Tfh And Breg Cells In Immunotherapy With Alternaria Alternata And Analysis Of The Endotypes In Alternaria-induced Asthma Patients

[Background]The prevalence of Alternaria-induced allergic asthma is increasing every year.Subcutaneous allergen-specific immunotherapy(SCIT)with a whole Alternaria crude extract is the only disease-modifying and long-lasting therapy for allergic asthma at this stage.However,little has been published on the efficacy of Alternaria immunotherapy around the world until now,and this extract exists non-standardization and poor safety.In the search for novel immunotherapy agents,Alt a 1,the major allergen produced by Alternata,and its recombinant protein has been widely used for molecular diagnosing in allergic diseases already because of its easy accessibility and stable quality,by contrast,the usage and efficacy of recombinant Alt a 1(rAlt a 1)in immunotherapy have not been reported so far.Currently,the immunologic mechanisms in immunotherapy are still unknown.T follicular helper cells(Tfh),a novel subset of T cells,are associated with immunoglobulin E(IgE)production in allergic diseases and immune response regulation in AIT.Regulatory B cell(Breg)and its interleukin 10(IL-10)act as suppressive regulatory mediators which control the immune tolerance of the body receiving immunotherapy.Whether Tfh and Breg cells are involved in and affected by the immunotherapeutic process of Alternaria and rAlt a 1 deserves to be explored.Recently,a new classification of asthma endotypes that differentiates patients with T2high and T2-low asthma emphasized the importance of T2 inflammation.Studies have identified a significant association between microbiota composition in bronchial and T2type asthma already,but there are no studies on the clinical features of patients with asthma sensitized by Alternaria in T2-high and T2-low endotypes.Therefore,this study will focus on evaluating the effect of immunotherapy with Alternaria and rAlt a 1,exploring the expression of Tfh and Breg cells in the process of immunotherapy,and analyzing the clinical characteristics of the endotypes in Alternariainduced asthma patients at the same time,with the aim of providing a foundation for the precise treatment of these patients.[Objective]1.To analyze and evaluate the efficacy of immunotherapy with Alternaria in allergic asthma patients.2.To record the changes of Tfh cell and its subsets,and Breg cells after accepting Alternaria immunotherapy.3.To clarify the correlation between asthmatic features and Tfh cell and its subsets,and Breg cells after SCIT.4.To explore the effective immunotherapeutic concentration of rAlt a 1 in an asthmatic mouse model,and to fully evaluate the efficacy of immunotherapy.5.To clarify the expression of Tfh and Breg cells after accepting immunotherapy.6.To investigate the variations of cytokine-related Tfh cells and IL-10+ Breg cells after accepting an effective concentration of rAlt a 1 for immunotherapy.7.To characterize the potential clinical characteristics of Alternaria sensitization asthma patients in T2-high and T2-low endotypes.[Methods]1.Evaluating the efficacy of immunotherapy with Alternaria in patients by delivering questionaries and detecting immunologic parameters,and lung function.2.Using multi-cytokines detection method to analyze the levels of cytokines in the serum.3.Characterizing the expression of cTfh cell and its subsets,and Breg cells by flow cytometry.4.Determining the correlation between asthmatic features and Tfh cell and its subsets,and Breg cells after SCIT by correlation analysis.5.Synthesize and purify rAlt a 1,then tests its purity and allergenic potency.6.Establishing asthmatic mice models and setting the different immunotherapeutic concentrations of rAlt a 1(5 μg,50 μg,100 μg,150 μg)for SCIT.7.Exploring the efficacy of rAlt a 1-SCIT by evaluating lung inflammation and airway resistance,the concentration of cytokines in BALF,and immunoglobulin and MCP1 levels in serum.8.Detecting the expressions of Tfh and Breg cells in different dosages of rAlt a 1-SCIT in the spleen by flow cytometry.9.Purify nAlt a 