Impaired Dendritic Cell Research Articles

Monocyte-derived dendritic cell recruitment and allergic TH2 responses after exposure to diesel particles are CCR2 dependent

The inhalation of diesel exhaust particles (DEPs) is associated with increased sensitization toward inhaled allergens. Dendritic cells (DCs) are important mediators in immune regulation. We previously showed that the inhalation of DEPs increased the accumulation of DCs in the lung and enhanced the T(H)2 response in the mediastinal lymph node. We hypothesized that CC chemokine receptors CCR2, CCR5, and CCR6 critically mediate the DC recruitment upon exposure to DEPs and that these CC chemokine receptors are important in the DEP-induced T(H)2 response. We exposed CCR2 knockout, CCR5 knockout, CCR6 knockout, and wild-type mice to DEPs and examined the pulmonary monocyte and DC accumulation. By an adoptive transfer experiment, we assessed the direct involvement of CCR2 and CCR6 in the recruitment of blood monocytes toward the lung upon exposure to DEPs. We also examined the T(H)2 cytokine production in the mediastinal lymph nodes of DEP-exposed CCR2 knockout and CCR6 knockout mice. We observed that the DEP-induced monocyte and monocyte-derived DC recruitment was completely abolished in CCR2 knockout mice. CCR6 knockout mice also showed impaired monocyte recruitment upon exposure to DEPs. In contrast, monocyte and DC recruitment was comparable between DEP-exposed wild-type and CCR5 knockout mice. The impaired monocyte-derived DC recruitment in DEP-exposed CCR2 knockout, not CCR6 knockout, mice resulted in an abolished T(H)2 response in the mediastinal lymph node. These data suggest that monocyte-derived DCs, recruited in a CCR2-dependent manner, are critical in inducing T(H)2 responses upon inhalation of DEPs.

Systemic Immune Activation in HIV Infection Is Associated with Decreased MDC Responsiveness to TLR Ligand and Inability to Activate Naive CD4 T-Cells

BackgroundHIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host's response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.MethodsFreshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.ResultsMDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.ConclusionThese results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.

The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D invitro and are delayed in reaching the draining lymph node (DLN) invivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.

Altered monocyte-derived dendritic cell function in patients on hemodialysis: a culprit for underlying impaired immune responses

Patients with end-stage renal disease (ESRD) are known to have impaired immune function. Dendritic cells (DCs) are the major antigen-presenting cells that initiate primary immune responses, linking innate and adaptive immunity. Although suboptimal immune responses to vaccination, as frequently observed in ESRD patients, might suggest the presence of impaired DC function, the precise nature of altered DC function is not fully understood. In the current study, we compared the maturation status, viability, and function of monocyte-derived DCs (moDCs) of patients on hemodialysis (HD) with healthy controls. Surface expression of major histocompatibility complex class II, CD83, and CD86, and chemokine receptor CCR7 in moDCs was not different between HD patients and healthy controls. No significant difference was detected in the viability of moDCs determined by expression of annexin V and propidium iodide between two groups. However, moDCs from HD patients produced significantly higher amounts of IL-6 when stimulated by cytokine cocktails compared to healthy controls. In addition, mature moDCs from HD patients showed significantly enhanced allogeneic T-cell proliferation compared to healthy controls. Our data demonstrate aberrant DC function in HD patients and suggest that this might contribute to impaired immune responses.

CD8+ T cells regulate bone tumor burden independent of osteoclast resorption.

