Effector memory–type regulatory T cells display phenotypic and functional instability

Regulatory T cells (T_reg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of T_reg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited T_reg cell products. Here, we examined different T_reg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each T_reg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory T_reg cell populations, including a hitherto unidentified naïve-like memory T_reg cell subset, outperformed late-differentiated effector memory–like T_reg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional T_reg cell products could be achieved by depleting the small effector memory–like T_reg cell subset before manufacturing. Considering T_reg cell subset composition appears critical to maintain lineage stability in the final cell product.