Abstract
Tregs play a central role in maintaining immune tolerance. Recent progress in the clinical application of Tregs underscores their potential for cell therapy. Nevertheless, a notable hurdle remains in producing functional Tregs in vitro. There is also a lack of detailed studies evaluating the function of Tregs during their ex vivo expansion process. Our prior investigation showed that the ex vivo expansion with oligonucleotides produces FoxP3(high)Helios(high) subsets. To investigate how oligonucleotides in culture media influence on gene expression and epigenetic states at single cell resolution, we sorted Tregs from healthy individuals and profiled in vitro oligonucleotide-expanded and non-expanded Tregs. We discovered a subset of Tregs, specifically enriched in expanded Tregs (seTregs), through oligonucleotide-induced expansion. seTregs showed an enhancement in both stem-like characteristics and functional attributes. Through analysis of histone modification data and gene regulatory networks, we elucidated IKZF2 (Helios) as a pivotal transcription factor in generating these cell subsets. We believe these findings offer insights into evaluating functional regulation of in vitro expanded Tregs aimed at manufacturing Treg-based cell therapies.