Verena Raker

Tolerogenic Dendritic Cells for Regulatory T Cell Induction in Man

Dendritic cells (DCs) are highly specialized professional antigen-presenting cells that regulate immune responses, maintaining the balance between tolerance and immunity. Mechanisms via which they can promote central and peripheral tolerance include clonal deletion, the inhibition of memory T cell responses, T cell anergy, and induction of regulatory T cells (Tregs). These properties have led to the analysis of human tolerogenic DCs as a therapeutic strategy for the induction or re-establishment of tolerance. In recent years, numerous protocols for the generation of human tolerogenic DCs have been developed and their tolerogenic mechanisms, including induction of Tregs, 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 DCs. Therefore, the scientific rationale for the use of tolerogenic DCs 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 DCs with focus on IL-10-modulated DCs as inducers of Tregs and discuss their clinical applications and challenges faced in further developing this form of immunotherapy.

Interferon-α suppresses cAMP to disarm human regulatory T cells

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(+)CD25(high)Foxp(+) 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.

Nutritional Wheat Amylase-Trypsin Inhibitors Promote Intestinal Inflammation via Activation of Myeloid Cells

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.

The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases

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.

IL-10–Modulated Human Dendritic Cells for Clinical Use: Identification of a Stable and Migratory Subset with Improved Tolerogenic Activity

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.

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4(-)CD8(-) double-negative T cells.

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization.

Myeloid cell populations and fibrogenic parameters in bleomycin- and HOCl-induced 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.

Non‐eosinophilic Airway Hyper‐reactivity in Mice, Induced by IFN‐γ Producing CD4+ and CD8+ Lung T cells, is Responsive to Steroid Treatment

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.

Tolerance through Education: How Tolerogenic Dendritic Cells Shape Immunity

Dendritic cells (DCs) are central players in the initiation and control of responses, regulating the balance between tolerance and immunity. Tolerogenic DCs are essential in the maintenance of central and peripheral tolerance by induction of clonal T cell deletion and T cell anergy, inhibition of memory and effector T cell responses, and generation and activation of regulatory T cells. Therefore, tolerogenic DCs are promising candidates for specific cellular therapy of allergic and autoimmune diseases and for treatment of transplant rejection. Studies performed in rodents have demonstrated the efficacy and feasibility of tolerogenic DCs for tolerance induction in various inflammatory diseases. In the last years, numerous protocols for the generation of human monocyte-derived tolerogenic DCs have been established and some first phase I trials have been conducted in patients suffering from autoimmune disorders, demonstrating the safety and efficiency of this cell-based immunotherapy. This review gives an overview about methods and protocols for the generation of human tolerogenic DCs and their mechanisms of tolerance induction with the focus on interleukin-10-modulated DCs. In addition, we will discuss the prerequisites for optimal clinical grade tolerogenic DC subsets and results of clinical trials with tolerogenic DCs in autoimmune diseases.

Regulation of IgE production and airway reactivity by CD4−CD8− regulatory T cells

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.