Real-time quantitative PCR analysis of EV-associated miRNAs from Human Treg cells

MicroRNAs (miRNAs) are small non-coding molecules targeting messenger RNAs and inhibiting protein translation. Regulated by dynamic micro-environmental cues, miRNAs in turn modulate key biological processes, including cell growth and development, energy utilization and homeostasis. In particular, miRNAs control the differentiation, survival and activation of both CD4+ T conventional (Tconv) and CD4+ T regulatory (Treg) cells, key players of the adaptive immunity; in particular, miRNA-mediated gene expression regulation contributes to the physiological response of those cells to infections and the pathological loss of immune homeostasis in autoimmunity. Upon T cell receptor (TCR) stimulation, Treg cells release a fingerprint of miRNAs in association with extracellular vesicles (EVs), which is specific and significantly different from that released by Tconv cell counterpart (GSE183713). In this study, we have highlithed how Treg-cell derived EVs, and their miRNA content, are able to block the proliferation and activation of Tconv cells, demonstrating a strong tolerogenic function. Human Treg cell-derived EVs were isolated from conditioned media using size exclusion chromatography (Exo-spin™ columns, Cell Guidance) according to manufacturer’s protocol. Isolated EV eluate was total RNA extracted using RNAqueous-4PCR Kit (Ambion, U.S.A.) and a fixed volume of eluted RNA sample was used as input for reverse transcription reaction by miRCURY-LNA RT Kit according to manufacturer’s instruction (Qiagen, U.S.A.). EV-associated miRNAs (n = 752) were profiled by using the complete human miRCURY LNA miRNA panel I+II (V5, Qiagen, U.S.A.).