Wojciech Dawicki

Regulatory Dendritic Cells for Immunotherapy in Immunologic Diseases

We recognize well the abilities of dendritic cells to activate effector T cell (Teff cell) responses to an array of antigens and think of these cells in this context as pre-eminent antigen-presenting cells, but dendritic cells are also critical to the induction of immunologic tolerance. Herein, we review our knowledge on the different kinds of tolerogenic or regulatory dendritic cells that are present or can be induced in experimental settings and humans, how they operate, and the diseases in which they are effective, from allergic to autoimmune diseases and transplant tolerance. The primary conclusions that arise from these cumulative studies clearly indicate that the agent(s) used to induce the tolerogenic phenotype and the status of the dendritic cell at the time of induction influence not only the phenotype of the dendritic cell, but also that of the regulatory T cell responses that they in turn mobilize. For example, while many, if not most, types of induced regulatory dendritic cells lead CD4+ naïve or Teff cells to adopt a CD25+Foxp3+ Treg phenotype, exposure of Langerhans cells or dermal dendritic cells to vitamin D leads in one case to the downstream induction of CD25+Foxp3+ regulatory T cell responses, while in the other to Foxp3− type 1 regulatory T cells (Tr1) responses. Similarly, exposure of human immature versus semi-mature dendritic cells to IL-10 leads to distinct regulatory T cell outcomes. Thus, it should be possible to shape our dendritic cell immunotherapy approaches for selective induction of different types of T cell tolerance or to simultaneously induce multiple types of regulatory T cell responses. This may prove to be an important option as we target diseases in different anatomic compartments or with divergent pathologies in the clinic. Finally, we provide an overview of the use and potential use of these cells clinically, highlighting their potential as tools in an array of settings.

Tolerogenic Dendritic Cells Induce CD4+CD25hiFoxp3+ Regulatory T Cell Differentiation from CD4+CD25−/loFoxp3− Effector T Cells

IL-10–differentiated dendritic cells (DC10) induce allergen tolerance in asthmatic mice, during which their lung Th2 effector T cells (Teffs) are displaced by activated CD4+CD25hiFoxp3+ T cells. Intestinal DCs promote oral tolerance by inducing Ag-naive T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs), but whether DCs can induce Teffs to differentiate into Tregs remains uncertain. In this study, we addressed this question in OVA-asthmatic mice that were treated with DC10. OVA-presenting DC10 treatment maximally activated lung Tregs in these animals at 3 wk posttreatment, as determined by upregulation of activation markers (ICOS, programmed cell death-1, glucocorticoid-induced TNFR-related protein, LAG3, and CTLA-4) and in functional assays. This in vitro regulatory activity was ≥90% reduced by treatment with anti–IL-10 but not anti–TGF-β Abs. In parallel cultures, OVA- but not house dust mite (HDM)-presenting DC10 induced ≈43% of CFSE-labeled CD25−/loFoxp3− Teffs from asthmatic OVA–TCR transgenic mice to differentiate into tolerogenic CD25hiFoxp3+ Tregs. We recapitulated this in vivo using OVA-asthmatic mice that were coinjected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4+CD25-/loFoxp3− Teffs (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teffs that were recovered from the lungs, lung-draining lymph nodes, or spleens of the OVA–DC10 recipients had differentiated into CD4+CD25hiFoxp3+ Tregs, whereas no CFSE-positive Tregs were recovered from the HDM–DC10-treated animals. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teffs to differentiate into CD4+CD25hiFoxp3+ Tregs.

