Inhibition Of Dendritic Cells Maturation Research Articles

Suppression of dendritic cell functions contributes to the anti-inflammatory action of granulocyte-colony stimulating factor in experimental stroke

Cerebral ischemia provokes an inflammatory cascade, which is assumed to secondarily worsen ischemic tissue damage. Linking adaptive and innate immunity dendritic cells (DCs) are key regulators of the immune system. The hematopoietic factor G-CSF is able to modulate DC-mediated immune processes. Although G-CSF is under investigation for the treatment of stroke, only limited information exists about its effects on stroke-induced inflammation. Therefore, we investigated the impact of G-CSF on cerebral DC migration and maturation as well as on the mediated immune response in an experimental stroke model in rats by means of transient middle cerebral artery occlusion (tMCAO). Immunohistochemistry and quantitative PCR were performed of the ischemic brain and flow cytometrical analysis of peripheral blood. G-CSF led to a reduction of the infarct size and an improved neurological outcome. Immunohistochemistry confirmed a reduced migration of DCs and mature antigen-presenting cells after G-CSF treatment. Compared to the untreated tMCAO group, G-CSF led to an inhibited DC activation and maturation. This was shown by a significantly decreased cerebral transcription of TLR2 and the DC maturation markers, CD83 and CD86, as well as by an inhibition of stroke-induced increase in immunocompetent DCs (OX62+OX6+) in peripheral blood. Cerebral expression of the proinflammatory cytokine TNF-α was reduced, indicating an attenuation of cerebral inflammation. Our data suggest an induction of DC migration and maturation under ischemic conditions and identify DCs as a potential target to modulate postischemic cerebral inflammation. Suppression of both enhanced DC migration and maturation might contribute to the neuroprotective action of G-CSF in experimental stroke.

A role for anti-CD45RB monoclonal antibody treatment upon dendritic cells

Selective interference with CD45RB isoform by monoclonal antibody (anti-CD45RBmAb) reliably induces donor-specific tolerance. Dendritic cells (DCs) are the most potent antigen-presenting cells that are capable of activating naïve T cells. The purposes of the present study were to investigate the roles of anti-CD45RBmAb on the phenotypes and functioning of DCs and to further illustrate the mechanism of anti-CD45RBmAb-inducing immunologic tolerance. DCs from C57BL/6 mice were cultured and treated with various doses of anti-CD45RB monoclonal antibody. Cell phenotype, cycle and phagocytic ability were detected by flow cytometry. The production of IL-10 and IL-12 in the supernatants of mature DCs was measured with ELISA. Exosomes (Dex) were recovered from the supernatant of DCs cultured for 6 days in depleted medium, and effects of DCs and Dex on the ability of T-cell proliferation were detected by mixed lymphocyte culture. Anti-CD45RBmAb could inhibit DCs maturation in a dose-dependent manner, and the effects of exosomes (Dex) on DCs enhance or inhibition proliferation of T cells were also in a dose-dependent manner. Anti-CD45RBmAb could profoundly inhibit the maturation and functioning of DCs and generate tolerogenic dendritic cells (tDCs) as well as Dex, suggesting mechanistic contributions to tolerance development from the DCs through interactions with T cells.

Regulation of Immunity via Multipotent Mesenchymal Stromal Cells

Immune cells responsible for inflammation development are involved in tissue damage caused by wounding and various pathologies. Control of immune cell activation could be of significant benefit for regenerative medicine and the treatment of patients with autoimmune and degenerative diseases. It is a proven fact that MCSs (multipotent mesenchymal stromal cells) are capable of suppressing immune responses via the inhibition of dendritic cell maturation and via the restraining of the T, B, and NK cell function in the course of autoimmune diseases and various forms of inflammation. MSCs can be isolated easily from almost every type of tissue or organ and subsequently expandedin vitro. These cells are self-renewable and can be differentiated into various cell types of mesenchymal lineage. The current review contains a collection and critical analysis of data regarding the molecular mechanisms responsible for cross-talk between immune cells and MSCs. Some of these mechanisms can be used for the development of new practical approaches for the treatment of autoimmune diseases.

Airway Activation of Formyl Peptide Receptors Inhibits Th1 and Th17 Cell Responses via Inhibition of Mediator Release from Immune and Inflammatory Cells and Maturation of Dendritic Cells

