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  1. Author Response:

    Reviewer #1 (Public Review):

    The authors extend their previous work on thymus epithelial cells (TECs) and antigen presenting cells (APCs), which focused on TLR signaling in TECs and monocyte-derived dendritic cells (mDCs), and now focus on medullary (m) TECs and ask whether different subsets of DCs can uniquely serve as APCs for tissue-restricted antigens (TRAs) expressed by mTECs.

    The approach used makes use several reporter transgenic mouse models to conditionally express a fluorescent reporter gene in mTECs (Defa6-cre) or in TECs more broadly (Foxn1-cre or Csnb-cre). Their findings show that restricted expression of reporter genes in a subset of mTECs, which typically expressed AIRE-dependent TRAs, are typically presented by a subset of DCs that express XCR1 and CCR7, which, together with mDCs, are able to perform cooperative antigen transfer (CAT) effectively. The authors also examined the ability of different DC subsets to acquire antigens from other DCs, and show that mDCs excel in this ability.

    Overall, the work is clearly presented and makes use of several elegant mouse models to further delineate the role of different DC subsets in T cell selection. However, as pointed out by the authors, it remains to be determined whether the differences in the ability of different DC subsets to perform CAT has any fundamental impact in the establishing self-tolerance, either by negative selection or induction of Treg differentiation.

    We thank Reviewer #1 for this positive comment. We concur that describing a direct effect of the preferential pairing in CAT between TECs and DCs on the mechanisms of central immune tolerance would be very insightful and beneficial for the scientific community. However, a better understanding of the rules underpinning the interactions of this dynamic thymic cellular network in its complex and physiological outcomes will require the development of novel cellular tools and organismal genetic models, which is beyond the scope of this current work.

    Reviewer #3 (Public Review):

    In this manuscript, Voboril et al. address the question of whether specific dendritic cell (DC) types in the thymus acquire antigens from distinct thymic epithelial cell subsets. It is well-documented that both medullary thymic epithelial cells (mTECs) and thymic DCs present self-antigens to thymocytes to induce central tolerance. mTECs express the majority of the proteome, including AIRE-dependent tissue restricted antigens (TRAs), and thymocytes must be tolerized against this diverse set of self-antigens to prevent autoimmunity. While some self-antigens are expressed abundantly in an AIRE-independent manner by mTECs, a given AIRE-dependent TRA is expressed at low levels by only 1-3% of mTECs, raising the question of how thymocytes encounter rare, sparse self-antigens during their residence in the medulla. Part of the answer to this comes from the fact that thymic DCs can acquire self-antigens from mTECs to improve the efficiency of display to thymocytes. As the authors point out, several recent studies have utilized single-cell transcriptional profiling to identify multiple distinct medullary thymic epithelial cell subsets. Furthermore, work from the authors' lab and others has demonstrated heterogeneity within the thymic DC compartment. Thus, the authors set out to address whether DC interactions with mTECs are promiscuous or whether specific DC cell types interact with different mTEC subsets to acquire self-antigens to induce tolerance to different types of self-antigens, such as Aire-dependent versus Aire-independent TRAs or ubiquitous antigens.

    Using mouse strains that express a fluorescent protein in TECs under the control of different promoters, the authors use flow cytometry to determine the relative ability of thymic DC subsets to acquire self-antigens, TdTomato in this case, from TEC subsets. Using linear regression modeling to compare the frequency of TEC subsets expressing TdTomato in the different reporter strains to the frequency of DC subsets that acquired TdTomato, the authors conclude that there is specificity to the interactions of different DC subsets with distinct mTECs, resulting in antigen acquisition by the interacting DCs. Specifically, they conclude that pDCs and macrophages acquire antigens from mTEClow cells, cDC1 and activated XCR1+ DCs acquire antigen from mTEChigh cells, activated XCR1+ and activated XCR1- DCs acquire antigen from pre-post-Aire mTECs, cDC2 exclusively acquire antigen from Post-Aire mTECs, and activated XCR1+ DCs acquire antigen from Tuft cells. It is well documented that activated XCR1+ DCs (Ardouin et al. 2016, Oh et al. 2018, Perry et al. 2018) and activated XCR1- DCs (Leventhal 2016) acquire and present Aire-dependent self-antigens from mTECs to induce thymic central tolerance. Thus, the most novel claim is that cDC2 acquire antigens from Post-Aire mTECs. However, given that the model is derived from the linear regression analysis of fluorescent reporter mice in which multiple TEC subsets express each reporter, and there are some known caveats to these analyses, this interesting conclusion is not adequately supported by the data. The authors cleverly use Foxn1-cre ConfettiBrainbow2.1 reporters to conclude that moDCs are particularly adept at acquiring antigen serially from different mTECs. Furthermore, they use mixed bone marrow congenic/reporter mice to demonstrate that moDC are particularly good at acquiring antigens from other DC subsets. These two conclusions are well-supported by the data, although it is notable that while moDC are efficient at these two processes, other DC subsets acquire antigens from DCs, and fluorescent reporter acquisition does not indicate the ability to process and present antigens to developing T cells to promote central tolerance, somewhat reducing the impact of the findings. Altogether, this is a promising study that cleverly uses a variety of mouse models with flow cytometric analysis of recently identified TEC and DC subsets to delve into whether specificity of interactions between TEC and DC subsets could enable distinct DC subsets to contribute differentially to central tolerance induction against distinct types of self-antigens. However, the conclusions could be strengthened by additional analyses.

