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IFN-α is an antineoplastic agent in the treatment of several solid and hematologic malignancies that exerts strong immune- and autoimmune-stimulating activity. However, the mechanisms of immune activation by IFN-α remain incompletely understood, particularly with regard to CD4+CD25highFoxp+ regulatory T cells (Treg). Here, we show that IFN-α deactivates the suppressive function of human Treg by downregulating their intracellular cAMP level. IFN-α–mediated Treg inactivation increased CD4+ effector T-cell activation and natural killer cell tumor cytotoxicity. Mechanistically, repression of cAMP in Treg was caused by IFN-α–induced MAP–ERK kinase (MEK)/extracellular signal-regulated kinase (ERK)–mediated phosphodiesterase 4 (PDE4) activation and accompanied by downregulation of IFN receptor (IFNAR)-2 and negative regulation of T-cell receptor signaling. IFN-α did not affect the anergic state, cytokine production, Foxp3 expression, or methylation state of the Treg-specific demethylated region (TSDR) within the FOXP3 locus associated with a stable imprinted phenotype of human Treg. Abrogated protection by IFN-α–treated Treg in a humanized mouse model of xenogeneic graft-versus-host disease confirmed IFN-α–dependent regulation of Treg activity in vivo. Collectively, the present study unravels Treg inactivation as a novel IFN-α activity that provides a conceivable explanation for the immune-promoting effect and induction of autoimmunity by IFN-α treatment in patients with cancer and suggests IFN-α for concomitant Treg blockade in the context of therapeutic vaccination against tumor antigens. Cancer Res; 73(18); 5647–56. ©2013 AACR.

PDF file, 251K, Supplemental Figure 1. CD56+CD16+ NK cells isolation from buffy coats. Suppelmental Figure 2. IFN-alpha treatment impairs STAT1 activation in Treg. Supplemental Figure 3. IFN-alpha did not affect Foxp3 expression in Treg. Supplemental Figure 4. Pharmacological repression of cAMP production in Treg does not affect ZAP-70 phosphorylation.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape., The acidic tumour microenvironment in melanoma drives immune evasion by cAMP in tumor-infiltrating monocytes. Here, the authors show that the release of an adenylate cyclase inhibitor from micelles restores antitumor immunity and, when combined with regulatory T cell depletion, leads to remission of established B16-F10-OVA tumors.

Introduction: Targeting the α7 nicotinic acetylcholine receptor (α7nAChR) has recently been suggested as a potential new treatment for fibrotic skin diseases. Here, we performed a genetic and pharmacologic approach to clarify the role of this receptor in the bleomycin (BLM) mouse model of skin fibrosis using α7nAChR KO mice. Methods: We analyzed the expression of extracellular matrix (ECM) components in murine skin using quantitative RT-PCR, pepsin digestion/SDS-PAGE of proteins and performed hydroxyproline assays as well as histological/immunohistochemical staining of skin sections. To identity the target cells of the α7nAChR agonist PHA-543613, we used murine dermal fibroblasts (MDF). We tested their response to the profibrotic cytokine transforming growth factor-β1 (TGF-β1) and utilized gene silencing to elucidate the role of the α7nAChR. Results: We confirmed our previous findings on C3H/HeJ mice and detected a suppressive effect of PHA-543613 on BLM-induced skin fibrosis in the mouse strain C57BL/6J. This antifibrotic effect of PHA-543613 was abrogated in α7nAChR-KO mice. Interestingly, α7nAChR-KO animals exhibited a basal profibrotic signature by higher RNA expression of ECM genes and hydroxyproline content than WT mice. In WT MDF, PHA-543613 suppressed ECM gene expression induced by TGF-β1. Gene silencing of α7nAChR by small interfering RNA neutralized the effects of PHA-543613 on TGF-β1-mediated ECM gene expression. Conclusion: In summary, we have identified the α7nAChR as the essential mediator of the antifibrotic effect of PHA-543613. MDF are directly targeted by PHA-543613 to suppress collagen synthesis. Our findings emphasize therapeutic exploitation of α7nAChR receptor agonists in fibrotic skin diseases.

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species–induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6Chigh monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2+ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6Clow monocytes by a nuclear receptor Nur77–agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12–deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

The cells and mechanisms involved in blood clot resorption are only partially known. We show that regulatory T cells (Tregs) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. We describe a clot Treg population that forms the matricellular acid– and cysteine-rich protein SPARC (secreted protein acidic and rich in cysteine) and show that SPARC enhances monocyte MMP activity and that SPARC+ Tregs are crucial for blood clot resorption. By comparing different treatment times, we define a therapeutic window of Treg expansion that accelerates clot resorption.

BackgroundHerpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells.MethodWe employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells.ResultsAstrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism.ConclusionsHSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection Background: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells. Method: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells. Results: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism. Conclusions: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

Wheat amylase trypsin inhibitors (ATI) are dietary non-gluten proteins that activate the toll-like receptor 4 on myeloid cells, promoting intestinal inflammation.We investigated the effects of dietary ATI on experimental allergic airway inflammation.Mice on a gluten and ATI-free diet (GAFD), sensitized with PBS or ovalbumin (OVA) and challenged with OVA, were compared to mice on a commercial standard chow, a gluten diet naturally containing ~ 0.75% of protein as ATI (G+AD), a gluten diet containing ~ 0.19% of protein as ATI (G-AD) and a GAFD with 1% of protein as ATI (AD). Airway hyperreactivity (AHR), inflammation in bronchoalveolar lavage (BAL) and pulmonary tissue sections were analyzed. Allergic sensitization was assessed ex vivo via proliferation of OVA-stimulated splenocytes.Mice on a GAFD sensitized with PBS did not develop AHR after local provocation with methacholine. Mice on a GAFD or on a G-AD and sensitized with OVA developed milder AHR compared to mice fed a G+AD or an AD. The increased AHR was paralleled by increased BAL eosinophils, IL-5 and IL-13 production, and an enhanced ex vivo splenocyte activation in the ATI-fed groups.Dietary ATI enhance allergic airway inflammation in OVA-challenged mice, while an ATI-free or ATI-reduced diet has a protective effect on AHR. Nutritional wheat ATI, activators of intestinal myeloid cells, may be clinically relevant adjuvants to allergic airway inflammation.

Background Scurfy mice have a functional defect in regulatory T cells (Treg), which leads to lethal multi-organ inflammation. The missing Treg function results in uncontrolled autoimmune cellular and humoral inflammatory responses. We and others have previously shown that during the course of disease scurfy mice develop severe skin inflammation and autoantibodies including anti-nuclear autoantibodies (ANA). Objective Autoimmune skin inflammation and ANA are hallmarks for the diagnosis of autoimmune connective tissue diseases; therefore we analyzed scurfy mice for typical signs of these diseases. Methods Indirect immunofluorescence was used to specify the ANA pattern in scurfy mice. Skin fibrosis was assessed by cutaneous collagen accumulation (Goldeners trichrome staining), collagen crosslinking/disorganization (Sirus red polarimetry) and quantitative PCR for fibrosis-related transcripts. The cellular components of the inflammatory infiltrates in scurfy skin were analyzed by flow cytometry and intracellular cytokine staining. Results The majority of scurfy mice developed ANA with a predominant AC-5 pattern typical for mixed connective tissue disease, especially scleroderma. Scurfy mice showed higher skin collagen content compared to WT controls with a significant tendency in upregulation of TIMP-1. CD3+CD4+ T cells in scurfy skin exhibited a strong Th2 deviation with a significant increase of IL-4, IL-5 and IL-13, and M2-polarized CD11b+MHCII+ macrophages compared to WT mice. Conclusion We show that Scurfy mice show a predominant AC-5 ANA pattern typical for mixed connective tissue disease as in scleroderma. The autoimmune inflammation in scurfy skin mainly consists of CD4+ T cells with Th2 differentiation and alternatively-activated (M2) macrophages as it is found in scleroderma with advanced fibrosis.

Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-β transcripts in HOCl animals, whereas IL-1β and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19+ B cells, CD4+ T cells, CD11c+ DC and CD11b+ myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b+ Ly6Clow-high CD64low-high cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2low MHCIIhigh ) and monocyte-derived DC (CCR2high MHCIIhigh ) predominated over less activated CD11b+ myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b+ MHCIIhigh cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.

Tolerance to environmental antigens that encounter the organism at interfaces like skin or gut prevents deleterious systemic immune responses. The aim of this study was to analyze whether and how low doses of haptens, by entry through the skin or gastrointestinal tract, affect the outcome of the predominantly Th1/Th17-mediated 2,4,6-trinitro-benzenesulfonic acid-induced colitis, which mimics an autoimmune bowl disease in man. Epicutaneous and oral applications of low doses of the allergen resulted in the induction of low-zone tolerance (LZT) and protected from colitis development, demonstrated by a significantly reduced inflammatory response of the gut in vivo. In line with this observation, we found a significantly diminished Th1/Th17-mediated T cell response and reduced T cell proliferation after both tolerance regimes, indicating that epicutaneous LZT is just as well efficient as oral tolerance in prevention of a gut-associated inflammatory immune response. Use of a second, unrelated hapten for LZT induction revealed an antigen-specific tolerance mechanism. Intriguingly, in the absence of hapten-activated CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10, epicutaneous and oral LZT failed to abrogate the development of the intestinal inflammation. In conclusion, this study highlights in particular epicutaneous immunotherapies in the form of LZT through activation of CD4(+)CD25(+)Foxp3(+) regulatory T cells as treatment strategies for inflammatory, allergic, or autoimmune diseases.

