Molecular mechanisms of allorecognition in a basal chordate

当免疫细胞与靶细胞相互作用，受体与其同源配体结合时， 做出反应或不反应的决定。我们的长期目标是理解这一决定的基础。 细胞如何监视细胞表面的事件？什么被计算，以及倍数是如何计算的 刺激性和抑制性信号整合？这与门槛值相比如何？ 在发展和教育过程中，细胞如何知道门槛是什么？和 一旦设定了这些门槛，就可以操纵了吗？这些问题中的每个都有重大的生物医学 意义，从理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。 我们有能力研究在一次免疫相互作用中这些过程是如何工作的。我们的 模式是组织相容的基础脊索白僵菌。白僵菌中的同种异体识别 由单个基因座(称为FUHC)控制，具有以下规则：共享一个或多个 两个等位基因都是相容的；而那些没有共同等位基因的等位基因是不相容的。歧视是基于 在检测一种自体等位基因时，这个系统的特异性是显著的：大约有1000个 Fuhc等位基因遍布世界各地，因此效应器系统可以从大量竞争中挑选出一个自体等位基因。 具体细节。然而，Botryllus没有任何重组或体细胞超突变 这种特异性依赖于生殖线编码的受体。 这项提议的重点是了解这种先天的生化机制。 同种异体识别特异性。Botryllus异种识别的独特性质使其成为理想的模型 在这些研究中：决定结果的单一基因，反应发生在体外 在大血管末端的上皮细胞之间，称为壶腹，和结果 是由只有两个受体的信号通路整合决定的，一个受体激活，一个激活 一种是抑制作用，两种都可以在体内进行操作。在目标1中，我们将使用一种新的荧光 本实验室新近发展起来的分离单个壶腹细胞并直接评估 专一性的基础。我们的工作假设是，这是由于特定于基因的替代。 一种名为Fester的受体的拼接，这将在这里直接进行测试。使用这种技术，我们 还发现壶腹是双功能的，可以可逆地去分化为血管 不表达同种异体识别蛋白的细胞，使我们能够表征可逆变化 在候选蛋白表达/选择性剪接方面，这将揭示特异性的基础。在AIM 2、我们将评估细胞外配体/受体在体内和体外的相互作用。我们将测试推定的 受体与FUHC编码的伴侣蛋白和支架蛋白的细胞内相互作用 这可能在产生受体复合体方面发挥作用，并有助于特异性。在《目标3》中，我们将 描述在Botryllus异种识别中使用的信号转导通路，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 ZAP-70、LCK、Sph-1/2和SHIP在壶腹中有表达。这些基因很早就有 进化起源，使我们假设同种识别的保守方面是 整合这些具有良好特性的激活和抑制信号转导的机制 小路。然而，与目标1的集成也将使我们能够公正地看待假设 信号转导基因。 完成拟议的目标将促进我们对基础机制的理解 先天同种异体识别系统中的教育和宽容。这些细胞内过程监控 细胞表面的结合事件，整合激活和抑制输入，并设置和维持 回答的门槛：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 免疫的进化变化特征-和Botryllus呈现出独特和高度的 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 肿瘤是对人类健康非常重要的领域。

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.