carboxymethytl pachymaram up-regulates dendritic cell function in hepatitis B virus transgenic mice

Carboxymethytl pachymaram (CMP) was administered to HBV transgenic mice through abdominal injection. Lymphocytes were extracted from the spleens. MTT method was used to detect cytotoxicity of CMP. Dendritic cells (DCs) were separated from lymphocytes and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Phenotypes of DC’s were assayed by flow cytometry (FCM). IL-12 released by DCs and IL-10 and IFN-γ produced by T cells in mixed lymphocyte reaction (MLR) were measured using ELISA. Results showed that CMP within the concentration of 0–500 μg/mL did not produce cytotoxicity to lymphocytes and could even increase DC phenotypes, and IL-12 level in HBV transgenic mice. It could also increase the secretion of IFN-γ, and inhibit the secretion of IL-10 inMLR. Thus it can up-regulate DC function. __________ Translated from Journal of Wuhan University (Natural Science Edition), 2006, 52(6): 778–782 [译自: 武汉大学学报(理学版)]     

The Cryptococcus neoformans cap10 and cap59 mutant strains, affected in glucuronoxylomannan synthesis, differentially activate human dendritic cells

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides: glucuronoxylomannan and galactoxylomannan. The presence of this polysaccharide capsule was previously shown to interfere with maturation of human dendritic cells (DCs), possibly by shielding cell-wall components from interacting with these host immune cells. Here we show that two mutant strains of C. neoformans , both lacking a visible capsule due to a defect in glucuronoxylomannan synthesis, differentially activate human monocyte-derived DCs. Cells from a cap59 mutant, but not of a cap10 mutant strain, induce maturation of DCs as indicated by an increase in the expression of the costimulatory molecules CD80 and CD86, and production of the cytokines interleukin (IL)-10, IL-12p40 and tumor necrosis factor α. Interestingly, cap59 mutant cells reassociated with a concentrated culture medium of wild-type C. neoformans had lost their capacity to induce DC maturation. Summarizing, our data suggest that glucuronoxylomannan confers properties to the capsule that protect the fungus against activation of DCs; however, the presence of intact glucuronoxylomannan is not an absolute requirement to prevent activation of DCs.     

Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell development

An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.     

Third generation dendritic cell vaccines for tumor immunotherapy

This review summarizes our studies of the past several years on the development of third generation dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3 days and, second, the DCs are matured with the help of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory capacity to polarize Th1 responses in CD4⁺ T cells, induce antigen-specific CD8⁺ CTL and activate natural killer cells, these young mDCs display all the important properties needed for initiating good antitumor responses in a vaccine setting.     

Cathepsin S inhibition in dendritic cells prevents Th17 cell differentiation in perivascular adipose tissues following vascular injury in diabetic rats

In the context of diabetes mellitus (DM), the circulating cathepsin S (CTSS) level is significantly higher in the cardiovascular disease group. Therefore, this study was designed to investigate the role of CTSS in restenosis following carotid injury in diabetic rats. To induce DM, 60 mg/kg of streptozotocin (STZ) in citrate buffer was injected intraperitoneally into Sprague-Dawley rats. After successful modeling of DM, wire injury of the rat carotid artery was performed, followed by adenovirus transduction. Levels of blood glucose and Th17 cell surface antigens including ROR-γt, IL-17A, IL-17F, IL-22, and IL-23 in perivascular adipose tissues (PVAT) were evaluated. For in vitro analysis, human dendritic cells (DCs) were treated with 5.6−25 mM glucose for 24 h. The morphology of DCs was observed using an optical microscope. CD4⁺ T cells derived from human peripheral blood mononuclear cells were cocultured with DCs for 5 days. Levels of IL-6, CTSS, ROR-γt, IL-17A, IL-17F, IL-22 and IL-23 were measured. Flow cytometry was conducted to detect DC surface biomarkers (CD1a, CD83, and CD86) and Th17 cell differentiation. The collected DCs presented a treelike shape and were positive for CD1a, CD83, and CD86. Glucose impaired DC viability at the dose of 35 mM. Glucose treatment led to an increase in CTSS and IL-6 expression in DCs. Glucose-treated DCs promoted the differentiation of Th17 cells. CTSS depletion downregulated IL-6 expression and inhibited Th17 cell differentiation in vitro and in vivo. CTSS inhibition in DCs inhibits Th17 cell differentiation in PVAT tissues from diabetic rats following vascular injury.     

OP9-DL1 cell co-culture enhances anti-tumour immunity of mouse bone marrow-derived dendritic cells

DCs (dendritic cells) are the strongest professional APCs (antigen-presenting cells) to initiate immune responses against pathogens, but they are usually incompetent in initiating efficient immune responses in the progress of solid tumours. We have shown that Notch signalling plays a pivotal role in DC-dependent anti-tumour immunity. Compared with the control DCs, OP9-DL1 (Delta-like1) cell co-cultured DCs gained increased tumour suppression activity when inoculated together with tumour cells. This was probably due to the activation of Notch signalling in DCs enhancing their ability to evoke anti-tumour immune responses in solid tumours. Indeed, the OP9-DL1 cell co-cultured DCs expressed higher levels of MHC I, MHC II, CXCR4 (CXC chemokine receptor 4), CCR7 (CC chemokine receptor 7), IL-6 (interleukin 6), IL-12 and TNFα (tumour necrosis factor α), and a lower level of IL-10 than control DCs, resulting in more efficient DC migration and T-cell activation in vivo and in vitro. T-cells stimulated by OP9-DL1 cells co-cultured DCs more efficiently; and were cytotoxic against tumour cells, in contrast with control DCs. These results indicated that up-regulation of Notch signalling in DCs by co-culturing with OP9-DL1 cells enhances DC-dependent anti-tumour immune reactions, making the Notch signalling pathway a target for the establishment of the DC-based anti-tumour immunotherapies.     