Formyl peptide receptors (FPRs) are chemoattractant receptors that mediate inflammatory cell responses to infection. Recent evidence indicates that noneosinophilic asthma phenotypes can be developed by both Th1 and Th17 cell responses when exposed to LPS-containing allergens. In this study, we evaluated the effects of airway activation of FPRs by their synthetic agonist, Trp-Lys-Tyr-Met-Val-D-Met (W-peptide), on the development of Th1 and Th17 cell responses in a noneosinophilic asthma mouse model. A noneosinophilic asthma mouse model was generated by intranasal sensitization with 10 μg of LPS plus 75 μg of OVA on days 0, 1, 2, and 7. Mice were then challenged with 50 μg of OVA alone on days 14, 15, 21, and 22. W-peptide was administered during the sensitization period, and immune and inflammatory responses were evaluated after OVA challenge. Lung inflammation after OVA challenge was partly abolished by airway activation of FPRs during sensitization. Maturation of dendritic cells (DCs) and migration of DCs from the lung to lung-draining lymph nodes were inhibited by FPR activation. In addition, airway activation of FPRs inhibited allergen-specific T cell proliferation in the lymph nodes. Production of IL-12 and IL-6 (Th1- and Th17-polarizing cytokines) from lung DCs was decreased by airway activation of FPRs. This effect resulted in the inhibition of allergen-specific Th1 and Th17 cell responses. Airway activation of FPRs during sensitization effectively prevents the development of Th1 and Th17 cell responses induced by LPS-containing allergens via multiple mechanisms, such as inhibition of DC maturation and migration and the production of Th1- and Th7-polarizing cytokines.

Aberrant elevated microRNA-146a in dendritic cells (DC) induced by human pancreatic cancer cell line BxPC-3-conditioned medium inhibits DC maturation and activation

It has been shown that the function of dendritic cell (DC) is suppressed in pancreatic cancer patients; however, the detailed mechanism involved in it remains unclear. Here, we used medium conditioned by a highly metastatic human pancreatic cancer cell line BxPC-3 [BxPC-3-conditioned medium (BxCM)] to culture human CD14+ monocyte-derived DCs in vitro. Both DC differentiation and antigen presentation function were inhibited by BxCM. The microRNA-146a (miRNA-146a) expression is aberrantly up-regulated in BxCM-treated DCs. In addition, inhibition of aberrant miRNA-146a expression partly rescues the BxCM-induced defects in differentiation and function of DCs, which may be through regulation of Smad4 expression. Taken together, our findings indicate that aberrant miRNA-146a expression is one of main factors responsible for inhibition of DC maturation and antigen presentation function, and this inhibitory effect on DCs may be due to the repression of Smad4 mediated signal pathway by BxCM.

Early exposure of interferon-γ inhibits signal transducer and activator of transcription-6 signalling and nuclear factor κB activation in a short-term monocyte-derived dendritic cell culture promoting ‘FAST’ regulatory dendritic cells

Interferon (IFN)-γ is a cytokine with immunomodulatory properties, which has been shown previously to enhance the generation of tolerogenic dendritic cells (DC) when administered early ex vivo in 7-day monocyte-derived DC culture. To generate tolerogenic DC rapidly within 48 h, human monocytes were cultured for 24 h with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence (IFN-γ-DC) or absence of IFN-γ (500 U/ml) (UT-DC). DC were matured for 24 h with TNF-α and prostaglandin E(2) (PGE(2) ). DC phenotype, signal transducer and activator of transcription-6 (STAT-6) phosphorylation and promotion of CD4(+) CD25(+) CD127(neg/low) forkhead box P3 (FoxP3)(hi) T cells were analysed by flow cytometry. DC nuclear factor (NF)-κB transcription factor reticuloendotheliosis viral oncogene homologue B (RELB) and IL-12p70 protein expression were also determined. Phenotypically, IFN-γ-DC displayed reduced DC maturation marker CD83 by 62% and co-stimulation molecules CD80 (26%) and CD86 (8%). IFN-γ treatment of monocytes inhibited intracellular STAT6, RELB nuclear translocation and IL-12p70 production. IFN-γ-DC increased the proportion of CD4(+) CD25(+) CD127(neg/low) foxp3(hi) T cells compared to UT-DC from 12 to 23%. IFN-γ-DC primed T cells inhibited antigen-specific, autologous naive T cell proliferation by 70% at a 1:1 naive T cells to IFN-γ-DC primed T cell ratio in suppression assays. In addition, we examined the reported paradoxical proinflammatory effects of IFN-γ and confirmed in this system that late IFN-γ exposure does not inhibit DC maturation marker expression. Early IFN-γ exposure is critical in promoting the generation of regulatory DC. Early IFN-γ modulated DC generated in 48 h are maturation arrested and promote the generation of antigen-specific regulatory T cells, which may be clinically applicable as a novel cellular therapy for allograft rejection.

