Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Cells must accurately replicate and segregate their DNA once per cell cycle in order to successfully transmit genetic information. During S phase in the presence of agents that cause replication stress, ATR-dependent checkpoints regulate origin firing and the replication machinery as well as prevent untimely mitosis. Here, we investigate the role of ATR during unperturbed growth in vertebrate cells. In the absence of ATR, individual replication forks progress more slowly, and an increased number of replication origins are activated. These cells also enter mitosis early and divide more rapidly, culminating in chromosome bridges and laggards at anaphase, failed cytokinesis, and cell death. Interestingly, cell death can be rescued by prolonging mitosis with partial inhibition of the mitotic cyclin-dependent kinase 1. Our data indicate that one of the essential roles of ATR during normal growth is to minimize the level of unreplicated DNA before the onset of mitosis.

15Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell 16 death and compromised organ function. This is first observed in the thymus, the primary 17 lymphoid organ that generates and selects T cells. However, the molecular and cellular 18 mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse 19 ageing leads to less efficient T cell selection, decreased self-antigen representation and 20 increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and 21 lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the 22 function of mature thymic epithelial cells is compromised only modestly. Specifically, an early-23 life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished 24 at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance 25 2 of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs 26 the core immunological functions of the thymus. 27 28