Interleukin-2 (IL-2) is a T cell growth factor particularly required in regulatory T cell maintenance and memory T cell responses. High-dose IL-2 treatment was the first FDA-approved immunotherapy for cancer, while low-dose IL-2 administration has shown promise in allograft rejection and autoimmune and inflammatory diseases. However, its pleiotropic nature and the existence of IL-2 receptors with different binding affinity limit its therapeutic application. For an improved clinical applicability of the cytokine, a targeted receptor assignment must, therefore, be achieved. Nanoparticles allow controlling the location and dose of immunomodulating compounds and to specifically address specific receptors through targeted drug binding. In this review article we discuss the IL-2 biology and current clinical application with regard to nanoparticle-based IL-2-mediated manipulation of T cell responses in autoimmunity, chronic inflammation, and cancer.

Systemic sclerosis (SSc) is one of the most complex systemic autoimmune diseases with multi-organ involvement and heterogeneous clinical manifestations. The exact etiology of SSc is still unknown. However, identified target structures are components of endothelial cells, the innate/adaptive immune systems and fibroblasts, resulting in the hallmarks of the disease in form of inflammation/autoimmunity, vasculopathy and fibrosis of the skin and internal organs. There has been a large body of evidence that the adaptive immune system with autoreactive T and B cells producing autoantibodies plays a central role in the pathogenesis of SSc but the role of earlier pathogenic processes involving the innate immune system, is far from being understood. There is strong evidence that a dysregulation of innate lymphoid cells and myeloid cells critically contributes to early pathogenic events in SSc. As disruption of vascular homeostasis and a fibroproliferative vasculopathy are hallmarks of early SSc, intravascular processes including platelet activation and interaction with neutrophils and monocytes, may even be upstream of innate immune deviation. Therefore, further studies of the dysregulated innate immune system may provide insights into novel and potentially curative treatments of SSc. In this review, we highlight the most relevant findings regarding the involvement of innate immune cells during the early stage of cutaneous fibrosis in SSc, with emphasis on the role of neutrophils, myeloid cells and innate lymphoid/NK cells in the pathogenesis of SSc and their potential as therapeutic targets for this difficult-to-treat autoimmune disease.

Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β-galactosidase (pFascin-βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to βGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4(+) T cells into Th2 and Th17 cells, pFascin-βGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-βGal mice revealed that CD4(+) and CD8(+) cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-βGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-βGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.

Interferon-α (IFN-α) is the only drug approved for adjuvant therapy of malignant melanoma and is also used in the treatment of hematological and solid tumors. Along with its proven clinical efficacy, IFN-α produces several side effects, particularly with regard to autoimmune disorders. Curious about symptoms of autoimmunity during IFN-α therapy, we asked whether IFN-α directly impacts on immune tolerance. We found that IFN-α does alter the function of tolerogenic dendritic cells (DC) as well as of induced and naturally occurring T-regulatory cells (nTregs). IFN-α blocks the tolerogenic phenotype of DC by inducing maturation and thus preventing the induction of inducible Tregs by DC. It also has direct effects on nTregs. IFN-α reduces cAMP in Tregs via ERK/phosphodiesterase-mediated pathways. Since cAMP is essentially involved in suppression by nTregs, the IFN-α-dependent reduction of cAMP levels abolishes the suppressive capacity of nTregs. Therefore, Tregs are incapable of suppressing the activity of effector T cells and natural killer cells, resulting in tumor rejection. Thus, IFN-α overcomes immunological tolerance processes, leading to an improved immunostimulation and efficient tumor rejection, but also increases the risk of autoimmunity.

