Stable Expression of FOXP3 and Core Tolerogenic Genes by Engineered Human Tregs

FOXP3 +Regulatory T cells (Tregs) play a critical role in the prevention of fatal autoimmunity and maintaining tissue homeostasis. The functional stability of Tregs remains critical in their contribution towards immune tolerance rather than runaway immunity, particularly for cell therapy whereby an inflammatory microenvironment may impact the expression of FOXP3 and associated tolerogenic genes. To address potential Treg instability, we generated human Engineered Tregs (EngTregs) through a gene editing approach of PBMC isolated CD4+ T cells, leading to stable FOXP3 expression and a rapamycin-activated signaling complex that provides tunable IL-2 signal, thereby effectively divorcing FOXP3 expression from existing regulatory elements known to promote Treg instability under inflammatory conditions. Using transcriptomic analysis, we evaluated the stability of cultured sorted Tregs (cTregs) compared to EngTregs. We found EngTregs expressed higher levels of key “Core Treg” and “FOXP3 Synergy” genes based on bulk and single cell RNA-seq analysis as confirmed by flow cytometry. Additionally, favorable expression pattern of key Treg stability markers, CD27, CD70 and IKZF4 (EOS) was observed in EngTregs compared to cTregs. Conversely, cTregs express higher levels of cytotoxic genes including GZMA and PERFORIN1 compared to EngTregs along with other inflammatory genes. Finally, we observed that these correlations have a functional implication as demonstrated by higher expression of key tolerogenic markers in EngTregs compared to cTregs. This work strongly supports EngTregs as stable, tolerogenic FOXP3+ T cells, thereby providing an invaluable asset for treating life-threatening autoimmune and autoinflammatory diseases.