Thymic macrophages consist of two populations with distinct localization and origin

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor (TF) SpiC with these cells. Single-cell RNA sequencing (scRNA-Seq) showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4⁺ cells were located in the cortex, while Cx3cr1⁺ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4⁺ thymic macrophages are of embryonic origin, while Cx3cr1⁺ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4⁺ cells underwent gradual attrition, while Cx3cr1⁺ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.