IFN-α is an antineoplastic agent in the treatment of several solid and hematologic malignancies that exerts strong immune- and autoimmune-stimulating activity. However, the mechanisms of immune activation by IFN-α remain incompletely understood, particularly with regard to CD4+CD25highFoxp+ regulatory T cells (Treg). Here, we show that IFN-α deactivates the suppressive function of human Treg by downregulating their intracellular cAMP level. IFN-α–mediated Treg inactivation increased CD4+ effector T-cell activation and natural killer cell tumor cytotoxicity. Mechanistically, repression of cAMP in Treg was caused by IFN-α–induced MAP–ERK kinase (MEK)/extracellular signal-regulated kinase (ERK)–mediated phosphodiesterase 4 (PDE4) activation and accompanied by downregulation of IFN receptor (IFNAR)-2 and negative regulation of T-cell receptor signaling. IFN-α did not affect the anergic state, cytokine production, Foxp3 expression, or methylation state of the Treg-specific demethylated region (TSDR) within the FOXP3 locus associated with a stable imprinted phenotype of human Treg. Abrogated protection by IFN-α–treated Treg in a humanized mouse model of xenogeneic graft-versus-host disease confirmed IFN-α–dependent regulation of Treg activity in vivo. Collectively, the present study unravels Treg inactivation as a novel IFN-α activity that provides a conceivable explanation for the immune-promoting effect and induction of autoimmunity by IFN-α treatment in patients with cancer and suggests IFN-α for concomitant Treg blockade in the context of therapeutic vaccination against tumor antigens. Cancer Res; 73(18); 5647–56. ©2013 AACR.

Background & Aims Wheat amylase-trypsin inhibitors (ATIs) are nutritional activators of innate immunity, via activation of the toll-like receptor 4 (TLR4) on myeloid cells. We aimed to characterize the biologic activity of ATIs in various foods and their effect on intestinal inflammation. Methods We selected 38 different gluten-containing and gluten-free products, either unprocessed (such as wheat, rye, barley, quinoa, amaranth, soya, lentils, and rice) or processed (such as pizza, pasta, bread, and biscuits). ATIs were extracted and their biological activities determined in TLR4-responsive mouse and human cell lines. Effects of oral ATIs on intestinal inflammation were determined in healthy C57BL/6 mice on a gluten-free or ATI-free diet and in mice given low-level polyinosinic:polycytidylic acid or dextran sodium sulfate to induce colitis. Parameters of innate and adaptive immune activation were determined in duodenum, ileum, colon, and mesenteric lymph nodes. Results Modern gluten-containing staples had levels of TLR4-activating ATIs that were as much as 100-fold higher than in most gluten-free foods. Processed or baked foods retained ATI bioactivity. Most older wheat variants (such as Emmer or Einkorn) had lower bioactivity than modern (hexaploid) wheat. ATI species CM3 and 0.19 were the most prevalent activators of TLR4 in modern wheat and were highly resistant to intestinal proteolysis. Their ingestion induced modest intestinal myeloid cell infiltration and activation, and release of inflammatory mediators—mostly in the colon, then in the ileum, and then in the duodenum. Dendritic cells became prominently activated in mesenteric lymph nodes. Concentrations of ATIs found in a normal daily gluten-containing diet increased low-level intestinal inflammation. Conclusions Gluten-containing cereals have by far the highest concentrations of ATIs that activate TLR4. Orally ingested ATIs are largely resistant to proteases and heat, and increase intestinal inflammation by activating gut and mesenteric lymph node myeloid cells.

Nucleotide signaling molecules contribute to the regulation of cellular pathways. In the immune system, cyclic adenosine monophosphate (cAMP) is well established as a potent regulator of innate and adaptive immune cell functions. Therapeutic strategies to interrupt or enhance cAMP generation or effects have immunoregulatory potential in autoimmune and inflammatory disorders. Here, we provide an overview of the cyclic AMP axis and its role as a regulator of immune functions and discuss the clinical and translational relevance of interventions with these processes.

Hintergrund: Die Sensibilisierung von Inzuchtmausen mit niedrigen Dosen eines Antigens (LD) fuhrt zu einer erheblichen Produktion von antigenspezifischen IgE-Antikorpern, welche bei Applikation von hohen Antigendosen (HD) ausbleibt. Wir haben erstmals gezeigt, dass in HD-Mausen CD4-CD8--doppelt-negative-T (dnT)-Zellen induziert werden, die regulatorische Funktionen im Rahmen der IgE-Immunantwort ausuben.

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. Methods Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naive mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Dendritic cells are (DC) highly specialized professional antigen-presenting cells (APC) that regulate immune responses, maintaining the balance between tolerance and immunity. Mechanisms via which they can promote central and peripheral tolerance include clonal deletion, inhibition of memory T cell responses, T cell anergy and induction of regulatory T cells. These properties have led to the analysis of human tolerogenic DC as a therapeutic strategy for induction or re-establishment of tolerance. In the recent years, numerous protocols for the generation of human tolerogenic DC have been developed and their tolerogenic mechanisms, including induction of regulatory T cells, are relatively well understood. Phase I trials have been conducted in autoimmune disease, with results that emphasize the feasibility and safety of treatments with tolerogenic DC. Therefore, the scientific rationale for the use of tolerogenic DC therapy in the fields of transplantation medicine and allergic and autoimmune diseases is strong. This review will give an overview on efforts and protocols to generate human tolerogenic DC with focus on IL-10-modulated DC as inducers of regulatory T cells and discuss their clinical applications and challenges faced in further developing this form of immunotherapy.