Comparison of Induced versus Natural Regulatory T Cells of the Same TCR Specificity for Induction of Tolerance to an Environmental Antigen

Recent evidence shows that natural CD25+Foxp3+ regulatory T cells (nTreg) and induced CD25+Foxp3+ regulatory T cells (iTreg) both contribute to tolerance in mouse models of colitis and asthma, but there is little evidence regarding their relative contributions to this tolerance. We compared the abilities of nTreg and iTreg, both from OVA-TCR–transgenic OTII mice, to mediate tolerance in OVA-asthmatic C57BL/6 mice. The iTreg were differentiated from Th2 effector T cells by exposure to IL-10–differentiated dendritic cells (DC10) in vitro or in vivo, whereas we purified nTreg from allergen-naive mice and exposed them to DC10 before use. Each Treg population was subsequently repurified and tested for its therapeutic efficacy in vitro and in vivo. DC10 engaged the nTreg in a cognate fashion in Forster (or fluorescence) resonance energy transfer assays, and these nTreg reduced in vitro OVA-asthmatic Th2 effector T cell responses by 41–56%, whereas the comparator iTreg reduced these responses by 72–86%. Neutralization of IL-10, but not TGF-β, eliminated the suppressive activities of iTreg but not nTreg. Delivery of 5 × 105 purified nTreg reduced allergen challenge–induced airway IL-4 (p ≤ 0.03) and IL-5 (p ≤ 0.001) responses of asthmatic recipients by ≤23% but did not affect airway hyperresponsiveness or IgE levels, whereas equal numbers of iTreg of identical TCR specificity reduced all airway responses to allergen challenge by 82–96% (p ≤ 0.001) and fully normalized airway hyperresponsiveness. These data confirm that allergen-specific iTreg and nTreg have active roles in asthma tolerance and that iTreg are substantially more tolerogenic in this setting.

Therapeutic induction of tolerance by IL-10-differentiated dendritic cells in a mouse model of house dust mite-asthma.

To cite this article: Lu M, Dawicki W, Zhang X, Huang H, Nayyar A, Gordon JR. Therapeutic induction of tolerance by IL‐10‐differentiated dendritic cells in a mouse model of house dust mite‐asthma. Allergy 2011; 66: 612–620.

Induction of prolonged asthma tolerance by IL-10-differentiated dendritic cells: differential impact on airway hyperresponsiveness and the Th2 immunoinflammatory response.

IL-10–differentiated dendritic cells (DC10s) can prevent allergen sensitization and reverse the asthma phenotype in mice with established disease. However, little is known about the time-frames over which this tolerance is effective. We report that at 2 wk after i.p. or transtracheal delivery of 1 × 106 OVA-, but not house dust mite- presenting, DC10s to OVA-asthmatic mice, significant diminution of airway hyperresponsiveness (AHR) was first apparent, whereas AHR was abrogated between 3 and 10 wk posttreatment. At 13 wk, AHR returned to pretreatment levels but could again be reversed by DC10 retreatment. The impact of a single DC10 treatment on airway eosinophil and Th2 cytokine responses to recall OVA challenge, and on OVA-specific IgE/IgG1 responses, was substantial at 3 wk posttreatment, but progressively increased thereafter, such that at 8 mo, airway eosinophil and Th2 responses to recall allergen challenge remained ∼85–95% suppressed relative to saline-treated asthmatic mice. Four biweekly DC10 treatments, whether transtracheal or i.p., reduced all asthma parameters to near background by 8 wk, whereas s.c. DC10 treatments did not affect AHR but did reduce the airway Th2 responses (i.v. DC10 had no discernible effects). Repeated challenge of the DC10-treated mice with aerosolized OVA (100 μg/ml) did not reverse tolerance, but treatment with the indoleamine-2,3-dioxygenase antagonist 1-methyltryptophan or neutralizing anti–IL-10R from days 12 to 21 after DC10 therapy partially reversed tolerance (Th2 cytokine responses, but not AHR). These findings indicate that DC10-induced Th2 tolerance in asthmatic animals is long lived, but that DC10s employ distinct mechanisms to affect AHR versus Th2 immunoinflammatory parameters.

