Human CD4⁺CD25⁺CD226^- Tregs Demonstrate Increased Purity, Lineage Stability, and Suppressive Capacity Versus CD4⁺CD25⁺CD127^lo/- Tregs for Adoptive Cell Therapy

Regulatory T cell (Treg) adoptive cell therapy (ACT) represents an emerging strategy for restoring immune tolerance in autoimmune diseases. Tregs are commonly purified using a CD4⁺CD25⁺CD127^lo/- gating strategy, which yields a mixed population: 1) cells expressing the transcription factors, FOXP3 and Helios, that canonically define lineage stable thymic Tregs and 2) unstable FOXP3⁺Helios^- Tregs. Our prior work identified the autoimmune disease risk-associated locus and costimulatory molecule, CD226, as being highly expressed not only on effector T cells but also, interferon-γ (IFN-γ) producing peripheral Tregs (pTreg). Thus, we sought to determine whether isolating Tregs with a CD4⁺CD25⁺CD226^- strategy yields a population with increased purity and suppressive capacity relative to CD4⁺CD25⁺CD127^lo/- cells. After 14d of culture, expanded CD4⁺CD25⁺CD226^- cells displayed a decreased proportion of pTregs relative to CD4⁺CD25⁺CD127^lo/- cells, as measured by FOXP3⁺Helios^- expression and the epigenetic signature at the FOXP3 Treg-specific demethylated region (TSDR). Furthermore, CD226^- Tregs exhibited decreased production of the effector cytokines, IFN-γ, TNF, and IL-17A, along with increased expression of the immunoregulatory cytokine, TGF-β1. Lastly, CD226^- Tregs demonstrated increased in vitro suppressive capacity as compared to their CD127^lo/- counterparts. These data suggest that the exclusion of CD226-expressing cells during Treg sorting yields a population with increased purity, lineage stability, and suppressive capabilities, which may benefit Treg ACT for the treatment of autoimmune diseases.