Blockade of osteoclast (OC) activity efficiently decreases tumor burden as well as associated bone erosion in immune-compromised animals bearing human osteolytic cancers. In this study, we showed that modulation of antitumor T-cell responses alters tumor growth in bone, regardless of OC status, by using genetic and pharmacologic models. PLCγ2(-/-) mice, with dysfunctional OCs and impaired dendritic cell (DC)-mediated T-cell activation, had increased bone tumor burden despite protection from bone loss. In contrast, Lyn(-/-) mice, with more numerous OCs and a hyperactive myeloid population leading to increased T-cell responses, had reduced tumor growth in bone despite enhanced osteolysis. The unexpected tumor/bone phenotype observed in PLCγ2(-/-) and Lyn(-/-) mice was transplantable, suggesting the involvement of an immune component. Consistent with this hypothesis, T-cell activation diminished skeletal metastasis whereas T-cell depletion enhanced it, even in the presence of zoledronic acid, a potent antiresorptive agent. Importantly, injection of antigen-specific wild-type cytotoxic CD8(+) T cells in PLCγ2(-/-) mice or CD8(+) T-cell depletion in Lyn(-/-) mice normalized tumor growth in bone. Our findings show the important contribution of CD8(+) T cells in the regulation of bone metastases regardless of OC status, thus including T cells as critical regulators of tumor growth in bone.

Different cytokines induce the maturation of human monocyte-derived dendritic cells

Objective To investigate the immune effect of different cytokines on immature dendritic cells (DC) in vitro. Methods The human monocyte-derived DCs were induced in the presence of recombinant human GM-CSF (rhGM-CSF, 1000 U/ml) and interleukin (IL)-4 (500 U/ml). The effects of tumor necrosis factor (TNF) -α (1000 U/ml) , lipopolysaccharide (LPS) (1 mg/L), the cocktail of cytokines (TNF-α, IL-6, IL-1β, PGE2) , and sodium butyrate (1 mmol/L) on the DCs were detected by flow cytometry (FCM), endocytic activity, T cells stimulatory proliferation capacity, and IL-12 production. Results The cocktail of cytokines could up-regulate the major histocompatibility complex (MHC) class Ⅱ and costimulatory molecules of DCs, decrease the endocytic activity 186. 74 ±38. 66, induce a stage of Tcell stimulation 18. 23 ± 2. 22 and promote the T helper cell type 1-skewing factor IL-12 production (656. 18 ±38. 52) ng/L. But the sodium butyrate induced reverse result on DCs compared to cocktail of cytokines. Conclusion The most effective method for inducing dendritic cells maturation was cocktail of cytokines. The sodium butyrate could inhibit the development of mature DCs, resulting in impaired DC function, and modify the outcome of the subsequent immune response. Key words: Dendritic cell; Cytokines; Sodium butyrate

Niacin inhibits skin dendritic cell mobilization in a GPR109A independent manner but has no impact on monocyte trafficking in atherosclerosis

High-dose niacin therapy in humans reduces mortality from cardiovascular disease and may also protect against death from other causes, with benefits apparent more than a decade beyond the therapeutic period. Niacin therapy modulates circulating lipids, raising HDL and lowering LDL, but has the unwanted side effect of inducing skin flushing in response to treatment. Skin flushing results from niacin-induced activation of GPR109A and subsequent release of prostaglandins that promote vasodilation. GPR109A may also mediate HDL elevation. Recent data suggest that high-dose niacin may have benefits beyond improved lipid profiles, such as quelling inflammation, suggesting a potential role in immune cell trafficking. To explore effects of niacin on immune cell trafficking independently of its effects on lipid profiles, we took advantage of the fact that niacin therapy does not raise HDL in wild-type or apoE−/− mouse strains. Wild-type and apoE−/− C57BL/6 mice were fed standard chow or high-fat diets supplemented or not with 1% niacin. Against our predictions, this treatment did not modulate monocyte recruitment to or retention within atherosclerotic plaques. By contrast, stimulating the skin of niacin-treated mice with a contact sensitizer revealed impaired dendritic cell accumulation in draining lymph nodes and associated impaired adaptive immunity. Surprisingly, niacin-mediated impaired dendritic cell mobilization could not be reversed by cyclooxygenase inhibitor treatment nor deletion of the niacin receptor GPR109A, suggesting that the effects of niacin on modulating the migration of dendritic cells are not directly linked to skin flushing. Overall, these data suggest the existence of novel pathways triggered by niacin that, through suppression of dendritic cell migration, might impact adaptive immune responses that participate in sustained therapeutic benefits independent of niacin's cardioprotective capabilities.