Effect of siRNA-mediated downregulation of VEGF in Tca8113 cells on the activity of monocyte-derived dendritic cells

Vascular endothelial growth factor (VEGF) is a tumor angiogenesis factor that is important in immune regulation. In our previous study, we found that VEGF expression in the peripheral blood and neoplasm nest from patients with oral squamous cell carcinoma (OSCC) was positively correlated with the course of disease, while an inverse correlation between VEGF expression and dendritic cells (DCs) was identified in the peripheral blood. Therefore, in the present study, we investigated whether inhibition of human VEGF in the human tongue carcinoma cell line Tca8113 had effects on the activity of monocyte-derived DCs. We knocked down the expression of human VEGF in Tca8113 cells using the small interfering RNA (siRNA) technique. Tca8113 cells pre-transfected with siRNA targeting VEGF were co-cultured with monocyte‑derived immature and mature DCs. Cell proliferation was evaluated by a WST-8 assay. Cell apoptosis, cell cycle and cell phenotypes were determined by flow cytometry. The data revealed that downregulation of the human VEGF significantly inhibited the proliferation of Tca8113 cells and increased apoptosis. Inhibition of human VEGF arrested the cell cycle of Tca8113 cells at the G0/G1 phase. Our results showed that the co-culture of DCs with Tca8113 cells markedly inhibited the expression of the mature markers of DCs including HLA-DR, CD80, CD86, CD40 and CD1a, as well as the immature marker CD83, while inhibition of human VEGF in Tca8113 cells significantly reversed these effects. Therefore, human VEGF in Tca8113 cells may not only regulate the cell proliferation and apoptosis of oral squamous cell carcinoma cells, but may also inhibit DC maturation.

Low Levels of 25-Hydroxyvitamin D Prior to Allogeneic Transplantation Correlate with the Development of Chronic Graft-Versus-Host Disease

Abstract 4063 Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. GVHD is mediated by the activation and expansion of donor derived alloreactive lymphocytes stimulated in part by residual host dendritic cells (DCs) and donor DCs with cross presentation of alloantigens. Vitamin D is a hormone essential for calcium homeostasis and exhibits potent immunomodulatory characteristics. We have demonstrated that vitamin D exposure inhibits DC maturation and DC mediated stimulation of allogeneic T cells. As such, we hypothesized that vitamin D deficiency may result in the enhanced capacity of DCs to induce GVHD. In this study, we performed a retrospective case-control study to evaluate whether monohydroxyvitamin D levels measured in serum samples obtained from patients prior to allogeneic transplantation were predictive of the risk for GVHD. Fifty-three patients aged 18–60 who underwent a myeloablative matched related or matched unrelated donor transplant between December 2000 and December 2009 at Dana Farber Cancer Institute (DFCI) were included in the analysis. 25-OH vitamin D concentrations were measured by an ELISA assay on serum samples that had been cryopreserved between one and three months prior to transplantation. All patients were alive and evaluable at day 100. The median 25-OH vitamin D level was 21.9 ng/mL (range 7.8–45.7). Patients were divided into two cohorts based on a vitamin D level: < 25 ng/mL (32 patients) or ≥25 ng/mL (21 patients). There was no difference between the groups with respect to patient or donor age, donor type (matched related versus unrelated donor), cell source (peripheral blood versus bone marrow), patient-donor sex matching, myeloid versus lymphoid malignancy, risk status, patient/donor CMV status, conditioning regimen, or GVHD prophylaxis regimen. The cumulative incidence of grades II-IV acute GVHD at 100 days was 53.1% in the group with vitamin D level <25 versus 33.3% in the group with vitamin D≥25 (p=0.13). Remarkably, the cumulative incidence (CI) of chronic GVHD (cGVHD) at 2 years in patients with 25-OH vitamin D <25 was 63.8%, compared to 23.8% in patients with vitamin D levels ≥ 25 (p=0.009). Similarly the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D levels <25 compared to those with ≥25 (54.5% versus 14.3%, p=0.005). This result is consistent in multivariable competing risks model for which patient and transplant related factors mentioned above were adjusted (HR 5.26, p=0.02). [Figure 1][1] represents the CI of cGVHD with death and relapse as a competing risk. Three-year progression-free survival and overall survival were similar between the two groups (51% vs. 47%, p=0.61; and 53% vs. 50%, p=0.57 respectively). In summary, vitamin D deficiency prior to allogeneic transplantation is associated with a significantly increased risk of chronic GVHD. A prospective clinical trial evaluating the use of vitamin D for the prevention of chronic GVHD is planned. ![Figure 1:][2] Figure 1: Cumulative Incidence of cGVHD with Death/Relapse as a Competing Risk Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes

CLINICAL EVALUATION OF DENDRITIC CELLS VACCINATION FOR ADVANCED CANCER PATIENTS AT FUKUSHIMA MEDICAL UNIVERSITY

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) that have attracted attention in recent years from the viewpoint of DC vaccine therapy against cancer. However, the existence of a strongly immunosuppressed state in cancer-bearing individuals inhibits DC maturation, which is one of the problems facing anti-cancer DC vaccine therapy. Isolated DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce protective and therapeutic anti-tumor immunity in experimental animals. In clinical trials of DC vaccination for cancer patients, induction of anti-tumor immune responses and tumor regression has been observed. In this study, eighty-one advanced cancer patients unsuccessfully treated by established treatment in individual cases were selected between January 2002 and May 2007 at Fukushima Medical University. The usefulness of DC therapy was investigated by intradermal injection of peptide pulsed DCs for an overall objective response rate of 28.0%. Furthermore, direct injection of immature DCs into tumor extracted an overall objective response rate of 35.7%, and especially 40.0% for advanced pancreatic cancer by using endoscopic ultrasound-guided fine-needle injection technique as a novel approach. These results indicate that DC-based vaccination could be a promising treatment modality for various cancers, however multiple hurdles must be cleared before the development of an affordable DC-based vaccination can be used worldwide.

