table_2_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。鉴于微RNA（miRNA）在CD28+和CD28- T细胞间的差异表达已被描述，我们提出miRNA表达的改变可能参与了与年龄相关的CD28表达下调。为避免体内T细胞在各个分化阶段比例随年龄相关变化所带来的混杂效应，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表达随时间变化的情况，该群体具有较低或较高的倍增数（PDs）。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。结果已在体外样本中得到验证。累积的PDs数量与CD28表达的降低相关，而并非T细胞克隆供体的年龄。通过对252个表达的miRNA进行主成分分析，发现无论克隆供体的年龄如何，都根据PDs的多少进行聚类。在PDs较高的克隆中观察到miR-9-5p和miR-34a-5p表达的增加，且miR-9-5p的表达与体外分离的健康受试者T细胞的CD28表达呈负相关。随后，我们考察了miR-9-5p、miR-34a-5p以及miR-23a~24-2簇成员在IL-15诱导T细胞CD28丢失中的作用，该簇中的所有成员均预测能与CD28的3′UTR结合。新鲜未成熟的CD28+ T细胞在存在IL-15的条件下培养，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。使用荧光素酶报告基因构建体研究了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相关的过程，即PD和CD28表达。