    We thank the Reviewer for the many comments and suggestions. We are well aware of the fact that our model is based on the linear regression analysis that brings about some known caveats, which have been now newly described in the Discussion section. As suggested by the Reviewer, to better resolve some discrepancies and strengthen our conclusions, we conducted a novel analysis of our data and made some vital changes to the manuscript.

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  2. Evaluation Summary:

    This manuscript will be of interest to immunologists studying mechanisms of thymic central tolerance. The study elegantly makes use of multiple genetic mouse models to generate data supporting the conclusion that different dendritic cell subsets in the thymus capture self-antigens from distinct subsets of thymic epithelial cells. However, some key conclusions are not entirely novel, and the final model, as currently presented, draws from only selective analyses and thus may not accurately reflect the antigen transfer between thymic epithelial cell and dendritic cell subsets that promote central tolerance.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors extend their previous work on thymus epithelial cells (TECs) and antigen presenting cells (APCs), which focused on TLR signaling in TECs and monocyte-derived dendritic cells (mDCs), and now focus on medullary (m) TECs and ask whether different subsets of DCs can uniquely serve as APCs for tissue-restricted antigens (TRAs) expressed by mTECs.

    The approach used makes use several reporter transgenic mouse models to conditionally express a fluorescent reporter gene in mTECs (Defa6-cre) or in TECs more broadly (Foxn1-cre or Csnb-cre). Their findings show that restricted expression of reporter genes in a subset of mTECs, which typically expressed AIRE-dependent TRAs, are typically presented by a subset of DCs that express XCR1 and CCR7, which, together with mDCs, are able to perform cooperative antigen transfer (CAT) effectively. The authors also examined the ability of different DC subsets to acquire antigens from other DCs, and show that mDCs excel in this ability.

    Overall, the work is clearly presented and makes use of several elegant mouse models to further delineate the role of different DC subsets in T cell selection. However, as pointed out by the authors, it remains to be determined whether the differences in the ability of different DC subsets to perform CAT has any fundamental impact in the establishing self-tolerance, either by negative selection or induction of Treg differentiation.

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  4. Reviewer #2 (Public Review):

    Medullary thymic epithelial cells (mTEC) are a heterogenous population of cells that are best known for their expression of genes that are typically limited to certain tissues. This promiscuous gene expression allows developing T cells to test their antigen receptors against the majority of self-peptides they may encounter when patrolling the peripheral organs to ensure those that may be autoreactive to self-antigens are rendered tolerant before they exit the thymus. The expression of any given tissue restricted antigen by mTEC is rare, and an important role for thymic dendritic cells in taking up and presenting antigen from mTEC has been suggested to broaden the presentation of these rare antigens. Further, it has previously been shown that thymic dendritic cells are also diverse and that the subsets are differentially cross-dressed with self-peptide:MHC complexes from mTECs, In addition, it has been suggested that certain dendritic cell populations are more efficient at cross-presenting secreted versus membrane bound antigen. Here, the authors expand upon this previous work and put forth an interesting hypothesis that there is preferential antigen exchange between specific mTEC and dendritic cell subsets.

    To test this hypothesis, the authors employ a number of mouse models in which a fluorescent protein is differentially expressed among mTEC subsets. They demonstrate, as expected, that mTEC expressed fluorescent protein can be taken up by thymic dendritic cells. Perhaps unexpectedly, their data are suggestive of biases in the interactions between different dendritic cell and mTEC subsets that lead to the biased exchange of antigen. The authors go on to suggest differences in the efficiency with which individual dendritic cells from different subsets take up antigen from multiple mTECs using a genetic cell-labeling technique to direct distinct fluorescent protein expression among individual mTECs. Lastly, the authors suggest that exchanges in dendritic cell antigen also occur using dendritic cell-directed fluorescent protein expression and bone marrow chimeras. Important controls are provided, and complementary assays are included that strengthen confidence in the authors observations.