The secretions products from invading cercariae of S. japonicum (0–3hRP) restrain mouse dendritic cells to mature

Schistosomiasis is a water-borne infection caused mainly by human schistosomes including Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum). In the infected host, immune events in the skin appear to have an important initiating role in determining the type, and guiding the magnitude, of the ensuing acquired immune response. The previous studies of S. mansoni showed that dendritic cells (DCs) prime Th2 response after activating with products released by schistosome larvae (material released in the first 3h after transformation, 0-3hRP). Therefore, it is interesting to know whether 0-3hRP from S. japonicum also activate DC and induce Th2 immune response. Here, we report 0-3hRP of S. japonicum failed to stimulate the maturation of bone marrow-derived DCs (BM-DCs), while soluble antigen of the body of cercariae preparation (SCAP) induced BM-DCs to mature which can be weakened by 0-3hRP. Moreover, using an in vitro ovalbumin peptide-restricted priming assay, DCs treated with 0-3hRP failed to induce T cells to secrete more cytokines than the negative control group by CD4(+) T cells from DO11.10 transgenic mice, while DCs treated with SCAP upregulated secretions of IFN-γ and IL-17A, but downregulated IL-4. Importantly, BM-DCs treated with 0-3hRP plus SCAP induced BM-DCs selective mature which drive Th2-type polarized response. Our in vitro results agree with the findings of in vivo studies by inoculation of DO11.10 mice with different stimulus-activated DCs pulsed with ovalbumin peptide. Our data demonstrate that the secretions from invading cercariae of S. japonicum (0-3hRP) impaired DCs to mature, which is potentially allowing the parasite to negotiate the immune recognition and attack.

Dendritic cell and macrophage infiltration in microsatellite-unstable and microsatellite-stable colorectal cancer

High level microsatellite instability (MSI-H) is a hallmark of Lynch syndrome-associated colorectal cancer (CRC). MSI-H CRC express immunogenic tumour antigens as a consequence of DNA mismatch repair deficiency-induced frameshift mutations. Consequently, frameshift antigen-specific immune responses are commonly observed in patients with Lynch syndrome-associated MSI-H CRC. Dendritic cells (DC) and macrophages play a crucial role in the induction and modulation of immune responses. We here analysed DC and macrophage infiltration in MSI-H and microsatellite-stable CRC. Sixty-nine CRC (MSI-H, n=33; microsatellite-stable, n=36) were examined for the density of tumour-infiltrating DC, Foxp3-positive regulatory T cells, and CD163-positive macrophages. In MSI-H lesions, S100-positive and CD163-positive cell counts were significantly higher compared to microsatellite-stable lesions (S100: epithelium P=0.018, stroma P=0.042; CD163: epithelium P<0.001, stroma P=0.046). Additionally, numbers of CD208-positive mature DC were significantly elevated in the epithelial compartment of MSI-H CRC (P=0.027). High numbers of tumour-infiltrating Foxp3-positive T cells were detected in tumours showing a low proportion of CD208-positive, mature DC among the total number of S100-positive cells. Our study demonstrates that infiltration with DC, mature DC, and macrophages is elevated in MSI-H compared to microsatellite-stable CRC. The positive correlation of Foxp3-positive Treg cell density with a low proportion of mature DC suggests that impaired DC maturation may contribute to local immune evasion in CRC. Our results demonstrate that DC and macrophages in the tumour environment likely play an important role in the induction of antigen-specific immune responses in Lynch syndrome. Moreover, impaired DC maturation might contribute to local immune evasion in CRC.

CD8α + dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice

Objective Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3 + regulatory T cells (T reg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient ( Ccr2 −/−) mice had impaired DCs migration and reduced CD8α + DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2 −/− mice, we tested the hypothesis that CD8α + DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α + DCs in Ccr2 −/− and SKG mice. Methods To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α + DCs. Results Flt3L-mediated expansion of endogenous CD8α + DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α + DCs ameliorated arthritis in Ccr2 −/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype. Conclusion CD8α + DCs were tolerogenic to the development of arthritis. CD8α + DCs deficiency heightened the sensitivity to arthritis in Ccr2 −/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2 −/− mice was T cell-independent.

