Isolation of Pure Stable Human Treg Cells Based on Expression of GPA33

ABSTRACT Adoptive cell therapy (ACT) with regulatory T (Treg) cells offers potential for treating immune‐mediated diseases. Ensuring the purity and stability of Treg cell products is critical for safe and effective therapies, particularly when targeting specific self‐antigens. The purest products are currently obtained using CD4⁺CD25⁺CD127 low/– CD45RA⁺ naïve (n)Treg cells. However, these still include cells lacking key transcription factors FOXP3 and Helios, able to produce inflammatory cytokines. Previously, we identified GPA33 as a surface marker for stable FOXP3⁺Helios⁺ human Treg cells and demonstrated that GPA33 high nTreg cell populations maintain higher purity during in vitro expansion compared with standard nTreg cells. However, the definition of the GPA33 high population among nTreg cells was arbitrary, and cell yields were low. Here, we show methods to unequivocally identify GPA33⁺ cells within the Treg cell fraction and generate Treg cell products that match nTreg cell populations in size and expansion capacity but exhibit superior FOXP3⁺Helios⁺ purity, lack effector cytokine production, and retain full suppressive function. Combining GPA33 with CD226 exclusion eliminates the need for CD127‐based gating. Post‐expansion, co‐expression of GPA33 with TIGIT reliably identifies lineage‐stable Treg cells. Thus, GPA33, alone or with CD226/TIGIT, is a robust marker for isolating Treg cells with enhanced therapeutic safety.