Thymus development, cell by cell: The human thymus is the organ responsible for the maturation of many types of T cells, which are immune cells that protect us from infection. However, it is not well known how these cells develop with a full immune complement that contains the necessary variation to protect us from a variety of pathogens. By performing single-cell RNA sequencing on more than 250,000 cells, Park et al. examined the changes that occur in the thymus over the course of a human life. They found that development occurs in a coordinated manner among immune cells and with their developmental microenvironment. These data allowed for the creation of models of how T cells with different specific immune functions develop in humans. Science , this issue p. eaay3224

T cells develop from circulating precursor cells, which enter the thymus and migrate through specialized subcompartments that support their maturation and selection¹. In humans, this process starts in early fetal development and is highly active until thymic involution in adolescence. To map the microanatomical underpinnings of this process in pre- and early postnatal stages, we established a quantitative morphological framework for the thymus—the Cortico-Medullary Axis—and used it to perform a spatially resolved analysis. Here, by applying this framework to a curated multimodal single-cell atlas, spatial transcriptomics and high-resolution multiplex imaging data, we demonstrate establishment of the lobular cytokine network, canonical thymocyte trajectories and thymic epithelial cell distributions by the beginning of the the second trimester of fetal development. We pinpoint tissue niches of thymic epithelial cell progenitors and distinct subtypes associated with Hassall’s corpuscles and identify divergence in the timing of medullary entry between CD4 and CD8 T cell lineages. These findings provide a basis for a detailed understanding of T lymphocyte development and are complemented with a holistic toolkit for cross-platform imaging data analysis, annotation and OrganAxis construction (TissueTag), which can be applied to any tissue.

In humans, high Notch activation promotes γδ T cell development, whereas lower levels promote αβ-lineage differentiation. How these different Notch signals are generated has remained unclear. We show that differential Notch receptor–ligand interactions mediate this process. Whereas Delta-like 4 supports both TCR-αβ and -γδ development, Jagged1 induces mainly αβ-lineage differentiation. In contrast, Jagged2-mediated Notch activation primarily results in γδ T cell development and represses αβ-lineage differentiation by inhibiting TCR-β formation. Consistently, TCR-αβ T cell development is rescued through transduction of a TCR-β transgene. Jagged2 induces the strongest Notch signal through interactions with both Notch1 and Notch3, whereas Delta-like 4 primarily binds Notch1. In agreement, Notch3 is a stronger Notch activator and only supports γδ T cell development, whereas Notch1 is a weaker activator supporting both TCR-αβ and -γδ development. Fetal thymus organ cultures in JAG2-deficient thymic lobes or with Notch3-blocking antibodies confirm the importance of Jagged2/Notch3 signaling in human TCR-γδ differentiation. Our findings reveal that differential Notch receptor–ligand interactions mediate human TCR-αβ and -γδ T cell differentiation and provide a mechanistic insight into the high Notch dependency of human γδ T cell development.

Given the high frequency of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL), inhibition of the γ-secretase complex remains an attractive target to prevent ligand-independent release of the cytoplasmic tail and oncogenic NOTCH1 signaling. However, four different γ-secretase complexes exist, and available inhibitors block all complexes equally. As a result, these cause severe “on-target” gastrointestinal tract, skin, and thymus toxicity, limiting their therapeutic application. Here, we demonstrate that genetic deletion or pharmacologic inhibition of the presenilin-1 (PSEN1) subclass of γ-secretase complexes is highly effective in decreasing leukemia while avoiding dose-limiting toxicities. Clinically, T-ALL samples were found to selectively express only PSEN1-containing γ-secretase complexes. The conditional knockout of Psen1 in developing T cells attenuated the development of a mutant NOTCH1-driven leukemia in mice in vivo but did not abrogate normal T cell development. Treatment of T-ALL cell lines with the selective PSEN1 inhibitor MRK-560 effectively decreased mutant NOTCH1 processing and led to cell cycle arrest. These observations were extended to T-ALL patient-derived xenografts in vivo, demonstrating that MRK-560 treatment decreases leukemia burden and increased overall survival without any associated gut toxicity. Therefore, PSEN1-selective compounds provide a potential therapeutic strategy for safe and effective targeting of T-ALL and possibly also for other diseases in which NOTCH signaling plays a role.