Fighting with the Enemys Weapons? The Role of Costimulatory Molecules in HIV

HIV infection is characterized by a number of abnormalities in several components of the immune system. For example, during HIV infection, a massive decrease of CD4(+) T cells is observed, as well as a progressive depletion of naïve CD8(+) T cells. Furthermore, elevated numbers of apoptotic B and T cells are present in HIV-infected patients, and a systemic immune activation results in T-cell exhaustion. Finally, HIV infection is characterized by the presence of functionally impaired dendritic cells, with decreased expression of maturation markers, decreased secretion of cytokines and defects in antigen processing and presentation. All these characteristics result in the occurrence of non-functional cytotoxic T lymphocytes, that fail to control HIV-replication in most individuals during progressive disease. Costimulatory and co-inhibitory molecules are involved in the activation, differentiation and survival of several cell-types of the immune system. Each costimulatory receptor (generally expressed on effector cells) can conjugate with one or more specific ligands (expressed on antigen-presenting cells), which leads to an activation of intracellular signaling pathways inside the cells on which they are expressed. HIV infection is characterized by an aberrant expression of these molecules on cells of the immune system. Many of the immune deficiencies mentioned in the previous paragraph can be explained by abnormal expression of costimulatory molecules, and could consequently be overcome by interfering with their interactions. In this review, we give an overview of the functions and expression patterns of the receptor/ligand pairs of the tumor necrosis factor and the B7 super-families of costimulatory and co-inhibitory molecules in HIV-infected patients. We will also discuss possibilities for manipulating their signaling as a therapeutic anti-HIV tool.

Impact of streptozotocin-induced diabetes on functional responses of dendritic cells and macrophages towardsBurkholderia pseudomallei

Diabetes mellitus is a documented risk factor for melioidosis, a tropical infection caused by Burkholderia pseudomallei. The increased susceptibility of diabetic individuals to infections with other pathogens has been associated with immune dysregulation. However, the impact of diabetes on the functional responses of dendritic cells (DC) and macrophages during B. pseudomallei infection has not been investigated. This study compared the responses of macrophages and DC towards B. pseudomallei using bone marrow-derived DC (BMDC) and peritoneal elicited macrophages (PEM) isolated from streptozotocin-induced diabetic C57BL/6 mice exhibiting hyperglycaemia for 9 days (acute) or 70 days (chronic) and age-matched nondiabetic C57BL/6 mice. Following coincubation of BMDC and PEM with a highly virulent B. pseudomallei isolate, maturation, bacterial internalization plus intracellular survival and cytokine gene expression profiles were assessed. No significant differences in functional responses of BMDC or PEM isolated from acute diabetic and nondiabetic mice were observed. However, significant differences in BMDC and PEM function were observed when chronic diabetic and nondiabetic mice were compared. This study demonstrates that diabetic mice with extended periods of uncontrolled hyperglycaemia have impaired DC and macrophage function towards B. pseudomallei, which may contribute to the high susceptibility observed in clinical practice.

Decreased numbers of peripheral blood dendritic cells in patients with coronary artery disease are associated with diminished plasma Flt3 ligand levels and impaired plasmacytoid dendritic cell function

