Tolerogenic Dendritic Cells Phenotype Research Articles

The CYP27B1 variant associated with an increased risk of autoimmune disease is underexpressed in tolerizing dendritic cells

Genome-wide association studies have identified a linkage disequilibrium (LD) block on chromosome 12 associated with multiple sclerosis (MS), type 1 diabetes and other autoimmune diseases. This block contains CYP27B1, which catalyzes the conversion of 25 vitamin D3 (VitD3) to 1,25VitD3. Fine-mapping analysis has failed to identify which of the 17 genes in this block is most associated with MS. We have previously used a functional approach to identify the causal gene. We showed that the expression of several genes in this block in whole blood is highly associated with the MS risk allele, but not CYP27B1. Here, we show that CYP27B1 is predominantly expressed in dendritic cells (DCs). Its expression in these cells is necessary for their response to VitD, which is known to upregulate pathways involved in generating a tolerogenic DC phenotype. Here, we utilize a differentiation protocol to generate inflammatory (DC1) and tolerogenic (DC2) DCs, and show that for the MS risk allele CYP27B1 is underexpressed in DCs, especially DC2s. Of the other Chr12 LD block genes expressed in these cells, only METT21B expression was as affected by the genotype. Another gene associated with autoimmune diseases, CYP24A1, catabolizes 1,25 VitD3, and is predominantly expressed in DCs, but equally between DC1s and DC2s. Overall, these data are consistent with the hypothesis that reduced VitD pathway gene upregulation in DC2s of carriers of the risk haplotype of CYP27B1 contributes to autoimmune diseases. These data support therapeutic approaches aimed at targeting VitD effects on DCs.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg

AbstractDenileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen–specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator–dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinicaltrials.gov, #NCT00056134.

Mycobacterium avium subspecies impair dendritic cell maturation

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

Alpha-melanocyte-stimulating hormone (a-MSH) inhibits the development and progression of autoimmune encephalomyelitis by generating functional regulatory T cells

Event Abstract Back to Event Alpha-melanocyte-stimulating hormone (α-MSH) inhibits the development and progression of autoimmune encephalomyelitis by generating functional regulatory T cells Nadine Sucker1*, Lars Klenner1, Sandra Laggies1, Christina Bürger1, Thomas A. Luger1 and Karin Loser1 1 University of Münster, Department of Dermatology, Germany The neuropeptide α-MSH is a potent immunomodulator capable of inducing immunosuppression and tolerance. Using the mouse model of experimental autoimmune encephalomyelitis (EAE) we systemically treated MOG-immunized mice with α-MSH before and after the onset of hind limb paralysis. Whereas control mice showed a significant weight loss and developed severe ascending paralysis, mice preemptively injected with α-MSH were resistant to EAE development. Notably, therapeutic treatment attenuated EAE progression and prevented mice from weight loss. Flow cytometry, immunofluorescence staining and gene expression analyses revealed the absence of pathogenic Th17 and Th1 cells from brain tissue of α-MSH-treated animals. This effect was mediated by up-regulated numbers of Foxp3+ regulatory T cells (Treg) in α-MSH-injected mice versus controls. Since α-MSH has been shown to expand Treg by the induction of tolerogenic dendritic cells (DC) we analyzed the DC phenotype at different stages of disease. DC from α-MSH-treated mice expressed increased levels of PD-L1 or IL 10 and down-regulated maturation markers pointing to the induction of a tolerogenic DC phenotype. Since signaling via melanocortin-1-receptor (MC-1R) mediates the immunomodulatory effects of α-MSH we induced EAE in MC-1R-deficient mice. Interestingly, upon α-MSH injection these mice developed hind limb paralysis similar to PBS treated controls demonstrating that binding to MC-1R is essential for the α MSH-mediated prevention of EAE. Together, these data indicate that α MSH induces tolerogenic DC and expands functional Treg in vivo. These Treg suppress pathogenic Th1 and Th17 cells during EAE development, suggesting α-MSH as potential therapeutic option for the treatment of patients with moderate multiple sclerosis. Keywords: α-MSH, protection, Experimental autoimmune encephalomyelitis, Treg, Th1 Cells, Th17 Cells Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Sucker N, Klenner L, Laggies S, Bürger C, Luger T and Loser K (2013). Alpha-melanocyte-stimulating hormone (α-MSH) inhibits the development and progression of autoimmune encephalomyelitis by generating functional regulatory T cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00112 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Nadine Sucker, University of Münster, Department of Dermatology, Münster, 48149, Germany, nadine.sucker@ukmuenster.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nadine Sucker Lars Klenner Sandra Laggies Christina Bürger Thomas A. Luger Karin Loser Google Nadine Sucker Lars Klenner Sandra Laggies Christina Bürger Thomas A. Luger Karin Loser Google Scholar Nadine Sucker Lars Klenner Sandra Laggies Christina Bürger Thomas A. Luger Karin Loser PubMed Nadine Sucker Lars Klenner Sandra Laggies Christina Bürger Thomas A. Luger Karin Loser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The Dark Side of Dendritic Cells: Development and Exploitation of Tolerogenic Activity That Favor Tumor Outgrowth and Immune Escape

