Multiple Dendritic Cells Research Articles

Activation of the Ancestral Polarity Regulator Protein Kinase Cζ at the Immunological Synapse Drives Polarization of Th Cell Secretory Machinery toward APCs

A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.

Identification of a novel antigen cross-presenting cell type in spleen

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely ‘L-DC’. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11cloCD11bhiMHC-II−CD8α− cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8+ T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4+ T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.

Mast Cells, Histamine, and IL-6 Regulate the Selective Influx of Dendritic Cell Subsets into an Inflamed Lymph Node

In response to bacterial stimuli, multiple dendritic cell (DC) populations accumulate within the draining lymph node, thus enhancing opportunities for effective T cell-DC interaction. DC subpopulations, such as plasmacytoid, CD8(+), and CD11b(+) subsets, have distinct roles in determining the nature of the immune response. The mechanisms whereby individual DC subpopulations are mobilized and the extent to which these processes are linked to increases in overall lymph node cellularity have not been determined. In the current study, the mechanisms of DC subset mobilization to the draining auricular lymph node were examined after intradermal injection of Staphylococcus aureus-derived peptidoglycan. Using mast cell-deficient mice and local mast cell reconstitution, plasmacytoid and CD8(+) DC responses were shown to be mast cell dependent, whereas the CD11b(+) DC response was not. A histamine H2 receptor-dependent, CXCL9-independent pathway controlled the selective influx of both plasmacytoid and CD11b(+) DC into the lymph node, but not lymph node cellularity. In contrast, IL-6 was important for the mobilization of CD8(+) and CD11b(+) DC. TNF and IL-1 receptor were dispensable for plasmacytoid, CD11b(+), and CD8(+) DC responses. These findings provide novel opportunities for the selective mobilization of specific DC subsets to lymph nodes and demonstrate critical roles for both histamine and IL-6 in this process.

Cross-Talk between Human Dendritic Cell Subsets Influences Expression of RNA Sensors and Inhibits Picornavirus Infection

Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs). In this study we analyzed the expression of PRRs responding to viral RNA in human monocyte-derived DCs (moDCs) under steady-state or pro-inflammatory conditions. We found that mRNA and protein levels for most PRRs were increased under pro-inflammatory conditions, with the most pronounced increases in the RIG-like helicase (RLH) family. Additionally, freshly isolated human plasmacytoid DCs (pDCs) displayed significantly higher levels of TLR7, RIG-I, MDA5 and PKR as compared to myeloid DCs and moDCs. Finally, we demonstrate for the first time that cross-talk between TLR-matured or virus-stimulated pDCs and moDCs leads to a type I interferon-dependent antiviral state in moDCs. This antiviral state was characterized by enhanced RLH expression and protection against picornavirus infection. These findings might represent a novel mechanism by which pDCs can preserve the function and viability of myeloid DCs that are attracted to a site with ongoing infection, thereby optimizing the antiviral immune response.

In Vitro Hematopoiesis Reveals a Novel denDritic-Like Cell Present in Murine Spleen

Dendritic cells (DC) are important antigen presenting cells (APC) which induce and control the adaptive immune response. In spleen alone, multiple DC subsets can be distinguished by cell surface marker phenotype. Most of these have been shown to develop from progenitors in bone marrow and to seed lymphoid and tissue sites during development. This study advances in vitro methodology for hematopoiesis of dendritic-like cells from progenitors in spleen. Since spleen progenitors undergo differentiation in vitro to produce these cells, the possibility exists that spleen represents a specific niche for differentiation of this subset. The fact that an equivalent cell subset has been shown to exist in spleen also supports that hypothesis. Studies have been directed at investigating the specific functional role of this novel subset as an APC accessible to blood-borne antigen, as well as the conditions under which hematopoiesis is initiated in spleen, and the type of progenitor involved.

Lung Dendritic Cell Expression of Maturation Molecules Increases with Worsening Chronic Obstructive Pulmonary Disease

Dendritic cells (DCs) have not been well studied in chronic obstructive pulmonary disease (COPD), yet their integral role in activating and differentiating T cells makes them potential participants in COPD pathogenesis. To determine the expression of maturation molecules by individual DC subsets in relationship to COPD stage and to expression of the acute activation marker CD69 by lung CD4(+) T cells. We nonenzymatically released lung leukocytes from human surgical specimens (n = 42) and used flow cytometry to identify three DC subsets (mDC1, mDC2, and pDC) and to measure their expression of three costimulatory molecules (CD40, CD80 and CD86) and of CD83, the definitive marker of DC maturation. Spearman nonparametric correlation analysis was used to identify significant correlations between expression of DC maturation molecules and COPD severity. Expression of CD40 by mDC1 and mDC2 and of CD86 by mDC2 was high regardless of GOLD stage, but CD80 and CD83 on these two DC subsets increased with disease progression. pDC also showed significant increases in expression of CD40 and CD80. Expression of all but one of the DC molecules that increased with COPD severity also correlated with CD69 expression on lung CD4(+) T cells from the same patients, with the exception of CD83 on mDC2. This cross-sectional study implies that COPD progression is associated with significant increases in costimulatory molecule expression by multiple lung DC subsets. Interactions with lung DCs may contribute to the immunophenotype of CD4(+) T cells in advanced COPD. Clinical trial registered with www.clinicaltrials.gov (NCT00281229).