We investigated whether activation of circulating DCs (dendritic cells) or levels of Flt3L (FMS-like tyrosine kinase 3 ligand) and GM-CSF (granulocyte/macrophage colony-stimulating factor), haematopoietic growth factors important for DC differentiation, could account for reduced blood DC numbers in CAD (coronary artery disease) patients. Concentrations of Flt3L and GM-CSF were measured in plasma from CAD patients (n = 15) and controls (n = 12). Frequency and phenotype of mDCs (myeloid dendritic cells) and pDCs (plasmacytoid dendritic cells) were analysed by multicolour flow cytometry in fresh blood, and after overnight incubation with TLR (Toll-like receptor)-4 or -7 ligands LPS (lipopolysaccharide) or IQ (imiquimod). DC function was measured by IL (interleukin)-12 and IFN (interferon)-α secretion. Circulating numbers of CD11c+ mDCs and CD123+ pDCs and frequencies of CD86+ and CCR-7+ (CC chemokine receptor type7) mDCs, but not pDCs, were declined in CAD. In addition, plasma Flt3L, but not GM-CSF, was lower in patients and positively correlated with blood DC counts. In response to LPS, mDCs up-regulated CD83 and CD86, but CCR-7 expression and IL-12 secretion remained unchanged, similarly in patients and controls. Conversely, pDCs from patients had lower CD83 and CCR-7 expression after overnight incubation and had a weaker IQ-induced up-regulation of CD83 and IFN-α secretion. In conclusion, our results suggest that reduced blood DC counts in CAD are, at least partly, due to impaired DC differentiation from bone marrow progenitors. Decreased levels of mDCs are presumably also explained by activation and subsequent migration to atherosclerotic plaques or lymph nodes. Although mDCs are functioning normally, pDCs from patients appeared to be both numerically and functionally impaired.

Mechanisms of alcohol-mediated hepatotoxicity in human-immunodeficiency-virus-infected patients

Clinical observations have demonstrated that excessive chronic alcohol use negatively affects human immunodeficiency virus (HIV) infection and contributes to the liver manifestations of the disease, even in HIV mono-infection. HIV/hepatitis C virus (HCV) co-infection is associated with increased progression of HVC liver disease compared to HCV infection alone, and both of these are negatively affected by alcohol use. Recent data suggest that alcohol use and HIV infection have common targets that contribute to progression of liver disease. Both HIV infection and chronic alcohol use are associated with increased gut permeability and elevated plasma levels of lipopolysaccharide; a central activator of inflammatory responses. Both alcoholic liver disease and HIV infection result in non-specific activation of innate immunity, proinflammatory cytokine cascade upregulation, as well as impaired antigen presenting cell and dendritic cell functions. Finally, alcohol, HIV and antiretroviral therapy affect hepatocyte functions, which contributes to liver damage. The common targets of alcohol and HIV infection in liver disease are discussed in this mini-review.

A Molecular Mechanism of Tumor Mediated Suppression of Dendritic Cell Differentiation

