CRISPR-Cas engineering of allogeneic regulatory T cells reveals an essential role for steroid receptor coactivator 3 (SRC-3) in maintaining FOXP3 expression and suppressive function

Genetically engineered allogeneic regulatory T cells (Tregs) represent a promising off‑the‑shelf therapy to prevent graft‑versus‑host disease and transplant rejection, but their clinical utility is constrained by immune rejection and alloreactivity. Using CRISPR-Cas editing, we generated hypoimmunogenic human Tregs through multiplex knockout of B2M (reducing HLA class I), TCRalpha/beta (eliminating receptor‑mediated alloreactivity), and SRC‑3 (a transcriptional coactivator linked to Treg stability). In prior murine studies, SRC‑3 disruption enhanced tumor clearance, but in human Tregs, its deletion impaired suppressive function, underscoring SRC‑3's role in maintaining lineage identity. Functionally, SRC‑3 knockout Tregs failed to suppress effector T cell proliferation, a defect mirrored in triple knockout (TCR+B2M+SRC‑3) cells. Co‑culture with unedited Tregs restored suppression, suggesting functional compensation. FOXP3 protein levels were markedly reduced in SRC‑3 and triple knockout Tregs, consistent with destabilized lineage commitment. These findings highlight SRC‑3, TCR, and B2M as central regulators of transcriptional and antigen‑presentation pathways shaping Treg identity. RNA sequencing revealed that triple knockout Tregs exhibited increased transcript diversity compared to controls. SRC‑1/NCOA1 and SRC‑2/NCOA2 were upregulated in SRC‑3 and triple knockout samples, while B2M and TRAC were strongly downregulated in triple knockouts. Chemokine transcripts were elevated in SRC‑3 and triple knockout Tregs, with triple knockouts also showing diverse cytokine upregulation. Collectively, these results demonstrate that while genome editing can enhance allogeneic compatibility, SRC‑3 is indispensable for human Treg stability and suppressive function. Effective engineering of universal Tregs must therefore balance hypoimmunogenic design with preservation of functional integrity for adoptive cell therapy.