Thymic development and MHC restriction of CD8aa TCRab intraepithelial lymphocytes

描述(由申请人提供)：来自小肠的上皮内淋巴细胞(IEL)构成了T细胞的异质性群体，形成了人体粘膜的第一道适应性免疫防御线，而黏膜是病原体的主要入口。虽然IEL是人体内最大的T细胞隔间之一，但我们对其发育和功能的了解尤其有限。近年来，我们的实验室对CD8的发展提出了一些最先进和最有意义的见解。TCR？？IEL表明，它们是在胸腺中产生的，它们的前体胸腺细胞经历了一种自我激动剂依赖的选择，也称为激动剂选择。这些观察是使用常见的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了来自传统选择的CD4或CD8的TCR？？识别已知抗原的T细胞。传统T细胞的TCR谱系很可能不同于CD8的寡克隆和可能的自我反应谱系，它们大多缺乏自身反应的特异性。TCR？？雅思。因此，我们认为这些转基因模型不适合于进一步分析和表征激动剂的选择和CD8的开发。TCR？？雅思。在这里，我们提出了一种原创性的方法，旨在阐明MHC的限制，并为CD8的分化途径提供新的见解。TCR？？雅思。首先，我们将从CD8？？生成一个TCR曲目序列数据库。TCR？？使用大规模并行测序的IEL。IEL将从多只表达固定TCR？的非共用小鼠中分离出来。(TCR V？？Tg模型)；因此每个单独的TCR？通过高通量测序获得的链序列与独特的TCRV？5链配对时将代表一个完整的克隆。然后，在曲目中高度代表的TCR克隆将被克隆，并在不同的MHC缺陷受体中逆转录病毒表达，以便评估和鉴定限制性MHC分子(目标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，这些克隆随后将被用于解决CD8的分化途径？？TCR？？雅思。我们将使用逆转录病毒系统来驱动TCR的时间调控表达吗？链条。用这种方法，TCR？利用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。对嵌合体的分析将使我们能够详细描述CD8的分化途径？？TCR？？体内ELs(目标2)。这两个目标是相辅相成的，并将导致a)解开CD8的MHC限制？TCR？？IEL和b)提供了关于激发型CD8胸腺发育的新见解？？TCR？？雅思。这项研究的完成将为我们提供大量关于CD8生物学的新的和重要的信息。TCR？？雅思。对肠道免疫系统的更好了解有望开发出一些肠道炎症性疾病的新治疗方法。 与公共卫生相关：鉴于肠道免疫系统在产生耐受性和保护性免疫方面发挥的深远作用，令人惊讶的是，该系统在临床实践中的潜力如此之少。非常规上皮内淋巴细胞在肠道中构成了大量的T细胞，但它们的作用仍然难以捉摸。我们开始解释它们是如何分化的，以及它们是如何识别外来分子的，这些信息对于了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。

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.