AbstractAbstract 1735The immune system plays a key role in preventing and controlling tumor growth. Cancer frequently induces a state of immune suppression in patients mediated, in part, through inhibition of dendritic cell (DC) differentiation. This results in the accumulation of actively immunosuppressive myeloid derived suppressor cells (MDSCs) and a loss of DCs (critical in the induction of T cell mediated immune responses), thus compromising the ability to initiate anti-tumor immune responses. This is mediated by tumor derived factors (TDFs)(e.g. VEGF) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling that inhibits DC differentiation, however, has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKC βII inhibition (signaling or expression) prevents DC differentiation. We've also found that PKC βII positively regulates its own expression and that, under certain circumstances, the PKCβ promoter is negatively regulated. These observations lead us to hypothesize that TDF activation of Stat3 inhibits DC differentiation by down regulating PKC βII expression. We've previously shown that culture in tumor conditioned media (TCM) decreased PKC βII protein levels and significantly reduced PKC βII mRNA transcript levels in KG1, a myeloid progenitor-like cell line long used to model DC differentiation. We've also previously seen that decreased PKC βII expression following culture in TCM significantly impaired DC differentiation, compared to cells grown in the absence of tumor conditions; however, the mechanism by which Stat3 signaling down regulated PKC βII expression remained unclear. We now show that culture in TCM reduced PKCβ promoter driven transcription 7-fold, compared to cells grown in normal media (p<0.01). Given the previously described importance of Stat3 hyperactivation in tumor-mediated suppression of DC differentiation, and since PKC βII down regulation appears to occur at the promoter level, we examined the role of Stat3 in regulating PKC βII expression. Culture in TCM rapidly (<5 min.) induced Stat3 phosphorylation, an indication of activation. By chromatin immunoprecipitation, we found that TCM treatment induces direct interaction between Stat3 and the PKCβ promoter, suggesting that Stat3 signaling may act to decrease PKC βII expression. To directly test the role of Stat3 signaling in regulation of PKC βII expression, we generated a series of clones stably expressing wild type (WT) or constitutive active (CA) Stat3 constructs in K562, a second DC progenitor-like cell line. We've previously seen that clones stably expressing the CA-Stat3 construct have decreased PKC βII protein levels and significantly decreased PKC βII mRNA levels, compared to the parental cell line and WT-Stat3 clones. We now show that this decrease in PKC βII expression was dependent on constitutive Stat3 signaling, as pharmacologic Stat3 inhibition restored PKC βII expression to levels seen in the parental cell line. Consistent with the proposed model and our previous work, decreased PKC βII expression in clones expressing CA-Stat3 resulted in significantly inhibited phorbol ester driven DC differentiation (p<0.05)(as measured by allogenic T cell proliferation, a key measure of DC differentiation). Interestingly, we've also found that PKC βII antagonizes Stat3 signaling: myeloid progenitor-like cells (KG1a) overexpressing PKC βII do not exhibit Stat3 activation in response to TCM: PKC βII overexpression or activation led to down regulation of the receptors for G-CSF, IL-6, and VEGF, TDFs demonstrated to inhibit myeloid progenitor to DC differentiation. These findings suggest a novel mechanism by which PKC βII negatively regulates the potential of a cell to respond to (tumor derived) inflammatory cytokines. This work demonstrates that tumor driven Stat3 hyperactivation down regulates PKCβ promoter activity, resulting in decreased PKC βII protein levels. In agreement with our previous work, this decrease in PKC βII expression impairs a cell's potential to undergo DC differentiation. Additionally, this work suggests that PKC βII signaling impairs a cell's potential to signal via Stat3 in response to TDFs, perhaps providing an avenue by which to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures:No relevant conflicts of interest to declare.

HDAC inhibition by LBH589 affects the phenotype and function of human myeloid dendritic cells

LBH589 is a novel pan-HDAC inhibitor which has potent antitumor activity in multiple myeloma and other hematologic malignancies. However, its impact on immune system has not been defined. We here evaluated the effects of LBH589 on human myeloid dendritic cells (DCs) at clinically relevant concentrations. Exposure to LBH589 affected the surface molecule expression on immature and mature DCs, associated with DC maturation (CD83↓), antigen presentation (HLA-ABC↓), and T cell co-stimulation (CD40↓ and CD86↑). LBH589 decreased both protein and polysaccharide antigen uptake capacities by DCs. Importantly, LBH589 impaired DCs function to stimulate antigen-specific immune responses, resulting in the significant reduction of invariant NKT cell (CD1d-restricted) and T cell (MHC-restricted) activation in innate and adaptive immunity. LBH589 also significantly repressed the production of IL-6, IL-10, IL-12p70, IL-23 and TNF-α by TLR3 and TLR4-induced DCs activation, indicating an important role of HDAC activity in immune regulation and inflammation. RelB, a component of NF-κB signaling pathway, was the key component regulated by HDAC inhibition in DCs. Together, our preclinical study demonstrates that LBH589 significantly impairs phenotype and function of DCs, indicating a need for monitoring the immune status in patients receiving HDAC inhibitor therapy. It also provides a rationale to evaluate LBH589 activity for the treatment of inflammation.

Age-associated impaired plasmacytoid dendritic cell functions lead to decreased CD4 and CD8 T cell immunity

