Thymic development and MHC restriction of CD8aa TCRab intraepithelial lymphocytes

CD8aa TCRab 上皮内淋巴细胞的胸腺发育和 MHC 限制

• 批准号: 8534699
• 负责人: Florence Lambolez
• 金额: $ 20.45万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534699
• 关键词: Address; Agonist; Antigens; Biology; Bone Marrow; Bone Marrow Cells; CD8B1 gene; Cells; Cellular biology; Chimera organism; Data; Databases; Development; Elements; Generations; Harvest; Histocompatibility Antigens Class II; Immune; Immune response; Immune system; Immunity; In Vitro; Individual; Inflammatory Bowel Diseases; Internal Ribosome Entry Site; Intestines; Knock-out; Lead; Lymphocyte; Major Histocompatibility Complex; Modality; Modeling; Mucous Membrane; Mus; Pathway interactions; Phenotype; Play; Population; Population Heterogeneity; Process; Retroviral Vector; Retroviridae; Role; Small Intestines; Specificity; Staging; System; T-Cell Antigen Receptor Specificity; T-Cell Receptor; T-Lymphocyte; Technology; Thymus Gland; Transgenes; Transgenic Mice; Transgenic Model; Transgenic Organisms; base; clinical practice; experience; in vivo; insight; intraepithelial; novel therapeutics; pathogen; precursor cell; thymocyte

DESCRIPTION (provided by applicant): Intraepithelial lymphocytes (IELs) from the small intestine constitute a heterogeneous population of T cells that form the first line of adaptive immune defense at the body's mucosa, the main entrance for pathogens. Although IELs constitute one of the largest T cell compartments of the body our understanding of their development and function is particularly limited. In recent years, our lab has made some of the most advanced and significant insights into the development of CD8?? TCR?? IELs, showing that they are generated in the thymus and that their precursor thymocytes undergo a self-agonist dependent selection also called agonist selection. These observations were made using commonly available TCR/cognate antigen dual transgenic mouse systems. Unfortunately, all of these models used TCRs derived from conventionally selected CD4 or CD8?? T cells that recognize known antigens. It is likely that the TCR repertoire of conventional T cells, which are mostly devoid of autoreactive specificities, differs from the oligoclonal and possibly self-reactiv repertoire of CD8?? TCR?? IELs. Therefore we believe that these transgenic models are not appropriate for further analysis and characterization of agonist selection and development of CD8?? TCR?? IELs. Here we propose an original approach aiming to elucidate the MHC restriction and providing new insights into differentiation pathway of CD8?? TCR?? IELs. First, we will generated a TCR repertoire sequence database from CD8?? TCR?? IELs using massively parallel sequencing. IEL will be isolated from multiple non-pooled mice expressing a fixed TCR? (TCR V?? Tg model); thus each individual TCR? chain sequences obtained by High Throughput sequencing will represent a complete clone when paired with the unique TCR V?5 chain. TCR clones that are highly represented within the repertoire will then be cloned and expressed retrovirally in different MHC-deficient recipients in order to assess and identify the restricting MHC molecules (Aim 1). This first approach will allow us rapidly and efficiently to screen a number of potential TCR clones that will subsequently be used to address the differentiation pathway of CD8?? TCR?? IELs. We will do this using a retroviral system that drives the temporally-regulated expression of the TCR? chain. With this approach, TCR? chains will start to be expressed specifically in CD4- expressing double positive thymocytes using a Cre-regulated retroviral vector. Analysis of the chimera should allow us to give a detail description of the differentiation pathway of CD8?? TCR?? IELs in vivo (Aim 2). Both aims are complementary and will lead to a) unravel the MHC restriction of CD8?? TCR?? IELs and b) provide with new insight regarding into the thymic development of agonistically selected CD8?? TCR?? IELs. Completion of this study will provide us with a tremendous amount of new and important information regarding the biology of CD8?? TCR?? IELs. A better understanding of the intestinal immune system holds promise of the development of novel therapeutic modalities for a number of intestinal inflammatory disorders.

描述（由申请人提供）：来自小肠的上皮内淋巴细胞 (IEL) 构成了异质 T 细胞群，它们构成了身体粘膜（病原体的主要入口）的第一道适应性免疫防御线。尽管 IEL 是人体最大的 T 细胞区室之一，但我们对其发育和功能的了解特别有限。近年来，我们实验室对CD8的发展取得了一些最先进、最重要的见解？ TCR？？ IEL，表明它们是在胸腺中产生的，并且它们的前体胸腺细胞经历自我激动剂依赖性选择，也称为激动剂选择。这些观察是使用常用的 TCR/同源抗原双转基因小鼠系统进行的。不幸的是，所有这些模型都使用源自传统选择的 CD4 或 CD8 的 TCR？识别已知抗原的 T 细胞。传统 T 细胞的 TCR 指令集（大多缺乏自身反应特异性）很可能不同于 CD8?? 的寡克隆和可能的自身反应指令集。 TCR？？ IEL。因此我们认为这些转基因模型不适合进一步分析和表征CD8??激动剂的选择和发育。 TCR？？ IEL。在这里，我们提出了一种原创方法，旨在阐明 MHC 限制并为 CD8?? 的分化途径提供新的见解。 TCR？？ IEL。首先，我们将从CD8生成TCR库序列数据库？ TCR？？使用大规模并行测序的 IEL。 IEL 将从表达固定 TCR 的多只非合并小鼠中分离出来？ (TCR V??Tg模型)；那么每个单独的TCR？当与独特的 TCR V?5 链配对时，通过高通量测序获得的链序列将代表完整的克隆。然后，在不同的 MHC 缺陷受体中克隆和逆转录病毒表达在库中高度代表性的 TCR 克隆，以评估和鉴定限制性 MHC 分子（目标 1）。第一种方法将使我们能够快速有效地筛选许多潜在的 TCR 克隆，这些克隆随后将用于解决 CD8?? 的分化途径。 TCR？？ IEL。我们将使用驱动 TCR 时间调节表达的逆转录病毒系统来做到这一点？链。通过这种方法，TCR？使用 Cre 调节的逆转录病毒载体，链将开始在表达 CD4 的双阳性胸腺细胞中特异性表达。对嵌合体的分析应该可以让我们详细描述CD8的分化途径？ TCR？？体内 IEL（目标 2）。这两个目标是互补的，并将导致 a) 解开 CD8 的 MHC 限制？ TCR？？ IEL 和 b) 提供了关于竞争性选择的 CD8?? 胸腺发育的新见解？ TCR？？ IEL。这项研究的完成将为我们提供大量有关 CD8?? 生物学的新的重要信息。 TCR？？ IEL。对肠道免疫系统的更好了解有望为许多肠道炎症性疾病开发新的治疗方式。