CD25 Plasticity in Monoclonal Regulatory T Cells: Implications for Regulatory T Cell Identity and Therapeutic Applications

Abstract

Regulatory T cells (Tregs) play a crucial role in immune homeostasis and suppression of autoimmunity. To investigate their therapeutic potential and gain deeper insight into their function and therapeutic potential, a monoclonal Treg population was generated through single-cell cloning. Peripheral blood mononuclear cells (PBMCs) were isolated from human donors, and CD4⁺ T cells were magnetically separated from CD8⁺ T cells before being expanded in culture with IL-2 and TGF-β. A limiting dilution method was used to obtain a monoclonal population. Flow cytometry analysis confirmed that the cloned Treg line (2G4) exhibited key markers of regulatory function, including CD4, FOXP3, and CD25, with an increased expression of CD25 compared to the parental polyclonal line (Cardio-1). Interestingly, CD25 expression within the 2G4 clone showed a bimodal distribution, suggesting that CD25^high and CD25^low states represent interconvertible phenotypes rather than fixed, terminally differentiated T-cell subsets. This plasticity reflects a dynamic response to environmental cues and challenges the traditional view of CD25 as a static Treg marker. These findings support the value of monoclonal Tregs as a controlled and reproducible model for studying regulatory function and advancing cell-based therapies.