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: 8285724
• 负责人: Florence Lambolez
• 金额: $ 26.1万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8285724
• 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. PUBLIC HEALTH RELEVANCE: Given the profound role the intestinal immune system plays in generation of tolerance and protective immunity, it is surprising how little the potential of this system has been utilized in clinical practice. Unconventional intraepithelial lymphocytes constitute a substantial population of T cells in the intestine, yet their role remains elusive. Hee we set out to explain how they differentiate and how they recognize foreign molecules, information that is critical in understanding how the intestinal immune lymphocytes are capable of eliciting an immune response against pathogens.

描述（由申请人提供）：来自小肠的上皮内淋巴细胞（IEL）构成异质性T细胞群体，其形成身体粘膜（病原体的主要入口）处的适应性免疫防御的第一线。虽然IEL构成了人体最大的T细胞区室之一，但我们对其发育和功能的理解特别有限。近年来，我们的实验室对CD 8？TCR？？IEL，表明它们在胸腺中产生，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。这些观察是使用通常可获得的TCR/同源抗原双重转基因小鼠系统进行的。不幸的是，所有这些模型都使用了来自传统选择的CD 4或CD 8？识别已知抗原的T细胞。这是可能的，传统的T细胞，这是大多缺乏自身反应特异性的TCR库，从寡克隆和可能的自我反应库的CD 8？TCR？？IELs。因此，我们认为，这些转基因模型是不适合进一步分析和表征的激动剂的选择和发展的CD 8？ TCR？？IELs。在这里，我们提出了一个新的方法，旨在阐明MHC的限制，并提供新的见解CD 8？TCR？？IELs。首先，我们将产生一个TCR库序列数据库从CD 8？？TCR？？IEL使用大规模平行测序。将从表达固定TCR的多只非合并小鼠中分离IEL？(TCR小维？Tg模型）;因此，每个个体TCR？通过高重复序列测序获得的链序列将代表一个完整的克隆时，与独特的TCR V？5链。然后将在库中高度代表的TCR克隆克隆并在不同的MHC缺陷型受体中逆转录病毒表达，以评估和鉴定限制性MHC分子（Aim 1）。这第一种方法将使我们能够快速，有效地筛选一些潜在的TCR克隆，随后将用于解决CD 8？？TCR？？IELs。我们将使用一种逆转录病毒系统来实现这一目标，该系统可以驱动TCR的临时调节表达。链在这种情况下，TCR？链将开始在表达CD 4的双阳性胸腺细胞中使用Cre调节的逆转录病毒载体特异性表达。嵌合体的分析应使我们能够详细描述的分化途径的CD 8？？TCR？？体内IEL（目标2）。这两个目标是互补的，并将导致a）解开CD 8？？TCR？？IEL和B）为激动性选择的CD 8？？TCR？？IELs。这项研究的完成将为我们提供大量关于CD 8？？TCR？？IELs。对肠道免疫系统的更好理解有望为许多肠道炎症性疾病开发新的治疗方式。 公共卫生相关性：考虑到肠道免疫系统在产生耐受性和保护性免疫中发挥的重要作用，令人惊讶的是，该系统在临床实践中的潜力是多么小。非常规的上皮内淋巴细胞构成了肠道中大量的T细胞，但它们的作用仍然难以捉摸。我们开始解释它们如何分化以及它们如何识别外来分子，这些信息对于理解肠道免疫淋巴细胞如何能够引发针对病原体的免疫反应至关重要。