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

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.