1,then establish asthmatic mice models that use nAlt a 1 and Alternaria as controls,the effective immunotherapeutic dosage of rAlt a 1 as an experiment control,and compare the efficacy of immunotherapy.10.Detecting the position and intensity of Bcl-6 in the spleen of mice by two-step immunochemistry.11.Clarifying the expressions of IL-4+ Tfh,IL-5+Tfh,IL-13+Tfh,and IL-17A+Tfh cells in the spleen and lymph gland,and IL-10+Breg cells in the spleen of mice again.12.Retrospective analysis of differences in age,sex,family history,inhaled corticosteroids,immunotherapy,history of pet exposure,and T-IgE in 582 Alternaria-induced asthma patients in both T2-high and T2-low endotypes.[Results]1.Patients’ symptoms and medication scores improved,and FEV1%values elevated after Alternaria-SCIT.2.The levels of IgE and eosinophils in the serum decreased without statistical significance after Alternaria-SCIT.3.A reduction of IL-4,IL-21,IL-5,IL-13,IL-17A levels,and an increase of IFN-γ were noted in the serum of the Alternaria-SCIT group,all have statistical significance.4.Alternaria-SCIT can lower the percentages of cTfh cells while increasing the levels of Breg cells in the blood of patients,cTfh2%had positive correlations with IL-5 and IL-13 in patients that accepted Alternaria-SCIT.5.The synthesized rAlt a 1 displayed a high purity and allergenic potency.6.An immunotherapy mouse model is established successfully,and following treatment with four different dosages of rAlt a 1,both lung and airway inflammations ameliorated,including lung pathology,airway resistance,inflammatory cells numbers,IL-4,IL-13 levels in BALF,and serum MCP-1 levels.7.Four different dosages of rAlt a 1-SCIT groups increased the expression of Alternaria-sIgG1,rAlt a 1-sIgG1,rAlt a 1-sIgG2a,and rAlt a 1-sIgG2b in serum.8.The number and percentage of Tfh cells were decreased in the rAlt a 1-SCIT groups.Meanwhile,the absolute numbers and proportions of Breg cells were evaluated after administration of different rAlt a 1 dosages.9.A positive correlation was observed between Tfh cells and inflammation grades,as well as a slightly strong positive relationship with IL-4 and IL-10 levels;Breg cells showed an opposite correlation with the grades of inflammation,along with negatively related to IL-4 and IL-10 levels.10.High purity and allergenic potency showed in synthesized nAlt a 1.11.The 5μg rAlt a 1-SCIT group was screened,the 5μg nAlt a 1-SCIT group and the 50μg Alternaria alternata-SCIT group acted as controls,the immunotherapeutic effects of Alt a 1 in the mouse model of asthma were verified again from lung tissue and airway inflammations,cytokine levels in BALF and immunoglobulin expressions,MCP-1 levels in the serum.12.The Tfh cells related transcription factor Bcl-6 located in the germinal center,which showed a weak expression in the model group,while attenuated after Alt a 1-SCIT.13.Alt a 1-SCIT decreased the levels of IL-4+Tfh,IL-5+Tfh,and IL-17A+Tfh cells in the spleen and lymph gland,and increased the percentages of IL-10+Breg cells in the spleen of mice.14.Lower age and higher number of male Alternaria-sensitized patients existed in the T2-high group,who pr-esented with higher levels of T-IgE and Alternaria sIgE.15.Patients in the T2-high group had better concordance of eosinophil values and T-IgE levels,and were also more prone to multiple sensitization,with a predominance of dust mite,birch and mugwort pollen in the sensitization profile.[Conclusion]1.Immunotherapy with Alternaria crude extracts in allergic asthma patients has immunotherapeutic effects,and declines the levels of cTfh2 cells,elevating the percentages of Breg cells at the same time.2.Immunotherapy with rAlt a 1 in asthmatic mice models has immunotherapeutic effects,which descends the proportions of Tfh,IL-4+Tfh,IL-5+Tfh,and IL-17A+Tfh cells while promoting the levels of IL-10+Breg cells.3.Both the Tfh and Breg cells are important for participating in the immunotherapy of Alternaria and Alt a 1.4.Alternaria-sensitized asthma patients show a great difference in age,the proportion of sex,IgE levels,and sensitization patterns.