Kazuko Miyazaki1,15, Kenta Horie2,15, Hitomi Watanabe3,15, Reiko Hidaka1,15, Rinako Hayashi1, Norihito Hayatsu4, Kentaro Fujiwara1, Rei Kuwata1, Takuya Uehata5, Yotaro Ochi6, Makoto Takenaka3, Risa Karakida Kawaguchi7, Koichi Ikuta8, Osamu Takeuchi5, Seishi Ogawa6,9,10, Katsuto Hozumi11, Georg A. Holländer12,13,14, Gen Kondoh3, Taishin Akiyama2 and Masaki Miyazaki1 1Laboratory of Immunology, Institute for Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; 2Laboratory for Immune Homeostasis, RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan; 3Laboratory of Integrative Biological Sciences, Institute for Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; 4Laboratory for Comprehensive Genomic Analysis, RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan; 5Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; 6Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; 7Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; 8Laboratory of Immune Regulation, Institute for Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; 9Institute for the Advanced Study of Human Biology (WPI ASHBi), Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; 10Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institute, Stockholm 171 77, Sweden; 11Department of Immunology, Tokai University School of Medicine, Kanagawa 259-1193, Japan; 12Department of Pediatrics, Institute of Developmental and Regenerative Medicine, University of Oxford, Oxford OX3 7TY, United Kingdom; 13Pediatric Immunology, Department of Biomedicine, University of Basel and University Children's Hospital Basel, Basel 4056, Switzerland; 14Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel 4056, Switzerland Corresponding author: mmiyazakiinfront.kyoto-u.ac.jp

↵15 These authors contributed equally to this work.

Abstract

External signals from the thymic microenvironment and the activities of lineage-specific transcription factors (TFs) instruct T-cell versus innate lymphoid cell (ILC) fates. However, mechanistic insights into how factors such as Notch1–Delta-like-4 (Dll4) signaling and E-protein TFs collaborate to establish T-cell identity remain rudimentary. Using multiple in vivo approaches and single-cell multiome analysis, we identified a feedback amplifier circuit that specifies fetal and adult T-cell fates. In early T progenitors (ETPs) in the fetal thymus, Notch signaling minimally lowered E-protein antagonist Id2 levels, and high Id2 abundance favored the differentiation of ETPs into ILCs. Conversely, in the adult thymus, Notch signaling markedly decreased Id2 abundance in ETPs, substantially elevating E-protein DNA binding and in turn promoting the activation of a T-cell lineage-specific gene expression program linked with V(D)J gene recombination and T-cell receptor signaling. Our findings indicate that, in the fetal versus the adult thymus, a simple feedback amplifier circuit dictated by Notch-mediated signals and Id2 abundance enforces T-cell identity and suppresses ILC development.

Received July 17, 2024. Accepted January 13, 2025.