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

Reviewer #1 (Public review):

Summary:

The current manuscript characterizes in detail the macrophages in the thymus. The authors identify two distinct populations of thymic macrophages and describe their surface marker expression and transcriptional signatures. They also explore their ontology and kinetics of settling and persistence in the thymus and find that the TIMD4+ macrophages are derived from embryonic progenitors and self-maintain in the thymus, while the TIMD4- macrophages are derived from monocytes. Most importantly, the authors test the functional importance of thymic macrophages for T cell development using an in vitro depletion system, from which they conclude that macrophages are important for one of the earliest selection steps in T cell development - the beta selection.

Strengths:

The authors use state-of-the-art techniques, such as multiple genetically modified mice, multi-color flow cytometry, single-cell RNA sequencing, genetic fate mapping, and fetal thymic organ culture (FTOC) combined with depletion. Their work is in good agreement with prior published studies on the subject, such as Tacke et al. (PMID: 26091486) and Zhou et al. (PMID: 36449334). In addition to reproducing prior knowledge, the authors uncover novel and unexpected facets of thymic macrophage biology, such as their SpiC independence and the fact that TIMD4- thymic macrophages depend on CCR2 (Tacke et al. have shown that the overall thymic macrophage compartment is normal in CCR2-/- mice). Most surprisingly, the authors claim that thymic macrophages control an early checkpoint in T cell development, the beta selection. This has not been reported before, as beta selection is usually considered a cell-autonomous process in thymocytes that does not require input from other cells.

Weaknesses:

The thymic macrophage depletion experiments are not well controlled, and the authors' interpretation of the results is a stretch. First, the treatment depletes other cell types, most notably dendritic cells (DCs), which have well-known roles in thymic selection (though not specifically in beta selection). The authors' reasoning that macrophages are abundant in the cortex, where beta selection occurs, while DCs are enriched in the medulla, seems questionable, as the embryonic thymus typically lacks (or has very small) medulla. A second salient point is that the authors haven't ruled out direct toxicity of the dimerizer drug AP20187 on thymocytes (specifically DN cells) in MAFIA mice.

Altogether, this is a solid manuscript that largely confirms the previously established ontogeny and heterogeneity of thymic macrophages. However, the participation of thymic macrophages in beta selection needs stronger evidence.

Reviewer #2 (Public review):

This manuscript from Zuniga-Pflucker laboratory describes that thymic macrophages are heterogeneous in flow cytometric and transcriptomic profiles, containing two major populations characterized by TIMD4 and CX3CR1 expression. These macrophage populations are both parenchymal in the thymus but are unequal in developmental ontogeny, Flt3 expression history, and CCR2 dependency. The manuscript further reports the interesting findings that the depletion of thymic macrophages impairs thymocyte development at the DN3 beta-selection checkpoint. These results provide an important advance for further understanding of thymus biology, especially in view of the contribution of heterogenous thymic macrophage subpopulations.

However, Zhou et al. previously reported essentially similar heterogeneity in thymic macrophages. It was demonstrated that TIMD4+ macrophages and CX3CR1+ macrophages have distinct origins and are different in developmental characteristics (27). The authors should better clarify what was previously demonstrated and what is newly described in this study. Zhou, et al. also demonstrated that TIMD4+ macrophages are localized in the cortex whereas CX3CR1+ macrophages distribute in the medullary region. Whether or not these previous findings are reproduced and supported in the present study is important in view of the new finding that thymic macrophages are important for beta-selection, which is presumed to occur in the thymic cortex. The authors may be able to suggest more strongly that TIMD4+ macrophages regulate beta-selection in the thymic cortex through phagocytic efferocytosis. (Indeed, the Figure 1 legend states that frozen thymic sections were used for immunofluorescent staining to identify the localization of thymic macrophages, without showing the results.)