Unravelling gene regulatory networks and driver TFs in CD4+ regulatory T cell development in the human thymus 2308489

Abstract Introduction CD4+ regulatory T cells (Tregs) are essential for maintaining immune homeostasis and preventing autoimmunity. Tregs primarily develop in the thymus, but can also arise from naïve CD4+ T cells in the periphery (pTregs) or be generated in vitro (iTregs). However, a major limitation of Treg-based therapies is the instability and plasticity of pTregs and iTregs. In contrast, tTregs have been shown to exhibit stable suppressive capacity, largely due to thymic-derived signals that epigenetically reinforce the Treg program. Elucidating the mechanisms governing Treg differentiation, stability, and function is therefore critical for improving Treg-based therapies. Methods We generated single-cell multiome data from human fetal and pediatric thymuses. We developed various analytical frameworks for unravelling gene regulatory networks (GRNs) involved in Treg lineage commitment. We identified candidate transcription factors (TFs) involved in thymic Treg differentiation and validated these TFs using a CRISPR-Cas9 KO system in primary human thymocytes. Results GRN analysis revealed key driver TFs, such as FOXP3, REL and IKZF2 within CD4+ Tregs. Comparison of GRNs between mature CD4+ Tregs and conventional CD4+ T cells further reveals TFs related to TCR signaling and other novel TFs. Finally, candidate TFs including IRF4, REL, FOXO1, BATF and others were validated utilizing a CRISPR-Cas9 KO system. Conclusion We generated a single cell multiome atlas of fetal and pediatric thymuses and unravel GRNs involved in Treg lineage-specific differentiation. We develop novel analytical frameworks to identify lineage-specific driver TFs in Tregs and validated these by KO of primary human thymocytes. This framework provides an important mapping of GRNs involved in thymic T cell differentiation, particularly focusing on Tregs and will serve as an important basis for understanding Treg biology and improving Treg-based therapies. Funding Source Creative-Pioneering Researchers Program (800-20230490) Seoul National University Topic Categories Hematopoiesis and Immune System Development (HEM)