Genomic Approaches to the T Cell Receptor Repertoire during Aging

描述（由申请人提供）：许多关于免疫衰老的研究都集中在T细胞上，主要是因为外周血中少量的初始T细胞是免疫衰老的第一个迹象[1]。此外，T细胞受体（TCR）库多样性的变化与衰老和免疫衰老有关[2]。除了T细胞更新和TCR库多样性的自然减少外，巨细胞病毒（CMV）感染对老年人T细胞的亚群分布、表型以及潜在功能具有深远影响[3-5]。然而，目前还不清楚CMV感染是否是T细胞免疫衰老的驱动因素，如果是，在多大程度上。尽管有这些有趣的和提示性的观察结果，缺乏适当的工具，使罕见的CMV特异性T细胞在CMV阴性个体在序列和功能水平的系统研究阻碍了寻求答案的上述问题。因此，我们建议开发：1）基于下一代测序的技术来分析TCR序列库; 2）四聚体染色和分离方法来检查TCR功能库;以及3）基于微流控芯片的单细胞定量PCR（qPCR）技术来剖析CMV特异性T细胞的功能能力。我们将应用这些技术来跟踪和表征老年和年轻献血者中的特定T细胞群，并将其与两组中CMV血清转换的时间相关联。 我们假设，由于异常的全球和CMV特异性T细胞库（孔和/或减少的频率和/或多样性的TCR序列和配体库）和/或缺陷的CMV特异性前体T细胞的功能能力，老年人易受CMV感染。此外，对病毒的无效免疫应答的延长时间可能导致脱敏的免疫应答，进一步驱动免疫衰老的发展。我们的具体目标是：目标1。使用高通量测序研究年轻和老年队列中的总体TCR 1和2链基因多样性差异。将T细胞受体序列库与配体库和从年轻和老年队列分离的CMV免疫显性表位特异性T细胞中的表型相关联。比较从年轻和老年队列中分离的CMV表位特异性T细胞的功能能力 公共卫生相关性：这项研究产生的数据将帮助我们了解TCR库如何老化以及CMV感染与免疫衰老之间的关系。这里开发的技术将有助于推进CMV感染和免疫衰老的临床诊断。

DESCRIPTION (provided by applicant): Much research on immune senescence has been focused on T-cells, mainly because a low numbers of naive T-cells in the peripheral blood are the first sign of immune senescence [1]. Furthermore, changes in the T cell receptor (TCR) repertoire diversity have been linked to aging and immune senescence [2]. Aside from the natural reduction in T-cell renewal and TCR repertoire diversity, cytomegalovirus (CMV) infection has a profound influence on subset distribution, phenotype and potentially also on the function of T cells in the elderly [3-5]. However, it is still unclear whether CMV infection is the driver of T cell immune senescence and if so, to what extent. Despite these interesting and suggestive observations, lacking proper tools that enable the systematic study of the rare CMV specific T cells in CMV negative individuals at both the sequence and functional level hinders the quest for an answer to the above questions. Therefore, we propose to develop: 1) a next-generation sequencing based technology to analyze the TCR sequence repertoire; 2) a tetramer staining and isolation method to examine the TCR functional repertoire; and 3) a microfluidic chip-based single cell quantitative PCR (qPCR) technology to dissect functional capabilities of CMV specific T cells. We will apply these technologies to follow and characterize specific T cell populations in older and younger blood donors and correlate this with the timing of CMV seroconversions in both groups. We hypothesize that due to abnormalities in the global and CMV specific T cell repertoires (holes and/or reduced frequency and/or diversity in both TCR sequence and ligand repertoire) and/or defects in the functional capacities of CMV specific precursor T cells, the elderly are predisposed to CMV infection. Furthermore, prolonged period of an inefficient immune response to the virus may result in desensitized immune responses that further drive the development of immune senescence. Our specific aims are: Aim 1. Study global TCR 1 and 2 chain gene diversity difference in young and elderly cohorts using high-throughput sequencing Aim 2. Correlate T cell receptor sequence repertoire with ligand repertoire and phenotype in T cells specific for CMV immunodominant epitopes isolated from young and elderly cohorts Aim 3. Compare functional capabilities of T cells specific for CMV epitopes isolated from young and elderly cohorts PUBLIC HEALTH RELEVANCE: Data generated from this study will help us understand how the TCR repertoire ages and what is the relation between CMV infection and immune senescence. Technologies developed here will be useful to advance clinical diagnostics in CMV infection and immune senescence.