Induction Of T Cell Anergy By High Dose Allergen In Allergic Mice And Underlying Mechanism

Asthma is a chronic inflammatory disorder of the airway caused by many inflammatory cells. Th1/Th2 paradigm is known as the main pathogenesis of asthma. Excess activation of Th2 cells evokes Th2 immune response, and it causes elevated Th2 cytokines. Th2 cytokines such as IL-4, IL-5 and IL-13 play a pivotal role on the onset and maintenance of airway allergic inflammation in asthma. More recently, it has been found that Th1/Th2 paradigm can not explain the immune dysfunction of asthma completely. Because of immune tolerance to harmless allergen, harmless allergens do not lead to excess activation of T cells in healthy person. But in asthmatic patients, harmless allergens induce excess activation and proliferation of T cells, and make T cells differentiate into Th2 cells. Therefore, defect of immune tolerance may be an important mechanism for asthma.Specific immunotherapy (SIT) has been the only therapy method for asthma base on pathology, but underlying mechanism is still not clearly understood. T cell tolerance can be induced by several mechanisms: deletion of antigen-responsive T cells, anergy (T cells that are intrinsically functionally inactivated but remain alive), or the differentiation of regulatory T cells. Tregs suppress Th1 and Th2 responses via cell–cell interactions and/or the production of suppressor cytokines. IL-10 and TGF-βsuppress IgE production and induce non-inflammatory Ig isotypes IgG4 and IgA, respectively. Furthermore, these two cytokines directly suppress allergic inflammation induced by effector cells such as mast cells, basophils and eosinophils.DCs are centrally involved in all mechanisms of peripheral tolerance. The differentiation state of DCs, the specific pathway of antigen uptake, signals from stromal cells, and the antigen dose are critical determinants for the outcome of tolerance. DCs that express low levels of MHC II and costimulatory molecules also induce T cell and B cell unresponsiveness to inhaled, harmless antigens by yet unknown mechanisms. DCs that originated from the plasmacytoid bone marrow precursor appear to be critical to down-regulate immune responses to harmless, inhaled antigens.Several recent studies indicate that Notch signaling can suppress immune responses by generating cells either capable of or required for regulation. They studied forced Notch activation through overexpression of either Notch ligand on the antigen presenting cell (APC) orNIC in the T cell. Cells capable of regulation can be induced by ligation with either Jagged or Dl family members or by transgenic expression of Notch3IC, and include both CD4+ and CD8+ cells responding to a variety of antigens.Early study showed that T cell anergy could be induced by high dose allergen in allergic mice. But the underlying mechanism is not clearly. In this study, we investigated the induction of T cell anergy by high dose allergen in allergic mice and the effects of high dose OVA on costimulatory molecules expression and cytokines secretion of DC, induction of Tregs, expression of Notch signaing, expression of T-bet and GATA3 in vitro. And we also observed the effect of DC transfected with Jagged1 on induction of Tregs.Methods:1. Spleen DC and T cells were separated from allergic mice and normal mice. The levels of IL-10 and TGF-β1 and Th1/Th2 cytokines produced by T cells treated with OVA at different concentrations in vitro were measured by ELISA and the correlations between IL-10 and TGF-β1 with Th1/Th2 cytokines were analyzed.2. The costimulate molecules on spleen DC were detected by FACS in allergic mice and normal mice and the effect of OVA at 10mg/ml was observed.3. CD4+CD25+Foxp3+ T cells were detected by FACS in allergic mice and normal mice and the effect of OVA at 10mg/ml was observed.4. The mRNA and protein levels of T-bet and GATA3 were detected by RT-PCR and Western blotting in allergic mice and normal mice. And the effect of OVA at 10mg/ml was observed.5. The expression of Notch ligands and receptors were measured in allergic mice and normal mice by RT-PCR and Western blotting. And the effects of OVA at 10mg/ml on that were observed.6. The mouse bone marrow cells were isolated and cultured in complete medium with recombinant murine GM-CSF, then the purity and phenotype of DC was analyzed by flow cytometry (FCM). 7. The vectors expressing Jagged1 were lipotransfected into DC and the transgene expression at both mRNA and protein level in transfected DC by RT-PCR and Western blotting.8. The splenic CD4+ T cells of allergic mice were isolated and cocultured with control DC, Jagged1-DC respectively, CD4+CD25+Foxp3+ T cells were detected by FACS.Results:1. Th1/Th2 cytokines produced by T cells were significantly inhibited by OVA at 10mg/ml in allergic mice. No significant changes were observed in normal mice.2. The levels of IL-10 and TGF-β1 were increased by dose-dependent means. No significant correlation was observed between IL-10 and TGF-β1 with Th1/Th2 cytokines.3. The levels of MHCII, CD80 and CD86 were higher in allergic mice than normal mice. And the levels of CD80 and CD86 decreased when treated with OVA at 10mg/ml.4. The amount of CD4+CD25+Foxp3+ T cells separated from spleen was significantly decreased in allergic mice and it would increase when treated with OVA at 10mg/ml.5. The mRNA and protein levels of T-bet were lower in allergic mice than normal mice. And the mRNA and protein levels of GATA3 were higher in allergic mice than normal mice.6. The mRNA and protein levels of T-bet and GATA3 were significantly increased in T cells when treated by OVA at 10mg/ml in allergic mice.7. The mRNA levels of Jagged1, Jagged2 and Delta1 in DC and the mRNA and protein levels of Notch1 and Notch3 in T cells were significantly decreased in allergic mice. And the mRNA and protein levels of Jagged1 and Notch3 were significantly increased in spleen DC and T cells treated by OVA at 10mg/ml in allergic mice.8. The protein levels of Jagged1 and Notch3 in DC and T cells were significantly decreased in allergic mice. After co-cultured with OVA at 10mg/ml, the protein levels of Jagged1 and Notch3 significantly increased.9. The transgene expression at both mRNA and protein level in Jagged1-DC could be detected by RT-PCR and Western blotting, It showed that Jagged1 gene could be expressed in transfected-DC.10. After co-cultured with Jagged1-DC in vitro, the amount of CD4+CD25+Foxp3+ T cells in allergic mice was significangtly increased. Conclusion:1. T cell anergy can be induced by high dose allergen in allergic mice in vitro. And the underlying mechanism related to the decrease of costimulatory molecules in DC and the increase of amount of CD4+CD25+Foxp3+ T cells.2. Expression of Notch ligands and receptors decreased significantly in DC and T cells in allergic mice. Expression of Jagged1 and Notch3 increased with high dose allergen. Jagged1-DC could increase the mount of CD4+CD25+Foxp3+ T cells. The results suggest that defect of Notch signaling could play an important role in deficit polarization of Tregs.Our results demonstrated that immunotolerance of sensitized T cells could be induced by high dose allergen and provided more theory evidence for immunotherapy with allergen vaccine.