table_1_Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells.PDF

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28− T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3′UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3′UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3′UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

CD28的缺失是T细胞衰老的典型特征，但其缺失的潜在机制尚不明确。鉴于CD28+和CD28- T细胞之间已描述了微RNA（miRNA）表达的差异表达，我们假设miRNA表达的改变是导致与年龄相关的CD28下调的年龄相关因素。为了避免体内T细胞分化各阶段比例随年龄相关变化所带来的混淆效应，本研究采用了一种实验模型系统，以研究单克隆T细胞群体中与CD28表达缺失相关的miRNA表达随时间的变化，这些群体在细胞倍增数（PDs）较低或较高。此方法使得在纵向模型中识别与年龄相关的miRNA表达变化成为可能。实验结果已在体外样本中得到验证。PDs的累积数与CD28表达的下调相关，而非T细胞克隆捐赠者的年龄。252个表达miRNA的原理成分分析表明，无论克隆捐赠者的年龄如何，均基于PDs的高低进行聚类。在PDs较高的克隆中观察到miR-9-5p和miR-34a-5p的表达增加，且miR-9-5p的表达与来自健康受试者的体外分离的T细胞的CD28表达呈负相关。随后，我们研究了miR-9-5p、miR-34a-5p以及miR-23a~24-2簇成员在IL-15诱导的T细胞CD28丢失中的作用，该簇成员均被预测与CD28的3'非编码区（3'UTR）结合。在存在IL-15的情况下培养新鲜未成熟的CD28+ T细胞导致CD28表达的逐渐丢失，同时miR-9-5p、miR-34a-5p和miR-23a~24-2簇成员的表达增加。利用荧光素酶报告基因结构研究了miR-9-5p、miR-34a-5p、miR-24-3p和miR-27-3p与CD28的3'UTR的结合。结果显示miR-24-3p和miR-27a-3p与3'UTR的功能性结合。我们的结果表明，特定的miRNA在T细胞中参与了与T细胞衰老的特定特征（即PD和CD28表达）相关的过程。