Human CD4+CD25+ Regulatory T Cells Deficient of CD226 Demonstrate Increased Purity and Lineage Stability Following Ex Vivo Expansion for Adoptive Treg Therapies

Regulatory T cell (Treg) therapy is an emerging strategy for restoring immune tolerance in autoimmune diseases. Tregs are commonly purified as CD4⁺CD25⁺CD127^lo/- T cells, yielding a population enriched in the expression of the canonical thymically-derived Treg (tTreg) transcription factors FOXP3 and Helios. Previous work has identified the type 1 diabetes risk-associated locus and costimulatory molecule CD226 as being highly expressed on both effector T cells and interferon-γ-producing peripheral Tregs (pTregs). Thus, we sought to determine whether isolating CD4⁺CD25⁺CD226^- Tregs yields a Treg population with increased purity and suppressive capacity relative to CD4⁺CD25⁺CD127^lo/- cells. After 14d of culture, expanded CD4⁺CD25⁺CD226^- Tregs displayed a decreased proportion of pTregs relative to CD4⁺CD25⁺CD127^lo/- cells as determined by the 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-α, and IL-17A, along with increased expression of the inhibitory cytokine TGF-β1 as well as a higher suppressive capacity in vitro compared to their CD127^lo/- counterparts. To corroborate the pathogenic role of CD226 in Treg stability, we conducted CRISPR-Cas9 gene knockouts (KOs) of CD226 on CD127^lo/- Tregs. Following expansion, CD226 KO Tregs demonstrated higher proportions of total Tregs and tTregs, decreased proportions of pTregs, as well as increased expression of CD25 and TIGIT compared to non-edited controls. Overall, these data suggest the elimination of CD226 signaling during in vitro expansion generates a tTreg population with increased purity, lineage stability, and suppressive capabilities for adoptive cell therapies for the treatment of autoimmune diseases.