Multiple Antigen-targeted Immunotherapy With α-Galactosylceramide–loaded and Genetically Engineered Dendritic Cells Derived From Embryonic Stem Cells

Numerous tumor-associated antigens (TAA) have been identified and their use in immunotherapy is considered to be promising. For TAA-based immunotherapy to be broadly applied as standard anticancer medicine, methods for active immunization should be improved. In the present study, we demonstrated the efficacy of multiple TAA-targeted dendritic cell (DC) vaccines and also the additive effects of loading alpha-galactosylceramide to DC using mouse melanoma models. On the basis of previously established methods to generate DC from mouse embryonic stem cells (ES-DC), 4 kinds of genetically modified ES-DC, which expressed the melanoma-associated antigens, glypican-3, secreted protein acidic and rich in cysteine, tyrosinase-related protein-2, or gp100 were generated. Anticancer effects elicited by immunization with the ES-DC were assessed in preventive and also therapeutic settings in the models of peritoneal dissemination and spontaneous metastasis to lymph node and lung. The in vivo transfer of a mixture of 3 kinds of TAA-expressing ES-DC protected the recipient mice from melanoma cells more effectively than the transfer of ES-DC expressing single TAA, thus demonstrating the advantage of multiple as compared with single TAA-targeted immunotherapy. Loading ES-DC with alpha-galactosylceramide further enhanced the anticancer effects, suggesting that excellent synergic effects of TAA-specific cytotoxic T lymphocytes and natural killer T cells against metastatic melanoma can be achieved by using genetically modified ES-DC. With the aid of advancing technologies related to pluripotent stem cells, induced pluripotent stem cells, and ES cells, clinical application of DC highly potent in eliciting anticancer immunity will be realized in the near future.

Signal Transduction Inhibition of APCs Diminishes Th17 and Th1 Responses in Experimental Autoimmune Encephalomyelitis

IL-17- and IFN-gamma-secreting T cells play an important role in autoimmune responses in multiple sclerosis and the model system experimental autoimmune encephalomyelitis (EAE). Dendritic cells (DCs) in the periphery and microglia in the CNS are responsible for cytokine polarization and expansion of this T cell subset. Our results indicate that in vivo administration of a signal transduction inhibitor that targets DCs to mice with EAE led to a decrease in CNS infiltration of pathogenic Ag-specific T cells. Since this approach does not target T cells directly, we assessed the effects on the APCs that are involved in generating the T cell responses. Since in EAE and multiple sclerosis, both microglia and peripheral DCs are likely to contribute to disease, we utilized a bone marrow chimera system to distinguish between these two populations. These studies show that peripheral DCs are the primary target but that microglia are also modestly affected by CEP-701, as numbers and activation states of the cells in the CNS are decreased after therapy. Our results also showed a decrease in secretion of TNF-alpha, IL-6, and IL-23 by DCs as well as a decrease in expression of costimulatory molecules. We further determined that levels of phospho-Stat1, Stat3, Stat5, and NF-kappaB, which are signaling molecules that have been implicated in these pathways, were decreased. Thus, use of this class of signal transduction inhibitors may represent a novel method to treat autoimmunity by dampening the autoreactive polarizing condition driven by DCs.

Increased expression of IL-12p70 and IL-23 by multiple dendritic cell and macrophage subsets in plaque psoriasis

Psoriasis is a chronic immune-mediated skin disease, in which interleukins 12 and 23 have been postulated to play a critical role. However, the cellular source of these cytokines in psoriatic lesions are still poorly defined and their relative contribution in inducing skin inflammation has been discussed controversially. To investigate immunoreactivity of the bioactive forms of IL-12 and IL-23 in plaque psoriasis and to characterize the dendritic cell (DC) and macrophage subsets responsible for the production of these cytokines. Immunohistochemistry was performed on normal skin (n=11) as well as non-lesional (n=11) and lesional (n=11) skin of patients with plaque psoriasis using monoclonal antibodies targeting the bioactive forms of IL-12 (IL-12p70) and IL-23 (IL-23p19/p40) on serial cryostat sections using the alkaline phosphatase-antialkaline phosphatase. Co-localization of IL-12 and IL-23 with different dendritic cells and macrophage cell markers (CD1a, CD11c, CD14, CD32, CD68, CD163, CD208/DC-LAMP) was performed using double immunofluorescence staining. Immunoreactivity for IL-12 and IL-23 was significantly enhanced in lesional psoriatic skin as compared to non-lesional and normal skin. No difference was observed between IL-12 and IL-23 immunoreactivity in any skin types. Both IL-12 and IL-23 immunoreactivity was readily detected mainly in CD11c+, CD14+, CD32+, CD68+ and some CD163+, DC-LAMP+ cells. IL-12 and occasionally IL-23 were also found in some CD1a+ dendritic cells. In addition, an enhanced expression mainly of IL-23 was observed in keratinocytes. Bioactive forms of IL-12 and IL-23 are highly expressed in various DC and macrophage subsets and their marked in situ production suggest that both cytokines have crucial pathogenic role in psoriasis.

