MicroRNA signatures of CD4+ T cell subsets in healthy and multiple sclerosis subjects determined by small RNA-sequencing

Diverse CD4+ T cell subsets with specialized functions operate at different phases of the immune response. Among these are naïve, central memory (CM), effector memory (CM), and regulatory (Treg) cells that have been phenotypically and functionally characterized. To expand current knowledge and identify highly subset-selective markers, we have profiled miRNAs using small RNA-sequencing in FACS-sorted CD4+ T cell subsets from healthy subjects and untreated patients with relapsing-remitting multiple sclerosis (RRMS). Genomic clustering and abundance of the miRNAs were also investigated. From the 60 most differentially expressed miRNAs, broad signatures and highly selective core signatures were identified for naïve and memory CD4+ T cells at homeostasis, while miR-146a-5p was strongly upregulated in Treg cells. In line with other studies, a 5-miRNA core signature was identified for naïve cells (miR-125b-5p, miR-99a-5p, miR-365a-3p, miR-365b-3p, miR-193b-3p). Supporting the progressive memory T cell differentiation model, a number of miRNAs were upregulated in CM and EM cells and, notably, at higher level in EM cells. This was particularly the case for a miRNA core of 8 miRNAs from miR-23a~27a~24-2, miR-23b~27b~24-1, miR-221~222 clusters, and miR-22-3p, miR-181c-5p, with highly abundant miR-24-3p. In addition, we detected activation of the large ChrXq27.3 miR-506~514 cluster in EM cells. Interestingly, most of the miRNAs that were enriched in EM cells were shown to negatively regulate cell proliferation and survival. Finally, we found that the miRNA core signatures of naïve and memory CD4+ T cells were conserved in RRMS patients. Only few miRNAs were quantitatively modified, among these, miR-1248 was downregulated and may be a candidate disease marker. Overall design: We have profiled miRNAs using small RNA-sequencing in FACS-sorted CD4+ T cell subsets from healthy subjects and untreated patients with relapsing-remitting multiple sclerosis (RRMS)

具备特殊功能的多样化CD4+ T细胞（CD4+ T cell）亚群，在免疫应答的不同阶段发挥调控作用。此类亚群包括表型与功能均已得到详尽表征的初始（naïve）T细胞、中枢记忆（central memory, CM）T细胞、效应记忆（effector memory, EM）T细胞以及调节性T细胞（regulatory T cell, Treg）。为拓展现有认知并鉴定具有高度亚群选择性的标志物，我们利用小RNA测序（small RNA-sequencing）技术，对来自健康受试者与未接受治疗的复发缓解型多发性硬化（relapsing-remitting multiple sclerosis, RRMS）患者的荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化的CD4+ T细胞亚群中的微小RNA（microRNA, miRNA）进行了表达谱分析。此外，我们还对miRNA的基因组聚类特征与表达丰度展开了研究。

从60个差异表达最为显著的miRNA中，我们鉴定出了稳态下初始与记忆性CD4+ T细胞的广谱特征及高度选择性核心特征，其中miR-146a-5p在Treg细胞中显著上调。与既往研究结果一致，我们为初始T细胞鉴定出包含5个miRNA的核心特征：miR-125b-5p、miR-99a-5p、miR-365a-3p、miR-365b-3p及miR-193b-3p。

为验证渐进式记忆T细胞分化模型，我们检测到多个miRNA在CM与EM细胞中呈上调表达，且在EM细胞中的上调程度更为显著。其中尤以源自miR-23a~27a~24-2、miR-23b~27b~24-1、miR-221~222基因簇以及miR-22-3p、miR-181c-5p的8个miRNA构成的核心特征最为突出，该簇中miR-24-3p的表达丰度极高。此外，我们还在EM细胞中检测到X染色体q27.3区域的大型miR-506~514基因簇被激活。

值得注意的是，多数在EM细胞中富集的miRNA被证实可负向调控细胞增殖与存活。最后我们发现，初始与记忆性CD4+ T细胞的miRNA核心特征在RRMS患者中仍保持保守，仅少数miRNA发生了定量表达改变，其中miR-1248呈下调趋势，有望成为候选疾病标志物。

整体实验设计：我们利用小RNA测序技术，对来自健康受试者与未接受治疗的复发缓解型多发性硬化（RRMS）患者的FACS纯化CD4+ T细胞亚群中的miRNA进行了表达谱分析。