High-dimensional profiling of B cells in food allergy

 描述(由申请者提供)：食物过敏影响了1500万美国人，并具有威胁生命的过敏反应的高风险。因此，确定其成因是长期风险管理的关键。过敏原特异性IgE抗体的产生是这些疾病的关键媒介。然而，作为IgE来源的B细胞群体以及如何控制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。这个项目的基础源于普氏-米尔斯和康明斯的初步观察，即一种与半乳糖-α-1，3-半乳糖(α)特异性Ig E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出现迟发性过敏反应。α-半乳糖是非灵长类哺乳动物的一种血型碳水化合物，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以前已经对肉类耐受多年，这表明对阿尔法-半乳糖的敏化发生在生活的后期。 与产生α-半乳糖特异性IgE有关。这些IgE抗体的主要原因是扁虱的叮咬，扁虱在胃肠道中含有α-半乳糖。因此，扁虱体内的α-半乳糖解释了扁虱暴露和对α-半乳糖致敏之间的关系，红肉过敏的发生是次要事件。在前期工作中，我们评估了体外B细胞对扁虱提取物的反应，发现过敏受试者的PBMC中记忆B细胞和浆细胞的频率增加，但不是健康对照组。我们进一步观察到，表达先前与记忆表型相关的标志物的类别切换B细胞的激活和增殖程度更高。基于这些观察，我们假设红肉过敏患者的阿尔法-半乳糖IgE来自记忆B细胞亚群，该亚群对扁虱叮咬和食用红肉后的阿尔法-半乳糖暴露有反应。然而，使用标准的细胞分析方法，如流式细胞术，解决包括记忆B细胞在内的外周B细胞的异质性一直受到限制。我们已经应用飞行时间(CyTOF)质量细胞术，它在单个样本中支持40个标记，用于对健康捐赠者的外周B细胞进行高维免疫图谱分析，并识别记忆B细胞中的不同亚群，这在个体之间是一致的。这表明有可能检测B细胞群中与过敏相关的变化，并使用细胞TOF确定人类外周血样本中IgE的细胞来源。在这项建议中，我们将在这些新发现的基础上，确定红肉过敏受试者的B细胞亚群，确定B细胞群与临床数据之间的关系，并确定过敏原暴露后对阿尔法-半乳糖特异性IgE反应的要求。总而言之，我们希望这些研究能为了解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，并更好地定义告知全局和过敏原特异性B细胞反应的免疫信号。

 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.