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（IELs）构成了一个异质的T细胞群，在人体粘膜（病原体的主要入口）形成了适应性免疫防御的第一道防线。尽管IELs是人体中最大的T细胞区室之一，但我们对它们的发育和功能的了解尤其有限。近年来，我们的实验室在CD8的发展方面取得了一些最先进和最重要的见解。TCR ? ?IELs，表明它们是在胸腺中产生的，它们的前体胸腺细胞经历了自我激动剂依赖的选择，也称为激动剂选择。这些观察结果是使用常用的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了传统选择的CD4或CD8衍生的tcr。识别已知抗原的T细胞。传统T细胞的TCR库大多缺乏自身反应特异性，可能与CD8的寡克隆和可能的自反应库不同。TCR ? ?IELs。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 ? ?IELs。在这里，我们提出了一种新颖的方法，旨在阐明MHC限制，并为CD8的分化途径提供新的见解。TCR ? ?IELs。首先，我们将从CD8?？TCR ? ?使用大规模并行测序的IELs。IEL将从表达固定TCR？(TCR V ? ?Tg模型);那么每个个体的TCR呢？高通量测序获得的链序列与唯一的TCR V？5链。然后，在保留库中高度代表性的TCR克隆将被克隆并在不同的MHC缺陷受体中逆转录表达，以评估和识别限制性MHC分子（目的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 ? ?IELs。我们将使用一种逆转录病毒系统来驱动TCR的临时调节表达。链。通过这种方法，TCR？链将开始在CD4表达双阳性胸腺细胞中特异性表达，使用cre调控的逆转录病毒载体。对嵌合体的分析应该允许我们对CD8的分化途径给出一个详细的描述。TCR ? ?体内IELs （Aim 2）。这两个目标是互补的，将导致a)解开CD8的MHC限制？？TCR ? ?IELs和b)为激动性选择CD8的胸腺发育提供了新的见解？？TCR ? ?IELs。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 ? ?IELs。更好地了解肠道免疫系统，有望为许多肠道炎症性疾病开发新的治疗方式。

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.