Simultaneous Epigenetic and Gene Expression Profiling at Single Cell Resolution Uncovers Stem-Like Treg Subsets Induced With Oligonucleotide Expansion in Humans.

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 . There is also a lack of detailed studies evaluating the function of Tregs during their expansion process. Our prior investigation showed that the expansion with oligonucleotides produces FoxP3Helios 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 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 (Helios) as a pivotal transcription factor in generating these cell subsets. We believe these findings offer insights into evaluating functional regulation of expanded Tregs aimed at manufacturing Treg-based cell therapies.