    One limitation of the authors' approach is the lack of mTEC subset restricted fluorescent reporters; in addition, several of the conclusions in the manuscript are therefore reliant on linear regression analysis to identify correlations in mTEC subset expression of fluorescent protein and dendritic cell subsets that preferentially take up the fluorescent antigen. The authors acknowledge some caveats to this approach. Regardless, the results are consistent with the idea that dendritic cell subsets are specialized to take up and present antigen from certain mTEC populations and set the stage for further testing of this hypothesis as well as the physiological impact of this mTEC-dendritic cell interaction bias awaits the development of additional tools.

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  5. Reviewer #3 (Public Review):

    In this manuscript, Voboril et al. address the question of whether specific dendritic cell (DC) types in the thymus acquire antigens from distinct thymic epithelial cell subsets. It is well-documented that both medullary thymic epithelial cells (mTECs) and thymic DCs present self-antigens to thymocytes to induce central tolerance. mTECs express the majority of the proteome, including AIRE-dependent tissue restricted antigens (TRAs), and thymocytes must be tolerized against this diverse set of self-antigens to prevent autoimmunity. While some self-antigens are expressed abundantly in an AIRE-independent manner by mTECs, a given AIRE-dependent TRA is expressed at low levels by only 1-3% of mTECs, raising the question of how thymocytes encounter rare, sparse self-antigens during their residence in the medulla. Part of the answer to this comes from the fact that thymic DCs can acquire self-antigens from mTECs to improve the efficiency of display to thymocytes. As the authors point out, several recent studies have utilized single-cell transcriptional profiling to identify multiple distinct medullary thymic epithelial cell subsets. Furthermore, work from the authors' lab and others has demonstrated heterogeneity within the thymic DC compartment. Thus, the authors set out to address whether DC interactions with mTECs are promiscuous or whether specific DC cell types interact with different mTEC subsets to acquire self-antigens to induce tolerance to different types of self-antigens, such as Aire-dependent versus Aire-independent TRAs or ubiquitous antigens.

    Using mouse strains that express a fluorescent protein in TECs under the control of different promoters, the authors use flow cytometry to determine the relative ability of thymic DC subsets to acquire self-antigens, TdTomato in this case, from TEC subsets. Using linear regression modeling to compare the frequency of TEC subsets expressing TdTomato in the different reporter strains to the frequency of DC subsets that acquired TdTomato, the authors conclude that there is specificity to the interactions of different DC subsets with distinct mTECs, resulting in antigen acquisition by the interacting DCs. Specifically, they conclude that pDCs and macrophages acquire antigens from mTEClow cells, cDC1 and activated XCR1+ DCs acquire antigen from mTEChigh cells, activated XCR1+ and activated XCR1- DCs acquire antigen from pre-post-Aire mTECs, cDC2 exclusively acquire antigen from Post-Aire mTECs, and activated XCR1+ DCs acquire antigen from Tuft cells. It is well documented that activated XCR1+ DCs (Ardouin et al. 2016, Oh et al. 2018, Perry et al. 2018) and activated XCR1- DCs (Leventhal 2016) acquire and present Aire-dependent self-antigens from mTECs to induce thymic central tolerance. Thus, the most novel claim is that cDC2 acquire antigens from Post-Aire mTECs. However, given that the model is derived from the linear regression analysis of fluorescent reporter mice in which multiple TEC subsets express each reporter, and there are some known caveats to these analyses, this interesting conclusion is not adequately supported by the data. The authors cleverly use Foxn1-cre ConfettiBrainbow2.1 reporters to conclude that moDCs are particularly adept at acquiring antigen serially from different mTECs. Furthermore, they use mixed bone marrow congenic/reporter mice to demonstrate that moDC are particularly good at acquiring antigens from other DC subsets. These two conclusions are well-supported by the data, although it is notable that while moDC are efficient at these two processes, other DC subsets acquire antigens from DCs, and fluorescent reporter acquisition does not indicate the ability to process and present antigens to developing T cells to promote central tolerance, somewhat reducing the impact of the findings. Altogether, this is a promising study that cleverly uses a variety of mouse models with flow cytometric analysis of recently identified TEC and DC subsets to delve into whether specificity of interactions between TEC and DC subsets could enable distinct DC subsets to contribute differentially to central tolerance induction against distinct types of self-antigens. However, the conclusions could be strengthened by additional analyses.

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