High-dimensional profiling of B cells in food allergy

 食物过敏影响1500万美国人，并具有危及生命的过敏反应的高风险。因此，确定其原因对长期风险管理至关重要。过敏原特异性IgE抗体的产生是这些疾病的关键介质。然而，作为IgE来源的B细胞群以及如何控制IgE的产生仍不清楚。该项目的总体目标是以食物过敏为模型，确定产生过敏原特异性IgE的人类B细胞的异质性。该项目的基础源于Platts-Mills和Commins的初步观察，即一种与半乳糖-α-1，3-半乳糖（α-半乳糖）特异性IgE抗体相关的新型食物过敏 gal）导致成人和儿童患者在食用红肉后的延迟过敏反应。α-半乳糖是非灵长类哺乳动物的血型碳水化合物，因此存在于携带这种碳水化合物的动物的肉中。值得注意的是，这些患者中的大多数人以前对肉类有多年的耐受性，这表明对α-半乳糖的敏感性发生在生活的后期， 产生α-半乳糖特异性IgE。这些IgE抗体的主要原因是蜱虫叮咬，胃肠道中含有α-gal。因此，蜱内的α-半乳糖解释了蜱暴露与α-半乳糖致敏之间的关系，红肉过敏的发展是次要事件。 在初步工作中，我们评估了体外B细胞对蜱提取物的反应，发现过敏受试者PBMC中记忆B细胞和浆细胞的频率增加，但健康对照组没有。我们进一步观察到表达以前与记忆表型相关的标记物的类别转换B细胞的更大活化和增殖。基于这些观察结果，我们假设红肉过敏患者的α-gal IgE来自记忆B细胞亚群，该亚群通过蜱叮咬和食用红肉后对α-gal暴露做出反应。然而，使用标准细胞分析方法如流式细胞术来解析外周B细胞（包括记忆B细胞）的异质性受到限制。我们已经应用了飞行时间质谱（CyTOF），其支持单个样品中的40种标志物，用于来自健康供体的外周B细胞的高维免疫谱分析，并鉴定了记忆B细胞内的异质亚群，其在个体间是一致的。这表明，它可能检测过敏相关的变化，B细胞群体和识别IgE的细胞来源，在人外周血样本中使用CyTOF。在本提案中，我们将建立在这些新的发现，以确定B细胞室在红肉过敏受试者，并确定B细胞群体和临床数据之间的关系，并确定过敏原暴露后的α-半乳糖特异性IgE反应的要求。总的来说，我们希望这些研究能够为了解IgE介导的食物过敏中不同B细胞亚群的免疫状态提供新的信息，并更好地定义免疫特征，从而为全球和过敏原特异性B细胞反应提供信息。

 DESCRIPTION (provided by applicant): Food allergies affect 15 million Americans and carry a high risk of life-threatening allergic reactions. Therefore, establishing its cause is critical to ong-term risk management. The production of allergen-specific IgE antibodies is a key mediator of these disorders. However, the B cell populations that are the source of IgE and how IgE production is controlled remain unclear. The overall goal of this project is to define the heterogeneity of human B cells that produce allergen-specific IgE using food allergy as a model. The basis for this project stems from initial observations by Platts-Mills and Commins that a novel form of food allergy related to IgE antibodies specific for galactose-α-1,3-galactose (alpha gal) results in delayed anaphylaxis in adult and pediatric patients after consumption of red meat. Alpha-gal is a blood group carbohydrate of nonprimate mammals and therefore is present in meat of animals that carry this carbohydrate. Remarkably, most of these patients had previously tolerated meat for years, suggesting that sensitization to alpha-gal occurred later in life leading to the production of alpha-gal specific IgE. The primary cause of these IgE antibodies is bites from ticks, which contain alpha-gal in the gastrointestinal tract. Thus, alpha-gal within ticks explains the relationship between tick exposure and sensitization to alpha-gal, with development of red meat allergy as a secondary event. In preliminary work, we assessed in vitro B cell responses to tick extract and found increased frequencies of memory B cells and plasma cells from PBMCs of allergic subjects but not healthy controls. We further observed greater activation and proliferation of class-switched B cells expressing markers previously associated with a memory phenotype. Based on these observations, we hypothesize that the alpha-gal IgE in patients with red meat allergy emerges from a memory B cell subset that responds to alpha-gal exposure via tick bites and after eating red meat. However, resolving the heterogeneity of peripheral B cells, including memory B cells, has been limited using standard cell analysis methods such as flow cytometry. We have applied mass cytometry by time-of-flight (CyTOF), which supports 40 markers in a single sample, for high- dimensional immune profiling of peripheral B cells from healthy donors and identified heterogeneous subsets within memory B cells that was consistent across individuals. This suggests that it may be possible to detect allergy-related changes in B cell populations and identify cellular sources of IgE in human peripheral blood samples using CyTOF. In this proposal we will build on these new findings to define the B cell compartment in red meat allergy subjects and determine the relationships between B cell populations and clinical data, and to determine the requirements for an alpha-gal specific IgE response following allergen exposure. Collectively, we expect these studies to yield new information for understanding the immune status of distinct B cell subsets in IgE-mediated food allergy, and better define immune signatures that inform global and allergen-specific B cell responses.