Dendritic cell maturation occurs through the inhibition of GSK-3β

Dendritic cell (DC) maturation results in changes in antigen processing and presentation, governing the fate of adaptive immunity. Understanding the intracellular signaling pathways governing DC maturation is therefore critical. In this study, we observed that the kinase, GSK-3β, is present in its active form in resting immature DCs isolated from the spleen and bone marrow of mice. Induction of DC maturation using GM-CSF, IL-4 and TNF-α resulted in GSK-3β inhibition, as reflected by increased phosphorylation of Serine 9 on the kinase, and concomitant stabilization of its substrate, β-catenin. Treatment of immature DCs with a GSK-3β inhibitor increased cell surface expression of CD80, CD86 and CD40 on DCs, enhancing their ability to present antigen and activating IL-2 secretion by T cells. GSK-3β inhibition also parallels dendritic cell maturation in vivo. Our results show that GSK-3β signaling controls DC maturation and suggest that this kinase could be manipulated to modulate adaptive immunity.     

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)-y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi-mature stage (smDCs) retaining the capacity to secret interleukin (IL)-12 and thus support cytolytic T-cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 ± 4% recovery, which was approximately 5-fold higher as the most frequently used adherence protocol (8 ± 1%), and a very good purity (92 ± 5%) of smDCs. Immune phenotype and IL-12 secretion (adherence: 1.4 ± 0.4; selection: 20 ± 0.6; depletion: 1 ±0.5; elutriation: 3.6 ± 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9.4 ± 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.     

登革病毒对人树突状细胞感染性的研究

探讨登革病毒对人树突状细胞(DC)的感染性。人外周新鲜血常规分离单核细胞,经细胞因子GMCSF、IL4诱导培养成DC,通过形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型(DV2)感染DC,于作用后6h、24h、48h、72h、96h分别收集上清液和细胞,甲基纤维素微量空斑试验测定病毒滴度,间接免疫荧光法检测细胞上病毒抗原表达,透射电镜观察病毒在细胞内的定位。病毒感染后6h即可在培养上清中测出病毒,病毒滴度在48h达到高峰,以后逐渐下降。间接免疫荧光法证明感染的DC胞浆及胞膜上携带病毒抗原。透射电镜下在病毒感染48h后DC胞浆内可见大量病毒颗粒。树突状细胞是登革病毒感染的靶细胞,病毒可感染DC并产生大量病毒颗粒,可能在其发病机制中起重要作用。     

Oncostatin M production by human dendritic cells in response to bacterial products

Oncostatin M (OSM) is a pleiomorphic cytokine that belongs to the IL-6 cytokine family. It is produced by activated T cells and monocytes/macrophages and plays an important role in the process of inflammatory responses. Although dendritic cells (DCs) have been shown to secrete a variety of cytokines, it is not elucidated whether DCs are able to produce OSM. To clarify this, using human DCs derived from peripheral blood cells, we measured the protein levels of OSM in the supernatants of DC cultures by ELISA and examined the expression of OSM mRNA by RT-PCR after stimulation with lipopolysaccharide (LPS) or fixed Staphylococcus aureus (SACS). Upon stimulation with bacterial products, DCs secreted a large amount of OSM protein in a dose- and time-dependent manner. Concomitantly, the expression of OSM mRNA by DCs was markedly up-regulated. Compared the ability of DCs to produce OSM with that of monocytes, which are major producers of OSM, DCs released significantly higher amounts of OSM protein in the culture supernatants than monocytes. These findings indicate for the first time that human monocyte-derived DCs can synthesize and secrete large amounts of OSM in response to bacterial products, suggesting that OSM produced by DCs at infectious sites may play a role in modulating inflammatory responses.     

Comparison of media and serum supplementation for generation of monophosphoryl lipid A/interferon-γ–matured type I dendritic cells for immunotherapy

Background aimsEx vivo–generated monocyte-derived dendritic cells (DCs) matured with monophosphoryl lipid A (MPLA) and interferon-γ (IFN-γ) can be used as cancer immunotherapy. MPLA/IFN-γ DCs induce Th1 T cell responses and have migratory capacity. Different culture regimens have been used for generation of immunotherapeutic DCs, with varying results. In the present study, culture conditions for MPLA/IFN-γ–matured type I DCs were optimized for clinical application.MethodsDCs were generated from monocytes in the clinical grade culture media CellGro DC, AIM V or X-VIVO 15 in the absence or presence of 2% human serum (HS) and matured with the use of MPLA/IFN-γ. DC yield and DC functionality were assessed. DC functionality was determined by means of analysis of cytokines in culture supernatant, migratory capacity, expression of co-stimulatory molecules, T cell stimulatory capacity of DCs and T helper cell (Th) polarization by the DCs.ResultsDCs generated in the presence of 2% HS produced low amounts of pro-inflammatory cytokines and could not migrate irrespective of the medium used. In the absence of HS, MPLA/IFN-γ DCs generated in X-VIVO did not migrate either. MPLA/IFN-γ DCs generated in AIM V have slightly lower capacity to induce Th1 cells than do DCs generated in CellGro or X-VIVO.ConclusionsAddition of HS to different GMP culture media is detrimental for pro-inflammatory DC maturation and migration. In the absence of serum, CellGro is the most optimal medium tested for generation of migratory and Th1-inducing MPLA/IFN-γ DCs for cancer immunotherapy.     

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.