Heterogeneity and ontogeny of mouse thymic macrophages reveal a requirement for <i>Csf1r</i>-expressing myeloid cells during early T cell development

Thymic macrophages (TMs) play a crucial role in maintaining tissue homeostasis by clearing apoptotic cells during T cell development. However, the relationship between TM heterogeneity and their developmental origin during thymocyte maturation is not fully understood. By employing flow-cytometry, single-cell transcriptomics, and genetic methods, researchers have identified two primary TM populations: TIMD4+ cortical and CX3CR1+ medullary/cortico-medullary macrophages.

TIMD4+ VCAM1+ TMs demonstrate a strong efferocytosis and apoptotic-cell-clearance function, while TIMD4- VCAM1+ TMs are enriched for antigen presentation and interferon response pathways. Fate mapping showed that progenitors contribute unequally to these populations. CCR2 deficiency selectively diminished TIMD4- VCAM1+ TMs and thymic monocytes, indicating a reliance on circulating precursors.

Through pseudotime analysis, a transcriptional continuum was observable as Ly6c2+ Ccr2+ monocytes evolved into macrophage states. Using MaFIA thymic organ cultures, the study found that AP20187-mediated depletion of Csf1r-expressing myeloid cells led to reduced CD4+ CD8+ thymocyte differentiation, an accumulation of DN3 cells, loss of DN4 cells, and decreased CD27 expression.

These findings establish that the DN3-to-DN4 transition is a developmental stage that necessitates a functional Csf1r-expressing myeloid compartment, thereby highlighting a previously unrecognized dependency on myeloid-cell support during the αβ T cell development checkpoint.