Jagged1-expressing adenovirus-infected dendritic cells induce expansion of Foxp3+ regulatory T cells and alleviate T helper type 2-mediated allergic asthma in mice

Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in directing T-cell responses. Regulatory T (Treg) cells possess an immunosuppressive ability to inhibit effector T-cell responses, and Notch ligand Jagged1 (Jag1) is implicated in Treg cell differentiation. In this study, we evaluated whether bone marrow-derived DCs genetically engineered to express Jag1 (Jag1-DCs) would affect the maturation and function of DCs in vitro and further investigated the immunoregulatory ability of Jag1-DCs to manipulate T helper type 2 (Th2) -mediated allergic asthma in mice. We produced Jag1-DCs by adenoviral transduction. Overexpression of Jag1 by ovalbumin (OVA) -stimulated Jag1-DCs exhibited increased expression of programmed cell death ligand 1 (PD-L1) and OX40L molecules. Subsequently, co-culture of these OVA-pulsed Jag1-DCs with allogeneic or syngeneic CD4⁺ T cells promoted the generation of Foxp3⁺ Treg cells, and blocking PD-L1 using specific antibodies partially reduced Treg cell expansion. Furthermore, adoptive transfer of OVA-pulsed Jag1-DCs to mice with OVA-induced asthma reduced allergen-specific immunoglobulin E production, airway hyperresponsiveness, airway inflammation, and secretion of Th2-type cytokines (interleukin-4, interleukin-5, and interleukin-13). Notably, an increased number of Foxp3⁺ Treg cells associated with enhanced levels of transforming growth factor-β production was observed in Jag1-DC-treated mice. These data indicate that transgenic expression of Jag1 by DCs promotes induction of Foxp3⁺ Treg cells, which ameliorated Th2-mediated allergic asthma in mice. Our study supports an attractive strategy to artificially generate immunoregulatory DCs and provides a novel approach for manipulating Th2 cell-driven deleterious immune diseases.     

Expansion of Foxp3+ T‐cell populations by Candida albicans enhances both Th17‐cell responses and fungal dissemination after intravenous challenge

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3⁺ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3⁺ Treg‐cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4⁺Foxp3⁺ population that correlated positively with fungal burden. Depletion from Foxp3^hCD2 reporter mice in vivo confirmed that Foxp3⁺ cells exacerbated fungal burden and inflammatory renal disease. The CD4⁺Foxp3⁺ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg‐cell subset, together with conversion of Foxp3^? cells to the induced Treg‐cell form, and to a cell type sharing effector Th17‐cell characteristics, expressing ROR‐γt, and secreting IL‐17A. The expanded Foxp3⁺ T cells inhibited Th1 and Th2 responses, but enhanced Th17‐cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17‐cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3⁺ T cells promotes Th17‐cell responses that drive pathology.     

Infiltration of IL-17-Producing T Cells and Treg Cells in a Mouse Model of Smoke-Induced Emphysema

Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease associated with the accumulation of activated T cells. To date, there is little information concerning the intrinsic association among Th17, Tc17, and regulatory T (Treg) cells in COPD. The objective of this study was to investigate the variation of lungs CD4⁺Foxp3⁺ Treg cells and IL-17-producing CD4 and CD8 (Th17 and Tc17) lymphocytes in mice with cigarette-induced emphysema. Groups of mice were exposed to cigarette smoke or room air. At weeks 12 and 24, mice were sacrificed to observe histological changes by HE stain. The frequencies of Th17 (CD4⁺IL-17⁺T), Tc17 (CD8⁺IL-17⁺T), and Treg (CD4⁺Foxp3⁺T) cells in lungs from these mice were analyzed by flow cytometry. The mRNA levels of orphan nuclear receptor ROR γt and Foxp3 were performed by real-time quantitative polymerase chain reaction. The protein levels of interleukin-17 (IL-17), IL-6, IL-10, and transforming growth factor-beta (TGF-β1) were measured by enzyme-linked immunosorbent assay. Cigarette smoke caused substantial enlargement of the air spaces accompanied by the destruction of the normal alveolar architecture and led to emphysema. The frequencies of Th17 and Tc17 cells, as well as the expressions of IL-6, IL-17, TGF-β1, and ROR γt were greater in the lungs of cigarette smoke (CS)-exposed mice, particularly in the 24-week CS-exposed mice. The frequencies of Treg cells and the expressions of IL-10 and Foxp3 were lower in CS-exposed mice compared to control group. More important, the frequencies of Tregs were negatively correlated with Th17 cells and with Tc17 cells. Interestingly, a significant portion of the cells that infiltrate the lungs was skewed towards a Tc17 phenotype. Our findings suggest the contribution of Th17, Tc17, and Treg cells in the pathogenesis of COPD. Rebalance of these cells will be helpful for developing and refining the new immunological therapies for COPD.     

