Molecular mechanisms of allorecognition in a basal chordate

基底脊索动物同种异体识别的分子机制

• 批准号: 9433671
• 负责人: Anthony W De Tomaso
• 金额: $ 29.06万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9433671
• 关键词: Alleles; Alternative Splicing; Area; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biochemical; Biological Assay; Blood Vessels; Bone Marrow Transplantation; Cell surface; Cells; Characteristics; Chordata; Clinical; Complex; Detection; Development; Discrimination; Dissection; Education; Epigenetic Process; Epithelial Cells; Event; Funding; Future; Genes; Genetic Recombination; Genotype; Goals; Health; Histocompatibility; Human; Immune; Immune system; Immunity; Immunoglobulin Somatic Hypermutation; Immunoprecipitation; Immunosuppressive Agents; In Vitro; Individual; Intervention; Label; Lead; Ligands; Link; Maintenance; Memory; Modeling; Molecular; Molecular Chaperones; Monitor; Monoclonal Antibodies; Natural Killer Cells; Natural regeneration; Organism; Outcome; Pathway interactions; Phenotype; Play; Population; Process; Property; Proteins; Proteome; Proteomics; RNA Splicing; Reaction; Reagent; Resolution; Role; Scaffolding Protein; Sea; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Stimulus; Study models; System; Techniques; Testing; Time; Transduction Gene; Transplantation; Transplanted tissue; Variant; Work; ZAP-70 Gene; ascidian; base; crosslink; extracellular; in vivo; in vivo evaluation; insight; mRNA sequencing; novel; novel strategies; prevent; protein expression; receptor; receptor binding; response; transdifferentiation; tumor

When an immune cell interacts with a target cell, and receptors bind their cognate ligands, a decision is made to react or not. Our long-range goal is to understand the basis of that decision. How does a cell monitor events at the cell surface? What gets counted, and how are multiple stimulatory and inhibitory signals integrated? How is that compared to a threshold value, and during development and education processes, how does a cell know what the threshold is? And once set, can these thresholds be manipulated? Each of these questions has major biomedical significance, from understanding the changes in reactivity that lead to autoimmunity, to inducing tolerance to a transplanted tissue. We have the ability to study how these processes work during a single immune interaction. Our model is histocompatibility in the basal chordate Botryllus schlosseri. Allorecognition in Botryllus is controlled by a single locus (called the fuhc) with the following rules: individuals that share one or both alleles are compatible; while those that share none are incompatible. Discrimination is based on the detection of a self-allele, and the specificity of this system is significant: there are ca. 1000 fuhc alleles world-wide, thus the effector system can pick out a self-allele from a sea of competing specificities. However, Botryllus does not have any recombination or somatic hypermutation machinery, and this specificity relies on germline-encoded receptors. This proposal is focused on understanding the biochemical mechanisms that underlie this innate allorecognition specificity. The unique properties of Botryllus allorecognition make it an ideal model for these studies: a single locus that determines outcome, the reaction occurs outside the body between epithelial cells on the tips of macroscopic blood vessel, called ampullae, and the outcome is determined by the integration of signaling pathways from only two receptors, one activating, and one inhibitory, both of which can be manipulated in vivo. In Aim 1, we will use a novel fluorescent labeling technique recently developed in our lab to isolate single ampullae cells and directly assess the basis of specificity. Our working hypothesis is that this is due to genotype-specific alternative splicing of a receptor called fester, and that will be directly tested here. Using this technique, we have also found that ampullae are bifunctional and can reversibly de-differentiate into vascular cells, which do not express allorecognition proteins, allowing us to characterize reversible changes in candidate protein expression/alternative splicing, which will reveal the basis of specificity. In Aim 2, we will assess extracellular ligand/receptor interactions in vivo and in vitro. We will test putative intracellular interactions between receptors and fuhc-encoded chaperones and scaffolding proteins that may play a role in creating receptor complexes and contribute to specificity. In Aim 3, we will characterize the signal transduction pathways used in Botryllus allorecognition, using a combination of FACS and proteomics. We have found that signal transduction molecules such as Zap-70, LCK, sph-1/2 and SHIP are expressed in ampullae. These genes have an early evolutionary origin, leading us to hypothesize that the conserved aspects of allorecognition are the mechanisms that integrate these well-characterized activating and inhibitory signal transduction pathways. However, integration with Aim 1 will also allow us take an unbiased view of putative signal transduction genes. Completion of the proposed aims will advance our understanding of the mechanisms that underlie education and tolerance in innate allorecognition systems. These intracellular processes monitor binding events at the cell surface, integrate activating and inhibitory inputs, and set and maintain the threshold for a response: the basis of specificity. We hypothesize that these are the conserved aspects of immunity, have an early evolutionary origin, and are responsible for the rapid evolutionary change characteristic of immunity- and Botryllus presents a unique and highly simplified model to study these processes. Understanding and manipulating threshold responses would be the building blocks of future clinical interventions, including inducing tolerance following transplantation, blocking autoimmune reactions, or overcoming the immunosuppressive strategies of tumors, areas of great importance for human health.

