Epigenetic signature induced by prolonged culture may impact therapeutic function of Tregs used in adoptive cell therapy

Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product, which may be informative for predicting the cellular state and function of the final Treg product. First generation (1st gen.) Treg products were generated from peripheral blood-derived starting material which is depleted of CD8+ cells and enriched for CD25-expressing cells using the CliniMACS system. The cells were expanded for 23 days with repetitive TCR stimulation (using antiCD3/CD28 microbeads, 8 stimulations in total) in the presence of IL2 and Rapamycin. Cells were harvested at multiple timepoints throughout expansion, and subjected to DNA methylation analysis with the Illumina MethylationEPIC Array.