Dendritic cells (DC) play a central role in the regulation of the immune responses by providing the information needed to decide between tolerance, ignorance, or active responses. For this reason different therapies aim at manipulating DC to obtain the desired response, such as enhanced cell-mediated toxicity against tumor and infected cells or the induction of tolerance in autoimmunity and transplantation. In the last decade studies performed in these settings have started to identify (some) molecules/factors involved in the acquisition of a tolerogenic DC phenotype as well as the underlying mechanisms of their regulatory function on different immune cell populations.

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323-339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.

Crosstalk of regulatory T cells and tolerogenic dendritic cells prevents contact allergy in subjects with low zone tolerance

Allergic contact dermatitis is one of the most common occupational diseases. Amain protective mechanism in those who do not develop allergic contact dermatitis is tolerance induction by repeated exposure to low doses of contact allergen, which is termed low zone tolerance (LZT). The mechanisms that determine the tolerance induction in subjects with LZT are still elusive. We performed analysis of the role of CD4(+)CD25(+) forkhead box protein 3 (FOXP3)-positive regulatory T (Treg) cells and dendritic cells (DCs) in mice with LZT. Mechanisms of tolerance induction were analyzed in a murine model of LZT by using FOXP3 and IL-10 reporter mice, as well as mice that allow the selective depletion of Treg cells or DCs. Depletion of CD4(+)CD25(+)FOXP3(+) Treg cells during tolerance induction completely abolishes the development of LZT, resulting in a pronounced contact hypersensitivity response. Adoptive transfer experiments, depletion studies, and use of cell type-specific deficient mice revealed that IL-10 production is critical for the suppressor function of Treg cells in mice with LZT and that tolerogenic CD8(+)CD11c(+) DCs located in the skin-draining lymph nodes are essential for LZT. In the absence of Treg cells, DCs did not develop tolerogenic functions, indicating that activated IL-10(+) Treg cells might imprint the tolerogenic DC phenotype. Cell communication analysis revealed that the education of tolerogenic DCs might involve a direct interaction with Treg cells mediated by gap junctions. Subsequently, induction of tolerogenic CD11c(+) DCs leads to the generation of hapten-specific CD8(+) Treg cells, which protect against contact hypersensitivity. Our data demonstrate critical interactions between CD4(+)CD25(+)FOXP3(+) Treg cells and tolerogenic CD8(+)CD11c(+) DCs during the induction of LZT.

α-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a well-known mediator of skin pigmentation. More recently, it has been shown that α-MSH also exerts a strong anti-inflammatory and immunosuppressive activity. To elucidate the mechanisms underlying α-MSH-induced immunosuppression, we investigated whether α-MSH affects dendritic cell/T cell communication, as especially this interaction has an important role in the regulation of immune responses. Here, we show that α-MSH, by binding to MC-1R, induced tolerogenic dendritic cells, which were capable of expanding CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in vitro, as well as in vivo. Notably, those α-MSH-induced Tregs were functional as they efficiently inhibited cutaneous contact allergy and ongoing psoriasis-like skin inflammation in mice. Furthermore, α-MSH induced tolerogenic dendritic cells capable of generating functional Tregs in human blood. Interestingly, human Tregs expanded via α-MSH-stimulated dendritic cells suppressed the proliferation and cytokine secretion of pathogenic T-helper-17 (Th17) cells from individuals with psoriasis. Taken together, these data indicate that α-MSH induced immunosuppressive Tregs in vitro and in vivo, which inhibited disease progression in a mouse model of psoriasis-like skin inflammation and suppressed the activation and proliferation of effector T cells from subjects with psoriasis.

Differential Protein Pathways in 1,25-Dihydroxyvitamin D3 and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)(2)D(3)), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)(2)D(3), dexamethasone (DEX), and a combination of both compounds (combi). Human CD14(+) monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)(2)D(3) and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3-7NL and 6-9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.

Interaction of Antithymocyte Globulins with Dendritic Cell Antigens

The polyclonal rabbit antithymocyte globulins (ATGs), Thymoglobulin and ATG-Fresenius S, are widely used for prevention and therapy of allograft rejection and graft versus host disease. Dendritic cells (DC) govern immune responses and thus the interaction of ATGs with these cells could potentially contribute to the clinical effects of ATG therapy. Currently there is little information on the DC-antigens targeted by ATGs. In this study we have used a new methodology to identify DC surface antigens recognized by ATGs. By screening an eukaryotic expression library generated from DC with ATGs we could identify several novel ATG antigens including CD81, CD82, CD98, CD99 and CD147. Furthermore, we engineered cells to express previously described ATG antigens and probed them with Thymoglobulin and ATG-Fresenius S. Our results demonstrated strong binding to some but not all of these molecules. We show that previously described antigens and antigens identified in this study account for around 80% of the DC reactivity of ATGs. Analysis of molecules induced by ATG-DC interaction are more in support for an activation of these cells by ATGs than for a specific induction of a tolerogenic DC phenotype.