The Role of Mycobacterium leprae Phenolic Glycolipid I (PGL-I) in Serodiagnosis and in the Pathogenesis of Leprosy

PGL-I (phenolic glycolipid I) emerged in the early 1980s on the one hand as part of intensive efforts to define the typing antigens of a host of Mycobacterium spp. and also from characterisation of the lipids of skin biopsies from highly bacillary positive lepromatous leprosy patients. PGL-I, despite its extreme lipophilicity due to its inherent phthiocerol dimycocerosyl component, is highly antigenic evoking high titre IgM antibodies in lepromatous leprosy patients, attributable largely to the unique 3,6-di-O-methyl-beta-D-glucosyl entity at the non-reducing terminus of its trisaccharide. PGL-I itself or in the form of semisynthetic neoglycoproteins containing the synthetic terminal disaccharide or the whole trisaccharide chemically conjugated to such as bovine or human serum albumin, has found its greatest utility in the serological diagnosis, confirmation and management of lepromatous leprosy. PGL-I has also been implicated in the tropism of M. leprae for Schwann cells, through specific binding to laminin, and to play an important role in downregulation of the inflammatory immune response and inhibition of dendritic cell maturation and activation, thereby facilitating the persistence of M. leprae/leprosy.

Zoledronic acid modulates maturation of human monocyte-derived dendritic cells

Zoledronic acid (ZA) is a drug of the bisphosphonate class, which is widely used for the treatment of both osteoporosis and skeletal metastasis. Besides its main bone antiresorptive activity, ZA displays antitumor properties, by triggering the expansion and activation of γδ T-cells, which exert an antitumor effect through dendritic cells (DCs). Several studies have reported the interaction between ZA and γδ T-cells, but the potential immunoregulatory activity of this drug on DCs has scarcely been investigated. Therefore, in this paper, we evaluated the effects of a therapeutic dose of ZA on the in vitro generation and maturation of DCs derived from peripheral blood monocytes of healthy adult donors. We demonstrate that ZA treatment did not affect DC differentiation, but inhibited DC maturation on lipopolysaccharide activation, as shown by the impaired expression of maturation surface markers and reduced ability to induce allogeneic T-cell proliferation. Interestingly, IL-10 secretion by mature DCs was significantly lower in ZA-treated cells than in controls. We conclude that ZA exerts its immunological in vitro activity also by modulating the maturation of DCs.

Superoxide Dismutase 3 Suppresses Hyaluronic Acid Fragments Mediated Skin Inflammation by Inhibition of Toll-Like Receptor 4 Signaling Pathway: Superoxide Dismutase 3 Inhibits Reactive Oxygen Species-Induced Trafficking of Toll-Like Receptor 4 to Lipid Rafts

Hyaluronic acid (HA) is a component of the extracellular matrix and has been extensively applied for cosmetic, therapeutic, and antiaging purposes. However, HA fragments (HAFs) cause adverse effects. Considering that UV-exposure produces HAF that accumulated on the skin, the role of HAF in skin inflammation and its precise mechanism needs to be clarified, and strategies for the prevention of skin inflammation are necessary. We found that extracellular superoxide dismutase (SOD), SOD3, suppresses HAF-mediated skin inflammation, while HAF mediated skin inflammation, macrophages and dendritic cells (DCs) dominantly infiltrate, up-regulating inflammatory cytokines and chemokines receptors. However, keratinocytes indirectly responded to HAF. Instead, epidermis containing keratinocytes were stimulated by secreted molecules from HAF-treated macrophages or DC and produced inflammatory molecules including chemokines, which, in turn, led to skin inflammation. This orchestrated inflammatory response was inhibited by SOD3. In addition, SOD3 inhibited DC maturation by suppressing the expression of major histocompatibility complex II, CD80, and CD86. Interestingly, these responses did not occur in Toll-like receptor 4 (TLR4) deficient mice. Similar to lipopolysaccharide (LPS), HAF promoted TLR4 translocation into the lipid rafts to initiate signaling. This trafficking was mediated, at least in part, by NAPDH oxidase-dependent reactive oxygen species (ROS) generation. Subsequently, nuclear factor kappa B (NFκB) subunit, p65, was recruited the promoters of genes encoding inflammatory molecules. This inflammatory machinery was blocked by SOD3. Thus, we propose that SOD3 might provide an effective strategy for the treatment of HAF-mediated skin inflammation.