Behavioral alterations in rat offspring following maternal immune activation and ELR-CXC chemokine receptor antagonism during pregnancy: Implications for neurodevelopmental psychiatric disorders

Research suggests that maternal immune activation (MIA) during pregnancy increases the risk of neurodevelopmental disorders including schizophrenia and autism in the offspring. Current theories suggest that inflammatory mediators including cytokines and chemokines may underlie the increased risk of these disorders in humans. For example, elevated maternal interleukin-8 (IL-8) during pregnancy is associated with increased risk of schizophrenia in the offspring. Given this association, the present experiments examined ELR-CXC chemokines CXCL1 and CXCL2, rodent homologues of human IL-8, and activation of their receptors (CXCR1 and CXCR2) in an established rodent model of MIA. Pregnant Long Evans rats were treated with the viral mimetic polyinosinic-polycytidylic acid (polyI:C; 4 mg/kg, i.v.) on gestational day 15. Protein analysis using multiplex assays and ELISA showed that polyI:C significantly increased maternal serum concentrations of interleukin-1β, tumor necrosis factor, and CXCL1 3h after administration. Subsequent experiments tested the role of elevated maternal CXCL1 on behavior of the offspring by administering a CXCR1/CXCR2 antagonist (G31P; 500 μg/kg, i.p.; 1h before, 48 and 96 h after polyI:C treatment). The male offspring of dams treated with polyI:C demonstrated subtle impairments in prepulse inhibition (PPI), impaired associative and crossmodal recognition memory, and altered behavioral flexibility in an operant test battery. While G31P did not completely reverse the behavioral impairments caused by polyI:C, it enhanced PPI during adolescence and strategy set-shifting and reversal learning during young adulthood. These results suggest that while polyI:C treatment significantly increases maternal CXCL1, elevations of this chemokine are not solely responsible for the effects of polyI:C on the behavior of the offspring.

Therapeutic reversal of food allergen sensitivity by mature retinoic acid–differentiated dendritic cell induction of LAG3+CD49b−Foxp3− regulatory T cells

Background: Anaphylaxis is a life‐threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients. Objective: We assessed whether DC immunotherapy could reverse food allergen sensitivity in mouse models to provide proof of concept relating to their use in the clinic. Methods: We generated and characterized mature retinoic acid–skewed dendritic cells (DC‐RAs) and assessed their abilities to reverse ovalbumin or peanut allergies in mouse models, as well as their operative mechanisms. Results: DC‐RAs displayed a mature yet tolerogenic phenotype, expressing IL‐10, TGF‐&bgr;, IL‐27, and aldehyde dehydrogenase 1A2 but not IL‐12 or IL‐35; IL‐10 and TGF‐&bgr; together drove their suppression of TH2 cell proliferation. Delivery of specific allergen‐presenting DC‐RAs to half‐maximally sensitized mice with ovalbumin or peanut allergy reduced anaphylactic responses to oral allergen challenge by 84% to 90%, as well as diarrhea, mast cell activation, and TH2 cytokine responses and serum allergen‐specific IgE/IgG1 levels. DC‐RA expression of IL‐27 was important to their induction of CD25+ lymphocyte activation gene 3 (LAG3)+, CD49b−, forkhead box P3 (Foxp3)− regulatory T cells in vitro, such that &bgr; subunit of IL‐27 (Ebi)−/− (ie, IL‐27–incompetent) DC‐RAs were ineffective in inducing food allergen tolerance. Conclusion: Our data indicate that regulatory DC immunotherapy can be effective for food allergies and suggest that induction of Foxp3− regulatory T cells might be a useful strategy for tolerance induction in this context.

Regulatory dendritic cell expression of MHCII and IL-10 are jointly requisite for induction of tolerance in a murine model of OVA-asthma

Allergen‐presenting dendritic cells differentiated with IL‐10 (DC10) reverse the asthma phenotype in mice by converting their Th2 cells to regulatory T cells (Tregs). DC10 express elevated levels of IL‐10, but substantially reduced levels of MHCII and costimulatory molecules, so the relationships between these factors with each other and tolerogenicity have not been clearly elucidated.

Rapamycin reduces fibroblast proliferation without causing quiescence and induces STAT5A/B-mediated cytokine production.

Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin’s potential clinical use against MDR cancers.