Migratory and Lymphoid-resident Dendritic Cells Cooperate in Murine Lymph Nodes for Efficient CD4 + T Cell Priming

RATIONALE: Multiple dendritic cell (DC) subsets exist in cutaneous lymph nodes capable of presenting cognate peptide antigen on MHC class II to naïve CD4 + T cells. Although all DC subsets present antigen efficiently in vitro, the requirement for each population in initiating the CD4 + T cell response in vivo remains unclear.

Unusual intraoral lipoma with S-100 protein-positive dendritic cell nodules

Lipoma has been recognized to be one of the most common soft-tissue tumors in the oral region. This case report presents a heretofore undescribed intraoral lesion that resembled mature lipoma but also featured multiple non-lipomatous dendritic cell nodules immunopositive for S-100 protein. We demonstrate histological and immunohistochemical evidence of cellular transitions between non-lipogenic S-100 protein-positive dendrocytes and adipocytes. Although it is likely that this tumor comprises part of the spectrum of what has been referred to in the literature as lipoma, the presence of S-100 protein-positive dendritic cells as an integral component is quite unique and worthy of reporting.

Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.

Multibands of negative refractive indexes in the left-handed metamaterials with multiple dendritic structures

We demonstrate that the multibands of negative refraction indexes in the left-handed metamaterials may be appeared based on multiple dendritic structures cells. Employing microwave transmission experiment, the medium exhibits simultaneous negative refraction at the frequencies of 9.50, 10.24, and 10.78GHz for the electromagnetic wave parallel incidence, and at the frequencies of 9.24, 9.98, and 10.78GHz for normal incidence. The multiple dendritic metamaterials here open a way to prepare the cloak suitable for multifrequencies, especially for the cloak of infrared or visible frequency.

Multiple Dendritic Cell Populations Activate CD4+ T Cells after Viral Stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8α DC play a prominent, and sometimes exclusive, role in driving amplification of CD8+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4+ and CD8+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8α DC populations in the amplification of CD8+ and CD4+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8+ T cells are dominated by presentation of antigen by CD8α DC but can involve non-CD8α DC. In contrast, CD4+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

Dendritic Cell Subtypes and In Vitro Generation of Dendritic Cells

Dendritic cells (DCs) are highly potent antigen-presenting cells crucial for the innate and adaptive immune response and for maintaining immune tolerance towards self-antigens. Although they share many common features, multiple DC subtypes with different immune functions have been identified. Originally, DCs were considered to be cells with purely myeloid origin. Recent studies have now demonstrated that DCs can also develop from lymphatic progenitors. Various cytokines and transcription factors are known to be responsible for the development of DC subpopulation. Depending on the subpopulation and the maturation state of these cells, they are either able to induce a broad cytotoxic immune response, and therefore represent a promising tool for anticancer vaccination therapies in humans or induce immune tolerance and are important within the context of autoimmunity. This review will focus on recent advances on the identification of different DC subpopulations including phenotypical and functional differences and on recent developments on protocols for IN VITRO generation of myeloid-derived DCs.

Chemotactic activity of extracellular nucleotideson human immune cells.

Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5–triphosphate (UTP) and uridine 5–diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation.

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

The Src Homology 2 Domain-Containing Leukocyte Protein of 76-kDa Adaptor Links Integrin Ligation with p44/42 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+ B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.

Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, in the steady state or following their mobilization in vivo, has not been characterized. Herein, generation of mouse bone marrow-derived DC (BMDC) in response to fms-like tyrosine kinase 3 ligand was inhibited by physiologically relevant concentrations of EtOH with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory molecules (CD40, CD80, CD86) but not that of coinhibitory CD274 (B7-H1) on resting or CpG-stimulated DC subsets. Interleukin (IL)-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed BMDC exhibited a reduced capacity to induce naïve, allogeneic T cell proliferation and impaired ability to prime T cells in vivo. DC subsets freshly isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in their low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of surface costimulatory molecules in response to CpG stimulation in vivo. These splenic DC elicited reduced naïve, allogeneic T cell proliferation in vitro, and the stimulatory capacity of resting but not CpG-activated liver DC was reduced by chronic EtOH administration. T cells from animals primed with EtOH-exposed DC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. Thus, EtOH impairs cytokine-driven differentiation and function of myeloid DC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are less affected than splenic DC, which exhibit impaired functional maturation following CpG stimulation. These results indicate a potential mechanism by which alcohol consumption is associated with immunosuppression.

Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-α. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.