Low Levels of 25-Hydroxyvitamin D Prior to Allogeneic Transplantation Correlate with the Development of Chronic Graft-Versus-Host Disease,

Abstract 4063Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. GVHD is mediated by the activation and expansion of donor derived alloreactive lymphocytes stimulated in part by residual host dendritic cells (DCs) and donor DCs with cross presentation of alloantigens. Vitamin D is a hormone essential for calcium homeostasis and exhibits potent immunomodulatory characteristics. We have demonstrated that vitamin D exposure inhibits DC maturation and DC mediated stimulation of allogeneic T cells. As such, we hypothesized that vitamin D deficiency may result in the enhanced capacity of DCs to induce GVHD. In this study, we performed a retrospective case-control study to evaluate whether monohydroxyvitamin D levels measured in serum samples obtained from patients prior to allogeneic transplantation were predictive of the risk for GVHD. Fifty-three patients aged 18–60 who underwent a myeloablative matched related or matched unrelated donor transplant between December 2000 and December 2009 at Dana Farber Cancer Institute (DFCI) were included in the analysis. 25-OH vitamin D concentrations were measured by an ELISA assay on serum samples that had been cryopreserved between one and three months prior to transplantation. All patients were alive and evaluable at day 100. The median 25-OH vitamin D level was 21.9 ng/mL (range 7.8–45.7). Patients were divided into two cohorts based on a vitamin D level: < 25 ng/mL (32 patients) or ≥25 ng/mL (21 patients). There was no difference between the groups with respect to patient or donor age, donor type (matched related versus unrelated donor), cell source (peripheral blood versus bone marrow), patient-donor sex matching, myeloid versus lymphoid malignancy, risk status, patient/donor CMV status, conditioning regimen, or GVHD prophylaxis regimen. The cumulative incidence of grades II-IV acute GVHD at 100 days was 53.1% in the group with vitamin D level <25 versus 33.3% in the group with vitamin D≥25 (p=0.13). Remarkably, the cumulative incidence (CI) of chronic GVHD (cGVHD) at 2 years in patients with 25-OH vitamin D <25 was 63.8%, compared to 23.8% in patients with vitamin D levels ≥ 25 (p=0.009). Similarly the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D levels <25 compared to those with ≥25 (54.5% versus 14.3%, p=0.005). This result is consistent in multivariable competing risks model for which patient and transplant related factors mentioned above were adjusted (HR 5.26, p=0.02). Figure 1 represents the CI of cGVHD with death and relapse as a competing risk. Three-year progression-free survival and overall survival were similar between the two groups (51% vs. 47%, p=0.61; and 53% vs. 50%, p=0.57 respectively). In summary, vitamin D deficiency prior to allogeneic transplantation is associated with a significantly increased risk of chronic GVHD. A prospective clinical trial evaluating the use of vitamin D for the prevention of chronic GVHD is planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Decreased Expression of Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells From Patients with Immune Thrombocytopenia Induces Impaired Regulatory T-Cell Development

