Uncovering the Heterogeneity and Ontogeny of Mouse Thymic Macrophages Reveals an Unexpected Early Checkpoint Role

Through single-cell RNA sequencing and transgenic reporter mice, we revealed two novel TM subsets characterized by distinct surface markers and transcriptional programs. TIMD4⁺ VCAM1⁺ CX3CR1⁻ TMs were enriched for phagocytic and apoptotic-cell–clearance pathways, whereas TIMD4⁻ VCAM1⁺ CX3CR1⁺ TMs exhibited elevated expression of antigen-presentation machinery and interferon‑response genes.

Pseudotime trajectory analysis indicated a potential lineage progression from TIMD4⁻ VCAM1⁺ CX3CR1⁺ precursors toward TIMD4⁺ VCAM1⁺ CX3CR1⁻ effectors. Functional interrogation using fetal thymic organ cultures and the MaFIA depletion mouse model demonstrated that acute loss of TMs severely impaired thymocyte development, particularly at the pre‑T cell receptor (TCR) β‑selection checkpoint. TM ablation led to marked reductions in post--selection thymocyte subsets, implicating TMs in both survival and differentiation signals required for early T cell development.