Asthma Prevention by Lactobacillus Rhamnosus in a Mouse Model is Associated With CD4+CD25+Foxp3+ T Cells

Purpose

Probiotic bacteria can induce immune regulation or immune tolerance in allergic diseases. The underlying mechanisms have been recently investigated, but are still unclear. The aim of this study was to evaluate the protective effects of the probiotic Lactobacillus rhamnosus (Lcr35) in a mouse model of asthma and to identify its mechanism of action.

Methods

Lcr35 was administered daily by the oral route at a dosage of 1×10⁹ CFU/mouse in BALB/c mice for 7 days before the first sensitization. Clinical parameters and regulatory T (Treg) cells were examined. The role of CD4⁺CD25⁺Foxp3⁺ Treg cells was analyzed using a Treg cell-depleting anti-CD25 monoclonal antibody (mAb).

Results

Airway hyperresponsiveness, total IgE production, pulmonary eosinophilic inflammation, and splenic lymphocyte proliferation were suppressed after Lcr35 treatment. Th1 (IFN-γ) and Th2 (IL-4, IL-5, and IL-13) cytokines in the serum were suppressed, and the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the spleen was significantly increased in the Lcr35 treatment group. Anti-CD25 mAb administration abolished the protective effects of Lcr35, indicating that CD4⁺ CD25⁺Foxp3⁺ Treg cells are essential in mediating the activity of Lcr35.

Conclusions

Oral administration of Lcr35 attenuated the features of allergic asthma in a mouse model and induced immune regulation by a CD4⁺CD25⁺Foxp3⁺ Treg cell-mediated mechanism.     

Transient attenuated Foxp3 expression on CD4⁺ T cells treated with 7D4 mAb contributes to the control of parasite burden in DBA / 2 mice infected with lethal Plasmodium chabaudi chabaudi AS

CD4⁺ CD25⁺ regulatory T (Treg) cells expressing Foxp3⁺ play a critical role in maintaining immune homoeostasis and controlling excessive immune responses. However, controversy about the immunoregulatory role of Treg cells exists in malaria studies. Given the role of maintenance of Foxp3 expression in Treg cells’ activities, we investigated whether anti‐CD25 mAb (7D4 clone) treatment affects Foxp3 expression in CD4⁺ T cells in DBA/2 mice infected with Plasmodium chabaudi chabaudi AS (P. c. chabaudi AS). We found that DBA/2 mice succumbed to P. c. chabaudi AS infection, which was accompanied by increased expression of Foxp3 in CD4⁺ T cells at the peak parasitemia. In contrast, Foxp3 expression was impaired in CD25‐depleted mice with 7D4 mAb treatment, leading to delayed parasitemia and extended survival of infected mice. Production of IFN‐γ, TNF‐α and IL‐6, as well as NO was significantly enhanced in CD25‐depleted mice. The majority of CD4⁺ CTLA‐4⁺ cells expressed high levels of Foxp3 (Foxp3^hi cells) in control mice with P. c. chabaudi AS infection. However, the number of CD4⁺ Foxp3^hiCTLA‐4⁺ cells was reduced in CD25‐depleted mice. Together, these data suggest that CD4⁺ Foxp3^hiCTLA‐4⁺ cells may be involved in regulating the intensity of pro‐inflammatory responses via CTLA‐4.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

Regulatory T cells but not T helper 17 cells are modulated in an animal model of Graves’ hyperthyroidism