Abstract 696▪▪This icon denotes a clinically relevant abstractRegulatory T cells (Tregs) are a subset of T cells involved in the maintenance of peripheral self-tolerance. Specifically, Tregs modulate the maturation and/or function of dendritic cells (DCs). In turn, along with their immunogenic role, DCs are also critical in maintaining tolerance to self-antigens by inducing Tregs via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1-expressing DCs may play a role in preventing the initiation of autoimmune disorders.Immune Thrombocytopenia (ITP) is an autoimmune disorder in which platelet surface proteins become antigenic and stimulate the immune system. However, in ITP, the interaction between DCs and Tregs has never been investigated, although decreased numbers of Tregs as well as altered DCs have been described. Therefore, in the present study, we investigated whether, in ITP: 1) IDO1 expression/activity is decreased in monocyte-derived DCs (Mo-DCs); 2) the mechanism of Tregs generation is impaired; 3) Mo-DCs maturation is abnormally modulated by Tregs.We studied 54 patients with active ITP. Nineteen patients were newly diagnosed and 35 patients had persistent (25 cases) or chronic (10 cases) ITP. At the time of samples collection, all patients with persistent or chronic ITP were out of any treatment for at least two months and none of the patients received previous treatment with rituximab or were splenectomized.We demonstrated that in mature Mo-DCs from ITP patients the absolute number of IDO1 transcripts was significantly reduced as compared with healthy donors (973.000±771.000 vs 2.409.000±1.592.000 (IDO1 copy number/ABL1 copy number)*10.000; p<0.04). Accordingly, when we evaluated kynurenine levels as an index of IDO1 enzyme activity, we found that kynurenine concentration in the supernatants from equal numbers of mature Mo-DCs was significantly lower in ITP patients (40.7±12.5 μM) as compared to healthy subjects (66.5±9.4 μM) (p<0.05). We therefore assessed whether in ITP Mo-DCs, which have IDO1 reduced expression and activity, are less efficient in generating Tregs in vitro. We found that after co-cultures of Mo-DCs from healthy subjects or ITP patients with autologous CD4+CD25− T cells, the mean percentage of FoxP3+ cells (gated on CD4+CD25high T cells) was significantly reduced in ITP patients (54.4±4.5%) in comparison with that of healthy individuals (88.3±5.8%; p<0.01). When we evaluated whether these in vitro generated Tregs possess suppressive activity, we demonstrated that the inhibition of the proliferative response of CD4+CD25− cells in coculture with autologous in vitro generated CD4+CD25+ T cells (100:1 ratio) was significantly lower in ITP patients (25±4%) as compared with healthy controls (47±6%; p<0.05). To evaluate whether Tregs induce functional differences in Mo-DCs, we first assessed the production of cytokines in the supernatants of normal immature Mo-DCs co-cultured with highly purified circulating CD4+CD25+ or CD4+CD25− T cells from ITP patients or healthy controls. We found that in ITP patients the concentration of IL-10 was significantly reduced in the supernatant of Mo-DCs plus CD4+CD25+ T cells as compared with the normal counterparts (99±26 pg/mL vs 218±19 pg/mL; p<0.01). However, since IL-10 plays a central role in immune tolerance and can be produced by either DCs or T cells, we performed the intracytoplasmic staining of IL-10 to evaluate whether its secretion was due to Mo-DCs upon co-culture and/or produced by Tregs. The mean percentage of IL-10-producing Tregs was significantly lower in ITP patients (19.3±8.0 %) in comparison with that of normal individuals (34.9±6.6 %; p<0.04).We therefore analyzed the capacity of highly purified circulating Tregs of inhibiting Mo-DCs maturation. We found that, at variance with healthy controls, highly purified CD4+CD25+ T cells from ITP patients failed to inhibit the expression of CD80 and CD86 molecules on Mo-DCs.In summary, this study demonstrates that in ITP low IDO1 expression/activity in DCs results in the reduced ability of generating Tregs. In turn, circulating Tregs with impaired IL-10 production show decreased capacity of inhibiting DCs maturation. Taken together these data suggest that in ITP the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role. As a consequence, DCs are less “tolerogenic” and Tregs are defective in their regulatory function. Disclosures:No relevant conflicts of interest to declare.

The effects of transforming growth factor β2 and prostaglandin E2 secreted by murine corneal stroma cells on maturating procedure of dendritic cells

Background Researches demonstrated that dendritic cells(DCs) are uniformly immature in the central cornea but mature in the peripheral region of cornea.So an important question is which factor impact the maturation of DCs,especially in terms of corneal transplant rejection and the known roles of DCs in the development and persistence of some corneal diseases.Objective This study aimed to examine whether corneal stroma cells (CSCs) inhibit DCs maturation through secreting transforming growth factor beta 2 (TGF-β2) and prostaglandin E2 (PGE2).Methods DCs,T cells and CSCs were isolated and cultured from clean BALB/c and C57BL/6 mice.The level of PGE2 and TGF-β2in CSCs culture supernatant and the fresh RPMI 1640 medium were then analyzed by enzyme linked immunosorbent assay (ELISA).During the DCs maturation stage,the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were added in the CSCs culture supernatant respectively.According to the different treatment,cultured cells were assigned to different groups as follows:control group,CSCs culture supernatant group,AH6809 group,TGF-β2 antibody group,AH6809 +TGF-β2 antibody group.Subsequently,the cellular surface markers for DCs,including CD11c,CD80,CD86,and MHC- Ⅱ,were analyzed by flow cytometry.The capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions,and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran(FITC) uptake.Results The data of ELISA showed a higher concentration of TGF-β2 and PGE2 in murine CSCs culture supernatant than in the fresh RPMI 1640 medium.Compared with the CSCs culture supernatant group,the expression of CD80,CD86,and MHC- Ⅱ was up-regulated ( P ＜ 0.05 ),the expression of dextran was down-regulated ( P ＜ 0.05 ),and the stimulate index was increased( P＜ 0.05 ) in the TGF-β2 antibody group; the expression of CD86,and MHC-Ⅱ was up-regulated (P＜0.05),the expression of dextran was down-regulated ( F =13.740,P =0.006 ),and the stimulate index was increased(P＜0.05) in the AH6809 group;the expression of MHC-Ⅱ was up-regulated and the stimulate index was increased with statistical difference in interaction(P＜0.05 ) in the AH6809+TGF-β2 antibody group.Compared with the control group,the expression of CD80 and CD86,and the stimulate index was still lower(P＜0.05 ).Conclusions TGF-β2 and PGE2 contribute to the inhibitory effects on DCs maturation mediated by murine CSCs in vitro and further have additive effect on the immunosuppression of DCs. Key words: Corneal stroma cell; Dendritic cell/maturation; Transforming growth factor beta 2; Prostaglandin E2

