Chemokine Receptor Chimeras by Synthetic Gene Library

描述（由申请人提供）：趋化因子对于指导免疫细胞的运输和组织至关重要。我们和其他人已经表明，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，它们与抗原呈递细胞（APC）形成长寿命的静态缀合物，而在其他时间，T细胞检测到外来抗原，但仍然继续迁移。了解这些不同表型的决定因素及其免疫学后果对于了解体内免疫应答的基本原理至关重要。我们以前已经表明，一些趋化因子提供主导信号，指导T细胞继续迁移过去的抗原承载的APC，而其他趋化因子提供从属信号，允许长期共轭形成。2种这样的趋化因子是SLC（CCL 21，显性）和SDF-1a（CXCL 12，从属），由它们各自在T细胞上的受体CCR 7和CXCR 4识别。在这里，我们建议设计嵌合受体轴承的配体特异性1的主导受体和细胞内的信号转导反应的从属受体。由于趋化因子受体是7-跨膜G蛋白偶联受体，它们的细胞外、跨膜和细胞内结构是紧密偶联的。为了发现具有功能性配体结合和适当的细胞内信号传导特性的嵌合体，我们建议产生受体嵌合体的组合文库，其中所有细胞外环属于1个受体，所有细胞内环属于另一个受体，并且跨膜结构域在2个之间变化。目的一：通过基因合成的方法，设计并构建跨膜重组的CCR 7/CXCR 4和CCR 7/CCR 5文库。在目标2和3中，我们将筛选该文库的所需趋化性显性/从属行为。在目标4中，我们将使用这些数据来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CXCR 3和CCR 5之间的新型嵌合体来测试这些原理。通过阐明免疫学“停止”和“开始”信号的层次结构，我们将揭示控制免疫功能的基本原理，这些原理与理解肿瘤、病毒和其他微生物有时如何逃避免疫清除以及免疫如何在自身免疫性疾病中失调有关。

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.