Chemokine Receptor Chimeras by Synthetic Gene Library

描述（由申请人提供）：趋化因子对指导免疫细胞的运输和组织至关重要。我们和其他人已经证明T细胞在淋巴器官内以高速迁移。在某些条件下，当迁移的T细胞遇到外来抗原时，它们会与抗原呈递细胞（APC）形成长期的静态偶联物，而在其他情况下，T细胞检测到外来抗原，但仍会继续迁移。了解这些不同表型的决定因素及其免疫学后果对于理解体内免疫反应的基本原理至关重要。我们之前已经证明，一些趋化因子传递显性信号，指导T细胞继续迁移过去携带抗原的apc，而其他趋化因子传递次要信号，允许长期缀合形成。2个趋化因子是SLC （CCL21，显性）和SDF-1a （CXCL12，从属），它们分别被T细胞上的受体CCR7和CXCR4识别。在这里，我们建议设计嵌合受体，其具有显性受体的配体特异性1和从属受体的细胞内信号反应。由于趋化因子受体是7-跨膜g蛋白偶联受体，其胞外、跨膜和胞内结构紧密偶联。为了发现具有配体结合功能和适当的细胞内信号传导特性的嵌合体，我们提出建立一个受体嵌合体组合文库，其中所有细胞外环属于一个受体，所有细胞内环属于另一个受体，跨膜结构域在两个受体之间变化。在目标1中，我们将通过基因合成方法设计和生产跨膜洗牌CCR7/CXCR4和CCR7/CCR5文库。在目标2和目标3中，我们将筛选该库以寻找所需的趋化显性/从属行为。在Aim 4中，我们将使用这些数据推导趋化因子受体跨膜信号传导的一般原理，并通过合理设计CXCR3和CCR5之间的新型嵌合体来测试这些原理。通过阐明免疫“停止”和“继续”信号的层次结构，我们将揭示控制免疫功能的基本原理，这将有助于理解肿瘤、病毒和其他微生物如何有时逃避免疫清除，以及免疫如何在自身免疫性疾病中变得失调。

DESCRIPTION (provided by applicant): Chemokines are critical for directing the traffic and organization of immune cells. We and others have shown that T cells migrate within lymphoid organs at high speeds. Under certain conditions, when migrating T cells encounter foreign antigen they form long-lived static conjugates with the antigen-presenting cell (APC), while at other times T cells detect foreign antigen but continue to migrate nonetheless. Understanding the determinants of these distinct phenotypes and their immunological consequences is critical for understanding the basic principles of the in vivo immune response. We have previously shown that some chemokines deliver dominant signals that directed T cells to continue migrating past antigen-bearing APCs, while other chemokines deliver subordinant signals that permit long-term conjugate formation. 2 such chemokines are SLC (CCL21, dominant) and SDF-1a (CXCL12, subordinate), recognized by their respective receptors on T cells, CCR7 and CXCR4. Here we propose to design chimeric receptors bearing the ligand-specificity 1 of a dominant receptor and the intracellular signaling response of a subordinate receptor. Because chemokine receptors are 7-transmembrane G-protein coupled receptors, their extracellular, transmembrane and intracellular structures are tightly coupled. In order to discover chimeras with functional ligand binding and appropriate intracellular signaling properties, we propose to generate a combinatorial library of receptor chimeras where all extracellular loops belong to 1 receptor, all intracellular loops belong to the other, and the transmembrane domains are varied between the 2. In Aim 1 we will design and produce the transmembrane-shuffled CCR7/CXCR4 and CCR7/CCR5 libraries by a gene-synthesis approach. In Aims 2 & 3 we will screen this library for desired chemotactic dominant/subordinate behavior. In Aim 4 we will use this data to derive general principles for chemokine receptor transmembrane signaling and test these principles by rational design of a novel chimera between CXCR3 and CCR5. By elucidating the hierarchies of immunological "Stop" and "Go" signals we will reveal basic principles governing immune function that will be relevant to understand how tumors, viruses, and other microbes can sometimes evade immune clearance, and how immunity becomes dysregulated in autoimmune diseases.