Stimulation of dendritic cells withHelicobacter pylorivacuolating cytotoxin negatively regulates their maturation via the restoration of E2F1

Helicobacter pylori induces an infiltration of dendritic cells (DCs) into the infected gastric mucosa. Although DCs play an important role in the regulation of inflammation, the effects of H. pylori vacuolating cytotoxin (VacA) on DC maturation process have not yet been elucidated. The role of VacA in DC maturation following co-exposure to Escherichia coli lipopolysaccharide (LPS) was investigated. The treatment of immature DCs with LPS up-regulated the expression of surface molecules [e.g. CD40, CD80, CD86 and major histocompatibility complex (MHC) class II], as well as the production of cytokines [e.g. interleukin (IL)-1β, IL-12p70 and tumour necrosis gactor (TNF)-α] compared with those of unstimulated controls. Co-stimulation with H. pylori VacA significantly reduced the up-regulated DC maturation markers induced by LPS. In addition, VacA sustained the immature state of DCs with high endocytosis and low migratory capacity. The LPS-induced down-regulation of E2F1 expression in DCs was recovered by co-stimulation with VacA. Moreover, suppression of E2F1 by small interfering RNA resulted in a significant recovery of the inhibited DC maturation by VacA. In contrast, VacA did not affect nuclear factor (NF)-κB responses to LPS and the NF-κB signal was not associated with VacA-induced inhibition of DC maturation. These results suggest that the exposure of DCs to H. pylori VacA negatively regulates DC maturation via the restoration of E2F1. The immunomodulatory action of VacA on DCs may contribute to the ability of VacA-producing H. pylori to establish a persistent infection in the gastric mucosa.

Pharmacological manipulation of dendritic cells in the pursuit of transplantation tolerance

Although long-term immune suppression remains the intervention of choice for the treatment of allograft rejection, transplantation tolerance would achieve graft survival with fewer inherent risks. Although the use of dendritic cells for the induction of tolerance might confer antigen specificity, factors determining the balance between tolerogenicity and immunogenicity remain uncertain, as does the stability of the functional phenotype. Here, we review recent studies suggesting that pharmacological agents may profoundly influence this delicate balance and outline the insights they provide into parameters that contribute to the tolerogenic state. Recent findings have revealed that the inhibition of dendritic cell maturation by pharmacological intervention is not a prerequisite for the acquisition of tolerogenicity, but that susceptibility to a tolerogenic phenotype may vary between dendritic cell subsets and depend on the nature of maturation stimuli to which the cells are exposed. Furthermore, such studies have highlighted the degree to which the maintenance of tolerogenicity is influenced by local environmental factors, such as the cytokine milieu. Although the rational design of tolerogenic dendritic cells for modulating the outcome of organ transplantation remains ambitious, the use of pharmacological agents to influence their functional phenotype continues to illuminate the basic biology of this critical cell type.

Cholera toxin B subunit linked to glutamic acid decarboxylase suppresses dendritic cell maturation and function

Dendritic cells are the largest population of antigen presenting cells in the body. One of their main functions is to regulate the delicate balance between immunity and tolerance responsible for maintenance of immunological homeostasis. Disruption of this delicate balance often results in chronic inflammation responsible for initiation of organ specific autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and type I diabetes. The cholera toxin B subunit (CTB) is a weak mucosal adjuvant known for its ability to stimulate immunity to antigenic proteins. However, conjugation of CTB to many autoantigens can induce immunological tolerance resulting in suppression of autoimmunity. In this study, we examined whether linkage of CTB to a 5kDa C-terminal protein fragment of the major diabetes autoantigen glutamic acid decarboxylase (GAD(35)), can block dendritic cell (DC) functions such as biosynthesis of co-stimulatory factor proteins CD86, CD83, CD80 and CD40 and secretion of inflammatory cytokines. The results of human umbilical cord blood monocyte-derived DC-GAD(35) autoantigen incubation experiments showed that inoculation of immature DCs (iDCs), with CTB-GAD(35) protein dramatically suppressed levels of CD86, CD83, CD80 and CD40 co-stimulatory factor protein biosynthesis in comparison with GAD(35) alone inoculated iDCs. Surprisingly, incubation of iDCs in the presence of the CTB-autoantigen and the strong immunostimulatory molecules PMA and Ionomycin revealed that CTB-GAD(35) was capable of arresting PMA+Ionomycin induced DC maturation. Consistent with this finding, CTB-GAD(35) mediated suppression of DC maturation was accompanied by a dramatic decrease in the secretion of the pro-inflammatory cytokines IL-12/23p40 and IL-6 and a significant increase in secretion of the immunosuppressive cytokine IL-10. Taken together, our experimental data suggest that linkage of the weak adjuvant CTB to the dominant type 1 diabetes autoantigen GAD strongly inhibits DC maturation through the down regulation of major co-stimulatory factors and inflammatory cytokine biosynthesis. These results emphasize the possibility that CTB-autoantigen fusion proteins enhance DC priming of naïve Th0 cell development in the direction of immunosuppressive T lymphocytes. The immunological phenomena observed here establish a basis for improvement of adjuvant augmented multi-component subunit vaccine strategies capable of complete suppression of organ-specific autoimmune diseases in vivo.

