3D genome organization by CTCF prevents exTreg differentiation to control autoimmunity peripheral tolerance and immunotherapy outcomes [bulkRNAseq_ATACseq_CUT_Tag]

Regulatory T cells (Tregs) can undergo transformation into exTregs by downregulating Foxp3 expression, yet the underlying mechanisms and functional implications remain incompletely understood (1, 2). Here, we show that PD-1 blockade induces significant alterations in chromatin accessibility at CTCF binding sites within tumor-infiltrating Tregs. CTCF plays a crucial role in maintaining Treg identity and preventing autoimmune disorders in mice through the three-dimensional organization of key genes regulating Treg stability. Moreover, acute deletion of Treg-specific CTCF enhances T cell responses and promotes the differentiation of CD40L-expressing exTregs within tumors, without disrupting overall immune homeostasis in murine models. The single-cell T cell receptor sequencing analysis of colitis tissues from melanoma patients treated with anti-PD-1 and anti-CTLA-4 antibodies reveals a notable emergence of exTregs, elucidating the paradoxical increase in both Tregs and cytotoxic CD8+ T cells (3) (3, 4). Our findings underscore the therapeutic potential of manipulating exTreg differentiation to enhance cancer immunotherapy and mitigate associated adverse effects.