PTU-036 The immunomodulatory effects of vitamin d on low-density cells and monocyte-derived dendritic cells from human peripheral blood in vitro

Introduction Emerging evidence suggests Vitamin D (VD) possesses a wide range of immunomodulatory properties. Epidemiological evidence also implicates VD insufficiency in Inflammatory Bowel Disease (IBD).1 Dendritic Cells (DC) are conductors of the immune system, uniquely capable of primary antigen presentation to naive T cells, determining either immune response or tolerance. Studies on laboratory-generated monocyte-derived dendritic cells (MoDC) demonstrate 1,25-dihydroxyvitaminD3 (1,25(OH)2D3), the biologically active form of VD, induces an immature and tolerogenic DC phenotype.2 In vivo studies on murine models of IBD suggest therapeutic potential for VD.3 Methods We determined the immunomodulatory effects of 1,25(OH)2D3 on maturation and functional phenotype of blood DC, in particular DC-enriched Low-Density Cells (LDC). Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll gradients from blood of healthy volunteers. LDC enriched for DC were isolated on a NycoPrep gradient from non-adherent cells after overnight PBMC culture. MoDC were obtained from magnetically sorted CD14+ monocytes of PBMC, incubated for 5 days with IL-4 and GM-CSF. LDC and MoDC were cultured for another 24 h in complete medium with and without 1,25(OH)2D3 and/or LPS. LDC and MoDC maturation was determined by flow cytometry using monoclonal antibodies to maturation markers including CD14, CD40, HLA-DR and ILT-3, which is an inhibitory receptor and a marker of DC immaturity. MoDC functional phenotype was determined by phagocytotic capacity (FITC-Dextran uptake) which is reduced in mature DC. Cell morphology was also assessed by electron microscopy (EM). Results VD-conditioned LDC demonstrated an immature phenotype compared with controls with significantly increased expression of monocyte marker CD14 ( Conclusion 1,25(OH)2D3 promotes an immature phenotype in both LDC and MoDC in vitro inducing a tolerogenic-type phenotype and monocyte-like state. These immunomodulatory effects suggest a potential therapeutic role for VD in IBD possibly by promotion of oral tolerance.

Impaired IL-10–dependent Induction of Tolerogenic Dendritic Cells by CD4+CD25hiCD127lo/−Natural Regulatory T Cells in Human Allergic Asthma

Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However, the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. The study was performed in a cohort of 21 subjects with allergic asthma, 21 healthy control subjects, and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. Dendritic cells cultured with natural regulatory T cells up-regulated IL-10, down-regulated costimulatory molecules, and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore, this defective function of natural regulatory T cells was associated with their decreased IL-10 expression, disease severity, and could be reversed by oral corticosteroid therapy. These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma.

Ovarian Cancer-derived Glycodelin Impairs In Vitro Dendritic Cell Maturation

Local immunosuppressive mechanisms shape the tumor microenvironment and contribute to carcinogenesis. In ovarian cancer such mechanisms have been shown to influence survival. Dendritic cells (DCs) are central immunity regulators and induce potent cytotoxic T-cell responses as well as peripheral tolerance depending on modulatory stimuli. Here, we show that ovarian cancer-derived glycodelin (Gd), a glycoprotein that physiologically modulates local immunity in early pregnancy, induces a tolerogenic DC phenotype. Gd was isolated with high performance liquid chromatography from the malignant ascites of ovarian cancer patients. DCs were generated from monocytes of healthy donors and exposed to Gd with or without an inflammatory stimulus (tumor necrosis factor-alpha and interleukin 1-beta). We investigated the effect of Gd on DC surface marker expression, endopinocytotic activity, cytokine profile, and lymphoproliferative activity. DCs that were exposed to Gd altered their phenotype as seen by a differential expression of costimulatory molecules, whereas expression of DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, a marker of an immature phenotype, was increased. Functional data provided further evidence for the immature/tolerogenic properties of Gd-pretreated DCs. Antigen uptake was retained, production of interleukin-10 was increased, and lymphoproliferative activity was reduced. This effect was reversible by adding Gd-blocking antibodies. Gd, which is found in the malignant ascites of ovarian cancer patients, induces a tolerogenic phenotype in DC, thereby shaping an immunodeficient tumor micromilieu.

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance.

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.