Cellular stratification of atopic inflammatory circuits

先天免疫细胞在食物过敏中发挥重要作用，抗原呈递细胞（APC）启动初始T细胞反应，而肥大细胞（或嗜碱性细胞）是过敏反应的主要效应细胞。在第一个资助期（FP），我们开发了一种嗜碱性粒细胞激活试验（BAT），用于研究参与者的常规测试。我们确定CDSENS（激活50%嗜碱性粒细胞反应的抗原浓度）具有很强的诊断能力，可以通过BAT试验预测食物过敏。CDSENS与口服食物刺激阈值剂量有很强的相关性，表明脱敏过程中反应阈值的评估是相关的。通过我们的BAT，我们还评估了浆细胞样细胞和经典树突状细胞（pDC和cDC）中S6的磷酸化，这在耐受和过敏个体之间显示出显著差异。为了评估在嗜碱性粒细胞激活过程中磷酸化模式、激活和抑制标记的诱导，我们使用了大量细胞术。我们观察到CD300a、CD33和CD32b在耐受和过敏供者之间的诱导有显著差异，这表明它们可以作为食物过敏的新生物标志物。最后，我们评估了过敏原刺激后3天APC的激活情况，从而确定了过敏供体中几种APC亚群的特应性极化谱，特别是关于可能在脱敏中发挥作用的调节受体（CD103, PDL1, CTLA4）的表达变化。我们将在第二个计划生育的扩展研究中继续这些现已建立的分析，以完成不断增长的数据体。此外，我们想确定这些特征是否存在于其他2型疾病中。质细胞BAT测定将发展为常规的流式细胞测定。此外，我们将基于我们对食物过敏原引起的APC极化的见解，并将评估不同的介质、mirna或短链脂肪酸是否会在过敏原刺激期间改变APC表型（与B5和C2合作），以及这些不同的APC亚群如何在功能上影响T细胞启动。在这个检测系统中，我们将使用一种称为PADRE的泛hla - dr肽，它允许广泛激活初始和记忆CD4+ T细胞。总之，我们的数据将阐明先天免疫细胞在过敏诱导、维持和耐受中的作用。

Innate immune cells play an important role in food allergy with antigen-presenting cells (APC) priming the initial T cell response, while mast cells (or basophils) being major effector cells in allergic reactions. In the 1st Funding Period (FP), we developed a Basophil Activation Test (BAT) for routine testing of the study participants. We determined a strong diagnostic power of the CDSENS, the antigen concentration evoking activation of 50% basophil response, to predict food allergy from the BAT assay. CDSENS correlated strongly with the oral food challenge threshold dose, indicating that the assessment of the reaction threshold during desensitization is relevant. With our BAT, we also assess phosphorylation of S6 in plasmacytoid and classical dendritic cells (pDC and cDC), which displayed significant differences between tolerant and allergic individuals.To assess the induction of phosphorylation patterns, activation and inhibition markers during basophil activation, we utilized mass cytometry. We observed a significant different induction of CD300a, CD33 and CD32b between tolerant and allergic donors, suggesting these as candidates as novel biomarkers in food allergy. Finally, we assessed the activation of APC three days following allergen stimulation, thereby identifying atopic polarization profiles of several APC subsets in allergic donors, especially regarding the expression changes of regulatory receptors (CD103, PDL1, CTLA4) that may play a role in desensitization. We will continue these now well-established assays in the extension studies in the 2nd FP to complete the growing data body. Furthermore, we want to determine whether these signatures are present in other type 2 disorders. The mass cytometric BAT assay will be developed to a conventional flow cytometry assay. In addition, we will build upon our insights into APC polarization by food allergens and will assess whether different mediators, miRNAs or short chain fatty acids modify APC phenotypes during allergen stimulation (in collaboration with B5 and C2), and how these different APC subsets can functionally affect T cell priming. In this assay system, we will make use of a Pan-HLA-DR-peptide called PADRE, that allows for broad activation of naive as well as memory CD4+ T cells. Altogether, our data will clarify the role of innate immune cells in allergy induction, maintenance and tolerization.