Graves’ disease (GD) involves auto-immunity against thyroid cell antigens, but the reasons for the induction of auto-immunity are uncertain. We wished to investigate the role of T helper 17 (Th17) and regulatory T cells (Treg) in a mouse model of Graves’ hyperthyroidism. The model was generated by immunizing mice with adenovirus expressing the autoantigen thyroid-stimulating hormone receptor (Ad-TSHR289). The frequencies of splenic Th17 and Treg were determined by flow cytometry. The levels of interleukin-17(IL-17), forkhead box P3 (Foxp3), and orphan retinoic acid nuclear receptor (RORγt) mRNA were determined by real-time PCR. The number of CD4⁺CD25⁺Foxp3⁺ T lymphocyte was significantly reduced in the Ad-TSHR289 group compared with the Ad-control (P < 0.05). mRNA level for Foxp3 was less abundant in Ad-TSHR289 group compared with Ad-control (P < 0.05). However, CD4⁺IL-17⁺ T-cell subpopulation and expression of RORγt mRNA did not differ significantly between Ad-TSHR289 and Ad-control groups (P > 0.05). Nevertheless, in Ad-TSHR289 group, a profound increase in the Th17/Treg ratios was found. The present study demonstrates that Th17 is not involved in promoting Graves’ hyperthyroidism, while Treg and the ratio of Th17/Treg might play a role in the pathogenesis of Graves’ hyperthyroidism.     

Glucocorticoid hormone differentially modulates the in vitro expansion and cytokine profile of thymic and splenic Treg cells

Objective

Functional disturbances in regulatory T cells (Treg) have been described in autoimmune diseases, and their potential therapeutic use is intensively studied. Our goal was to investigate the influence of glucocorticoid hormone on the in vitro differentiation of Treg cells from thymic and splenic CD4⁺ T cells under different conditions to establish methods for generating stable and functionally suppressive iTregs for future use in adoptive transfer experiments.

Induction of immune tolerance in asthmatic mice by vaccination with DNA encoding an allergen-cytotoxic T lymphocyte-associated antigen 4 combination

Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen–cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA_3.1) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed and transfected into COS-7 cells to obtain the fusion protein OVA-CTLA-4, which was able to bind the B7 ligand on dendritic cells (DCs), and induced CD25⁺ Foxp3⁺ regulatory T (Treg) cells by the coculture of naive CD4⁺ T cells with DCs in vitro. In an animal study, BALB/c mice were sensitized and challenged with OVA to establish the asthmatic model. Vaccination with a high dose of OVA-CTLA-4-pcDNA_3.1 significantly decreased interleukin-4 (IL-4) and IL-5 levels and eosinophil counts and prevented OVA-induced reduction of the gamma interferon level in the bronchoalveolar lavage fluid. In addition, these mice suffered less severe airway inflammation and had lower levels of OVA-specific IgE and IgG1 titers in serum. Also, high-dose OVA-CTLA-4-pcDNA_3.1 vaccination inhibited the development of airway hyperreactivity and prevented OVA-induced reduction of the percentages of Foxp3⁺ Treg cells in the spleen. Our results indicate that a high dose of allergen-CTLA-4-encoding DNA vaccine was more effective in preventing an allergen-induced Th2-skewed immune response through the induction of Treg cells and may be a new alternative therapy for asthma.     

Loss of regulatory characteristics in CD4+CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4⁺CD25⁺ T cells compared to controls, whereas the CD4⁺CD25⁺ and CD4⁺CD25^hi T cells were enriched with Th1‐ and Th17‐like cells but not Foxp3‐expressing Treg cells and evidenced by significantly higher T‐bet, interferon (IFN)‐γ, and interleukin (IL)‐17 expression but lower Foxp3 and transforming growth factor (TGF)‐β expression. Regarding the CD4⁺CD25^hi T‐cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF‐β and an enrichment in T‐bet, RORγt, IFN‐γ, and IL‐17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14⁺ monocytes and CD19⁺ B cells, the levels of IFN‐γ and IL‐17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF‐β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF‐β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3⁺ Treg cells associated with impaired TGF‐β secretion.     

