CD8+ Regulatory T Cells in the human thymus: chromatin accessibility and transcription factor networks uncover a role for BRD2 and BCL2L1 2946

Abstract Description CD8+ regulatory T cells (Tregs) are increasingly recognized as relevant players in immune regulation, with roles in cancer and autoimmune/inflammatory diseases. Although a thymic origin is well-established, the mechanisms driving CD8 Treg differentiation and lineage commitment remain elusive. To investigate the regulation of CD8 Tregs in the human thymus, we performed ATAC-seq on double-positive (DP), CD4 single-positive (CD4SP), and CD8 single-positive (CD8SP) Tregs. We analyzed chromatin accessibility to identify lineage-specific transcription factor (TF) occupancy in cis-regulatory elements. AI-driven modeling identified gene regulatory modules (GRMs) unique to CD8SP Tregs, distinguishing them from DP and CD4SP Tregs. ATAC-seq from conventional thymocytes (Tconvs) further contrasted the transcriptional landscape of CD8SP Tregs. This revealed regions of open chromatin (ROC) specific to DP and CD8SP Tregs. scRNA-seq integration (Yayon Kedlian & Boehme et al., Nature 2024, GSE271304) confirmed active transcription in these regions, highlighting TFs binding CD8 Treg-specific genes, including BRD2 and BCL2L1, as key pathways for differentiation. To assess the functional significance of these findings, we used pharmacological inhibitors of BRD2 and BCL2L1 to modulated human thymocytes in vitro, evaluating their survival, differentiation, and function with spectral flow cytometry. Overall, BRD2 and BCL2L1 emerged as novel target genes implicated in human CD8 Treg biology. Funding Sources Supported by PAC-PRECISE-LISBOA-01-0145-FEDER-016394 and LISBOA-01-0145-FEDER-007391, by FEDER through POR Lisboa 2020 -and Fundação para a Ciência e a Tecnologia (FCT); PTDC/MED-IMU/0938/2020, by the Portuguese government through FCT; GenomePT PINFRA/22184/2016/POCI-01-0145-FEDER-022184; Marie Sklowdowska-Curie-COFUND Action MSCA-ITN-2015-675395 - H2020; and CEECIND/01474/2017 (FCT). Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)