当免疫细胞与靶细胞相互作用，并且受体结合其同源配体时， 决定是否作出反应。我们的长期目标是了解这一决定的基础。 细胞如何监测细胞表面的事件？什么被计算，以及如何多个 刺激和抑制信号的整合？这与阈值相比如何， 在发育和教育过程中，细胞如何知道阈值是多少？和 一旦设定，这些阈值是否可以操纵？每一个问题都有重要的生物医学 重要性，从了解导致自身免疫的反应性变化，到诱导 对移植组织的耐受性。 我们有能力研究这些过程如何在单一的免疫相互作用中发挥作用。我们 模型在基底脊索动物Schlosseri中具有组织相容性。葡萄球菌的异源识别 受单一基因座（称为fuhc）控制，具有以下规则：共享一个或多个基因座的个体 两个等位基因都是相容的;而那些没有共享的等位基因是不相容的。歧视是基于 在检测自身等位基因上，该系统的特异性是显著的：存在ca. 1000 fuhc等位基因在世界范围内，因此效应系统可以从竞争的海洋中挑选出一个自我等位基因。 特殊性然而，Botryllus没有任何重组或体细胞超突变 这种特异性依赖于种系编码的受体。 这项建议的重点是了解这种先天性的生化机制， 同种识别特异性葡萄孢菌独特的识别特性使其成为一种理想的识别模型 对于这些研究：一个决定结果的单一位点，反应发生在体外， 在肉眼可见的血管尖端（称为壶腹）的上皮细胞之间， 是由来自两个受体的信号通路的整合决定的，一个是激活的， 一种是抑制性的，两种都可以在体内操纵。在目标1中，我们将使用一种新的荧光 本实验室最近开发了一种标记技术，用于分离单个壶腹细胞并直接评估 特异性的基础。我们的工作假设是，这是由于基因型特异性的替代 一种叫做“溃烂”的受体的剪接，这将在这里直接进行测试。利用这项技术，我们 还发现壶腹是双功能的，可以可逆地去分化为血管， 细胞，不表达同种异体识别蛋白，使我们能够表征可逆的变化 在候选蛋白表达/选择性剪接，这将揭示特异性的基础。在Aim中 2，我们将在体内和体外评估细胞外配体/受体相互作用。我们将测试假定的 受体与fuhc编码的分子伴侣和支架蛋白之间的细胞内相互作用 这可能在产生受体复合物中起作用并有助于特异性。在目标3中，我们 特征的信号转导通路中使用的葡萄球菌同种异体识别，使用 流式细胞仪和蛋白质组学的结合。我们已经发现，信号转导分子，如 壶腹部表达Zap-70、LCK、sph-1/2和SHIP。这些基因有一个 进化起源，导致我们假设，同种异体识别的保守方面是 整合这些充分表征的激活和抑制信号转导的机制 途径。然而，与目标1的整合也将使我们能够对假定的 信号转导基因 完成拟议的目标将促进我们对作为其基础的机制的理解 先天同种异体识别系统中的教育和耐受性。这些细胞内过程监测 在细胞表面的结合事件，整合激活和抑制输入，并设置和维持 反应的阈值：特异性的基础。我们假设这些是保守的 免疫方面，有一个早期的进化起源，并负责快速 进化的变化特点的免疫-和葡萄球菌提出了一个独特的和高度 简化模型来研究这些过程。理解和操纵阈值反应 将是未来临床干预的基石，包括诱导耐受性， 移植，阻断自身免疫反应，或克服免疫抑制策略 肿瘤，对人类健康非常重要的领域。