Genomic Approaches to the T Cell Receptor Repertoire during Aging

描述（由申请人提供）：很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幼稚t细胞数量低是免疫衰老的第一个迹象。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。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、表型以及潜在的功能都有深远的影响[3-5]。然而，目前尚不清楚巨细胞病毒感染是否是T细胞免疫衰老的驱动因素，如果是，在多大程度上驱动。尽管有这些有趣且具有启发性的观察结果，但缺乏适当的工具能够在序列和功能水平上对CMV阴性个体中罕见的CMV特异性T细胞进行系统研究，这阻碍了对上述问题的答案的探索。因此，我们建议开发：1)基于新一代测序技术的TCR序列库分析；2)采用四聚体染色和分离方法检测TCR功能库；3)基于微流控芯片的单细胞定量PCR （qPCR）技术分析CMV特异性T细胞的功能。我们将应用这些技术来跟踪和表征老年和年轻献血者的特定T细胞群，并将其与两组中巨细胞病毒血清转化的时间联系起来。我们假设，由于全局和CMV特异性T细胞库的异常（TCR序列和配体库的空洞和/或频率和/或多样性降低）和/或CMV特异性前体T细胞功能的缺陷，老年人易患CMV感染。此外，长时间的低效率免疫反应可能导致免疫反应脱敏，进一步推动免疫衰老的发展。我们的具体目标是：目标1。利用高通量测序技术研究青年和老年人群TCR 1和2链基因多样性差异将T细胞受体序列库与从年轻人和老年人中分离的CMV免疫显性表位特异性T细胞中的配体库和表型相关联。比较从年轻和老年人群中分离的CMV表位特异性T细胞的功能能力

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.