Composition and structure of antibody receptors at the surface of primary human cells during immune activation

项目摘要/摘要 Fc g受体(FcgRs)在与Fc G受体相互作用后触发细胞介导的细胞毒机制 免疫球蛋白G分子聚集在目标分子的表面，保护宿主。广泛的帖子- 这种相互作用需要FcgRs和Ig G上的翻译修饰(PTM)，包括 天冬酰胺连接的糖基化(N-糖链)。免疫球蛋白N-糖链对压力和疾病的反应不同 N-葡聚糖的组成变化可以改变FcgRIIIa(CD16A)的亲和力，其亲和力可高达8倍于 体外培养。与免疫球蛋白相比，FcgRs含有2-7个N-糖链和一些其他已知的糖链，其修饰程度更高 修饰，Barb实验室证明CD16A N-糖链组成对亲和力的影响高达 100倍。因此，FcgR N-葡聚糖对亲和力的影响大于Ig G N-葡聚糖。然而，几乎没有什么是 已知人体内的FcgR成分，成分如何随压力变化，或 成分如何影响效应细胞本身的结构和功能(不包括初步描述 CD16a和CD16b)。本提案中概述的实验的成功完成将提供 关于FcgRs修改的前所未有的详细信息以及这些修改如何影响 免疫系统。这些结果还将强调从内源性组织中确定PTM的重要性 用于功能研究，并提供克服限制研究的常见障碍的技术 内源性受体。 在目标1.1中，我们将定义FcgR PTM的组成，包括：来自巨噬细胞的CD16A，CD32a 来自中性粒细胞、单核细胞、巨噬细胞、血小板和嗜碱性粒细胞；CD32b来自B细胞和嗜碱性粒细胞；以及 CD64来源于单核细胞和巨噬细胞。在Aim 1.2中，我们将分离出慢性粒细胞白血病患者的NK细胞和单核细胞。 镰状细胞性贫血、重症肌无力和视神经脊髓炎，以测定这些细胞的细胞毒性和 定义CD16A和CD64的组成。在目标2中，我们将描述免疫的结构 突触和特定受体和辅助受体的位置，细胞杀伤的动力学，以及 患者NK细胞和单核细胞突触形成和受体运动的动力学。这些 结果将与在目标1中完成的细胞上的PTMS的特征进行比较。目标2还将研究 特定的FcgR PTM如何通过培养细胞影响免疫细胞功能。来自两个来源的多个数据源 将使用单一捐赠者的样本收集AIMS，以确定捐赠者群体的可变性。通过 通过比较收集到的大量数据，我们期望确定PTM组成的具体特征 影响免疫细胞功能，代表新的治疗靶点。

Project Summary/Abstract Fc g receptors (FcgRs) trigger cell-mediated cytotoxic mechanisms following interactions with immunoglobulin G molecules clustered on the surface of a target molecule, protecting the host. Extensive post- translational modifications (PTMs) on both the FcgRs and IgG are required for this interaction, including asparagine-linked glycosylation (N-glycans). IgG N-glycans vary in response to stress and disease and compositional variability of the N-glycan can modify the affinity for FcgRIIIa (CD16a) by as much as 8-fold in vitro. FcgRs are more heavily modified than IgG with 2-7 N-glycans as well as a handful of other known modifications, and the Barb lab demonstrated that CD16a N-glycan composition impacts affinity by as much as 100-fold. Thus, FcgR N-glycans impact affinity more than the IgG N-glycans. However, almost nothing is known regarding FcgR composition within the human body, how composition changes in response to stress, or how composition impacts the structure and function of the effector cell itself (excluding preliminary descriptions of CD16a and CD16b). The successful completion of the experiments outlined in this proposal will provide unprecedented detail regarding the modification of FcgRs and how these modifications impact function of the immune system. These results will also emphasize the importance of defining PTMs from endogenous tissue for functional studies as well as provide techniques to overcome common barriers that limit studies of endogenous receptors. In Aim 1.1, we will define the composition of FcgR PTMs, including: CD16a from macrophages, CD32a from neutrophils, monocytes, macrophages, platelets and basophils; CD32b from B cells and basophils; and CD64 from monocytes and macrophages. In Aim 1.2, we will isolate NK cells and monocytes from patients with sickle cell anemia, myasthenia gravis and neuromyelitis optica to measure the cytotoxicity of these cells and define the composition of CD16a and CD64. In Aim 2, we will characterize the structure of the immune synapse and the locations of specific receptors and co-receptors, the kinetics of cell killing, as well as the kinetics of synapse formation and receptor motion using NK cells and monocytes from the patients. These results will be compared to the characterization of PTMs on the cells completed in Aim 1. Aim 2 will also study how specific FcgR PTMs impact immune cell function using cultured cells. Multiple sources of data from both Aims will be collected using samples from a single donor to identify variability in the donor population. By comparing the large amount of data collected, we expect to identify specific features of PTM composition that impact immune cell function and that represent novel therapeutic targets.