Increased susceptibility to infections, particularly respiratory viral infections, is a hallmark of advancing age. The underlying mechanisms are not well understood, and there is a scarcity of information regarding the contribution of the innate immune system, which is the first line of defense against infections. In the present study, we have investigated the effect of advancing age on plasmacytoid dendritic cell (PDC) function because they are critical in generating a robust antiviral response via the secretion of interferons (IFN). Our results indicate that PDCs from the aged are impaired in their capacity to secrete IFN-I in response to influenza virus and CPG stimulation. Additionally, we observed a severe reduction in the production of IFN-III, which plays an important role in defense against viral infections at respiratory mucosal surfaces. This reduction in IFN-I and IFN-III were a result of age-associated impaired phosphorylation of transcription factor, IRF-7. Furthermore, aged PDCs were observed to be impaired in their capacity to induce perforin and granzyme in CD8 T cells. Comparison of the antigen-presenting capacity of aged PDC with young PDC revealed that PDCs from aged subjects display reduced capacity to induce proliferation and IFN-gamma secretion in CD4 and CD8 T cells as compared with PDCs from young subjects. In summary, our study demonstrates that advancing age has a profound effect on PDC function at multiple levels and may therefore, be responsible for the increased susceptibility to infections in the elderly.

Mechanisms by which chronic ethanol feeding limits the ability of dendritic cells to stimulate T-cell proliferation.

As initiators of immune responses, dendritic cells (DCs) are required for antigen (Ag)-specific activation of naïve T cells in the defense against infectious agents. The increased susceptibility to and severity of infection seen in chronic alcoholics could be because of impaired DCs initiation of naïve T-cell responses. Specifically, these DCs may not provide adequate Signals 1 (Ag presentation), 2 (costimulation), or 3 (cytokine production) to these T cells. Using the Meadows-Cook murine model of chronic alcohol abuse, the ability of ethanol (EtOH)-exposed DCs to stimulate T-cell proliferation, acquire and process Ag, express costimulatory molecules, and produce inflammatory cytokines was assessed. Normal naïve T cells primed by EtOH-exposed DCs showed decreased proliferation in vitro and in vivo, compared to water-fed control mice. These EtOH-exposed DCs, after activation by CpG or tumor necrosis factor alpha (TNFα), were less able to upregulate costimulatory molecules CD40, CD80, or CD86, and produced less IL-12 p40, TNFα, and IFNα than DCs from water-fed mice. TLR9 and TNF receptor expression were also reduced in/on EtOH-exposed DCs. No evidence of defective Ag acquisition or processing as a result of EtOH feeding was identified. Inadequate proliferation of normal T cells following stimulation by EtOH-exposed DCs is likely a result of diminished Signal 2 and Signal 3. Lack of adequate inflammatory stimulation of EtOH-exposed DCs because of diminished receptors for inflammatory mediators appears to be at least partially responsible for their dysfunction. These findings provide a mechanism to explain increased morbidity and mortality from infectious diseases in alcoholics and suggest targets for therapeutic intervention.

Mammalian Target of Rapamycin Controls Dendritic Cell Development Downstream of Flt3 Ligand Signaling

Dendritic cells (DCs) comprise distinct functional subsets including CD8⁻ and CD8(+) classical DCs (cDCs) and interferon-secreting plasmacytoid DCs (pDCs). The cytokine Flt3 ligand (Flt3L) controls the development of DCs and is particularly important for the pDC and CD8(+) cDC and their CD103(+) tissue counterparts. We report that mammalian target of rapamycin (mTOR) inhibitor rapamycin impaired Flt3L-driven DC development in vitro, with the pDCs and CD8(+)-like cDCs most profoundly affected. Conversely, deletion of the phosphoinositide 3-kinase (PI3K)-mTOR negative regulator Pten facilitated Flt3L-driven DC development in culture. DC-specific Pten targeting in vivo caused the expansion of CD8(+) and CD103(+) cDC numbers, which was reversible by rapamycin. The increased CD8(+) cDC numbers caused by Pten deletion correlated with increased susceptibility to the intracellular pathogen Listeria. Thus, PI3K-mTOR signaling downstream of Flt3L controls DC development, and its restriction by Pten ensures optimal DC pool size and subset composition.

PP-151 Increased PD-L1 expression and PD-L1/CD86 ratio on dendritic cells were inversely associated with impaired dendritic cells function in HCV chronic infection

PP-151 Increased PD-L1 expression and PD-L1/CD86 ratio on dendritic cells were inversely associated with impaired dendritic cells function in HCV chronic infection