Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the β-selection checkpoint.

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the β-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.