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

项目概要/摘要 Fc g受体（FcgR）在与细胞因子相互作用后触发细胞介导的细胞毒性机制。 免疫球蛋白G分子聚集在靶分子的表面上，保护宿主。广泛的后- 这种相互作用需要FcgR和IgG上的翻译修饰（PTM），包括 天冬酰胺连接的糖基化（N-聚糖）。IgG N-聚糖响应于压力和疾病而变化， N-聚糖的组成可变性可以将对FcgRIIIa（CD 16 a）的亲和力改变多达8倍， 体外FcgR比IgG更重地修饰，具有2-7个N-聚糖以及少数其他已知的N-聚糖。 Barb实验室证明，CD 16 a N-聚糖组成对亲和力的影响高达 一百倍因此，FcgR N-聚糖比IgG N-聚糖更影响亲和力。然而，几乎没有什么是 已知关于人体内FcgR组成、组成如何响应于应激而变化，或 组合物如何影响效应细胞本身的结构和功能（不包括初步描述 CD 16 a和CD 16 b）。成功完成本提案中概述的实验将提供 关于FcgR的修饰以及这些修饰如何影响细胞的功能的前所未有的细节 免疫系统这些结果也将强调从内源性组织中定义PTM的重要性。 功能研究，并提供技术，以克服共同的障碍，限制研究 内源性受体 在目的1.1中，我们将定义FcgR PTM的组成，包括：来自巨噬细胞的CD 16 a，来自巨噬细胞的CD 32a。 来自嗜中性粒细胞、单核细胞、巨噬细胞、血小板和嗜碱性粒细胞; 来自单核细胞和巨噬细胞的CD 64。在目标1.2中，我们将从患有以下疾病的患者中分离NK细胞和单核细胞： 镰状细胞性贫血、重症肌无力和视神经肌无力，以测量这些细胞的细胞毒性， 定义了CD 16 a和CD 64的组成。在目标2中，我们将描述免疫系统的结构 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 使用来自患者的NK细胞和单核细胞的突触形成和受体运动的动力学。这些 将结果与目标1中完成的电池上PTM的表征进行比较。Aim 2还将研究 使用培养的细胞，特异性FcgR PTM如何影响免疫细胞功能。来自两个数据源的多个数据源 将使用来自单个供体的样本收集目的，以确定供体群体的变异性。通过 通过对收集到的大量数据进行比较，我们希望确定PTM成分的具体特征， 影响免疫细胞功能并代表新治疗靶点。

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.