Schistosoma mansoni Larvae Do Not Expand or Activate Foxp3+ Regulatory T Cells during Their Migratory Phase

Foxp3⁺ regulatory T (Treg) cells play a key role in suppression of immune responses during parasitic helminth infection, both by controlling damaging immunopathology and by inhibiting protective immunity. During the patent phase of Schistosoma mansoni infection, Foxp3⁺ Treg cells are activated and suppress egg-elicited Th2 responses, but little is known of their induction and role during the early prepatent larval stage of infection. We quantified Foxp3⁺ Treg cell responses during the first 3 weeks of murine S. mansoni infection in C57BL/6 mice, a time when larval parasites migrate from the skin and transit the lungs en route to the hepatic and mesenteric vasculature. In contrast to other helminth infections, S. mansoni did not elicit a Foxp3⁺ Treg cell response during this early phase of infection. We found that the numbers and proportions of Foxp3⁺ Treg cells remained unchanged in the lungs, draining lymph nodes, and spleens of infected mice. There was no increase in the activation status of Foxp3⁺ Treg cells upon infection as assessed by their expression of CD25, Foxp3, and Helios. Furthermore, infection failed to induce Foxp3⁺ Treg cells to produce the suppressive cytokine interleukin 10 (IL-10). Instead, only CD4⁺ Foxp3⁻ IL-4⁺ Th2 cells showed increased IL-10 production upon infection. These data indicate that Foxp3⁺ Treg cells do not play a prominent role in regulating immunity to S. mansoni larvae and that the character of the initial immune response invoked by S. mansoni parasites contrasts with the responses to other parasitic helminth infections that promote rapid Foxp3⁺ Treg cell responses.     

The transcriptional repressor Bcl6 controls the stability of regulatory T cells by intrinsic and extrinsic pathways

Foxp3⁺ regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6^−/−) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6^−/− mice, increased numbers of Foxp3-negative ‘exTreg’ cells were found, particularly in the CD25⁺ population. ExTreg cells from Bcl6^−/− mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6^−/− mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6^Foxp3−/−) mice were analysed. Bcl6^Foxp3−/− mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6^−/− mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6^Foxp3−/− mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6⁺ Treg cells. Bcl6^Foxp3−/− mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Transient Treg‐cell depletion in adult mice results in persistent self‐reactive CD4+ T‐cell responses

Depletion of Foxp3⁺CD4⁺ regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg‐cell depletion on self‐reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4⁺ T‐cell responses to endogenous self‐antigens. Short‐term ablation of Treg cells in mice resulted in rapid activation of CD4⁺ T cells, increased percentage of IFN‐γ⁺ and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self‐reactive responses, we analyzed the activation of naïve gastric‐specific CD4⁺ T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric‐specific T cells in the stomach draining LNs of Treg‐cell‐depleted mice, compared with untreated mice, either during Treg‐cell depletion or after Treg‐cell reconstitution. Moreover, the hyperproliferation of gastric‐specific T cells in the Treg‐cell‐ablated mice was predominantly antigen‐dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg‐cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg‐cell depletion results in ongoing antigen‐driven self‐reactive T‐cell responses and emphasize the continual requirement for an intact Treg‐cell population.     

Tregs are dysfunctional in vivo in a spontaneous murine model of Crohn's disease

Although regulatory T cells (Tregs) have been implicated in inflammatory bowel disease, Tregs from Crohn’s disease (CD) patients are increased in number and function normally in vitro. To clarify this disparity, we studied Treg function in vivo using a spontaneous model of CD-like ileitis. We first administered anti-CD25-depleting antibodies to SAMP1/YitFc (SAMP) mice to assess ileitis; mesenteric lymph node cells were then transferred into SCID (severe combined immunodeficient) recipients to induce colitis. CD25 depletion increased the severity of both spontaneous ileitis and adoptively transferred colitis. Interestingly, a second transfer of CD4⁺CD25⁺ cells from untreated AKR control mice was able to ameliorate the induced colitis, whereas CD4⁺CD25⁺ cells from untreated SAMP mice were not, suggesting a functional abnormality in SAMP Tregs. Anti-CD25 treatment in SAMP mice also induced proliferation of CD25⁻Foxp3⁺ Tregs, which had a proinflammatory intestinal T helper type 1/ T helper type 2 (Th1/Th2) effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis.