Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium

Reviewer #1 (Public Review):

The work by Ohigashi and colleagues addresses the developmental and lineage relationship of a newly characterized thymus epithelial cell (TEC) progenitor subset. The authors take advantage of an elegant and powerful set of experimental approaches to demonstrate that CCL21-expressing TECs appear early in thymus organogenesis and that these cells, which are centrally located, go on to give rise to medullary (m)TECs. What makes the findings intriguing is that these CCL21-expressing mTECs are a distinct subset, which do not express RANK or AIRE, and transcriptomic and lineage tracing approaches point to these cells as potential mTEC progenitor-like cells. Of note, using in vitro and in vivo precursor-product cell transfer experiments, the authors show that this subset has a developmental potential to give rise to AIRE+ self-antigen-displaying mTECs, revealing that CCL21-expressing mTECs can give rise to distinct mTEC subsets. This functional duality provides an attractive rationale for the necessary function of mTECs, which is to attract CCR7+ thymocytes that have just undergone positive selection in the thymus cortex to enter the medulla to undergo tolerance-induction against self-antigen-displaying mTECs. Overall, the work is well supported and offers new insights into the diverse functions of the medullary compartment, and how two distinct subsets of mTECs can achieve it.

Reviewer #2 (Public Review):

Summary:

The authors set out to discover a developmental pathway leading to functionally diverse mTEC subsets. They show that Ccl21 is expressed early during thymus ontogeny in the medullary area. Fate-mapping gives evidence for the Ccl21 positive history of Aire positive mTECs as well as of thymic tuft cells and postnatally of a certain percentage of cTECs. Therefore, the differentiation potential of Ccl21+ TECs is tested in reaggregate thymus experiments - using embryonic or postnatal Ccl21+ TECs. From these experiments, the authors conclude that at least embryonic mTECs in large part pass through a Ccl21 positive stage prior to differentiation towards an Aire expressing or tuft cell stage.

The authors are using Ccl21a as a marker for a bipotent progenitor that is detectable in the embryonic thymus and is still present at the adult stage mainly giving rise to mTECs. The choice of this marker gene is very interesting since Ccl21 expression can directly be linked to an important aspect in thymus biology: the expression of Ccl21 by cells in the thymic medulla allows trafficking of T cells into the medulla in order to undergo T cell selection.

Making use of the Ccl21 detection, the authors can nicely show that cells actively expressing Ccl21 are localized throughout the medulla at an embryonic stage but also in adult thymus tissue. This suggests, that this progenitor is not accumulating at a specific area inside the medulla. This is a new finding.

Moreover, the finding that a Ccl21+ progenitor population plays a functional role in thymocyte trafficking towards the medulla has not been described. Thus, Ccl21 expression may be used to localize a late bipotent progenitor in the thymic lobes.
In addition, in Fig.8, the authors provide evidence that these progenitor cells have the potential to self-maintain as well as to differentiate in reaggregate experiments at E17 (not at 4 weeks of age). The first point is of great interest and importance since these cells in theory can be of therapeutic use.

Overall assessment:

The authors highlight a developmental pathway starting from a Ccl21-expressing TEC progenitor that contributes to a functionally diverse mTEC repertoire. This is a welcome addition to current knowledge of TEC differentiation.

Reviewer #3 (Public Review):

In this manuscript, the authors define the developmental trajectory resulting in a diverse mTEC compartment. Using a variety of approaches, including a novel CCL21-fate mapping model, data is presented to argue that embryonic CCL21-expressing thymocyte attracting mTECs naturally convert to into self-antigen displaying mTEC subsets, including Aire+ mTECs and thymic tuft cells. Perhaps somewhat surprisingly, a large fraction of cTECs were also marked for having expressed CCL21, suggesting that there exists some conversion of mTEC (progenitors) into cTEC, a developmentally interesting observation that could be followed up later. Overall, the experimental setup, writing, and conclusions, are all outstanding.

Author Response

We thank the editors and reviewers for their supportive comments onto our manuscript. We will revise the manuscript according to their helpful recommendations.