Dendritic cells (DCs) are key regulators of protective immune responses and tolerance to (self-)Ags. Therefore, the scientific rationale for the use of tolerogenic DC therapy in the fields of allergies, autoimmunity, and transplantation medicine is strong. In this study, we analyzed the tolerogenic capacity of IL-10–modulated DC (IL-10DC) subpopulations to identify a DC subset that combines potent immunosuppressive activities with valuable immune properties for clinical implementation. IL-10DCs consist of two phenotypically distinct subpopulations: CD83highCCR7+ IL-10DCs and CD83lowCCR7− IL-10DCs. Suppressor assays with activated effector T cells revealed that CD4+ regulatory T cells generated by CD83high IL-10DCs (iTreg+) exhibited a significantly higher suppressive capacity compared with CD4+ regulatory T cells generated by CD83low IL-10DCs (iTreg−). In this context, iTreg+ displayed a more activated phenotype (proliferation, cytokine production) compared with iTreg−. In contrast to CD83low IL-10DCs, CD83high IL-10DCs exerted a strong migratory capacity toward the secondary lymphoid organ chemokine CCL21 and retained a functionally stable phenotype under inflammatory conditions. In addition, CD83high IL-10DCs expressed significantly higher levels of surface and soluble CD25. Functional analysis demonstrated that IL-10DC–related soluble CD25 efficiently inhibited the proliferation of activated T cells and that blockade of CD25 function abolished the induction of regulatory T cells by IL-10DCs, indicating a critical role for IL-10DC–related CD25 in shifting the immune response toward an iTreg− controlled tolerance reaction. In conclusion, the selective use of the CD83high IL-10DC subset may result in a higher efficacy of tolerance induction in vivo and may support the development of novel DC vaccination strategies for transplantations, as well as for allergic and autoimmune diseases.

The mechanisms of tolerance induction occurring in the course of allergen-specific immunotherapy have not been elucidated in full detail. Our study aimed to characterize high zone tolerance in mouse models of type I allergy and of allergic airway inflammation induced by subcutaneous sensitization of mice with high doses of the model allergen ovalbumin (OVA) without the use of adjuvant. Mice were immunized by subcutaneous injection of high doses (HD) of OVA or, for comparison, low doses (LD) of OVA in saline. HD-mice showed lower specific IgE, but augmented IgG in sera than LD-mice. Pre-treatment of mice with HD-OVA antigen-specifically inhibited IgE production subsequently induced by LD-OVA. OVA-restimulated splenocytes from HD-mice revealed hypoproliferation and impaired production of Th2-associated cytokines. HD-mice exhibited lower airway reactivity, goblet cell hyperplasia and mucus production, as well as IL-5 and IL-13 production in the lungs than LD-mice following local provocation. Recruitment of inflammatory cells into the airways was comparable, while the number of eosinophils in the bronchoalveolar lavage was substantially higher in HD-mice. Adoptive transfer of dnTC from HD-mice into naïve mice, which were subsequently sensitized with LD-OVA, suppressed IgE production in the recipients. The number of dnTC was higher in the spleens of HD-mice than LD-mice. In conclusion, our study demonstrates that subcutaneous sensitization of mice with high doses of allergen in the absence of adjuvant results in attenuated airway reactivity as compared with LD-sensitization and induces CD4(-)CD8(-) dnTC with regulatory function on IgE production.

Gene gun-mediated delivery of allergen-encoding plasmid DNA has been in focus for many years now as being a needle-free alternative to the protein-based desensitization regimen used in specific immunotherapy. Biolistic immunization with the Helios gene gun has proven to be potent in the induction of antigen-specific CD4(+) and CD8(+) T cells. Here we describe biolistic vaccination in experimental mouse models of IgE-mediated type I allergy as well as allergen-induced airway inflammation.

Gene gun-mediated delivery of allergen-encoding plasmid DNA has been in focus for many years now as being a needle-free alternative to the protein-based desensitization regimen used in specific immunotherapy. Biolistic immunization with the Helios gene gun has proven to be potent in the induction of antigen-specific CD4(+) and CD8(+) T cells. Here we describe biolistic vaccination in experimental mouse models of IgE-mediated type I allergy as well as allergen-induced airway inflammation.