Defining the immunoglobulin (Ig) switch to IgE in glycan-specific B cell responses using the model disease of anaphylaxis to galactose-alpha-1,3-galactose

到目前为止，对碳水化合物特异性免疫反应的了解还不够充分。在本提案的范围内，我们使用模型抗原半乳糖- α -1,3-半乳糖（“α -gal”）研究碳水化合物特异性B细胞反应的免疫学机制。α -是普遍表达的，除了旧世界的猴子和人类，所有哺乳动物都表达这种碳水化合物。因此，α -gal被后者识别为非自我，并且已知在蜱虫叮咬后，α -gal特异性IgE抗体可被诱导，在接触表达α -gal的药物或红肉时可能引发危及生命的过敏反应。有趣的是，大量的α -半乳糖特异性IgM和IgG存在于所有人类中，独立于这种类型的α -半乳糖致敏，被认为有助于防止寄生虫感染，属于来自B-1细胞的“天然”抗体池。为了进行更详细的研究，我们建立了一个小鼠模型，其中α -半乳糖特异性IgE抗体和α -半乳糖经皮致敏后暴露后的过敏反应。然而，使用该模型，我们可以显示α -半乳糖特异性B-2细胞和生发中心B细胞的扩增，其中IgG1占主导地位，但缺乏IgE表达，这首次表明碳水化合物特异性IgE反应是通过涉及IgG1中间阶段的顺序类转换产生的。根据这些观察结果，我们将对α -半乳糖特异性B细胞进行深入分析，并通过将其转移到B细胞缺陷小鼠中来破译特定B细胞群的作用。在小鼠模型以及α -半乳糖过敏患者和对照组中，B细胞受体库分析和不同同型的离体类转换方法将用于阐明通过IgG1中间阶段产生α -半乳糖IgE。在提案的第二部分，我们将首次研究2型免疫在碳水化合物抗原（在我们的案例中是α -gal）诱导的IgE类转换中的作用。为此，我们将使用IL-4报告小鼠使用单细胞RNA测序来破译2型免疫级联。滤泡和2型辅助性T细胞在α -半乳糖特异性IgE反应和过敏反应中的作用将通过T细胞亚群过继转移到T细胞缺陷小鼠中进行研究。总之，该项目将帮助我们破译到目前为止尚未充分理解的高亲和力碳水化合物特异性体液免疫反应的免疫学机制

Up to now, carbohydrate-specific immune responses are insufficiently understood. In the scope of this proposal, we investigate the immunological mechanisms of carbohydrate-specific B cell responses using the model antigen galactose-alpha-1,3-galactose ("alpha-gal"). Alpha-is ubiquitously expressed and all mammals except of old-world monkeys and humans express this carbohydrate. Consequently, alpha-gal is recognized as non-self by the latter and it is known that after tick bites, alpha-gal specific IgE antibodies can be induced potentially eliciting life-threatening allergic reactions upon contact with alpha-gal expressing drugs or red meat. Interestingly, a substantial amount of alpha-gal specific IgM and IgG is present in all humans independent of this type of alpha-gal sensitization and is believed to contribute to the protection e.g. against parasite infections and to belong to the pool of ‘natural’ antibodies derived from B-1 cells. For more detailed investigations, we established a mouse model in which alpha-gal specific IgE antibodies and anaphylaxis after alpha-gal exposure develop upon percutaneous sensitization to alpha-gal. Using this model, however, we could show an expansion of alpha-gal specific B-2 cells as well as germinal center B cells with dominant IgG1 but absent IgE expression, indicating for the first time that carbohydrate-specific IgE responses arise through sequential class switching involving an IgG1 intermediate stage. Following up on these observations, we will perform in-depth analysis of alpha-gal specific B cells and, among others, decipher the role of specific B cell populations using transfer into B cell deficient mice. B cell receptor repertoire analyses and ex vivo class switching approaches of different isotypes in the mouse model as well as in alpha-gal allergic patients and controls will be used to elucidate the generation of alpha-gal IgE via an IgG1 intermediate stage. In the second part of the proposal, we will for the first time investigate the role of type 2 immunity in carbohydrate antigen, in our case alpha-gal, induced IgE class switching. To this end, we will use IL-4 reporter mice to decipher the type 2 immune cascade using single-cell RNA sequencing. The role of follicular and type 2 helper T cells in alpha-gal specific IgE responses and anaphylaxis will be investigated using adoptive transfer of T cell subsets into T cell deficient mice. In summary, this project will help us to decipher the so far insufficiently understood immunological mechanisms underlying high affinity carbohydrate-specific humoral immune responses