The neuropeptide calcitonin gene‐related peptide affects allergic airway inflammation by modulating dendritic cell function

The neuropeptide calcitonin gene-related peptide (CGRP) is released in the lung by sensory nerves during allergic airway responses. Pulmonary dendritic cells (DC) orchestrating the allergic inflammation could be affected by CGRP. To determine the immunomodulatory effects of CGRP on DC function and its impact on the induction of allergic airway inflammation. CGRP receptor expression on lung DC was determined by RT-PCR and immunofluorescence staining. The functional consequences of CGRP receptor triggering were evaluated in vitro using bone marrow-derived DC. DC maturation and the induction of ovalbumin (OVA)-specific T cell responses were analysed by flow cytometry. The in vivo relevance of the observed DC modulation was assessed in a DC-transfer model of experimental asthma. Mice were sensitized by an intrapharyngeal transfer of OVA-pulsed DC and challenged with OVA aerosol. The impact of CGRP pretreatment of DC on airway inflammation was characterized by analysing differential cell counts and cytokines in bronchoalveolar lavage fluid (BALF), lung histology and cytokine responses in mediastinal lymph nodes. RT-PCR, immunofluorescence and cAMP assay demonstrated the expression of functionally active CGRP receptors in lung DC. RT-PCR revealed a transcriptional CGRP receptor down-regulation during airway inflammation. CGRP specifically inhibited the maturation of in vitro generated DC. Maturation was restored by blocking with the specific antagonist CGRP(8-37) . Consequently, CGRP-pretreated DC reduced the activation and proliferation of antigen-specific T cells and induced increased the numbers of T regulatory cells. The transfer of CGRP-pretreated DC diminished allergic airway inflammation in vivo, shown by reduced eosinophil numbers and increased levels of IL-10 in BALF. CGRP inhibits DC maturation and allergen-specific T cell responses, which affects the outcome of the allergic airway inflammation in vivo. This suggests an additional mechanism by which nerve-derived mediators interfere with local immune responses. Thus, CGRP as an anti-inflammatory mediator could represent a new therapeutic tool in asthma therapy.

Abstract 3646: The role of myeloid-derived suppressor cells in tumor development

Abstract Myeloid-derived suppressor cells (MDSC) play an important role in regulation of immune response in cancer. They also have been implicated into promotion of tumor metastases and angiogenesis. We asked what role if any these myeloid cells can play in tumor formation associated in inflammation. To address this question we used previously described S100A9 deficient and S100A9 transgenic mice. S100A9, also known as calgranulin B or myeloid related protein 14 (MRP14), is a member of the S100 family of Ca2+ binding proteins. Previous studies have established that up-regulation of this protein in hematopoietic progenitor cells results in inhibition of dendritic cell maturation and accumulation of MDSC. We tested the role of MDSC in skin tumor formation using TgAC transgenic mice. These mice have activated k-ras mutation in keratinocytes. Upon topical treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) they develop skin papillomas. We generated double transgenic mice and demonstrated that in mice overexpressing S100A9 the frequency and number of papillomas increased dramatically comparing with wild-type TgAC mice. The formation of papillomas was also significantly accelerated. To confirm the critical role of myeloid cells in the observed effect we used bone marrow transfer from wild-type or S100A9 Tg mice into lethally irradiated TgAC mice. Transfer of S100A9 Tg bone marrow cells resulted in significantly more rapid and more extensive formation of papillomas in TPA treated mice than transfer of control bone marrow. To assess effect of TPA treatment on cell infiltrate into skin wild-type or S100A9Tg mice were treated with TPA for 3 weeks. Skin from S100A9Tg mice had significantly higher number of Gr-1+ myeloid cells and CD4+ T cells than skin from wild-type mice. No differences in F4/80 macrophages and CD11c+ dendritic cells were seen. To further evaluate the possible role of MDSC in tumor formation we treat control and S100A9 knockout (KO) mice with combination of carcinogen DMBA and TPA using accelerated skin carcinogenesis protocol. The absence of S100A9 substantially delayed the onset of papilloma and decreased the number of lesions as compared with wild-type mice. These results suggest that MDSC may play an important role in inflammation associated tumoregenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3646. doi:10.1158/1538-7445.AM2011-3646