Epitope validation of fungal allergens in severe asthma

项目总结 在这项应用中，我们将表征对真菌变应原曲霉菌表位起反应的CD4T细胞 烟曲霉菌(ASP)和交链孢霉(ALT)，以了解重症哮喘的分子基础。 致敏和/或暴露于来自真菌物种的抗原(孢子)，如天冬氨酸转氨酶和丙氨酸氨基转移酶 与一些与哮喘严重程度相关的不良后果的风险密切相关。在此基础上 前提下，我们将检验以下假设：(I)真菌过敏原特异性的CD4T细胞更具致病性 与吸入非真菌的变应原特异性CD4T细胞相比，质量上的不同 过敏原，如屋尘螨(HDM)、蟑螂(CR)、老鼠(MO)或潜在的口服过敏原，如 牛奶(CM)。(Ii)重症哮喘患者真菌变应原表位特异性CD4T细胞 病原学特征明显优于轻度哮喘患者。我们将利用在三个大的 这些研究的哮喘队列。 在目标1中，我们将定义曲霉属(Asp)和交链孢属(Alternaria)致敏哮喘患者所识别的表位 根据疾病的严重程度进行分类。预测的天冬氨酸转氨酶和丙氨酸氨基转移酶表位的反应性将在20年内测试 有轻度和重度哮喘的致敏受试者和20名无哮喘的非致敏受试者进行评估 过敏反应与健康反应的本质。通过比较三组人群的表位反应性，我们发现 将定义每个亚组共有的或特定的表位：重度哮喘、轻度哮喘和非哮喘 致敏的非哮喘患者。在目标2中，我们将确定真菌(天冬氨酸转氨酶和丙氨酸氨基转移酶)特异性的分子图谱 不同程度哮喘患者外周血中CD4T细胞的变化我们将利用真菌表位巨型池来定义 天冬氨酸转氨酶和丙氨酸氨基转移酶表位特异性CD4T细胞的表型和转录水平(散体和单细胞水平) 10-20例真菌致敏的重度和轻中度哮喘过敏性受试者的反应 支气管肺曲霉菌病(ABPA)和无哮喘的对照组。同时，我们还将评估 哮喘患者呼吸道真菌特异性CD4T细胞的分子图谱。要确定是否 真菌过敏原特异性CD4T细胞的分子特征与过敏原特异性CD4T细胞不同 针对接种疫苗(百日咳和破伤风)诱导的HDM、CR、MO、CM或抗原特异性Th2细胞，我们 将对这些细胞进行额外的转录分析，并通过综合数据管理 专注于生物信息学的CORE将数据集与项目1和项目2进行交叉比较。 在不同哮喘严重程度的受试者中进行的表位验证研究将提供对CD4T细胞本质的洞察 推动严重哮喘发病的细胞反应。

PROJECT SUMMARY In this application, we will characterize CD4 T cells reactive to epitopes of the fungal allergens Aspergillus fumigatus (ASP) and Alternaria alternata (ALT) to understand the molecular basis of severe asthma. Sensitization and/or exposure to antigens (spores) from fungal species such as ASP and ALT have been strongly associated with risk for a number of asthma-severity-related adverse outcomes. Based on this premise, we will test the hypotheses that (i) Fungal allergen-specific CD4 T cells are more pathogenic and qualitatively different when compared to allergen-specific CD4 T cells directed towards non-fungal inhaled allergens such as house dust mite (HDM), cockroach (CR), mouse (MO) or potential oral allergens such as cow milk (CM). (ii) Fungal allergen-epitope-specific CD4 T cells in patients with severe asthma have more pathogenic features than those from mild asthmatics. We will capitalize on subjects enrolled in three large asthma cohorts for these studies. In Aim 1, we will define epitopes recognized by Aspergillus (ASP) and Alternaria (ALT)-sensitized asthmatics categorized based on disease severity. The reactivity of predicted ASP and ALT epitopes will be tested in 20 sensitized subjects with mild and severe asthma and in 20 non-sensitized subjects without asthma to assess the nature of allergic versus healthy responses. By comparing the epitope-reactivity of the three cohorts, we will define epitopes that are common or specific to each subgroup: severe asthma, mild asthma and non- sensitized non-asthmatics. In Aim 2, we will determine the molecular profile of fungal (ASP and ALT)-specific CD4 T cells in patients with different asthma severity. We will utilize fungal epitope mega-pools to define the phenotype and transcriptional profile (at bulk and single-cell level) of ASP and ALT epitope-specific CD4 T cell responses in 10-20 fungal-sensitized subjects with severe and mild-to-moderate asthma, allergic bronchopulmonary aspergillosis (ABPA) and in control subjects without asthma. In parallel, we will also assess the molecular profile of fungal-specific CD4 T cells present in the airways of asthmatic subjects. To determine if the molecular features of fungal allergen-specific CD4 T cells are different from allergen-specific CD4 T cells directed to HDM, CR, MO, CM or antigen-specific Th2 cells induced by vaccination (pertussis and tetanus), we will perform additional transcriptomic analysis of these cells, and through the integrated Data Management Core specializing in bioinformatics cross compare data sets with Project 1 and 2. Together these fungal epitope validation studies in subjects with varying asthma severity will provide insights into the nature of CD4 T cell responses that drive pathogenesis of severe asthma.